PTH regulates bone marrow progenitor fate Nature.com New research published in Cell Metabolism reveals an important mechanism underlying the anabolic effects of parathyroid hormone (PTH) on bone. Mice with conditional deletion of the gene encoding the PTH 1 receptor (PTH1R) in bone marrow progenitors ...

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Back to Browse Journals Journal of Neurorestoratology Volume 5

Zhijian Cheng, Xijing He

Department of Orthopedics, The Second Affiliated Hospital of Xian Jiaotong University, Xian, Shaanxi, Peoples Republic of China

Abstract: Spinal cord injury (SCI) is a traumatic event that involves not just an acute physical injury but also inflammation-driven secondary injury. Macrophages play a very important role in secondary injury. The effects of macrophages on tissue damage and repair after SCI are related to macrophage polarization. Stem cell transplantation has been studied as a promising treatment for SCI. Recently, increasing evidence shows that stem cells, including mesenchymal stem, neural stem/progenitor, and embryonic stem cells, have an anti-inflammatory capacity and promote functional recovery after SCI by inducing macrophages M1/M2 phenotype transformation. In this review, we will discuss the role of stem cells on macrophage polarization and its role in stem cell-based therapies for SCI.

Keywords: stem cells, macrophages, spinal cord injury, polarization

This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution - Non Commercial (unported, v3.0) License. By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms.

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Ten-year-old Boon Kye Feng prances around the living room in furry purple pants that match the lion's head he is wearing.

He lifts the head and moves it from side to side to a beat only he can hear.

Even when the little lion gets thirsty, he drinks water through the opening in the head.

Seeing him at play, it may be difficult for strangers to tell that he has spent almost half his life battling leukaemia.

His family fought it along with him, gifting two transplants - cord blood from his baby sister and stem cells from his mother - to keep him alive.

MIRACLE BOY

I believe Kye Feng is a 'miracle'. We have all learnt a lot from him, not only in the science of managing the disease and the doctor-patient relationship, but also in his love of life, and his fearlessness and resilience, despite the years of pain and suffering.

ASSOCIATE PROFESSOR TAN POH LIN, from the paediatric haematology- oncology division of NUH.

Despite the intensive treatment, his parents said he had remained positive and playful.

It had started in late 2011 when Kye Feng developed spots and bruises which his parents thought were sandfly bites.

When the spots appeared a second time, his mother, Mrs Celine Boon, decided to take him for a check-up.

Doctors found that his white blood cell count was very high and told the family he could have leukaemia (cancer of the blood).

It was diagnosed as juvenile myelomonocytic leukaemia (JMML), a rare form of the disease.

But Mrs Boon, 38, was not too surprised.

This was because Kye Feng and his twin brother, Kye Teck, had previously developed juvenile xanthogranuloma (JXG), a skin disorder that is usually benign and self-limiting.

They also have an older sister, now 16, who was unaffected.

While reading up on JXG earlier, Mrs Boon had come across a potential link to JMML.

She said: "Still, I had never expected that it would happen to my son. I was quite alarmed."

JMML is so rare that blood samples had to be sent to Germany to confirm the diagnosis.

Kye Feng began chemotherapy at KK Women's and Children's Hospital (KKH) in 2012 to control the condition while waiting for a bone marrow transplant.

Although KKH doctors had not seen a JMML case in about 10 years, they did the transplant as there were few other options.

His father, Mr Roy Boon, 46, said: "It was all trial and error. There's no exact treatment for JMML."

Mrs Boon was then pregnant with their fourth child and doctors said the baby girl's cord blood could be used for the transplant as there is a 25 per cent chance of a match between siblings.

Juvenile myelomonocytic leukaemia (JMML) is a very rare form of childhood leukaemia. The hallmark symptom of the disease is the increased number of white blood cells known as monocytes.

Normal monocytes protect the body from infections, but those in patients with this leukaemia are cancerous and reproduce uncontrollably. The monocytes may then infiltrate organs such as the liver, spleen, lungs, lymph nodes and even skin.

In Western countries, one in a million children are afflicted with the disease each year. Based on Singapore population statistics last year, there is an average of one case every three years.

For the majority of JMML patients, a haematopoietic - or blood forming - stem cell transplant (HSCT) is the only curative option.

Stem cells are cells that have the potential for self-renewal and differentiation. They can develop into different forms, including white blood cells, red blood cells and platelets. Such a transplant can help patients develop new and healthy blood cells.

Stem cells can be found in the bone marrow, blood, fat tissue and placenta. They are abundant in the bone marrow but, even so, make up only 1 per cent of all cells there.

They can be "harvested" directly from the bone marrow or from the blood, whether they are from an adult volunteer or from umbilical cord blood.

The bone marrow must be stimulated to coax or force the stem cells into the peripheral blood system, but techniques are well-tested and safe.

After undergoing HSCT, 50 per cent of the patients will go on to become long-term survivors.

Abigail Ng

Source: Associate Professor Tan Poh Lin, senior consultant at the division of paediatric haematology-oncology, National University Hospital.

Thankfully, it was a full match for Kye Feng, who had the transplant and recovered well.

He looked forward to starting Primary 1 with his brother.

But before the March holidays of his first year in school, doctors noticed that the percentage of donor cells in him was beginning to fall, signalling that there could be a problem.

When it became clear that the cancer had returned, Mrs Boon said she broke down and cried.

"I was shocked. There weren't any physical symptoms. Why did it happen so quickly? It wasn't even one year after the transplant and things had looked so promising," she said.

A SECOND CHANCE

The family sought a second opinion from the National University Hospital (NUH) and entered into the care of Associate Professor Tan Poh Lin from the paediatric haematology-oncology division.

While doctors from both hospitals suggested a second transplant for Kye Feng, there was more bad news.

His illness was mutating into mixed-phenotype acute leukaemia, a combination of two forms of cancer.

He also faced a life-threatening infection that caused high fever and bloating.

Besides beginning palliative care to improve his quality of life, the family continued to push for treatment, including natural killer-cell therapy and the removal of Kye Feng's enlarged spleen in a complicated seven-hour operation.

Even though the test results showed that leukaemic cells remained in his bone marrow, Kye Feng had a second transplant in September 2015, this time using stem cells from his mother.

Doctors usually recommend transplants only when patients register no leukaemic cells.

Mrs Boon said: "If he didn't have the transplant, he would have only six months more. With the transplant, he would at least have a chance of recovery.

"He was fighting hard. If I didn't give him the chance, I would never know if he could have survived."

Kye Feng responded well to his mother's stem cells.

Dr Tan said: "I believe Kye Feng is a 'miracle'. We have all learnt a lot from him, not only in the science of managing the disease and the doctor-patient relationship, but also in his love of life, and his fearlessness and resilience, despite the years of pain and suffering."

The crucial three months after the transplant passed by without issue, but the boy developed a graft versus host disease (GVHD) one year later.

Still, his parents were relieved that it was not a second relapse.

He was put on medication for GVHD and will recover completely.

In the meantime, the family is treasuring the time they can spend together.

Mrs Boon said: "We will relax and go with the flow, as long as Kye Feng is happy."

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Genetic profiling can guide stem cell transplantation for patients with ... Science Daily A single blood test and basic information about a patient's medical status can indicate which patients with myelodysplastic syndrome (MDS) are likely to benefit ... and more »

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Ally Little described the past month of her life as a nightmare from which she just cant wake up.

On Dec. 22, the University of Southern Maine soccer player learned she had a rare and life-threatening disease in which her bone marrow stops producing healthy blood cells. However, the words severe aplastic anemia meant nothing to Little at the time.

Its really hard because I didnt know what this was before I had it, said Little, a 20-year-old sophomore from Stoneham, Massachusetts. No one has really heard of aplastic anemia or what the treatment is.

A bone marrow transplant is the cure for this disease, and Little has yet to find a matching donor. Littles teammates have organized a bone marrow donor registry drive from 9 a.m. to 1 p.m. Wednesday at Abromson Mezzanine at the USM Portland campus and from 2:30 to 5:30 p.m. at Costello Complex at the Gorham campus.

Diagnosed during winter break, Little broke the news to her teammates on social media.

It hit home, said USM womens soccer coach Lisa Petruccelli. This is really the first time someone their age at this juncture is struggling with something like this.

Littles initial symptoms didnt seem serious. She started getting pounding headaches around Thanksgiving, but she had gotten headaches before. Physical activities such as skiing or working out for soccer became unusually exhausting, which Little attributed to dehydration. She didnt go to her doctor until she noticed blood in her stool.

(Aplastic anemia) is believed to be an autoimmune system gone wrong, said Paul Scribner, Senior Director of Patient Advocacy Programs with the Aplastic Anemia and MDS International Foundation (AAMDS). The disease usually results from the destruction of bone marrow stem cells by the immune system. Other symptoms include infections and the tendency to bruise and bleed easily. With such innocuous warning signs, Scribner said a lot of people find out when they go to their doctor because theyre feeling run down.

After bloodwork, Little was told that her results were very abnormal. She spent the next few days in the hospital undergoing tests while doctors prepared her for the worst case scenario leukemia.

That was obviously horrifying, Little said. We didnt find out until about three days later that it was severe aplastic anemia.

Aplastic anemia is rare and can occur at any age. In the United States, about 600 to 900 people are diagnosed each year, according to AAMDS. The disease is considered severe when all three types of blood cells red blood cells (carry oxygen), white blood cells (fight infections) and platelets (help blood to clot) are very low in number.

I was kind of relieved it wasnt cancer, Little said. Then, doctors explained to me that its really not that good. It was devastating.

Little couldnt go back to school. With her compromised immune system, crowds are off limits. She cant play contact sports or do anything that could put her at risk of internal bleeding. She gets blood transfusions every week, and she can tell when shes due for another by the dizziness and headaches she gets. The long-term risk of too many transfusions, Scribner said, is iron overload.

Most days, I feel OK, Little said. I dont really feel sick, which is good. But its hard to remember I cant do certain things.

Little is buying time until she can get a bone marrow transplant. Bone marrow is the spongy tissue inside of the bones that produces the bodys blood cells. She didnt find a match among her family or with Be The Match a national bone marrow registry that contains 22.5 million adult donors.

Registering at the drive is simple. Potential donors must be between 18-44 years old and fill out basic paperwork and get their cheek swabbed to have their tissue type added to the registry a process that takes just a few minutes. After that, they will remain registered until age 61, unless they withdraw.

However for those in need of bone marrow finding a perfect match is not so easy.

Think about Megabucks and how hard it is to match that, said Jackie McLoon, Assistant Account Executive with Rhode Island Blood Center as well as a bone marrow donor. McLoon, a representative with Be the Match, has helped the USM soccer team organize its drive. Everyday, there are donors getting added to the database. Hopefully, her match shows up one of these days.

Only 30 percent of patients in need of a marrow transplant have a matching donor in their family. Be The Match helps the 14,000 patients a year who suffer from leukemia, lymphoma or a variety of bone marrow functioning diseases. McLoon said a protein called human leukocyte antigen (HLA) is used to match patients with donors, and potential matches will then undergo bloodwork to determine if they would be a good fit. Only about 1 in 500 registrants go on to actually donate marrow.

There are 10 things that they are supposed to match, Little said. They think one of my 10 is very rare.

But her teammates are optimistic. On Saturday, they attended a home basketball game clad in T-shirts adorned with the phrase: All for Ally. They gushed about Littles kind personality and reminisced about all the times they crashed in her room.

Shes the best teammate ever. Shes so sweet oh my god, I love her, said Jessica Preble, a sophomore on the team. If you ask anything of her, shell drop everything and do it.

This team is kind of used to bad things happening to our girls, said Dayna Staffiere, noting that one of their teammates lost her dad at sea last season when the cargo ship El Faro sank after encountering Hurricane Joaquin. It just brings us all closer.

When Little isnt at the hospital, shes usually working on her online classes or walking her dog. She said the support from family, friends and her soccer team is what keeps her going.

Were the ones who are supposed to be strong for her, but shes so strong for us, Preble said. Just a cheek swab and some paperwork could help save her life.

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Bone Marrow Stem Cells Comments Off on USM women’s soccer players organize bone marrow drive for teammate with rare disease – Press Herald | February 8th, 2017

Stem cells are often in the news. These days its usually about some advance in research. Sometimes the controversy about using embryonic stem cells resurfaces. Despite all the coverage (pro or con) stem cells are not well understood. What are they and why are they important?

In more ways than one, its the potential of stem cells that makes them important. At the moment most of the work with stem cells is still in the laboratory; but thats changing. Within the next few years stem cells, in one form or another, will be at work in medical applications such as repairing a damaged pancreas or a heart. In fact, stem cells will be used to repair or even re-grow tissues all over the body skin, liver, lungs, bone marrow. The production of stem cells, their delivery, and procedures for using them will become the basis of an industry. In the not too distant future stem cells, or the knowledge we gain from working with them, will be used in sophisticated repair of the brain and as part of the development of replacement organs. The potential is enormous.

What are stem cells?

Stem cells are found in most multicellular creatures and come in different varieties; all have an important ability: They can fully reproduce themselves almost indefinitely. For example, in mammals like human beings, blood stem cells (hematopoietic stem cells) are active all our lives in the marrow of bones, where they continually produce the many different kinds of blood cells. Therein is another key property for most stem cells; they can become other kinds of cells. The word for this process is differentiate; blood stem cells can differentiate into red blood cells, white blood cells, blood platelets and so forth. The ability to produce different kinds of cells is why stem cells may be used, for example, to repair or replace damaged heart cells something mature heart cells cannot do on their own.

Stem cell jargon

When you read about stem cells, there are a number of words that jump out jargon, yes, but still descriptive. Stem cells are classified by their potency, that is, what other kinds of cells they can become, or put another way, their ability to differentiate into other cells. There is a rank order from more to less potent:

Totipotent sometimes also called omnipotent stem cells can construct a complete and viable organism. In short, they are the same as a cell created by the fusion of the egg and a sperm (an embryonic cell). Totipotent cells can become any type of cell.

Pluripotent stem cells are derived from totipotent cells and are nearly as versatile. They can become any type of cell, except embryonic.

Multipotent stem cells can become a wide variety of cells, but only those of a close family, for example blood stem cells (hematopoietic cells) can become any of the blood cells, but not other kinds of cells.

Oligopotent stem cells are limited to becoming specific types of cells, such as endoderm, ectoderm, and mesoderm.

Unipotent stem cells can only produce cells of their own type, for example skin cells. They can renew themselves (replicate indefinitely), which distinguishes them from non-stem cells.

To a certain extent the potency of a stem cell relates to its usefulness. In one view of an ideal (lab) world, only totipotent stem cells would be used because they can become any other kind of cell. The real world (lab or otherwise) doesnt work that way. For one thing, stem cells of lesser versatility than totipotent cells are valuable for use in specific applications. Even unipotent stem cells, lowest on the potency poll, are arguably better suited for some targeted uses than more generic stem cells. Most importantly, for many uses, especially for medical purposes, pluripotent stem cells are extremely versatile and less controversial.

Avoiding embryonic stem cells

The true totipotent stem cell is a fertilized egg one embryonic cell. To obtain it means detecting and collecting the cell shortly after fertilization and before it begins to divide. Collecting embryonic stem cells one at a time is very difficult and very expensive. Also, in some parts of the world, using embryonic stem cells is highly controversial, usually on religious grounds. Collecting embryonic stem cells can be considered abortion, since the procedure means the cell(s) will not become an embryo. The label abortion is also applied to collecting embryonic stem cells (by gastrulation) shortly after the first fertilized cell begins to divide. These cells, obviously more numerous, are pluripotent and have been the mainstay of stem cell research.

The history of opposition to the use of embryonic stem cells goes back to the 1990s, when stem cell research was in its own infancy. At that time the only source of viable laboratory stem cells was from in vitro living donors. Most of these were harvested from fertilization clinics. They were so difficult to acquire that only a few stem cell lines (painstakingly cultivated generations of embryonic stem cells) were available. Even those were controversial. The United States banned the taking of embryonic stem cells except for 23 grandfathered lines. (This ban was lifted in 2009.)

The controversy over embryonic stem cells can be avoided primarily in two ways. One way is to use adult stem cells. The word adult is a bit misleading since the cells may be derived from fetuses, newborns, and children, which is why theyre sometimes called somatic stem cells. It means that these stem cells come from relatively mature tissue, cells that are already differentiated to a certain degree. Thats why adult stem cells are almost always classified as multipotent, oligopotent, or unipotent. The other way is to transform adult stem cells into pluripotent stem cells. Many approaches to this transformation are being explored in labs all over the world. Some approaches are derived from fetal/newborn substances such as amniotic fluid and placental or umbilical tissue. Other approaches use mature (differentiated) stem cells, such as those from skin, and genetically modify them until they become pluripotent. Such cells are called induced pluripotent stem cells, often abbreviated as iPSC.

At the moment, it is not possible to say which approaches to stem cell production and application will be the most effective. Even some that seem unlikely (stem cells from skin cells?) may turn out to be the most economical and useful. Still, this is where the payoff for stem cell research lies both in terms of scientific knowledge and in profits for medical applications. Consequently the amount of research work in progress is substantial, and often competitive.

Stem Cell Tourism

Because experimental medical techniques and human desperation can add up to big money, there is a developing market for stem cell applications for a variety of medical disorders. Unfortunately, at least for now, with the exception of blood cell transplants and skin cell treatments, most of these applications are either fraudulent or based on shaky experimental results. In general, most stem cell treatments are at best unethical and often illegal; however, their status around the world is a patchwork quilt of laws and regulations (or their absence). It is a near ideal situation for scam artists to lure desperate people into traveling long distances for stem cell treatment that is illegal in their own country. Hence the name: stem cell tourism.

Tracking the Impact of Stem Cell Research

In relative terms, stem cell research is just getting started. Researchers have been at it since the 1950s; but one of the most important discoveries so far induced pluripotent stem cells dates back to only 2006. This means that stem cells are: a. Not yet well understood and b. Their use in medicine is largely experimental and tentative. Heres a useful listing of what the National Institute of Health (U.S. NIH) considers some of the major open questions about adult stem cells:

How many kinds of adult stem cells exist, and in which tissues do they exist? How do adult stem cells evolve during development and how are they maintained in the adult? Are they leftover embryonic stem cells, or do they arise in some other way? Why do stem cells remain in an undifferentiated state when all the cells around them have differentiated? What are the characteristics of their niche that controls their behavior? Do adult stem cells have the capacity to transdifferentiate, and is it possible to control this process to improve its reliability and efficiency? If the beneficial effect of adult stem cell transplantation is a trophic effect, what are the mechanisms? Is donor cell-recipient cell contact required, secretion of factors by the donor cell, or both? What are the factors that control adult stem cell proliferation and differentiation? What are the factors that stimulate stem cells to relocate to sites of injury or damage, and how can this process be enhanced for better healing? [Source: U.S. National Institute of Health]

SciTechStory Impact Area: Stem Cells

Theres not much debate on the importance of stem cell research. It has already had major impact on our understanding of cell biology, and it will provide more. It is just beginning to have an impact on medicine, with much more to come. In fact, news about stem cell research already occurs once or twice a week (on average) that pace is likely to increase. As a matter of keeping up, its necessary to attempt sorting lab work from practical application, which is to say sorting promise from delivery. Even at that it will be difficult to select which stem cell stories are significant.

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Louisville, Ky. man saves stranger's life

Julia Rose, WHAS 1:17 PM. EST February 07, 2017

Eric Gurevich and Ron Dreben

LOUISVILLE (WHAS11) -- It's hard to believe that up until a few months ago, Eric Gurevich and Ron Dreben were total strangers.

I got to see him for the first time and I just gave him this big bear hug and he was crying and his wife was there and his 81-year-old mother was there, Gurevich said.

That bear hug was years in the making but the two were bonded long before as blood brothers.

Because he received those, he was kind of given a new lease on life, Gurevich said.

Gurevich lives in Louisville. He donated his stem cells to Dreben who lives in Washington D.C. in 2014, one, quick decision that changed the lives of two people. Gurevich remembers the call from the organization Gift of Life like it was yesterday.

They said that there was a 54-year-old man with MDS and his life was dire and I am the only potential match, Gurevich said.

Without hesitation, he hopped on a plane from Louisville to D.C. and a week later started the donation process. Despite some concerns from family members who weren't totally sold on the idea of him undergoing the major medical procedure for a man he didn't even know, Gurevich says his decision was a no-brainer.

You're a little kid and you dream of being a superhero or helping someone or saving someone's life and you get this call. You get an opportunity. How could you not? Gurevich said.

He says donating 1.5 billion stem cells is painless, just like the cheek swab he did back in 2008, the reason he was even a possible match for Dreben.

Didn't think anything of it, got my cheek swabbed and then forgot about it pretty much the next day, Gurevich said.

That cheek swab was taken on his Birthright trip to Israel, a once in a lifetime opportunity that he never expected to lead to another once in a life time journey.

Gurevichs stem cell donation saved Dreben's life and for a full year after the transplant, they wondered about one another. By law, they weren't allowed to know each other's identities but that didn't stop them from exchanging cards and small gifts.

Eric Gurevich donated his stem cells to Dreben who lives in Washington D.C. in 2014, one, quick decision that changed the lives of two people.

He sent me a magnet that says 'life is a journey not a destination' and I have it right on my fridge and I think about him just about every day, Gurevich said.

Finally, after more than a year, the pair met face to face in Miami in November, a moment captured in a picture and forever captured in their hearts.

When you donate to a stranger you always wonder, you know who is on the other side and I was just so grateful that it was him, Gurevich said.

Gurevich says he and Dreben text each other often, sending pictures of their families back and forth and they plan to meet up again in the future.

If you're interested in becoming a potential stem cell or bone marrow donor with Gift of Life, you can find more information here: http://www.giftoflife.org.

There are also two local organizations dedicated to stem cell research and transplants: sharingamericasmarrow.com & nationalstemcellfoundation.org.

( 2017 WHAS)

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The 2017 Australian of the Year award went to Professor Alan Mackay-Sim for his significant career in stem cell science.

The prize was linked to barbeque-stopping headlines equating his achievements to the scientific equivalent of the moon landing and paving the road to recovery for people with spinal cord injuries.

Such claims in the media imply that there is now a scientifically proven stem cell treatment for spinal cord injury. This is not the case.

For now, any clinic or headline claiming miracle cures should be viewed with caution, as they are likely to be trading on peoples hope.

Put simply, injury to the spinal cord causes damage to the nerve cells that transmit information between the brain and the rest of the body.

Depending on which part of the spine is involved, the injury can affect the nerves that control the muscles in our legs and arms; those that control bowel and bladder function and how we regulate body temperature and blood pressure; and those that carry the sensation of being touched. This occurs in part because injury and subsequent scarring affect not just the nerves but also the insulation that surrounds and protects them. The insulation the myelin sheath is damaged and the body cannot usually completely replace or regenerate this covering.

Stem cells can self-reproduce and grow into hundreds of different cell types, including nerves and the cells that make myelin. So the blue-sky vision is that stem cells could restore some nerve function by replacing missing or faulty cells, or prevent further damage caused by scarring.

Studies in animals have applied stem cells derived from sources including brain tissue, the lining of the nasal cavity, tooth pulp, and embryos (known as embryonic stem cells).

Dramatic improvements have been shown on some occasions, such as rats and mice regaining bladder control or the ability to walk after injury. While striking, such improvement often represents only a partial recovery. It holds significant promise, but is not direct evidence that such an approach will work in people, particularly those with more complex injuries.

The translation of findings from basic laboratory stem cell research to effective and safe treatments in the clinic involves many steps and challenges. It needs a firm scientific basis from animal studies and then careful evaluation in humans.

Many clinical studies examining stem cells for spinal repair are currently underway. The approaches fit broadly into two categories:

using stem cells as a source of cells to replace those damaged as a result of injury

applying cells to act on the bodys own cells to accelerate repair or prevent further damage.

One study that has attracted significant interest involves the injection of myelin-producing cells made from human embryonic stem cells. Researchers hoped that these cells, once injected into the spinal cord, would mature and form a new coating on the nerve cells, restoring the ability of signals to cross the spinal cord injury site. Preliminary results seem to show that the cells are safe; studies are ongoing.

Other clinical trials use cells from patients own bone marrow or adipose tissue (fat), or from donated cord blood or nerves from fetal tissue. The scientific rationale is based on the possibility that when transplanted into the injured spinal cord, these cells may provide surrounding tissue with protective factors which help to re-establish some of the connections important for the network of nerves that carry information around the body.

The field as it stands combines years of research, and tens of millions of dollars of investment. However, the development of stem cell therapies for spinal cord injury remains a long way from translating laboratory promise into proven and effective bedside treatments.

Each case is unique in people with spinal cord injury: the level of paralysis, and loss of sensation and function relate to the type of injury and its location. Injuries as a result of stab wounds or infection may result in different outcomes from those incurred as a result of trauma from a car accident or serious fall. The previous health of those injured, the care received at the time of injury, and the type of rehabilitation they access can all impact on subsequent health and mobility.

Such variability means caution needs to accompany claims of man walking again particularly when reports relate to a single individual.

In the case that was linked to the Australian of the Year award, the actual 2013 study focused on whether it was safe to take the patients own nerves and other cells from the nose and place these into the damaged region of the spine. While the researchers themselves recommended caution in interpreting the results, accompanying media reports focused on the outcome from just one of the six participants.

While the outcome was significant for the gentleman involved, we simply do not know whether recovery may have occurred for this individual even without stem cells, given the type of injury (stab wounds), the level of injury, the accompanying rehabilitation that he received or a combination of these factors. It cannot be assumed a similar outcome would be the case for all people with spinal injury.

Finding a way to alleviate the suffering of those with spinal cord injury, and many other conditions, drives the work of thousands of researchers and doctors around the globe. But stem cells are not a silver bullet and should not be immune from careful evaluation in clinical trials.

Failure to proceed with caution could actually cause harm. For example, a paraplegic woman who was also treated with nasal stem cells showed no clinical improvement, and developed a large mucus-secreting tumour in her spine. This case highlights the need for further refinement and assessment in properly conducted clinical trials before nasal stem cells can become part of mainstream medicine.

Its also worth noting that for spinal cord injury, trials for recovery of function are not limited to the use of stem cells but include approaches focused on promoting health of surviving nerves (neuroprotection), surgery following injury, nerve transfers, electrical stimulation, external physical supports known as exoskeletons, nanotechnology and brain-machine interfaces.

Ultimately, determining which of these approaches will improve the lives of people with spinal injury can only be done through rigorous, ethical research.

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Yes there's hope, but treating spinal injuries with stem cells is not a reality yet - The Conversation AU

Bone Marrow Stem Cells Comments Off on Yes there’s hope, but treating spinal injuries with stem cells is not a reality yet – The Conversation AU | February 7th, 2017

By filling out a form, and swabbing his mouth, Harlee O'Watch could save a life.

"To find a match, because the list of donors is so low, is really unlikely," said the 22-year-old.

O'Watch is one of four young adults from Carry the Kettle First Nation who registered with the OneMatch program, which connects donors with people in need of bone marrow or stem cell transplants.

A problem for the 14 indigenous people currently waiting for a match is that, out of the 17,000 people on the Canadian registry, fewer than one per cent are indigenous.

"It doesn't give me much hope if I ever get sick and need a blood transfusion or bone marrow transplant, said OWatch.

It doesn't give me much hope because, if there's no potential matches, I'm going to die, bottom line, and I don't want to die."

Robyn Henwood works for Canadian Blood Services, which runs OneMatch. She covers Alberta to Northern Ontario and the Northwest Territories, including the Prairies, and visited Carry the Kettle to recruit. A match requires a genetic twin and indigenous people are only in Canada.

"It does get more complicated [with] these different ethnic backgrounds. . . even within First Nations that get brought into it, said Henwood.

The chances of finding a match becomes that much more difficult."

This means someone who is Cree cannot donate to someone who is Mohawk, she said.

In the past year, Canadian Blood Services has visited less than 12 reserves to help find matches for indigenous people. Carry the Kettle is Henwoods third community.

"We have been leaving messages and voicemails, not getting a lot of response back, she said.

I'm hoping a new technique will work. Things like this, this is so important to spread our message."

According to Indigenous and Northern Affairs Canada, more than 50 per cent of indigenous people live in urban centres. And yet, Henwood says finding indigenous donors in cities is also a struggle.

"Trying to get someone to sign up and commit for the next 30 to 40 years, to potentially save a stranger's life is not an easy thing to do," she said.

Henwood says informing indigenous people about one match will empower more to donate. Until then, the chance of survival for those waiting on the registry is low.

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Program seeks to boost bone marrow, stem cell donations from indigenous people - CTV News

Bone Marrow Stem Cells Comments Off on Program seeks to boost bone marrow, stem cell donations from indigenous people – CTV News | February 7th, 2017

Human skin can be morphed into genetically modified, cancer-killing brain stem cells, according to a new study. This latest advance has only been tested in mice but eventually, its possible that it could be translated into a personalized treatment for people with a deadly form of brain cancer.

The study builds on an earlier discovery that brain stem cells have a weird affinity for cancers. So researchers, led by Shawn Hingtgen, a professor at University of North Carolina at Chapel Hill, created genetically engineered brain stem cells out of human skin. Then they armed the stem cells with drugs to squirt directly onto the tumors of mice that had been given a human form of brain cancer. The treatment shrank the tumors and extended survival of the mice, according to results recently published in the journal Science Translational Medicine.

The treatment shrank the tumors and extended survival

Usually we think about stem cell therapy in the context of rebuilding or regrowing a broken body part like a spinal cord. But if they could be modified to become cancer-fighting homing missiles, it would give patients with a deadly and incurable brain cancer called glioblastoma a better chance at survival. Glioblastomas typically affect adults, and are highly fatal because they send out a web of cancerous threads. Even when the main mass is removed, those threads remain despite chemotherapy and radiation treatment. This cancer has caused a number of high-profile deaths including Senator Edward (Ted) Kennedy in 2009, and possibly Beau Biden more recently. Approximately 12,000 new cases of glioblastoma are estimated to be diagnosed in 2017.

We really have no drugs, no new treatment options in years to even decades, Hingtgen says. [We] just really want to create new therapy that can stand a chance against this disease.

But theres a problem: brain stem cells arent exactly easy to get. Brain stem cells, more properly known as neural stem cells, hang out in the walls of the brains irrigation canals areas filled with cerebrospinal fluid, called ventricles. They generate the cells of the nervous system, like neurons and glial cells, throughout our lives.

They could be modified to become cancer-fighting homing missiles

A research group at the City of Hope in California conducted a clinical trial to make sure it was safe to treat glioblastoma patients with genetically engineered neural stem cells. But they used a neural stem cell line that theyd obtained from fetal tissue. Since the stem cells werent the patients own, people who were genetically more likely to reject the cells couldnt receive the treatment at all. For the people who could, treatment with the neural stem cells turned out to be relatively safe although at this phase of clinical trials, it hasnt been particularly effective.

More personalized treatments have been held up by the challenge of getting enough stem cells out of the patients own brains, which is virtually impossible, says stem cell scientist Frank Marini at the Wake Forest School of Medicine, who was not involved in this study. You cant really generate a bank of neural stem cells from anybody because you have to go in and resect the brain.

So instead, Hingtgen and his colleagues figured out a way to generate neural stem cells from skin which in the future, could let them make neural stem cells personalized to each patient. For this study, though, Hingtgen and his colleagues extracted the skin cells from chunks of human flesh leftover as surgical waste. That really is the magic piece here, Marini says. Now, all of a sudden we have a neural stem cell that can be used as a tumor-homing vehicle.

That really is the magic piece here.

Using a disarmed virus to infect the cells with a cocktail of new genes, the researchers morphed the skin cells into something in between a skin cell and a neural stem cell. People have turned skin cells back into a more generalized type of stem cell before. But then turning those basic stem cells into stem cells for a certain organ like the brain takes another couple of steps, which takes more time. Thats something that people with glioblastoma dont have.

The breakthrough here is that Hingtgens team figured out how to go straight from skin cells to something resembling a neural stem cell in just four days. The researchers then genetically engineered these induced neural stem cells to arm them with one of two different weapons: One group was equipped with an enzyme that could convert an anti-fungal drug into chemotherapy, right at the cancers location. The other was armed with a protein that binds to the cancer cells and makes them commit suicide in an orderly process called apoptosis.

The researchers tested these engineered neural stem cells in mice that had been injected with human glioblastoma cells, which multiplied out of control to create a human cancer in a mouse body. Both of the weaponized stem cell groups were able to significantly shrink the tumors and keep the mice alive by about an extra 30 days (for scale, mice usually live an average of two years).

Were working as fast as we can.

But injecting the cells directly into the tumor doesnt really reflect how the therapy would be used in humans. Its more likely that a person with glioblastoma would get the bulk of the tumor surgically removed. Then, the idea is that these neural stem cells, generated from the patients own skin, will be inserted into the hole left in the brain. So, the researchers tried this out in mice, and the tumors that regrew after surgery were more than three times smaller in the mice treated with the neural stem cells.

Its a promising start, but it could take a few years still before its in the clinic, Hingtgen says. He and his colleagues started a company called Falcon Therapeutics to drive this new therapy forward. Were working as fast as we can, Hingtgen says. We probably cant help the patients today. Hopefully in a year or two, well be able to help those patients.

One of the things theyll have to figure out first is whether the neural stem cells can travel the much bigger distances in human brains, and whether theyll be able to eliminate every remaining cancer cell. The caveats on this are that, of course, its a mouse study, and whether or not that directly converts to humans is unclear, Marini says. Still, he adds, Theres a very high probability in this case.

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The next weapon against brain cancer may be human skin - The Verge

Skin Stem Cells Comments Off on The next weapon against brain cancer may be human skin – The Verge | February 7th, 2017

One of the current challenges for stem cell researchers is to understand how all the skin appendages are regenerated. This could lead to improved treatments for burn patients, or others with severe skin damage.

Researchers are also working to identify new ways to grow skin cells in the lab. Epidermal stem cells are currently cultivated on a layer of cells from rodents, called murine cells. These cell culture conditions have been proved safe, but it would be preferable to avoid using animal products when cultivating cells that will be transplanted into patients. So, researchers are searching for effective cell culture conditions that will not require the use of murine cells.

Scientists are also working to treat genetic diseases affecting the skin. Since skin stem cells can be cultivated in laboratories, researchers can genetically modify the cells, for example by inserting a missing gene. The correctly modified cells can be selected, grown and multiplied in the lab, then transplanted back onto the patient. Epidermolysis Bullosa is one example of a genetic skin disease that might benefit from this approach. Work is underway to test the technique.

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Skin stem cells: where do they live and what can they do ...

Skin Stem Cells Comments Off on Skin stem cells: where do they live and what can they do … | January 26th, 2017

CCR5 Identifiers Aliases CCR5, CC-CKR-5, CCCKR5, CCR-5, CD195, CKR-5, CKR5, CMKBR5, IDDM22, C-C motif chemokine receptor 5 (gene/pseudogene) External IDs OMIM: 601373 MGI: 107182 HomoloGene: 37325 GeneCards: CCR5 Targeted by Drug aplaviroc, cenicriviroc, maraviroc, vicriviroc[1] Orthologs Species Human Mouse Entrez Ensembl UniProt RefSeq (mRNA) RefSeq (protein) Location (UCSC) Chr 3: 46.37 46.38 Mb Chr 9: 124.12 124.15 Mb PubMed search [2] [3] Wikidata View/Edit Human View/Edit Mouse

C-C chemokine receptor type 5, also known as CCR5 or CD195, is a protein on the surface of white blood cells that is involved in the immune system as it acts as a receptor for chemokines. This is the process by which T cells are attracted to specific tissue and organ targets. Many forms of HIV, the virus that causes AIDS, initially use CCR5 to enter and infect host cells. Certain individuals carry a mutation known as CCR5-32 in the CCR5 gene, protecting them against these strains of HIV.

In humans, the CCR5 gene that encodes the CCR5 protein is located on the short (p) arm at position 21 on chromosome 3. Certain populations have inherited the Delta 32 mutation resulting in the genetic deletion of a portion of the CCR5 gene. Homozygous carriers of this mutation are resistant to M-tropic strains of HIV-1 infection.[4][5][6][7][8][9]

The CCR5 protein belongs to the beta chemokine receptors family of integral membrane proteins.[10][11] It is a G proteincoupled receptor[10] which functions as a chemokine receptor in the CC chemokine group.

CCR5's cognate ligands include CCL3, CCL4 (also known as MIP 1 and 1, respectively), and CCL3L1.[12][13] CCR5 furthermore interacts with CCL5 (a chemotactic cytokine protein also known as RANTES).[12][14][15]

CCR5 is predominantly expressed on T cells, macrophages, dendritic cells, eosinophils and microglia. It is likely that CCR5 plays a role in inflammatory responses to infection, though its exact role in normal immune function is unclear. Regions of this protein are also crucial for chemokine ligand binding, functional response of the receptor, and HIV co-receptor activity.[16]

HIV-1 most commonly uses the chemokine receptors CCR5 and/or CXCR4 as co-receptors to enter target immunological cells.[17] These receptors are located on the surface of host immune cells whereby they provide a method of entry for the HIV-1 virus to infect the cell.[18] The HIV-1 envelope glycoprotein structure is essential in enabling the viral entry of HIV-1 into a target host cell.[18] The envelope glycoprotein structure consists of two protein subunits cleaved from a Gp160 protein precursor encoded for by the HIV-1 env gene: the Gp120 external subunit, and the Gp41 transmembrane subunit.[18] This envelope glycoprotein structure is arranged into a spike-like structure located on the surface of the virion and consists of a trimer of three Gp120-Gp41 hetero-dimers.[18] The Gp120 envelope protein is a chemokine mimic.[17] It lacks the unique structure of a chemokine, however it is still capable of binding to the CCR5 and CXCR4 chemokine receptors.[17] During HIV-1 infection, the Gp120 envelope glycoprotein subunit binds to a CD4 glycoprotein and a HIV-1 co-receptor expressed on a target cell- forming a heterotrimeric complex.[17] The formation of this complex stimulates the release of a fusogenic peptide inducing the fusion of the viral membrane with the membrane of the target host cell.[17] Because binding to CD4 alone can sometimes result in gp120 shedding, gp120 must next bind to co-receptor CCR5 in order for fusion to proceed. The tyrosine sulfated amino terminus of this co-receptor is the "essential determinant" of binding to the gp120 glycoprotein.[19] Co-receptor recognition also include the V1-V2 region of gp120, and the bridging sheet (an antiparallel, 4-stranded sheet that connects the inner and outer domains of gp120). The V1-V2 stem can influence "co-receptor usage through its peptide composition as well as by the degree of N-linked glycosylation." Unlike V1-V2 however, the V3 loop is highly variable and thus is the most important determinant of co-receptor specificity.[19] The normal ligands for this receptor, RANTES, MIP-1, and MIP-1, are able to suppress HIV-1 infection in vitro. In individuals infected with HIV, CCR5-using viruses are the predominant species isolated during the early stages of viral infection,[20] suggesting that these viruses may have a selective advantage during transmission or the acute phase of disease. Moreover, at least half of all infected individuals harbor only CCR5-using viruses throughout the course of infection.

CCR5 is the primary co-receptor used by gp120 sequentially with CD4. This bind results in gp41, the other protein product of gp160, to be released from its metastable conformation and insert itself into the membrane of the host cell. Although it hasn't been finalized as a proven theory yet, binding of gp120-CCR5 involves two crucial steps: 1) The tyrosine sulfated amino terminus of this co-receptor is an "essential determinant" of binding to gp120 (as stated previously) 2) Following step 1., there must be reciprocal action (synergy, intercommunication) between gp120 and the CCR5 transmembrane domains [19]

CCR5 is essential for the spread of the R5-strain of the HIV-1 virus.[21] Knowledge of the mechanism by which this strain of HIV-1 mediates infection has prompted research into the development of therapeutic interventions to block CCR5 function.[22] A number of new experimental HIV drugs, called CCR5 receptor antagonists, have been designed to interfere with the associative binding between the Gp120 envelope protein and the HIV co-receptor CCR5.[21] These experimental drugs include PRO140 (CytoDyn), Vicriviroc (Phase III trials were cancelled in July 2010) (Schering Plough), Aplaviroc (GW-873140) (GlaxoSmithKline) and Maraviroc (UK-427857) (Pfizer). Maraviroc was approved for use by the FDA in August 2007.[21] It is the only one thus far approved by the FDA for clinical use, thus becoming the first CCR5 inhibitor.[19] A problem of this approach is that, while CCR5 is the major co-receptor by which HIV infects cells, it is not the only such co-receptor. It is possible that under selective pressure HIV will evolve to use another co-receptor. However, examination of viral resistance to AD101, molecular antagonist of CCR5, indicated that resistant viruses did not switch to another coreceptor (CXCR4) but persisted in using CCR5, either through binding to alternative domains of CCR5, or by binding to the receptor at a higher affinity. However, because there is still another co-receptor available, this indicates that lacking the CCR5 gene doesn't make one immune to the virus; it simply implies that it would be more challenging for the individual to contract it. Also, the virus still has access to the CD4. Unlike CCR5, which the body apparently doesn't really need due to those still living healthy lives even with the lack of/or absence of the gene (as a result of the delta 32 mutation), CD4 is critical in the bodies defense system (fighting against infection).[23] Even without the availability of either co-receptors (even CCR5), the virus can still invade cells if gp41 were to go through an alteration (including its cytoplasmic tail), resulting in the independence of CD4 without the need of CCR5 and/or CXCR4 as a doorway.[24]

CCR5-32 (or CCR5-D32 or CCR5 delta 32) is an allele of CCR5.[25][26]

CCR5 32 is a 32-base-pair deletion that introduces a premature stop codon into the CCR5 receptor locus, resulting in a nonfunctional receptor.[27][28] CCR5 is required for M-tropic HIV-1 virus entry.[29] Individuals homozygous for CCR5 32 do not express functional CCR5 receptors on their cell surfaces and are resistant to HIV-1 infection, despite multiple high-risk exposures.[29] Individuals heterozygous for the mutant allele have a greater than 50% reduction in functional CCR5 receptors on their cell surfaces due to dimerization between mutant and wild-type receptors that interferes with transport of CCR5 to the cell surface.[30] Heterozygote carriers are resistant to HIV-1 infection relative to wild types and when infected, heterozygotes exhibit reduced viral loads and a 2-3-year-slower progression to AIDS relative to wild types.[27][29][31] Heterozygosity for this mutant allele also has shown to improve one's virological response to anti-retroviral treatment.[32] CCR5 32 has an (heterozygote) allele frequency of 10% in Europe, and a homozygote frequency of 1%.

The CCR5 32 allele is notable for its recent origin, unexpectedly high frequency, and distinct geographic distribution,[33] which together suggest that (a) it arose from a single mutation, and (b) it was historically subject to positive selection.

Two studies have used linkage analysis to estimate the age of the CCR5 32 deletion, assuming that the amount of recombination and mutation observed on genomic regions surrounding the CCR5 32 deletion would be proportional to the age of the deletion.[26][34] Using a sample of 4000 individuals from 38 ethnic populations, Stephens et al. estimated that the CCR5-32 deletion occurred 700 years ago (275-1875, 95% confidence interval). Another group, Libert et al. (1998), estimated the age of the CCR5 32 mutation is based on the microsatellite mutations to be 2100 years (700-4800, 95% confidence interval). On the basis of observed recombination events, they estimated the age of the mutation to be 2250 years (900-4700, 95% confidence interval).[34] A third hypothesis relies on the north-to-south gradient of allele frequency in Europe which shows that the highest allele frequency occurred in Nordic regions such as Iceland, Norway and Sweden and lowest allele frequency in the south. Because the Vikings historically occupied these countries, it may be possible that the allele spread throughout Europe was due to the Viking dispersal in the 8th to 10th century.[35] Vikings were later replaced by the Varangians in Russia, which migrated East which may have contributed to the observed east-to-west cline of allele frequency.[33][35]

HIV-1 was initially transmitted from chimpanzees (Pan troglodytes) to humans in the early 1900s in Southeast Cameroon, Africa,[36] through exposure to infected blood and body fluids while butchering bushmeat.[37] However, HIV-1 was effectively absent from Europe until the late 1980s.[38] Therefore, given the average age of roughly 1000 years for the CCR5-32 allele, it can be established that HIV-1 did not exert selection pressure on the human population for long enough to achieve the current frequencies.[33] Hence, other pathogens have been suggested agents of positive selection for CCR5 32. The first major one being bubonic plague (Yersinia pestis), and later, smallpox (Variola major). Other data suggest that the allele frequency resulted as a negative selection pressure as a result of pathogens that became more widespread during Roman expansion.[39] The idea that negative selection played a role in its low frequency is also supported by experiments using knockout mice and Influenza A, which demonstrated that the presence of the CCR5 receptor is important for efficient response to a pathogen.[40][41]

Several lines of evidence suggest that the CCR5 32 allele evolved only once.[33] First, CCR5 32 has a relatively high frequency in several different Caucasian populations but is comparatively absent in Asian, Middle Eastern and American Indian populations,[26] suggesting that a single mutation occurred after divergence of Caucasians from their African ancestor).[26][27][42] Second, genetic linkage analysis indicates that the mutation occurs on a homogenous genetic background, implying that inheritance of the mutation occurred from a common ancestor.[34] This was demonstrated by showing that the CCR5 32 allele is in strong linkage disequilibrium with highly polymorphic microsatellites. More than 95% of CCR5 32 chromosomes also carried the IRI3.1-0 allele, while 88% carried the IRI3.2 allele. By contrast, the microsatellite markers IRI3.1-0 and IRI3.2-0 were found in only 2 or 1.5% of chromosomes carrying a wild-type CCR5 allele.[34] This evidence of linkage disequilibrium supports the hypothesis that most, if not all, CCR5 32 alleles arose from a single mutational event. Finally, the CCR5 32 allele has a unique geographical distribution indicating a single Northern origin followed by migration. A study measuring allele frequencies in 18 European populations found a North-to-South gradient, with the highest allele frequencies in Finnish and Mordvinian populations (16%), and the lowest in Sardinia (4%).[34]

In the absence of selection, a single mutation would take an estimated 127,500 years to rise to a population frequency of 10%.[26] Estimates based on genetic recombination and mutation rates place the age of the allele between 1000 and 2000 years. This discrepancy is a signature of positive selection.

It is estimated that HIV-1 entered the human population in Africa in the early 1900s,[36] symptomatic infections were not reported until the 1980s. The HIV-1 epidemic is therefore far too young to be the source of positive selection that drove the frequency of CCR5 32 from zero to 10% in 2000 years. In 1998, Stephens et al. suggested that bubonic plague (Yersinia pestis) had exerted positive selective pressure on CCR5 32.[26] This hypothesis was based on the timing and severity of the Black Death pandemic, which killed 30% of the European population of all ages between 1346 and 1352.[43] After the Black Death, there were less severe, intermittent, epidemics. Individual cities experienced high mortality, but overall mortality in Europe was only a few percent.[43][44][45] In 1655-1656 a second pandemic called the "Great Plague" killed 15-20% of Europes population.[43][46] Importantly, the plague epidemics were intermittent. Bubonic plague is a zoonotic disease, primarily infecting rodents and spread by fleas and only occasionally infecting humans.[47] Human-to-human infection of bubonic plague does not occur, though it can occur in pneumonic plague, which infects the lungs.[48] Only when the density of rodents is low are infected fleas forced to feed on alternative hosts such as humans, and under these circumstances a human epidemic may occur.[47] Based on population genetic models, Galvani and Slatkin (2003) argue that the intermittent nature of plague epidemics did not generate a sufficiently strong selective force to drive the allele frequency of CCR5 32 to 10% in Europe.[25]

To test this hypothesis, Galvani and Slatkin (2003) modeled the historical selection pressures produced by plague and smallpox.[25] Plague was modeled according to historical accounts,[49][50] while age-specific smallpox mortality was gleaned from the age distribution of smallpox burials in York (England) between 1770 and 1812.[44] Smallpox preferentially infects young, pre-reproductive members of the population since they are the only individuals who are not immunized or dead from past infection. Because smallpox preferentially kills pre-reproductive members of a population, it generates stronger selective pressure than plague.[25] Unlike plague, smallpox does not have an animal reservoir and is only transmitted from human to human.[51][52] The authors calculated that if plague were selecting for CCR5 32, the frequency of the allele would still be less than 1%, while smallpox has exerted a selective force sufficient to reach 10%.

The hypothesis that smallpox exerted positive selection for CCR5 32 is also biologically plausible, since poxviruses, like HIV, are viruses that enter white blood cells by using chemokine receptors.[53] By contrast, Yersinia pestis is a bacterium with a very different biology.

Although Caucasians are the only population with a high frequency of CCR5 32, they are not the only population that has been subject to selection by smallpox, which had a worldwide distribution before it was declared eradicated in 1980. The earliest unmistakable descriptions of smallpox appear in the 5th century A.D. in China, the 7th century A.D. in India and the Mediterranean, and the 10th century A.D. in southwestern Asia.[52] By contrast, the CCR5 32 mutation is found only in European, West Asian, and North African populations.[54] The anomalously high frequency of CCR5 32 in these populations appears to require both a unique origin in Northern Europe and subsequent selection by smallpox.

Research has not yet revealed a cost of carrying the CCR5 null mutation that is as dramatic as the benefit conferred in the context of HIV-1 exposure. In general, research suggests that the CCR5 32 mutation protects against diseases caused by certain pathogens but may also play a deleterious role in postinfection inflammatory processes, which can injure tissue and create further pathology.[55] The best evidence for this proposed antagonistic pleiotropy is found in flavivirus infections. In general many viral infections are asymptomatic or produce only mild symptoms in the vast majority of the population. However, certain unlucky individuals experience a particularly destructive clinical course, which is otherwise unexplained but appears to be genetically mediated. Patients homozygous for CCR5 32 were found to be at higher risk for a neuroinvasive form of tick-borne encephalitis (a flavivirus).[56] In addition, functional CCR5 may be required to prevent symptomatic disease after infection with West Nile virus, another flavivirus; CCR5 32 was associated with early symptom development and more pronounced clinical manifestations after infection with West Nile virus.[57]

This finding in humans confirmed a previously-observed experiment in an animal model of CCR5 32 homozygosity. After infection with West Nile Virus, CCR5 32 mice had markedly increased viral titers in the central nervous system and had increased mortality[58] compared with that of wild-type mice, thus suggesting that CCR5 expression was necessary to mount a strong host defense against West Nile virus.

CCR5 32 can be beneficial to the host in some infections (e.g., HIV-1, possibly smallpox), but detrimental in others (e.g., tick-borne encephalitis, West Nile virus). Whether CCR5 function is helpful or harmful in the context of a given infection depends on a complex interplay between the immune system and the pathogen.

A genetic approach involving intrabodies that block CCR5 expression has been proposed as a treatment for HIV-1 infected individuals.[59] When T-cells modified so they no longer express CCR5 were mixed with unmodified T-cells expressing CCR5 and then challenged by infection with HIV-1, the modified T-cells that do not express CCR5 eventually take over the culture, as HIV-1 kills the non-modified T-cells. This same method might be used in vivo to establish a virus-resistant cell pool in infected individuals.[59]

This hypothesis was tested in an AIDS patient who had also developed myeloid leukemia, and was treated with chemotherapy to suppress the cancer. A bone marrow transplant containing stem cells from a matched donor was then used to restore the immune system. However, the transplant was performed from a donor with 2 copies of CCR5-32 mutation gene. After 600 days, the patient was healthy and had undetectable levels of HIV in the blood and in examined brain and rectal tissues.[5][60] Before the transplant, low levels of HIV X4, which does not use the CCR5 receptor, were also detected. Following the transplant, however, this type of HIV was not detected either, further baffling doctors.[5] However, this is consistent with the observation that cells expressing the CCR5-32 variant protein lack both the CCR5 and CXCR4 receptors on their surfaces, thereby conferring resistance to a broad range of HIV variants including HIV X4.[61] After over six years, the patient has maintained the resistance to HIV and has been pronounced cured of the HIV infection.[6]

Enrollment of HIV-positive patients in a clinical trial was started in 2009 in which the patients' cells were genetically modified with a zinc finger nuclease to carry the CCR5-32 trait and then reintroduced into the body as a potential HIV treatment.[62][63] Results reported in 2014 were promising.[9]

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CCR5 - Wikipedia

Bone Marrow Stem Cells Comments Off on CCR5 – Wikipedia | January 13th, 2017

Session Tracks

Conference Series invites all the participants from all over the world to attend"8th European Immunology Conference, June 29-July 01, 2017 Madrid, Spain, includesprompt keynote presentations, Oral talks, Poster presentations and Exhibitions.

European ImmunologyConferenceis to gathering people in academia and society interested inimmunologyto share the latest trends and important issues relevant to our field/subject area.Immunology Conferencesbrings together the global leaders in Immunology and relevant fields to present their research at this exclusive scientific program. TheImmunology Conferencehosting presentations from editors of prominent refereed journals, renowned and active investigators and decision makers in the field of Immunology.European Immunology ConferenceOrganizing Committee also invites Young investigators at every career stage to submit abstracts reporting their latest scientific findings in oral and poster sessions.

Track:1Cellular Immunology

The study of the molecular and cellular components that comprise the immune system, including their function and interaction, is the central science ofimmunology. The immune system has been divided into a more primitive innate immune system and, in vertebrates, an acquired oradaptive immune system

The field concerning the interactions among cells and molecules of the immunesystem,and how such interactions contribute to the recognition and elimination of pathogens. Humans possess a range of non-specific mechanical and biochemical defences against routinely encountered bacteria, parasites, viruses, and fungi. The skin, for example, is an effective physical barrier to infection. Basic chemical defences are also present in blood, saliva, and tears, and on mucous membranes. True protection stems from the host's ability to mount responses targeted to specific organisms, and to retain a form of memory that results in a rapid, efficient response to a given organism upon a repeat encounter. This more formal sense of immunity, termed adaptive immunity, depends upon the coordinated activities of cells and molecules of the immune system.

RelatedImmunology Conferences|Immunologists Meetings|Conference Series LLC:

9thworld congress & expo on Immunology, Oct 02-04, 2017, Toronto, Canada; 3rdAntibodies and Bio Therapeutics Congress, November 02-03, 2017 Las Vegas, USA; Molecular Immunology & Immunogenetics Congress, March 20-21, 2017 Rome, Italy; Annual Meeting on Immunology and Immunologist, July 03-05, 2017 Malyasia, Kuala lumpur; 3rd International Congress on Neuroimmunology and Therapeutics, September 18-19, 2017 Philadelphia, USA; 2nd Autoimmunity Conference, Nov 9-10, 2017 Madrid, Spain; Integrating Metabolism and Immunity , May 29 - June 2, 2017 | Dublin, Ireland

Track: 2Inflammatory/Autoimmune Diseases

Autoimmune diseasescan affect almost any part of the body, including the heart, brain, nerves, muscles, skin, eyes, joints, lungs, kidneys, glands, the digestive tract, and blood vessels.

The classic sign of an autoimmune disease is inflammation, which can cause redness, heat, pain, and swelling. How an autoimmune disease affects you depends on what part of the body is targeted. If the disease affects the joints, as inrheumatoid arthritis, you might have joint pain, stiffness, and loss of function. If it affects the thyroid, as in Graves disease and thyroiditis, it might cause tiredness, weight gain, and muscle aches. If it attacks the skin, as it does in scleroderma/systemic sclerosis, vitiligo, andsystemic lupus erythematosus(SLE), it can cause rashes, blisters, and colour changes. Many autoimmune diseases dont restrict themselves to one part of the body. For example, SLE can affect the skin, joints, kidneys, heart, nerves, blood vessels, and more. Type 1 diabetes can affect your glands, eyes, kidneys, muscles, and more.

RelatedImmunology Conferences|Immunologists Meetings|Conference Series LLC:

9thworld congress & expo on Immunology, Oct 02-04, 2017, Toronto, Canada; 3rdAntibodies and Bio Therapeutics Congress, November 02-03, 2017 Las Vegas, USA; Molecular Immunology & Immunogenetics Congress, March 20-21, 2017 Rome, Italy; 3nd International Congress on Neuroimmunology and Therapeutics, September 18-19, 2017 Philadelphia, USA; 18th International Conference on Immunology (ICI) Dec 12-13, 2016, Bangkok, Thailand; Annual Meeting on Immunology and Immunologist, July 03-05, 2017 Malyasia, Kuala lumpur; British Society for Immunology Congress, Dec 06-09, 2016, Liverpool, United Kingdom; 7thInternational Conference on Allergy, Asthma and Clinical Immunology

Track: 3T-Cells and B-Cells

T cell: A type of white blood cell that is of key importance to the immune system and is at the core of adaptive immunity, the system that tailors the body's immune response to specific pathogens. The T cells are like soldiers who search out and destroy the targeted invaders. Immature T cells (termed T-stem cells) migrate to the thymus gland in the neck, where they mature and differentiate into various types of mature T cells and become active in the immune system in response to a hormone called thymosin and other factors. T-cells that are potentially activated against the body's own tissues are normally killed or changed ("down-regulated") during this maturational process.There are several different types of mature T cells. Not all of their functions are known. T cells can produce substances called cytokines such as the interleukins which further stimulate the immune response. T-cell activation is measured as a way to assess the health of patients withHIV/AIDSand less frequently in other disorders. T cell are also known as T lymphocytes. The "T" stands for "thymus" -- the organ in which these cells mature. As opposed to B cells which mature in the bone marrow.B cells, also known asBlymphocytes, are a type of white bloodcellof the lymphocyte subtype. They function in thehumoral immunitycomponent of the adaptive immune system by secreting antibodies. Many B cells mature into what are called plasma cells that produce antibodies (proteins) necessary to fight off infections while other B cells mature into memory B cells. All of the plasma cells descended from a single B cell produce the same antibody which is directed against the antigen that stimulated it to mature. The same principle holds with memory B cells. Thus, all of the plasma cells and memory cells "remember" the stimulus that led to their formation. The maturation of B cells takes place in birds in an organ called the bursa of Fabricus. B cells in mammals mature largely in the bone marrow. The B cell, or B lymphocyte, is thus an immunologically important cell. It is not thymus-dependent, has a short lifespan, and is responsible for the production ofimmunoglobulins.It expresses immunoglobulins on its surface.

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Molecular Immunology & Immunogenetics Congress, March 20-21, 2017 Rome, Italy; 3nd International Congress on Neuroimmunology and Therapeutics, September 18-19, 2017 Philadelphia, USA; 18thInternational Conference on Immunology (ICI) Dec 12-13, 2016, Bangkok, Thailand; Annual Meeting on Immunology and Immunologist, July 03-05, 2017 Malyasia, Kuala lumpur; 19thInternational Conference on Immunology (ICI) Sept 14-17, 2017, Berlin, Germany; Modelling Viral Infections and Immunity (E1) , May 1 - 4, 2017 | Estes Park, Colorado, USA; 7thInternational Conference on Allergy, Asthma and Clinical Immunology

Track: 4Cancer and Tumor Immunobiology

The tumour is an important aspect of cancer biology that contributes to tumour initiation, tumour progression and responses to therapy. Cells and molecules of the immune system are a fundamental component of the tumour microenvironment. Importantly,therapeutic strategies for cancer treatmentcan harness the immune system to specifically target tumour cells and this is particularly appealing owing to the possibility of inducing tumour-specific immunological memory, which might cause long-lasting regression and prevent relapse in cancer patients.The composition and characteristics of the tumour microenvironment vary widely and are important in determining the anti-tumour immune response.Immunotherapyis a new class ofcancer treatmentthat works to harness the innate powers of the immune system to fight cancer. Because of the immune system's unique properties, these therapies may hold greater potential than current treatment approaches to fight cancer more powerfully, to offer longer-term protection against the disease, to come with fewer side effects, and to benefit more patients with more cancer

RelatedImmunology Conferences|Immunologists Meetings|Conference Series LLC:

9thworld congress & expo on Immunology, Oct 02-04, 2017, Toronto, Canada; 3rdAntibodies and Bio Therapeutics Congress, November 02-03, 2017 Las Vegas, USA; Molecular Immunology & Immunogenetics Congress, March 20-21, 2017 Rome, Italy; 3nd International Congress on Neuroimmunology and Therapeutics, September 18-19, 2017 Philadelphia, USA; 18th International Conference on Immunology (ICI) Dec 12-13, 2016, Bangkok, Thailand; Annual Meeting on Immunology and Immunologist, July 03-05, 2017 Malyasia, Kuala lumpur; British Society for Immunology Congress, Dec 06-09, 2016, Liverpool, United Kingdom; 7thInternational Conference on Allergy, Asthma and Clinical Immunology

Track: 5 Vaccines

A vaccine is a biological preparation that improves immunity to a particular disease. A vaccine typically contains an agent that resembles a disease-causing microorganism, and is often made from weakened or killed forms of the microbe, its toxins or one of its surface proteins. The agent stimulates the body's immune system to recognize the agent as foreign, destroy it, and "remember" it, so that the immune system can more easily recognize and destroy any of these microorganisms that it later encounters. There are two basictypes of vaccines: live attenuated and inactivated. The characteristics of live and inactivatedvaccinesare different, and these characteristics determine how thevaccineis used. Liveattenuatedvaccinesare produced by modifying a disease-producing (wild) virus or bacteria in a laboratory.

RelatedImmunology Conferences|Immunologists Meetings|Conference Series LLC:

Molecular Immunology & Immunogenetics Congress, March 20-21, 2017 Rome, Italy; 3nd International Congress on Neuroimmunology and Therapeutics, September 18-19, 2017 Philadelphia, USA; 18thInternational Conference on Immunology (ICI) Dec 12-13, 2016, Bangkok, Thailand; Annual Meeting on Immunology and Immunologist, July 03-05, 2017 Malyasia, Kuala lumpur; 19thInternational Conference on Immunology (ICI) Sept 14-17, 2017, Berlin, Germany; Modelling Viral Infections and Immunity (E1) , May 1 - 4, 2017 | Estes Park, Colorado, USA; 7thInternational Conference on Allergy, Asthma and Clinical Immunology

Track: 6Immunotherapy

Immunotherapy,also called biologic therapy, is a type of cancer treatment designed to boost the body's natural defences to fight the cancer. It uses materials either made by the body or in a laboratory to improve, target, or restore immune system function. Immunotherapy is treatment that uses certain parts of a persons immune system to fight diseases such as cancer. This can be done in a couple of ways:1)Stimulating your own immune system to work harder or smarter to attack cancer cells2)Giving you immune system components, such as man-made immune system proteins. Some types of immunotherapy are also sometimes called biologic therapy or biotherapy.

In the last few decadesimmunotherapyhas become an important part of treating some types of cancer. Newer types of immune treatments are now being studied, and theyll impact how we treat cancer in the future. Immunotherapy includes treatments that work in different ways. Some boost the bodys immune system in a very general way. Others help train the immune system to attack cancer cells specifically. Immunotherapy works better for some types of cancer than for others. Its used by itself for some of these cancers, but for others it seems to work better when used with other types of treatment.

Many different types of immunotherapy are used to treat cancer. They include:Monoclonal antibodies,Adoptive cell transfer,Cytokines, Treatment Vaccines, BCG,

RelatedImmunology Conferences|Immunologists Meetings|Conference Series LLC:

9thworld congress & expo on Immunology, Oct 02-04, 2017, Toronto, Canada; 3rdAntibodies and Bio Therapeutics Congress, November 02-03, 2017 Las Vegas, USA; Molecular Immunology & Immunogenetics Congress, March 20-21, 2017 Rome, Italy; Annual Meeting on Immunology and Immunologist, July 03-05, 2017 Malyasia, Kuala lumpur; 3rd International Congress on Neuroimmunology and Therapeutics, September 18-19, 2017 Philadelphia, USA; 2nd Autoimmunity Conference, Nov 9-10, 2017 Madrid, Spain; Integrating Metabolism and Immunity , May 29 - June 2, 2017 | Dublin, Ireland; American Academy of Allergy, Asthma & Immunology (AAAAI) Annual Meeting, March 03-06, 2017, Atlanta, Georgia

Track: 7Neuro Immunology

Neuroimmunology, a branch of immunologythat deals especially with the inter relationships of the nervous system and immune responses andautoimmune disorders. It deals with particularly fundamental and appliedneurobiology,meetings onneurology,neuropathology, neurochemistry,neurovirology, neuroendocrinology, neuromuscular research,neuropharmacologyand psychology, which involve either immunologic methodology (e.g. immunocytochemistry) or fundamental immunology (e.g. antibody and lymphocyte assays).

RelatedImmunology Conferences|Immunologists Meetings|Conference Series LLC:

Molecular Immunology & Immunogenetics Congress, March 20-21, 2017 Rome, Italy; 3nd International Congress on Neuroimmunology and Therapeutics, September 18-19, 2017 Philadelphia, USA; 18thInternational Conference on Immunology (ICI) Dec 12-13, 2016, Bangkok, Thailand; Annual Meeting on Immunology and Immunologist, July 03-05, 2017 Malyasia, Kuala lumpur; 19thInternational Conference on Immunology (ICI) Sept 14-17, 2017, Berlin, Germany; Modelling Viral Infections and Immunity (E1) , May 1 - 4, 2017 | Estes Park, Colorado, USA; 7thInternational Conference on Allergy, Asthma and Clinical Immunology; 18thInternational Conference on Immunology (ICI) Dec 12-13, 2016, Bangkok, Thailand

Track: 8Infectious Diseases and Immune System

Infectious diseases are caused by pathogenic microorganisms, such as bacteria, viruses, parasites or fungi; the diseases can be spread, directly or indirectly, from one person to another.Zoonotic diseasesare infectious diseases of animals that can cause disease when transmitted to humans. Some infectious diseases can be passed from person to person. Some are transmitted by bites from insects or animals. And others are acquired by ingesting contaminated food or water or being exposed to organisms in the environment. Signs and symptoms vary depending on the organism causing the infection, but often include fever and fatigue. Mild complaints may respond to rest and home remedies, while some life-threatening infections may require hospitalization.

Many infectious diseases, such as measles andchickenpox, can be prevented by vaccines. Frequent and thorough hand-washing also helps protect you from infectious diseases

There are four main kinds of germs:

RelatedImmunology Conferences|Immunologists Meetings|Conference Series LLC:

Molecular Immunology & Immunogenetics Congress, March 20-21, 2017 Rome, Italy; 3nd International Congress on Neuroimmunology and Therapeutics, September 18-19, 2017 Philadelphia, USA; 18thInternational Conference on Immunology (ICI) Dec 12-13, 2016, Bangkok, Thailand; Annual Meeting on Immunology and Immunologist, July 03-05, 2017 Malyasia, Kuala lumpur; 19thInternational Conference on Immunology (ICI) Sept 14-17, 2017, Berlin, Germany; Modelling Viral Infections and Immunity (E1) , May 1 - 4, 2017 | Estes Park, Colorado, USA; 7thInternational Conference on Allergy, Asthma and Clinical Immunology; 18thInternational Conference on Immunology (ICI) Dec 12-13, 2016, Bangkok, Thailand

Track: 9Reproductive Immunology,

Reproductive immunologyrefers to a field of medicine that studies interactions (or the absence of them) between the immune system and components related to thereproductivesystem, such as maternal immune tolerance towards the fetus, orimmunologicalinteractions across the blood-testis barrier. The immune system refers to all parts of the body that work to defend it against harmful enemies. In people with immunological fertility problems their body identifies part of reproductive function as an enemy and sendsNatural Killer (NK) cellsto attack. A healthy immune response would only identify an enemy correctly and attack only foreign invaders such as a virus, parasite, bacteria, ect.

The concept of reproductive immunology is not widely accepted by all physicians.Those patients who have had repeated miscarriages and multiple failed IVF's find themselves exploring it's possibilities as the reason. With an increased amount of success among treating any potential immunological factors, the idea of reproductive immunology can no longer be overlooked.The failure to conceive is often due to immunologic problems that can lead to very early rejection of the embryo, often before the pregnancy can be detected by even the most sensitive tests. Women can often produce perfectly healthy embryos that are lost through repeated "mini miscarriages." This most commonly occurs in women who have conditions such asendometriosis, an under-active thyroid gland or in cases of so called "unexplained infertility." It has been estimated that an immune factor may be involved in up to 20% of couples with otherwiseunexplained infertility. These are all conditions where abnormalities of the womans immune system may play an important role.

RelatedImmunology Conferences|Immunologists Meetings|Conference Series LLC:

9thworld congress & expo on Immunology, Oct 02-04, 2017, Toronto, Canada; 3rdAntibodies and Bio Therapeutics Congress, November 02-03, 2017 Las Vegas, USA; Molecular Immunology & Immunogenetics Congress, March 20-21, 2017 Rome, Italy; 3nd International Congress on Neuroimmunology and Therapeutics, September 18-19, 2017 Philadelphia, USA; 18th International Conference on Immunology (ICI) Dec 12-13, 2016, Bangkok, Thailand; Annual Meeting on Immunology and Immunologist, July 03-05, 2017 Malyasia, Kuala lumpur; British Society for Immunology Congress, Dec 06-09, 2016, Liverpool, United Kingdom; 7thInternational Conference on Allergy, Asthma and Clinical Immunology; Cancer Immunology and Immunotherapy: Taking a Place in Mainstream Oncology (C7), March 19 - 23, 2017, Whistler, British Columbia, Canada

Track:10Auto Immunity,

Autoimmunityis the system ofimmuneresponses of an organism against its own cells and tissues. Any disease that results from such an aberrantimmuneresponse is termed an autoimmune disease.

Autoimmunity is present to some extent in everyone and is usually harmless. However, autoimmunity can cause a broad range of human illnesses, known collectively as autoimmune diseases. Autoimmune diseases occur when there is progression from benign autoimmunity to pathogenicautoimmunity. This progression is determined by genetic influences as well as environmental triggers. Autoimmunity is evidenced by the presence of autoantibodies (antibodies directed against the person who produced them) and T cells that are reactive with host antigens.

Autoimmune disorders

An autoimmune disorder occurs whenthe bodys immune systemattacks and destroys healthy body tissue by mistake. There are more than 80 types of autoimmune disorders.

Causes

The white blood cells in the bodys immune system help protect against harmful substances. Examples include bacteria, viruses,toxins,cancercells, and blood and tissue from outside the body. These substances contain antigens. The immune system producesantibodiesagainst these antigens that enable it to destroy these harmful substances. When you have an autoimmune disorder, your immune system does not distinguish between healthy tissue and antigens. As a result, the body sets off a reaction that destroys normal tissues. The exact cause of autoimmune disorders is unknown. One theory is that some microorganisms (such as bacteria or viruses) or drugs may trigger changes that confuse the immune system. This may happen more often in people who have genes that make them more prone toautoimmune disorders.

An autoimmune disorder may result in:

A person may have more than one autoimmune disorder at the same time. Common autoimmune disorders include:

RelatedImmunology Conferences|Immunologists Meetings|Conference Series LLC:

9thworld congress & expo on Immunology, Oct 02-04, 2017, Toronto, Canada; 3rdAntibodies and Bio Therapeutics Congress, November 02-03, 2017 Las Vegas, USA; Molecular Immunology & Immunogenetics Congress, March 20-21, 2017 Rome, Italy; 3nd International Congress on Neuroimmunology and Therapeutics, September 18-19, 2017 Philadelphia, USA; 18th International Conference on Immunology (ICI) Dec 12-13, 2016, Bangkok, Thailand; Annual Meeting on Immunology and Immunologist, July 03-05, 2017 Malyasia, Kuala lumpur; British Society for Immunology Congress, Dec 06-09, 2016, Liverpool, United Kingdom; 7thInternational Conference on Allergy, Asthma and Clinical Immunology; Cancer Immunology and Immunotherapy: Taking a Place in Mainstream Oncology (C7), March 19 - 23, 2017, Whistler, British Columbia, Canada

Track: 11Costimmulatory pathways in multiple sclerosis

Costimulatory moleculescan be categorized based either on their functional attributes or on their structure. The costimulatory molecules discussed in this review will be divided into (1)positive costimulatory pathways:promoting T cell activation, survival and/or differentiation; (2)negative costimulatory pathways:antagonizing TCR signalling and suppressing T cell activation; (3) as third group we will discuss themembers of the TIM family, a rather new family of cell surface molecules involved in the regulation of T cell differentiation and Treg function.Costimulatory pathways have a critical role in the regulation of alloreactivity. A complex network of positive and negative pathways regulates T cell responses. Blocking costimulation improves allograft survival in rodents and non-human primates. The costimulation blocker belatacept is being developed asimmunosuppressivedruginrenal transplantation.

RelatedImmunology Conferences|Immunologists Meetings|Conference Series LLC:

3rdAntibodies and Bio Therapeutics Congress, November 02-03, 2017 Las Vegas, USA; Molecular Immunology & Immunogenetics Congress, March 20-21, 2017 Rome, Italy; Annual Meeting on Immunology and Immunologist, July 03-05, 2017 Malyasia, Kuala lumpur; 3rd International Congress on Neuroimmunology and Therapeutics, September 18-19, 2017 Philadelphia, USA; 2nd Autoimmunity Conference, Nov 9-10, 2017 Madrid, Spain; Integrating Metabolism and Immunity , May 29 - June 2, 2017 | Dublin, Ireland; American Academy of Allergy, Asthma & Immunology (AAAAI) Annual Meeting, March 03-06, 2017, Atlanta, Georgia

Track: 12Autoimmunity and Therapathies

Autoimmunityis the system ofimmuneresponsesof an organism against its own cells and tissues. Any disease that results from such an aberrantimmuneresponse is termed an autoimmune disease.

Autoimmunity is present to some extent in everyone and is usually harmless. However, autoimmunity can cause a broad range of human illnesses, known collectively as autoimmune diseases.Autoimmune diseasesoccur when there is progression from benign autoimmunity to pathogenic autoimmunity. This progression is determined by genetic influences as well as environmental triggers. Autoimmunity is evidenced by the presence of autoantibodies (antibodies directed against the person who produced them) and T cells that are reactive with host antigens.

Current treatments for allergic and autoimmune disease treat disease symptoms or depend on non-specific immune suppression. Treatment would be improved greatly by targeting the fundamental cause of the disease, that is the loss of tolerance to an otherwise innocuous antigen in allergy or self-antigen in autoimmune disease (AID). Much has been learned about the mechanisms of peripheral tolerance in recent years. We now appreciate that antigen presenting cells (APC) may be either immunogenic or tolerogenic, depending on their location, environmental cues and activation state

RelatedImmunology Conferences|Immunologists Meetings|Conference Series LLC:

3rdAntibodies and Bio Therapeutics Congress, November 02-03, 2017 Las Vegas, USA; Molecular Immunology & Immunogenetics Congress, March 20-21, 2017 Rome, Italy; Annual Meeting on Immunology and Immunologist, July 03-05, 2017 Malyasia, Kuala lumpur; 3rd International Congress on Neuroimmunology and Therapeutics, September 18-19, 2017 Philadelphia, USA; 2nd Autoimmunity Conference, Nov 9-10, 2017 Madrid, Spain; Integrating Metabolism and Immunity , May 29 - June 2, 2017 | Dublin, Ireland; American Academy of Allergy, Asthma & Immunology (AAAAI) Annual Meeting, March 03-06, 2017, Atlanta, Georgia

Track: 13DiagnosticImmunology

Diagnostic Immunology. Immunoassays are laboratory techniques based on the detection of antibody production in response to foreign antigens. Antibodies, part of the humoral immune response, are involved in pathogen detection and neutralization.

Diagnostic immunology has considerably advanced due to the development of automated methods.New technology takes into account saving samples, reagents, and reducing cost.The future of diagnosticimmunologyfaces challenges in the vaccination field for protection against HIV and asanti-cancer therapy. Modern immunology relies heavily on the use of antibodies as highly specific laboratory reagents. The diagnosis of infectious diseases, the successful outcome of transfusions and transplantations, and the availability of biochemical and hematologic assays with extraordinary specificity and sensitivity capabilities all attest to the value of antibody detection.Immunologic methods are used in the treatment and prevention ofinfectious diseasesand in the large number of immune-mediated diseases. Advances in diagnostic immunology are largely driven by instrumentation, automation, and the implementation of less complex and more standardized procedures.

Examples of such processes are as follows:

These methods have facilitated the performance of tests and have greatly expanded the information that can be developed by a clinical laboratory. The tests are now used for clinical diagnosis and the monitoring of therapies and patient responses. Immunology is a relatively young science and there is still so much to discover. Immunologists work in many different disease areas today that include allergy, autoimmunity, immunodeficiency, transplantation, and cancer.

RelatedImmunology Conferences|Immunologists Meetings|Conference Series LLC:

3nd International Congress on Neuroimmunology and Therapeutics, September 18-19, 2017 Philadelphia, USA; 18thInternational Conference on Immunology (ICI) Dec 12-13, 2016, Bangkok, Thailand; Annual Meeting on Immunology and Immunologist, July 03-05, 2017 Malyasia, Kuala lumpur; 19thInternational Conference on Immunology (ICI) Sept 14-17, 2017, Berlin, Germany; Modelling Viral Infections and Immunity (E1) , May 1 - 4, 2017 | Estes Park, Colorado, USA; 7thInternational Conference on Allergy, Asthma and Clinical Immunology; 18thInternational Conference on Immunology (ICI) Dec 12-13, 2016, Bangkok, Thailand

Track: 14Allergy and Therapathies

Although medications available for allergy are usually very effective, they do not cure people of allergies. Allergenimmunotherapyis the closest thing we have for a "cure" for allergy, reducing the severity of symptoms and the need for medication for many allergy sufferers. Allergen immunotherapy involves the regular administration of gradually increasing doses of allergen extracts over a period of years. Immunotherapy can be given to patients as an injection or as drops or tablets under the tongue (sublingual).Allergen immunotherapy changes the way the immune system reacts to allergens, by switching off allergy. The end result is that you become immune to the allergens, so that you can tolerate them with fewer or no symptoms. Allergen immunotherapy is not, however, a quick fix form of treatment. Those agreeing to allergen immunotherapy need to be committed to 3-5 years of treatment for it to work, and to cooperate with your doctor to minimize the frequency of side effects.Allergen immunotherapyis usually recommended for the treatment of potentially life threatening allergic reactions to stinging insects. Published data on allergen immunotherapy injections shows that venom immunotherapy can reduce the risk of a severe reaction in adults from around 60 % per sting, down to less than 10%. In Australia and New Zealand,venom immunotherapyis currently available for bee and wasp allergy. Jack Jumper Ant immunotherapy is available in Tasmania for Tasmanian residents. Allergen immunotherapy is often recommended for treatment ofallergic rhinitis

RelatedImmunology Conferences|Immunologists Meetings|Conference Series LLC:

Molecular Immunology & Immunogenetics Congress, March 20-21, 2017 Rome, Italy; 3nd International Congress on Neuroimmunology and Therapeutics, September 18-19, 2017 Philadelphia, USA; 18thInternational Conference on Immunology (ICI) Dec 12-13, 2016, Bangkok, Thailand; Annual Meeting on Immunology and Immunologist, July 03-05, 2017 Malyasia, Kuala lumpur; 19thInternational Conference on Immunology (ICI) Sept 14-17, 2017, Berlin, Germany; Modelling Viral Infections and Immunity (E1) , May 1 - 4, 2017 | Estes Park, Colorado, USA; 7thInternational Conference on Allergy, Asthma and Clinical Immunology; 18thInternational Conference on Immunology (ICI) Dec 12-13, 2016, Bangkok, Thailand

Track: 15Technological Innovations inImmunology

Immunology is the branch of biomedical sciences concerned with all aspects of the immune system in all multicellular organisms. Immunology deals with physiological functioning of the immune system in states of both health and disease as well as malfunctions of the immune system in immunological disorders like allergies, hypersensitivities, immune deficiency, transplant rejection andautoimmune disorders.

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9thworld congress & expo on Immunology, Oct 02-04, 2017, Toronto, Canada; 3rdAntibodies and Bio Therapeutics Congress, November 02-03, 2017 Las Vegas, USA; Molecular Immunology & Immunogenetics Congress, March 20-21, 2017 Rome, Italy; Annual Meeting on Immunology and Immunologist, July 03-05, 2017 Malyasia, Kuala lumpur; 3rd International Congress on Neuroimmunology and Therapeutics, September 18-19, 2017 Philadelphia, USA; 2nd Autoimmunity Conference, Nov 9-10, 2017 Madrid, Spain; Integrating Metabolism and Immunity , May 29 - June 2, 2017 | Dublin, Ireland; American Academy of Allergy, Asthma & Immunology (AAAAI) Annual Meeting, March 03-06, 2017, Atlanta, Georgia

Track:16Antigen Processing

Antigen processingis an immunologicalprocessthat prepares antigensfor presentation to special cells of the immune system called T lymphocytes. It is considered to be a stage ofantigenpresentation pathways. The process by which antigen-presenting cells digest proteins from inside or outside the cell and display the resulting antigenic peptide fragments on cell surface MHC molecules for recognition by T cells is central to the body's ability to detect signs of infection or abnormal cell growth. As such, understanding the processes and mechanisms of antigen processing and presentation provides us with crucial insights necessary for the design ofvaccines and therapeutic strategiesto bolster T-cell responses.

RelatedImmunology Conferences|Immunologists Meetings|Conference Series LLC:

3rdAntibodies and Bio Therapeutics Congress, November 02-03, 2017 Las Vegas, USA; Molecular Immunology & Immunogenetics Congress, March 20-21, 2017 Rome, Italy; Annual Meeting on Immunology and Immunologist, July 03-05, 2017 Malyasia, Kuala lumpur; 3rd International Congress on Neuroimmunology and Therapeutics, September 18-19, 2017 Philadelphia, USA; 2nd Autoimmunity Conference, Nov 9-10, 2017 Madrid, Spain; Integrating Metabolism and Immunity , May 29 - June 2, 2017 | Dublin, Ireland; American Academy of Allergy, Asthma & Immunology (AAAAI) Annual Meeting, March 03-06, 2017, Atlanta, Georgia

Track: 17Immunoinformatics and Systems Immunology

Immunoinformaticsis a branch ofbioinformaticsdealing with in silico analysis and modelling of immunological data and problems Immunoinformatics includes the study and design of algorithms for mapping potential B- andT-cell epitopes, which lessens the time and cost required for laboratory analysis of pathogen gene products. Using this information, an immunologist can explore the potential binding sites, which, in turn, leads to the development of newvaccines. This methodology is termed reversevaccinology and it analyses the pathogen genome to identify potential antigenic proteins.This is advantageous because conventional methods need to cultivate pathogen and then extract its antigenic proteins. Although pathogens grow fast, extraction of their proteins and then testing of those proteins on a large scale is expensive and time consuming. Immunoinformatics is capable of identifying virulence genes and surface-associated proteins.

RelatedImmunology Conferences|Immunologists Meetings|Conference Series LLC:

9thworld congress & expo on Immunology, Oct 02-04, 2017, Toronto, Canada; 3rdAntibodies and Bio Therapeutics Congress, November 02-03, 2017 Las Vegas, USA; Molecular Immunology & Immunogenetics Congress, March 20-21, 2017 Rome, Italy; 3nd International Congress on Neuroimmunology and Therapeutics, September 18-19, 2017 Philadelphia, USA; 18th International Conference on Immunology (ICI) Dec 12-13, 2016, Bangkok, Thailand; Annual Meeting on Immunology and Immunologist, July 03-05, 2017 Malyasia, Kuala lumpur; British Society for Immunology Congress, Dec 06-09, 2016, Liverpool, United Kingdom; 7thInternational Conference on Allergy, Asthma and Clinical Immunology; Cancer Immunology and Immunotherapy: Taking a Place in Mainstream Oncology (C7), March 19 - 23, 2017, Whistler, British Columbia, Canada

Track: 18Rheumatology

Rheumatology represents a subspecialty in internal medicine and pediatrics, which is devoted to adequate diagnosis andtherapy of rheumatic diseases(including clinical problems in joints, soft tissues, heritable connective tissue disorders, vasculitis and autoimmune diseases). This field is multidisciplinary in nature, which means it relies on close relationships with other medical specialties.The specialty of rheumatology has undergone a myriad of noteworthy advances in recent years, especially if we consider the development of state-of-the-art biological drugs with novel targets, made possible by rapid advances in the basic science of musculoskeletal diseases and improved imaging techniques.

RelatedImmunology Conferences|Immunologists Meetings|Conference Series LLC:

Molecular Immunology & Immunogenetics Congress, March 20-21, 2017 Rome, Italy; 3nd International Congress on Neuroimmunology and Therapeutics, September 18-19, 2017 Philadelphia, USA; 18thInternational Conference on Immunology (ICI) Dec 12-13, 2016, Bangkok, Thailand; Annual Meeting on Immunology and Immunologist, July 03-05, 2017 Malyasia, Kuala lumpur; 19thInternational Conference on Immunology (ICI) Sept 14-17, 2017, Berlin, Germany; Modelling Viral Infections and Immunity (E1) , May 1 - 4, 2017 | Estes Park, Colorado, USA; 7thInternational Conference on Allergy, Asthma and Clinical Immunology; 18thInternational Conference on Immunology (ICI) Dec 12-13, 2016, Bangkok, Thailand

Track: 19Nutritional Immunology

Nutritional immunologyis an emerging discipline that evolved with the study of the detrimental effect of malnutrition on the immune system. The clinical and public health importance of nutritional immunology is also receiving attention. Immune system dysfunctions that result from malnutrition are, in fact, NutritionallyAcquired Immune Deficiency Syndromes(NAIDS). NAIDS afflicts millions of people in the Third World, as well as thousands in modern centers, i.e., patients with cachexia secondary to serious disease, neoplasia or trauma. The human immune system functions to protect the body against foreign pathogens and thereby preventing infection and disease. Optimal functioning of the immune system, both innate and adaptive immunity, is strongly influenced by an individuals nutritional status, with malnutrition being the most common cause of immunodeficiency in the world. Nutrient deficiencies result in immunosuppression and dysregulation of the immune response including impairment of phagocyte function and cytokine production, as well as adversely affecting aspects of humoral and cell-mediated immunity. Such alterations in immune function and the resulting inflammation are not only associated with infection, but also with the development of chronic diseases including cancer, autoimmune disease, osteoporosis, disorders of the endocrine system andcardiovascular disease.

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8 th European Immunology Conference June 29-July 01, 2017 ...

Bone Marrow Stem Cells Comments Off on 8 th European Immunology Conference June 29-July 01, 2017 … | January 11th, 2017

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The Journal of Cell Science & Therapy is an Open Access, peer-reviewed, academic journal with a wide range of fields within the discipline creates a platform for the authors to publish their comprehensive and most reliable source of information on the discoveries and current developments in the mode of original articles, review articles, case reports, short communications, etc, making them freely available through online without any restrictions or any other subscriptions to researchers worldwide.

The journal is using Editorial Manager System for quality in peer review process. Editorial Manager is an online manuscript submission, review and tracking systems. Review processing is performed by the editorial board members of Journal of Cell Science & Therapy or outside experts; at least two independent reviewers approval followed by editor approval is required for acceptance of any citable manuscript. Authors may submit manuscripts and track their progress through the system, hopefully to publication. Reviewers can download manuscripts and submit their opinions to the editor. Editors can manage the whole submission/review/revise/publish process.

Journal of Cell Science & Therapy is a peer reviewed scientific journal known for rapid dissemination of high-quality research. This Cell Science journal with highest impact factor offers an Open Access platform to the authors in academia and industry to publish their novel research. It serves the International Scientific Community with its standard research publications.

Cells are small compartments that hold the biological equipment necessary to keep an organism alive and successful. Living things may be unicellular or multicellular such as a human being. According to cell theory, cells are the fundamental unit of structure and function in all living organisms and come from preexisting cells, and that all cells contain the hereditary information necessary for regulating cell functions and for transmitting information to the next generation of cells.

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The cytokines produced by expression from suitable cloning vectors containing the desired cytokine gene, can be expressed in yeast (Saccharomyces cerevisiae expression system), bacteria (Escherichia coli expression system), mammalian cells (BHK, CHO, COS, Namalwa), or insect cell systems. Cytokines are designed for demanding applications such as cell culture, differentiation studies, and functional assays mainly in the fields of immunology, neurology, and stem cell research.

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Hematology is the investigation of blood, the blood-framing organs, and blood diseases in which the specialists deal with the diagnosis, treatment and overall management of people with blood disorders ranging from anemia to blood cancer. Some of the diseases treated by haematologists include Iron deficiency anaemia, Sickle cell anemia, Polycythemia or excess production of red blood cells, Myelofibrosis, Leukemia, hemophilia, myelodysplastic syndromes, Malignant lymphomas, Blood transfusion and bone marrow stem cell transplantation

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Cell biology (cytology) is a branch of biology that studies cells their physiological properties, their structure, the organelles they contain, interactions with their environment, their life cycle, division, death and cell function. Research in cell biology is closely related to genetics, biochemistry, molecular biology, immunology, and developmental biology.

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A hair follicle is part of the skin that grows hair by packing old cells together. Attached to the follicle is a sebaceous gland, a tiny sebum-producing gland found everywhere except on the palms, lips and soles of the feet. The follicle cells that extrude hairs from just below the surface of the skin are simply too hard to bring back to life, and even preventative therapies didnt seem to be able to do much to keep them alive. But research on inducing stem cells to grow into follicle cells could change that forever.

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Mesenchymal stem cells (MSCs), the major stem cells for cell therapy. From animal models to clinical trials, MSCs have afforded promise in the treatment of numerous diseases, mainly tissue injury and immune disorders. Cell sources for MSC administration in clinical applications, and provide an overview of mechanisms that are significant in MSC-mediated therapies. Although MSCs for cell therapy have been shown to be safe and effective, there are still challenges that need to be tackled before their wide application in the clinical research field.

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The external effects of degenerative processes inside the body which manifest especially in the face, hands, dcollet, and by hair loss are also psychically stressful. There are promising therapeutic approaches with stem cells and growth factors for both skin regeneration and hair growth regeneration. To dispense with hair transplants and surgical procedures such as facelifts and eyelid correction, in which the skin is pulled back and the excess tissue is excised. To treat the root cause and restore lost volume in a tissue-conserving, natural manner and regenerate both the subcutaneous tissue and the skin.

Related Journals of Skin Cell Therapy

Single Cell Biology, Genetic Syndromes & Gene Therapy, Cell Science & Therapy, Cell Biology: Research & Therapy, Journal of immunotherapy, Photo-dermatology, Case Reports in Dermatology, Current Stem Cell Research and Therapy, Dermatologic Therapy

Somatic cell therapy is viewed as a more conservative, safer approach because it affects only the targeted cells in the patient, and is not passed on to future generations. Somatic gene therapy represents mainstream basic and clinical research, in which therapeutic DNA (either integrated in the genome or as an external episome or plasmid) is used to treat disease. Most focus on severe genetic disorders, including immunodeficiencies, haemophilia, thalassaemia and cystic fibrosis. Such single gene disorders are good candidates for somatic cell therapy.

Related Journals of Somatic Cell Therapy

Cell Science & Therapy, Insights in Cell Science, Cellular and Molecular Biology, Cell Biology: Research & Therapy, Hematology/Oncology and Stem Cell Therapy, Journal of Cosmetic and Laser Therapy, Cancer Biology and Therapy, Cancer Gene Therapy, Cytotherapy

Rejuvenation and regeneration are two key processes that define cell therapy. Cellular Therapy is a form of non-toxic, holistic medicine in which the entire organism is being treated. Cellular Therapies are an integral part of complimentary treatment regimens. They are extremely versatile and can be used for a wide range of disorders.

Related Journals of Live Cell Therapy

Cell & Developmental Biology, Archives in Cancer Research,Cancer Clinical Trials, Cancer Science & Therapy, Cancer Biology and Therapy, Cancer Gene Therapy, Cytotherapy, Journal of Cancer Science and Therapy, Stem Cell Research and Therapy

Dendritic cells (DCs) cells are the most potent antigen-producing cells, represent unique antigen-producing cells capable of sensitizing T cells to both new and recall antigens. Dendritic Cell Vaccines, or Dendritic cell therapy, is another Alternative Cancer Therapy or newly emerging and potent form of immune therapy used to treat cancer.

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Clinical & Experimental Neuroimmunology, Immunochemistry & Immunopathology: Open Access, Clinical & Cellular Immunology, Immunooncology, Dendrobiology, Genes and Cancer, International Journal of Cancer, Journal of Cancer Science and Therapy, Molecular Cancer Research, Molecular Cancer Therapeutics

The cells are most commonly immune-derived, with the goal of transferring immune functionality and characteristics along with the cells. Transferring autologous cells minimizes GVHD issues. The adaptive transfer of autologous tumor infiltrating lymphocytes (TIL) or genetically re-directed peripheral blood mononuclear cells has been used to treat patients with advanced solid tumors, including melanoma and colorectal carcinoma, as well as patients with CD19-expressing hematologic malignancies. As of 2015 the technique had expanded to treat cervical cancer, lymphoma, leukemia, bile duct cancer and neuroblastoma.

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Cell Signaling, Cellular & Molecular Pathology, Cell Biology: Research & Therapy, Stem Cell Research & Therapy, Antiviral Chemistry and Chemotherapy, Cancer Biotherapy and Radiopharmaceuticals, Cancer Biology and Therapy, Cytotherapy, Japanese Journal of Cancer and Chemotherapy, Oncology and Stem Cell Therapy

The ability to convert one cell type into another has caused great excitement in the stem cell field. iPS Reprogramming and transdifferentiation are the two approaches which makes cells in to another type of cells. In iPS procedure, it make possible to convert essentially any cell type in the body back into pluripotent stem cells that are almost identical to embryonic stem cells. And another approach uses transcription factors to convert a given cell type directly into another specialized cell type, without first forcing the cells to go back to a pluripotent state.

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Cell Science & Therapy, Cell Biology: Research & Therapy, Insights in Cell Science, Cellular and Molecular Biology, Hematology/Oncology and Stem Cell Therapy, Annals of Cancer Research and Therapy, Annals of Cancer Research and Therapy

Autologous stem cell transplants are done using peripheral blood stem cell transplantation (PBSCT). With PBSCT, the stem cells are taken from blood. The growth factor G-CSF may be used to stimulate the growth of new stem cells so they spill over into the blood.

Related Journals of Autologous Cell

Cellular and Molecular Biology, Single Cell Biology, Molecular Biology, Stem Cell Research & Therapy, Insights in Stem Cells, Current Stem Cell Research and Therapy, Journal of Stem Cells, Journal of Stem Cells and Regenerative Medicine, Stem Cell Research, Stem Cell Research and Therapy, Stem Cells

Advance Cell & Gene Thearpy practical, experienced guidance in development, GMP/GTP manufacturing, and regulatory compliance, as well as comprehensive scientific and technical strategic analysis of business opportunities in cell therapy, gene therapy and tissue therapies.

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Cell Science & Therapy, Insights in Cell Science, Cell Biology: Research & Therapy, Cytology & Histology, Archives of Surgical Oncology, Annals of Cancer Research and Therapy, Cancer Biology and Therapy, Oncology and Stem Cell Therapy, Chinese Journal of Cancer Biotherapy, Current Cancer Therapy Reviews

Immunotherapy involves engineering patients own immune cells to recognize and attack their tumors. And although this approach, called adoptive cell transfer (ACT), has been restricted to small clinical trials so far, treatments using these engineered immune cells have generated some remarkable responses in patients with advanced cancer. .Adoptive T cell therapy for cancer is a form of transfusion therapy consisting of the infusion of various mature T cell subsets with the goal of eliminating a tumor and preventing its recurrence.

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Clinical & Cellular Immunology, Immunooncology, Molecular Immunology, Advances in Cancer Prevention, Cytotherapy, Journal of Acquired Immune Deficiency Syndromes, Advances in Neuroimmune Biology, Cancer Biology and Therapy, Cancer Immunology, Immunotherapy

Commercialization of the first cell-based therapeutics, including cartilage repair products; tissue-engineered skin; and the first personalized, cellular immunotherapy for cancer. Production, storage, and delivery of living cell-based pharmaceuticals presents several unique challenges. Novel, innovative technologies and strategies will be required to bring cell therapies to commercial success.

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Bioprocessing & Biotechniques, Cytology & Histology, Cell Biology: Research & Therapy , Molecular Biology, BioProcess International, Biotechnology and Bioprocess Engineering, Food and Bioprocess Technology, Industrial Bioprocessing

Cellular therapy products include cellular immunotherapies, and other types of both autologous and allogeneic cells for certain therapeutic indications, including adult and embryonic stem cells. Human gene therapy refers to products that introduce genetic material into a persons DNA to replace faulty or missing genetic material, thus treating a disease or abnormal medical condition.

Related Journals of Cell Therapy Products

Pharmacognosy & Natural Products, Natural Products Chemistry & Research, Stem Cell Research & Therapy, Cell Science & Therapy, Surgical Products, International Journal of Applied Research in Natural Products, Molecular Diagnosis and Therapy, Molecular Therapy, Molecular Therapy - Nucleic Acids

Journal of Cell Science and Therapy is associated with our international conference "6th World Congrss on Cell & Stem Cell Research" during Feb 29- March 2, 2016 Philadelphia, USA with a theme "Novel Therapies in Cell Science and Stem Cell Research. Stem Cell Therapy-2016 will encompass recent researches and findings in stem cell technologies, stem cell therapies and transplantations, current understanding of cell plasticity in cancer and other advancements in stem cell research and cell science.

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Cell Science & Therapy - omicsonline.org

IPS Cell Therapy Comments Off on Cell Science & Therapy – omicsonline.org | January 10th, 2017

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Neurology - Spinal Cord Introduction - YouTube

Spinal Cord Stem Cells Comments Off on Neurology – Spinal Cord Introduction – YouTube | January 6th, 2017

In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. This process occurs in all living organisms and is the basis for biological inheritance. DNA is made up of a double helix of two complementary strands. During replication, these strands are separated. Each strand of the original DNA molecule then serves as a template for the production of its counterpart, a process referred to as semiconservative replication. Cellular proofreading and error-checking mechanisms ensure near perfect fidelity for DNA replication.[1][2]

In a cell, DNA replication begins at specific locations, or origins of replication, in the genome.[3] Unwinding of DNA at the origin and synthesis of new strands results in replication forks growing bi-directionally from the origin. A number of proteins are associated with the replication fork to help in the initiation and continuation of DNA synthesis. Most prominently, DNA polymerase synthesizes the new strands by adding nucleotides that complement each (template) strand. DNA replication occurs during the S-stage of interphase.

DNA replication can also be performed in vitro (artificially, outside a cell). DNA polymerases isolated from cells and artificial DNA primers can be used to initiate DNA synthesis at known sequences in a template DNA molecule. The polymerase chain reaction (PCR), a common laboratory technique, cyclically applies such artificial synthesis to amplify a specific target DNA fragment from a pool of DNA.

DNA usually exists as a double-stranded structure, with both strands coiled together to form the characteristic double-helix. Each single strand of DNA is a chain of four types of nucleotides. Nucleotides in DNA contain a deoxyribose sugar, a phosphate, and a nucleobase. The four types of nucleotide correspond to the four nucleobases adenine, cytosine, guanine, and thymine, commonly abbreviated as A,C, G and T. Adenine and guanine are purine bases, while cytosine and thymine are pyrimidines. These nucleotides form phosphodiester bonds, creating the phosphate-deoxyribose backbone of the DNA double helix with the nuclei bases pointing inward (i.e., toward the opposing strand). Nucleotides (bases) are matched between strands through hydrogen bonds to form base pairs. Adenine pairs with thymine (two hydrogen bonds), and guanine pairs with cytosine (stronger: three hydrogen bonds).

DNA strands have a directionality, and the different ends of a single strand are called the 3 (three-prime) end and the 5 (five-prime) end. By convention, if the base sequence of a single strand of DNA is given, the left end of the sequence is the 5 end, while the right end of the sequence is the 3 end. The strands of the double helix are anti-parallel with one being 5 to 3, and the opposite strand 3 to 5. These terms refer to the carbon atom in deoxyribose to which the next phosphate in the chain attaches. Directionality has consequences in DNA synthesis, because DNA polymerase can synthesize DNA in only one direction by adding nucleotides to the 3 end of a DNA strand.

The pairing of complementary bases in DNA (through hydrogen bonding) means that the information contained within each strand is redundant. Phosphodiester (intra-strand) bonds are stronger than hydrogen (inter-strand) bonds. This allows the strands to be separated from one another. The nucleotides on a single strand can therefore be used to reconstruct nucleotides on a newly synthesized partner strand.[4]

DNA polymerases are a family of enzymes that carry out all forms of DNA replication.[6] DNA polymerases in general cannot initiate synthesis of new strands, but can only extend an existing DNA or RNA strand paired with a template strand. To begin synthesis, a short fragment of RNA, called a primer, must be created and paired with the template DNA strand.

DNA polymerase adds a new strand of DNA by extending the 3 end of an existing nucleotide chain, adding new nucleotides matched to the template strand one at a time via the creation of phosphodiester bonds. The energy for this process of DNA polymerization comes from hydrolysis of the high-energy phosphate (phosphoanhydride) bonds between the three phosphates attached to each unincorporated base. Free bases with their attached phosphate groups are called nucleotides; in particular, bases with three attached phosphate groups are called nucleoside triphosphates. When a nucleotide is being added to a growing DNA strand, the formation of a phosphodiester bond between the proximal phosphate of the nucleotide to the growing chain is accompanied by hydrolysis of a high-energy phosphate bond with release of the two distal phosphates as a pyrophosphate. Enzymatic hydrolysis of the resulting pyrophosphate into inorganic phosphate consumes a second high-energy phosphate bond and renders the reaction effectively irreversible.[Note 1]

In general, DNA polymerases are highly accurate, with an intrinsic error rate of less than one mistake for every 107 nucleotides added.[7] In addition, some DNA polymerases also have proofreading ability; they can remove nucleotides from the end of a growing strand in order to correct mismatched bases. Finally, post-replication mismatch repair mechanisms monitor the DNA for errors, being capable of distinguishing mismatches in the newly synthesized DNA strand from the original strand sequence. Together, these three discrimination steps enable replication fidelity of less than one mistake for every 109 nucleotides added.[7]

The rate of DNA replication in a living cell was first measured as the rate of phage T4 DNA elongation in phage-infected E. coli.[8] During the period of exponential DNA increase at 37C, the rate was 749 nucleotides per second. The mutation rate per base pair per replication during phage T4 DNA synthesis is 1.7 per 108.[9]

DNA replication, like all biological polymerization processes, proceeds in three enzymatically catalyzed and coordinated steps: initiation, elongation and termination.

For a cell to divide, it must first replicate its DNA.[10] This process is initiated at particular points in the DNA, known as origins, which are targeted by initiator proteins.[3] In E. coli this protein is DnaA; in yeast, this is the origin recognition complex.[11] Sequences used by initiator proteins tend to be AT-rich (rich in adenine and thymine bases), because A-T base pairs have two hydrogen bonds (rather than the three formed in a C-G pair) and thus are easier to strand separate.[12] Once the origin has been located, these initiators recruit other proteins and form the pre-replication complex, which unzips the double-stranded DNA.

DNA polymerase has 5-3 activity. All known DNA replication systems require a free 3 hydroxyl group before synthesis can be initiated (note: the DNA template is read in 3 to 5 direction whereas a new strand is synthesized in the 5 to 3 directionthis is often confused). Four distinct mechanisms for DNA synthesis are recognized:

The first is the best known of these mechanisms and is used by the cellular organisms. In this mechanism, once the two strands are separated, primase adds RNA primers to the template strands. The leading strand receives one RNA primer while the lagging strand receives several. The leading strand is continuously extended from the primer by a DNA polymerase with high processivity, while the lagging strand is extended discontinuously from each primer forming Okazaki fragments. RNase removes the primer RNA fragments, and a low processivity DNA polymerase distinct from the replicative polymerase enters to fill the gaps. When this is complete, a single nick on the leading strand and several nicks on the lagging strand can be found. Ligase works to fill these nicks in, thus completing the newly replicated DNA molecule.

The primase used in this process differs significantly between bacteria and archaea/eukaryotes. Bacteria use a primase belonging to the DnaG protein superfamily which contains a catalytic domain of the TOPRIM fold type.[13] The TOPRIM fold contains an / core with four conserved strands in a Rossmann-like topology. This structure is also found in the catalytic domains of topoisomerase Ia, topoisomerase II, the OLD-family nucleases and DNA repair proteins related to the RecR protein.

The primase used by archaea and eukaryotes, in contrast, contains a highly derived version of the RNA recognition motif (RRM). This primase is structurally similar to many viral RNA-dependent RNA polymerases, reverse transcriptases, cyclic nucleotide generating cyclases and DNA polymerases of the A/B/Y families that are involved in DNA replication and repair. In eukaryotic replication, the primase forms a complex with Pol .[14]

Multiple DNA polymerases take on different roles in the DNA replication process. In E. coli, DNA Pol III is the polymerase enzyme primarily responsible for DNA replication. It assembles into a replication complex at the replication fork that exhibits extremely high processivity, remaining intact for the entire replication cycle. In contrast, DNA Pol I is the enzyme responsible for replacing RNA primers with DNA. DNA Pol I has a 5 to 3 exonuclease activity in addition to its polymerase activity, and uses its exonuclease activity to degrade the RNA primers ahead of it as it extends the DNA strand behind it, in a process called nick translation. Pol I is much less processive than Pol III because its primary function in DNA replication is to create many short DNA regions rather than a few very long regions.

In eukaryotes, the low-processivity enzyme, Pol , helps to initiate replication because it forms a complex with primase.[15] In eukaryotes, leading strand synthesis is thought to be conducted by Pol ; however, this view has recently been challenged, suggesting a role for Pol .[16] Primer removal is completed Pol [17] while repair of DNA during replication is completed by Pol .

As DNA synthesis continues, the original DNA strands continue to unwind on each side of the bubble, forming a replication fork with two prongs. In bacteria, which have a single origin of replication on their circular chromosome, this process creates a theta structure (resembling the Greek letter theta: ). In contrast, eukaryotes have longer linear chromosomes and initiate replication at multiple origins within these.>[18]

The replication fork is a structure that forms within the nucleus during DNA replication. It is created by helicases, which break the hydrogen bonds holding the two DNA strands together. The resulting structure has two branching prongs, each one made up of a single strand of DNA. These two strands serve as the template for the leading and lagging strands, which will be created as DNA polymerase matches complementary nucleotides to the templates; the templates may be properly referred to as the leading strand template and the lagging strand template.

DNA is always synthesized in the 5 to 3 direction. Since the leading and lagging strand templates are oriented in opposite directions at the replication fork, a major issue is how to achieve synthesis of nascent (new) lagging strand DNA, whose direction of synthesis is opposite to the direction of the growing replication fork.

The leading strand is the strand of nascent DNA which is being synthesized in the same direction as the growing replication fork. A polymerase reads the leading strand template and adds complementary nucleotides to the nascent leading strand on a continuous basis.

The lagging strand is the strand of nascent DNA whose direction of synthesis is opposite to the direction of the growing replication fork. Because of its orientation, replication of the lagging strand is more complicated as compared to that of the leading strand. As a consequence, the DNA polymerase on this strand is seen to lag behind the other strand.

The lagging strand is synthesized in short, separated segments. On the lagging strand template, a primase reads the template DNA and initiates synthesis of a short complementary RNA primer. A DNA polymerase extends the primed segments, forming Okazaki fragments. The RNA primers are then removed and replaced with DNA, and the fragments of DNA are joined together by DNA ligase.

As helicase unwinds DNA at the replication fork, the DNA ahead is forced to rotate. This process results in a build-up of twists in the DNA ahead.[19] This build-up forms a torsional resistance that would eventually halt the progress of the replication fork. Topoisomerases are enzymes that temporarily break the strands of DNA, relieving the tension caused by unwinding the two strands of the DNA helix; topoisomerases (including DNA gyrase) achieve this by adding negative supercoils to the DNA helix.[20]

Bare single-stranded DNA tends to fold back on itself forming secondary structures; these structures can interfere with the movement of DNA polymerase. To prevent this, single-strand binding proteins bind to the DNA until a second strand is synthesized, preventing secondary structure formation.[21]

Clamp proteins form a sliding clamp around DNA, helping the DNA polymerase maintain contact with its template, thereby assisting with processivity. The inner face of the clamp enables DNA to be threaded through it. Once the polymerase reaches the end of the template or detects double-stranded DNA, the sliding clamp undergoes a conformational change that releases the DNA polymerase. Clamp-loading proteins are used to initially load the clamp, recognizing the junction between template and RNA primers.[2]:274-5

At the replication fork, many replication enzymes assemble on the DNA into a complex molecular machine called the replisome. The following is a list of major DNA replication enzymes that participate in the replisome:[22]

Replication machineries consist of factors involved in DNA replication and appearing on template ssDNAs. Replication machineries include primosotors are replication enzymes; DNA polymerase, DNA helicases, DNA clamps and DNA topoisomerases, and replication proteins; e.g. single-stranded DNA binding proteins (SSB). In the replication machineries these components coordinate. In most of the bacteria, all of the factors involved in DNA replication are located on replication forks and the complexes stay on the forks during DNA replication. These replication machineries are called replisomes or DNA replicase systems. These terms are generic terms for proteins located on replication forks. In eukaryotic and some bacterial cells the replisomes are not formed.

Since replication machineries do not move relatively to template DNAs such as factories, they are called a replication factory.[24] In an alternative figure, DNA factories are similar to projectors and DNAs are like as cinematic films passing constantly into the projectors. In the replication factory model, after both DNA helicases for leading stands and lagging strands are loaded on the template DNAs, the helicases run along the DNAs into each other. The helicases remain associated for the remainder of replication process. Peter Meister et al. observed directly replication sites in budding yeast by monitoring green fluorescent protein(GFP)-tagged DNA polymerases . They detected DNA replication of pairs of the tagged loci spaced apart symmetrically from a replication origin and found that the distance between the pairs decreased markedly by time.[25] This finding suggests that the mechanism of DNA replication goes with DNA factories. That is, couples of replication factories are loaded on replication origins and the factories associated with each other. Also, template DNAs move into the factories, which bring extrusion of the template ssDNAs and nascent DNAs. Meisters finding is the first direct evidence of replication factory model. Subsequent research has shown that DNA helicases form dimers in many eukaryotic cells and bacterial replication machineries stay in single intranuclear location during DNA synthesis.[24]

The replication factories perform disentanglement of sister chromatids. The disentanglement is essential for distributing the chromatids into daughter cells after DNA replication. Because sister chromatids after DNA replication hold each other by Cohesin rings, there is the only chance for the disentanglement in DNA replication. Fixing of replication machineries as replication factories can improve the success rate of DNA replication. If replication forks move freely in chromosomes, catenation of nuclei is aggravated and impedes mitotic segregation.[25]

Eukaryotes initiate DNA replication at multiple points in the chromosome, so replication forks meet and terminate at many points in the chromosome; these are not known to be regulated in any particular way. Because eukaryotes have linear chromosomes, DNA replication is unable to reach the very end of the chromosomes, but ends at the telomere region of repetitive DNA close to the ends. This shortens the telomere of the daughter DNA strand. Shortening of the telomeres is a normal process in somatic cells. As a result, cells can only divide a certain number of times before the DNA loss prevents further division. (This is known as the Hayflick limit.) Within the germ cell line, which passes DNA to the next generation, telomerase extends the repetitive sequences of the telomere region to prevent degradation. Telomerase can become mistakenly active in somatic cells, sometimes leading to cancer formation. Increased telomerase activity is one of the hallmarks of cancer.

Termination requires that the progress of the DNA replication fork must stop or be blocked. Termination at a specific locus, when it occurs, involves the interaction between two components: (1) a termination site sequence in the DNA, and (2) a protein which binds to this sequence to physically stop DNA replication. In various bacterial species, this is named the DNA replication terminus site-binding protein, or Ter protein.

Because bacteria have circular chromosomes, termination of replication occurs when the two replication forks meet each other on the opposite end of the parental chromosome. E. coli regulates this process through the use of termination sequences that, when bound by the Tus protein, enable only one direction of replication fork to pass through. As a result, the replication forks are constrained to always meet within the termination region of the chromosome.[26]

Within eukaryotes, DNA replication is controlled within the context of the cell cycle. As the cell grows and divides, it progresses through stages in the cell cycle; DNA replication takes place during the S phase (synthesis phase). The progress of the eukaryotic cell through the cycle is controlled by cell cycle checkpoints. Progression through checkpoints is controlled through complex interactions between various proteins, including cyclins and cyclin-dependent kinases.[27] Unlike bacteria, eukaryotic DNA replicates in the confines of the nucleus.[28]

The G1/S checkpoint (or restriction checkpoint) regulates whether eukaryotic cells enter the process of DNA replication and subsequent division. Cells that do not proceed through this checkpoint remain in the G0 stage and do not replicate their DNA.

Replication of chloroplast and mitochondrial genomes occurs independently of the cell cycle, through the process of D-loop replication.

In vertebrate cells, replication sites concentrate into positions called replication foci.[25] Replication sites can be detected by immunostaining daughter strands and replication enzymes and monitoring GFP-tagged replication factors. By these methods it is found that replication foci of varying size and positions appear in S phase of cell division and their number per nucleus is far smaller than the number of genomic replication forks.

P. Heun et al.(2001) tracked GFP-tagged replication foci in budding yeast cells and revealed that replication origins move constantly in G1 and S phase and the dynamics decreased significantly in S phase.[25] Traditionally, replication sites were fixed on spatial structure of chromosomes by nuclear matrix or lamins. The Heuns results denied the traditional concepts, budding yeasts dont have lamins, and support that replication origins self-assemble and form replication foci.

By firing of replication origins, controlled spatially and temporally, the formation of replication foci is regulated. D. A. Jackson et al.(1998) revealed that neighboring origins fire simultaneously in mammalian cells.[25] Spatial juxtaposition of replication sites brings clustering of replication forks. The clustering do rescue of stalled replication forks and favors normal progress of replication forks. Progress of replication forks is inhibited by many factors; collision with proteins or with complexes binding strongly on DNA, deficiency of dNTPs, nicks on template DNAs and so on. If replication forks stall and the remaining sequences from the stalled forks are not replicated, the daughter strands have nick obtained un-replicated sites. The un-replicated sites on one parents strand hold the other strand together but not daughter strands. Therefore, the resulting sister chromatids cannot separate from each other and cannot divide into 2 daughter cells. When neighboring origins fire and a fork from one origin is stalled, fork from other origin access on an opposite direction of the stalled fork and duplicate the un-replicated sites. As other mechanism of the rescue there is application of dormant replication origins that excess origins dont fire in normal DNA replication.

Most bacteria do not go through a well-defined cell cycle but instead continuously copy their DNA; during rapid growth, this can result in the concurrent occurrence of multiple rounds of replication.[29] In E. coli, the best-characterized bacteria, DNA replication is regulated through several mechanisms, including: the hemimethylation and sequestering of the origin sequence, the ratio of adenosine triphosphate (ATP) to adenosine diphosphate (ADP), and the levels of protein DnaA. All these control the binding of initiator proteins to the origin sequences.

Because E. coli methylates GATC DNA sequences, DNA synthesis results in hemimethylated sequences. This hemimethylated DNA is recognized by the protein SeqA, which binds and sequesters the origin sequence; in addition, DnaA (required for initiation of replication) binds less well to hemimethylated DNA. As a result, newly replicated origins are prevented from immediately initiating another round of DNA replication.[30]

ATP builds up when the cell is in a rich medium, triggering DNA replication once the cell has reached a specific size. ATP competes with ADP to bind to DnaA, and the DnaA-ATP complex is able to initiate replication. A certain number of DnaA proteins are also required for DNA replication each time the origin is copied, the number of binding sites for DnaA doubles, requiring the synthesis of more DnaA to enable another initiation of replication.

Researchers commonly replicate DNA in vitro using the polymerase chain reaction (PCR). PCR uses a pair of primers to span a target region in template DNA, and then polymerizes partner strands in each direction from these primers using a thermostable DNA polymerase. Repeating this process through multiple cycles amplifies the targeted DNA region. At the start of each cycle, the mixture of template and primers is heated, separating the newly synthesized molecule and template. Then, as the mixture cools, both of these become templates for annealing of new primers, and the polymerase extends from these. As a result, the number of copies of the target region doubles each round, increasing exponentially.[31]

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DNA replication Wikipedia IPS Cell Therapy IPS Cell ...

IPS Cell Therapy Comments Off on DNA replication Wikipedia IPS Cell Therapy IPS Cell … | December 17th, 2016

Induced stem cells (iSC) are stem cells derived from somatic, reproductive, pluripotent or other cell types by deliberate epigenetic reprogramming. They are classified as either totipotent (iTC), pluripotent (iPSC) or progenitor (multipotentiMSC, also called an induced multipotent progenitor celliMPC) or unipotent(iUSC) according to their developmental potential and degree of dedifferentiation. Progenitors are obtained by so-called direct reprogramming or directed differentiation and are also called induced somatic stem cells.

Three techniques are widely recognized:[1]

In 1895 Thomas Morgan removed one of a frog's two blastomeres and found that amphibians are able to form whole embryos from the remaining part. This meant that the cells can change their differentiation pathway. In 1924 Spemann and Mangold demonstrated the key importance of cellcell inductions during animal development.[20] The reversible transformation of cells of one differentiated cell type to another is called metaplasia.[21] This transition can be a part of the normal maturation process, or caused by an inducement.

One example is the transformation of iris cells to lens cells in the process of maturation and transformation of retinal pigment epithelium cells into the neural retina during regeneration in adult newt eyes. This process allows the body to replace cells not suitable to new conditions with more suitable new cells. In Drosophila imaginal discs, cells have to choose from a limited number of standard discrete differentiation states. The fact that transdetermination (change of the path of differentiation) often occurs for a group of cells rather than single cells shows that it is induced rather than part of maturation.[22]

The researchers were able to identify the minimal conditions and factors that would be sufficient for starting the cascade of molecular and cellular processes to instruct pluripotent cells to organize the embryo. They showed that opposing gradients of bone morphogenetic protein (BMP) and Nodal, two transforming growth factor family members that act as morphogens, are sufficient to induce molecular and cellular mechanisms required to organize, in vivo or in vitro, uncommitted cells of the zebrafish blastula animal pole into a well-developed embryo.[23]

Some types of mature, specialized adult cells can naturally revert to stem cells. For example, "chief" cells express the stem cell marker Troy. While they normally produce digestive fluids for the stomach, they can revert into stem cells to make temporary repairs to stomach injuries, such as a cut or damage from infection. Moreover, they can make this transition even in the absence of noticeable injuries and are capable of replenishing entire gastric units, in essence serving as quiescent "reserve" stem cells.[24] Differentiated airway epithelial cells can revert into stable and functional stem cells in vivo.[25]

After injury, mature terminally differentiated kidney cells dedifferentiate into more primordial versions of themselves and then differentiate into the cell types needing replacement in the damaged tissue[26] Macrophages can self-renew by local proliferation of mature differentiated cells.[27][28] In newts, muscle tissue is regenerated from specialized muscle cells that dedifferentiate and forget the type of cell they had been. This capacity to regenerate does not decline with age and may be linked to their ability to make new stem cells from muscle cells on demand.[29]

A variety of nontumorigenic stem cells display the ability to generate multiple cell types. For instance, multilineage-differentiating stress-enduring (Muse) cells are stress-tolerant adult human stem cells that can self-renew. They form characteristic cell clusters in suspension culture that express a set of genes associated with pluripotency and can differentiate into endodermal, ectodermal and mesodermal cells both in vitro and in vivo.[30][31][32][33][34]

Other well-documented examples of transdifferentiation and their significance in development and regeneration were described in detail.[35][36]

Induced totipotent cells can be obtained by reprogramming somatic cells with somatic-cell nuclear transfer (SCNT). The process involves sucking out the nucleus of a somatic (body) cell and injecting it into an oocyte that has had its nucleus removed[3][5][37][38]

Using an approach based on the protocol outlined by Tachibana et al.,[3] hESCs can be generated by SCNT using dermal fibroblasts nuclei from both a middle-aged 35-year-old male and an elderly, 75-year-old male, suggesting that age-associated changes are not necessarily an impediment to SCNT-based nuclear reprogramming of human cells.[39] Such reprogramming of somatic cells to a pluripotent state holds huge potentials for regenerative medicine. Unfortunately, the cells generated by this technology, potentially are not completely protected from the immune system of the patient (donor of nuclei), because they have the same mitochondrial DNA, as a donor of oocytes, instead of the patients mitochondrial DNA. This reduces their value as a source for autologous stem cell transplantation therapy, as for the present, it is not clear whether it can induce an immune response of the patient upon treatment.

Induced androgenetic haploid embryonic stem cells can be used instead of sperm for cloning. These cells, synchronized in M phase and injected into the oocyte can produce viable offspring.[40]

These developments, together with data on the possibility of unlimited oocytes from mitotically active reproductive stem cells,[41] offer the possibility of industrial production of transgenic farm animals. Repeated recloning of viable mice through a SCNT method that includes a histone deacetylase inhibitor, trichostatin, added to the cell culture medium,[42] show that it may be possible to reclone animals indefinitely with no visible accumulation of reprogramming or genomic errors[43] However, research into technologies to develop sperm and egg cells from stem cells raises bioethical issues.[44]

Such technologies may also have far-reaching clinical applications for overcoming cytoplasmic defects in human oocytes.[3][45] For example, the technology could prevent inherited mitochondrial disease from passing to future generations. Mitochondrial genetic material is passed from mother to child. Mutations can cause diabetes, deafness, eye disorders, gastrointestinal disorders, heart disease, dementia and other neurological diseases. The nucleus from one human egg has been transferred to another, including its mitochondria, creating a cell that could be regarded as having two mothers. The eggs were then fertilised and the resulting embryonic stem cells carried the swapped mitochondrial DNA.[46] As evidence that the technique is safe author of this method points to the existence of the healthy monkeys that are now more than four years old and are the product of mitochondrial transplants across different genetic backgrounds.[47]

In late-generation telomerase-deficient (Terc/) mice, SCNT-mediated reprogramming mitigates telomere dysfunction and mitochondrial defects to a greater extent than iPSC-based reprogramming.[48]

Other cloning and totipotent transformation achievements have been described.[49]

Recently some researchers succeeded to get the totipotent cells without the aid of SCNT. Totipotent cells were obtained using the epigenetic factors such as oocyte germinal isoform of histone.[50] Reprogramming in vivo, by transitory induction of the four factors Oct4, Sox2, Klf4 and c-Myc in mice, confers totipotency features. Intraperitoneal injection of such in vivo iPS cells generates embryo-like structures that express embryonic and extraembryonic (trophectodermal) markers.[51]

iPSc were first obtained in the form of transplantable teratocarcinoma induced by grafts taken from mouse embryos.[52] Teratocarcinoma formed from somatic cells.[53]Genetically mosaic mice were obtained from malignant teratocarcinoma cells, confirming the cells' pluripotency.[54][55][56] It turned out that teratocarcinoma cells are able to maintain a culture of pluripotent embryonic stem cell in an undifferentiated state, by supplying the culture medium with various factors.[57] In the 1980s, it became clear that transplanting pluripotent/embryonic stem cells into the body of adult mammals, usually leads to the formation of teratomas, which can then turn into a malignant tumor teratocarcinoma.[58] However, putting teratocarcinoma cells into the embryo at the blastocyst stage, caused them to become incorporated in the inner cell mass and often produced a normal chimeric (i.e. composed of cells from different organisms) animal.[59][60][61] This indicated that the cause of the teratoma is a dissonance - mutual miscommunication between young donor cells and surrounding adult cells (the recipient's so-called "niche").

In August 2006, Japanese researchers circumvented the need for an oocyte, as in SCNT. By reprograming mouse embryonic fibroblasts into pluripotent stem cells via the ectopic expression of four transcription factors, namely Oct4, Sox2, Klf4 and c-Myc, they proved that the overexpression of a small number of factors can push the cell to transition to a new stable state that is associated with changes in the activity of thousands of genes.[7]

Reprogramming mechanisms are thus linked, rather than independent and are centered on a small number of genes.[62] IPSC properties are very similar to ESCs.[63] iPSCs have been shown to support the development of all-iPSC mice using a tetraploid (4n) embryo,[64] the most stringent assay for developmental potential. However, some genetically normal iPSCs failed to produce all-iPSC mice because of aberrant epigenetic silencing of the imprinted Dlk1-Dio3 gene cluster.[18]

An important advantage of iPSC over ESC is that they can be derived from adult cells, rather than from embryos. Therefore, it became possible to obtain iPSC from adult and even elderly patients.[9][65][66]

Reprogramming somatic cells to iPSC leads to rejuvenation. It was found that reprogramming leads to telomere lengthening and subsequent shortening after their differentiation back into fibroblast-like derivatives.[67] Thus, reprogramming leads to the restoration of embryonic telomere length,[68] and hence increases the potential number of cell divisions otherwise limited by the Hayflick limit.[69]

However, because of the dissonance between rejuvenated cells and the surrounding niche of the recipient's older cells, the injection of his own iPSC usually leads to an immune response,[70] which can be used for medical purposes,[71] or the formation of tumors such as teratoma.[72] The reason has been hypothesized to be that some cells differentiated from ESC and iPSC in vivo continue to synthesize embryonic protein isoforms.[73] So, the immune system might detect and attack cells that are not cooperating properly.

A small molecule called MitoBloCK-6 can force the pluripotent stem cells to die by triggering apoptosis (via cytochrome c release across the mitochondrial outer membrane) in human pluripotent stem cells, but not in differentiated cells. Shortly after differentiation, daughter cells became resistant to death. When MitoBloCK-6 was introduced to differentiated cell lines, the cells remained healthy. The key to their survival, was hypothesized to be due to the changes undergone by pluripotent stem cell mitochondria in the process of cell differentiation. This ability of MitoBloCK-6 to separate the pluripotent and differentiated cell lines has the potential to reduce the risk of teratomas and other problems in regenerative medicine.[74]

In 2012 other small molecules (selective cytotoxic inhibitors of human pluripotent stem cellshPSCs) were identified that prevented human pluripotent stem cells from forming teratomas in mice. The most potent and selective compound of them (PluriSIn #1) inhibits stearoyl-coA desaturase (the key enzyme in oleic acid biosynthesis), which finally results in apoptosis. With the help of this molecule the undifferentiated cells can be selectively removed from culture.[75][76] An efficient strategy to selectively eliminate pluripotent cells with teratoma potential is targeting pluripotent stem cell-specific antiapoptotic factor(s) (i.e., survivin or Bcl10). A single treatment with chemical survivin inhibitors (e.g., quercetin or YM155) can induce selective and complete cell death of undifferentiated hPSCs and is claimed to be sufficient to prevent teratoma formation after transplantation.[77] However, it is unlikely that any kind of preliminary clearance,[78] is able to secure the replanting iPSC or ESC. After the selective removal of pluripotent cells, they re-emerge quickly by reverting differentiated cells into stem cells, which leads to tumors.[79] This may be due to the disorder of let-7 regulation of its target Nr6a1 (also known as Germ cell nuclear factor - GCNF), an embryonic transcriptional repressor of pluripotency genes that regulates gene expression in adult fibroblasts following micro-RNA miRNA loss.[80]

Teratoma formation by pluripotent stem cells may be caused by low activity of PTEN enzyme, reported to promote the survival of a small population (0,1-5% of total population) of highly tumorigenic, aggressive, teratoma-initiating embryonic-like carcinoma cells during differentiation. The survival of these teratoma-initiating cells is associated with failed repression of Nanog as well as a propensity for increased glucose and cholesterol metabolism.[81] These teratoma-initiating cells also expressed a lower ratio of p53/p21 when compared to non-tumorigenic cells.[82] In connection with the above safety problems, the use iPSC for cell therapy is still limited.[83] However, they can be used for a variety of other purposes - including the modeling of disease,[84] screening (selective selection) of drugs, toxicity testing of various drugs.[85]

It is interesting to note that the tissue grown from iPSCs, placed in the "chimeric" embryos in the early stages of mouse development, practically do not cause an immune response (after the embryos have grown into adult mice) and are suitable for autologous transplantation[86] At the same time, full reprogramming of adult cells in vivo within tissues by transitory induction of the four factors Oct4, Sox2, Klf4 and c-Myc in mice results in teratomas emerging from multiple organs.[51] Furthermore, partial reprogramming of cells toward pluripotency in vivo in mice demonstrates that incomplete reprogramming entails epigenetic changes (failed repression of Polycomb targets and altered DNA methylation) in cells that drive cancer development.[87]

Determining the unique set of cellular factors that is needed to be manipulated for each cell conversion is a long and costly process that involved much trial and error. As a result, this first step of identifying the key set of cellular factors for cell conversion is the major obstacle researchers face in the field of cell reprogramming. An international team of researchers have developed an algorithm, called Mogrify(1), that can predict the optimal set of cellular factors required to convert one human cell type to another. When tested, Mogrify was able to accurately predict the set of cellular factors required for previously published cell conversions correctly. To further validate Mogrify's predictive ability, the team conducted two novel cell conversions in the laboratory using human cells and these were successful in both attempts solely using the predictions of Mogrify.[89][90][91] Mogrify has been made available online for other researchers and scientists.

By using solely small molecules, Deng Hongkui and colleagues demonstrated that endogenous "master genes" are enough for cell fate reprogramming. They induced a pluripotent state in adult cells from mice using seven small-molecule compounds.[17] The effectiveness of the method is quite high: it was able to convert 0.02% of the adult tissue cells into iPSCs, which is comparable to the gene insertion conversion rate. The authors note that the mice generated from CiPSCs were "100% viable and apparently healthy for up to 6 months". So, this chemical reprogramming strategy has potential use in generating functional desirable cell types for clinical applications.[92][93]

In 2015th year a robust chemical reprogramming system was established with a yield up to 1,000-fold greater than that of the previously reported protocol. So, chemical reprogramming became a promising approach to manipulate cell fates.[94]

The fact that human iPSCs capable of forming teratomas not only in humans but also in some animal body, in particular in mice or pigs, allowed to develop a method for differentiation of iPSCs in vivo. For this purpose, iPSCs with an agent for inducing differentiation into target cells are injected to genetically modified pig or mouse that has suppressed immune system activation on human cells. The formed teratoma is cut out and used for the isolation of the necessary differentiated human cells[95] by means of monoclonal antibody to tissue-specific markers on the surface of these cells. This method has been successfully used for the production of functional myeloid, erythroid and lymphoid human cells suitable for transplantation (yet only to mice).[96] Mice engrafted with human iPSC teratoma-derived hematopoietic cells produced human B and T cells capable of functional immune responses. These results offer hope that in vivo generation of patient customized cells is feasible, providing materials that could be useful for transplantation, human antibody generation and drug screening applications. Using MitoBloCK-6[74] and/or PluriSIn # 1 the differentiated progenitor cells can be further purified from teratoma forming pluripotent cells. The fact, that the differentiation takes place even in the teratoma niche, offers hope that the resulting cells are sufficiently stable to stimuli able to cause their transition back to the dedifferentiated (pluripotent) state and therefore safe. A similar in vivo differentiation system, yielding engraftable hematopoietic stem cells from mouse and human iPSCs in teratoma-bearing animals in combination with a maneuver to facilitate hematopoiesis, was described by Suzuki et al.[97] They noted that neither leukemia nor tumors were observed in recipients after intravenous injection of iPSC-derived hematopoietic stem cells into irradiated recipients. Moreover, this injection resulted in multilineage and long-term reconstitution of the hematolymphopoietic system in serial transfers. Such system provides a useful tool for practical application of iPSCs in the treatment of hematologic and immunologic diseases.[98]

For further development of this method animal in which is grown the human cell graft, for example mouse, must have so modified genome that all its cells express and have on its surface human SIRP.[99] To prevent rejection after transplantation to the patient of the allogenic organ or tissue, grown from the pluripotent stem cells in vivo in the animal, these cells should express two molecules: CTLA4-Ig, which disrupts T cell costimulatory pathways and PD-L1, which activates T cell inhibitory pathway.[100]

See also: US 20130058900 patent.

In the near-future, clinical trials designed to demonstrate the safety of the use of iPSCs for cell therapy of the people with age-related macular degeneration, a disease causing blindness through retina damaging, will begin. There are several articles describing methods for producing retinal cells from iPSCs[101][102] and how to use them for cell therapy.[103][104] Reports of iPSC-derived retinal pigmented epithelium transplantation showed enhanced visual-guided behaviors of experimental animals for 6 weeks after transplantation.[105] However, clinical trials have been successful: ten patients suffering from retinitis pigmentosa have had their eyesight restoredincluding a woman who had only 17 percent of her vision left.[106]

Chronic lung diseases such as idiopathic pulmonary fibrosis and cystic fibrosis or chronic obstructive pulmonary disease and asthma are leading causes of morbidity and mortality worldwide with a considerable human, societal and financial burden. So there is an urgent need for effective cell therapy and lung tissue engineering.[107][108] Several protocols have been developed for generation of the most cell types of the respiratory system, which may be useful for deriving patient-specific therapeutic cells.[109][110][111][112][113]

Some lines of iPSCs have the potentiality to differentiate into male germ cells and oocyte-like cells in an appropriate niche (by culturing in retinoic acid and porcine follicular fluid differentiation medium or seminiferous tubule transplantation). Moreover, iPSC transplantation make a contribution to repairing the testis of infertile mice, demonstrating the potentiality of gamete derivation from iPSCs in vivo and in vitro.[114]

The risk of cancer and tumors creates the need to develop methods for safer cell lines suitable for clinical use. An alternative approach is so-called "direct reprogramming" - transdifferentiation of cells without passing through the pluripotent state.[115][116][117][118][119][120] The basis for this approach was that 5-azacytidine - a DNA demethylation reagent - can cause the formation of myogenic, chondrogenic and adipogeni clones in the immortal cell line of mouse embryonic fibroblasts[121] and that the activation of a single gene, later named MyoD1, is sufficient for such reprogramming.[122] Compared with iPSC whose reprogramming requires at least two weeks, the formation of induced progenitor cells sometimes occurs within a few days and the efficiency of reprogramming is usually many times higher. This reprogramming does not always require cell division.[123] The cells resulting from such reprogramming are more suitable for cell therapy because they do not form teratomas.[120] For example, Chandrakanthan et al., & Pimanda describe the generation of tissue-regenerative multipotent stem cells (iMS cells) by treating mature bone and fat cells transiently with a growth factor (platelet-derived growth factorAB (PDGF-AB)) and 5-Azacytidine. These authors claim that: "Unlike primary mesenchymal stem cells, which are used with little objective evidence in clinical practice to promote tissue repair, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner without forming tumors" and so "has significant scope for application in tissue regeneration."[124][125][126]

Originally only early embryonic cells could be coaxed into changing their identity. Mature cells are resistant to changing their identity once they've committed to a specific kind. However, brief expression of a single transcription factor, the ELT-7 GATA factor, can convert the identity of fully differentiated, specialized non-endodermal cells of the pharynx into fully differentiated intestinal cells in intact larvae and adult roundworm Caenorhabditis elegans with no requirement for a dedifferentiated intermediate.[127]

The cell fate can be effectively manipulated by epigenome editing. In particular, by directly activating of specific endogenous gene expression with CRISPR-mediated activator. When dCas9 (that has been modified so that it no longer cuts DNA, but still can be guided to specific sequences and to bind to them) is combined with transcription activators, it can precisely manipulate endogenous gene expression. Using this method, Wei et al., enhanced the expression of endogenous Cdx2 and Gata6 genes by CRISPR-mediated activators, thus directly converted mouse embryonic stem cells into two extraembryonic lineages, i.e., typical trophoblast stem cells and extraembryonic endoderm cells.[128] An analogous approach was used to induce activation of the endogenous Brn2, Ascl1, and Myt1l genes to convert mouse embryonic fibroblasts to induced neuronal cells.[129] Thus, transcriptional activation and epigenetic remodeling of endogenous master transcription factors are sufficient for conversion between cell types. The rapid and sustained activation of endogenous genes in their native chromatin context by this approach may facilitate reprogramming with transient methods that avoid genomic integration and provides a new strategy for overcoming epigenetic barriers to cell fate specification.

Another way of reprogramming is the simulation of the processes that occur during amphibian limb regeneration. In urodele amphibians, an early step in limb regeneration is skeletal muscle fiber dedifferentiation into a cellulate that proliferates into limb tissue. However, sequential small molecule treatment of the muscle fiber with myoseverin, reversine (the aurora B kinase inhibitor) and some other chemicals: BIO (glycogen synthase-3 kinase inhibitor), lysophosphatidic acid (pleiotropic activator of G-protein-coupled receptors), SB203580 (p38 MAP kinase inhibitor), or SQ22536 (adenylyl cyclase inhibitor) causes the formation of new muscle cell types as well as other cell types such as precursors to fat, bone and nervous system cells.[130]

The researchers discovered that GCSF-mimicking antibody can activate a growth-stimulating receptor on marrow cells in a way that induces marrow stem cells that normally develop into white blood cells to become neural progenitor cells. The technique[131] enables researchers to search large libraries of antibodies and quickly select the ones with a desired biological effect.[132]

Schlegel and Liu[133] demonstrated that the combination of feeder cells[134][135][136] and a Rho kinase inhibitor (Y-27632) [137][138] induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro. This process occurs without the need for transduction of exogenous viral or cellular genes. These cells have been termed "Conditionally Reprogrammed Cells (CRC)". The induction of CRCs is rapid and results from reprogramming of the entire cell population. CRCs do not express high levels of proteins characteristic of iPSCs or embryonic stem cells (ESCs) (e.g., Sox2, Oct4, Nanog, or Klf4). This induction of CRCs is reversible and removal of Y-27632 and feeders allows the cells to differentiate normally.[133][139][140] CRC technology can generate 2106 cells in 5 to 6 days from needle biopsies and can generate cultures from cryopreserved tissue and from fewer than four viable cells. CRCs retain a normal karyotype and remain nontumorigenic. This technique also efficiently establishes cell cultures from human and rodent tumors.[133][141][142]

The ability to rapidly generate many tumor cells from small biopsy specimens and frozen tissue provides significant opportunities for cell-based diagnostics and therapeutics (including chemosensitivity testing) and greatly expands the value of biobanking.[133][141][142] Using CRC technology, researchers were able to identify an effective therapy for a patient with a rare type of lung tumor.[143] Engleman's group[144] describes a pharmacogenomic platform that facilitates rapid discovery of drug combinations that can overcome resistance using CRC system. In addition, the CRC method allows for the genetic manipulation of epithelial cells ex vivo and their subsequent evaluation in vivo in the same host. While initial studies revealed that co-culturing epithelial cells with Swiss 3T3 cells J2 was essential for CRC induction, with transwell culture plates, physical contact between feeders and epithelial cells is not required for inducing CRCs and more importantly that irradiation of the feeder cells is required for this induction. Consistent with the transwell experiments, conditioned medium induces and maintains CRCs, which is accompanied by a concomitant increase of cellular telomerase activity. The activity of the conditioned medium correlates directly with radiation-induced feeder cell apoptosis. Thus, conditional reprogramming of epithelial cells is mediated by a combination of Y-27632 and a soluble factor(s) released by apoptotic feeder cells.[145]

Riegel et al.[146] demonstrate that mouse ME cells isolated from normal mammary glands or from mouse mammary tumor virus (MMTV)-Neuinduced mammary tumors, can be cultured indefinitely as conditionally reprogrammed cells (CRCs). Cell surface progenitor-associated markers are rapidly induced in normal mouse ME-CRCs relative to ME cells. However, the expression of certain mammary progenitor subpopulations, such as CD49f+ ESA+ CD44+, drops significantly in later passages. Nevertheless, mouse ME-CRCs grown in a three-dimensional extracellular matrix gave rise to mammary acinar structures. ME-CRCs isolated from MMTV-Neu transgenic mouse mammary tumors express high levels of HER2/neu, as well as tumor-initiating cell markers, such as CD44+, CD49f+ and ESA+ (EpCam). These patterns of expression are sustained in later CRC passages. Early and late passage ME-CRCs from MMTV-Neu tumors that were implanted in the mammary fat pads of syngeneic or nude mice developed vascular tumors that metastasized within 6 weeks of transplantation. Importantly, the histopathology of these tumors was indistinguishable from that of the parental tumors that develop in the MMTV-Neu mice. Application of the CRC system to mouse mammary epithelial cells provides an attractive model system to study the genetics and phenotype of normal and transformed mouse epithelium in a defined culture environment and in vivo transplant studies.

A different approach to CRC is to inhibit CD47a membrane protein that is the thrombospondin-1 receptor. Loss of CD47 permits sustained proliferation of primary murine endothelial cells, increases asymmetric division and enables these cells to spontaneously reprogram to form multipotent embryoid body-like clusters. CD47 knockdown acutely increases mRNA levels of c-Myc and other stem cell transcription factors in cells in vitro and in vivo. Thrombospondin-1 is a key environmental signal that inhibits stem cell self-renewal via CD47. Thus, CD47 antagonists enable cell self-renewal and reprogramming by overcoming negative regulation of c-Myc and other stem cell transcription factors.[147] In vivo blockade of CD47 using an antisense morpholino increases survival of mice exposed to lethal total body irradiation due to increased proliferative capacity of bone marrow-derived cells and radioprotection of radiosensitive gastrointestinal tissues.[148]

Differentiated macrophages can self-renew in tissues and expand long-term in culture.[27] Under certain conditions macrophages can divide without losing features they have acquired while specializing into immune cells - which is usually not possible with differentiated cells. The macrophages achieve this by activating a gene network similar to one found in embryonic stem cells. Single-cell analysis revealed that, in vivo, proliferating macrophages can derepress a macrophage-specific enhancer repertoire associated with a gene network controlling self-renewal. This happened when concentrations of two transcription factors named MafB and c-Maf were naturally low or were inhibited for a short time. Genetic manipulations that turned off MafB and c-Maf in the macrophages caused the cells to start a self-renewal program. The similar network also controls embryonic stem cell self-renewal but is associated with distinct embryonic stem cell-specific enhancers.[28]

Hence macrophages isolated from MafB- and c-Maf-double deficient mice divide indefinitely; the self-renewal depends on c-Myc and Klf4.[149]

Indirect lineage conversion is a reprogramming methodology in which somatic cells transition through a plastic intermediate state of partially reprogrammed cells (pre-iPSC), induced by brief exposure to reprogramming factors, followed by differentiation in a specially developed chemical environment (artificial niche).[150]

This method could be both more efficient and safer, since it does not seem to produce tumors or other undesirable genetic changes and results in much greater yield than other methods. However, the safety of these cells remains questionable. Since lineage conversion from pre-iPSC relies on the use of iPSC reprogramming conditions, a fraction of the cells could acquire pluripotent properties if they do not stop the de-differentation process in vitro or due to further de-differentiation in vivo.[151]

A common feature of pluripotent stem cells is the specific nature of protein glycosylation of their outer membrane. That distinguishes them from most nonpluripotent cells, although not white blood cells.[152] The glycans on the stem cell surface respond rapidly to alterations in cellular state and signaling and are therefore ideal for identifying even minor changes in cell populations. Many stem cell markers are based on cell surface glycan epitopes including the widely used markers SSEA-3, SSEA-4, Tra 1-60 and Tra 1-81.[153] Suila Heli et al.[154] speculate that in human stem cells extracellular O-GlcNAc and extracellular O-LacNAc, play a crucial role in the fine tuning of Notch signaling pathway - a highly conserved cell signaling system, that regulates cell fate specification, differentiation, leftright asymmetry, apoptosis, somitogenesis, angiogenesis and plays a key role in stem cell proliferation (reviewed by Perdigoto and Bardin[155] and Jafar-Nejad et al.[156])

Changes in outer membrane protein glycosylation are markers of cell states connected in some way with pluripotency and differentiation.[157] The glycosylation change is apparently not just the result of the initialization of gene expression, but perform as an important gene regulator involved in the acquisition and maintenance of the undifferentiated state.[158]

For example, activation of glycoprotein ACA,[159] linking glycosylphosphatidylinositol on the surface of the progenitor cells in human peripheral blood, induces increased expression of genes Wnt, Notch-1, BMI1 and HOXB4 through a signaling cascade PI3K/Akt/mTor/PTEN and promotes the formation of a self-renewing population of hematopoietic stem cells.[160]

Furthermore, dedifferentiation of progenitor cells induced by ACA-dependent signaling pathway leads to ACA-induced pluripotent stem cells, capable of differentiating in vitro into cells of all three germ layers.[161] The study of lectins' ability to maintain a culture of pluripotent human stem cells has led to the discovery of lectin Erythrina crista-galli (ECA), which can serve as a simple and highly effective matrix for the cultivation of human pluripotent stem cells.[162]

Cell adhesion protein E-cadherin is indispensable for a robust pluripotent phenotype.[163] During reprogramming for iPS cell generation, N-cadherin can replace function of E-cadherin.[164] These functions of cadherins are not directly related to adhesion because sphere morphology helps maintaining the "stemness" of stem cells.[165] Moreover, sphere formation, due to forced growth of cells on a low attachment surface, sometimes induces reprogramming. For example, neural progenitor cells can be generated from fibroblasts directly through a physical approach without introducing exogenous reprogramming factors.

Physical cues, in the form of parallel microgrooves on the surface of cell-adhesive substrates, can replace the effects of small-molecule epigenetic modifiers and significantly improve reprogramming efficiency. The mechanism relies on the mechanomodulation of the cells' epigenetic state. Specifically, "decreased histone deacetylase activity and upregulation of the expression of WD repeat domain 5 (WDR5)a subunit of H3 methyltranferaseby microgrooved surfaces lead to increased histone H3 acetylation and methylation". Nanofibrous scaffolds with aligned fibre orientation produce effects similar to those produced by microgrooves, suggesting that changes in cell morphology may be responsible for modulation of the epigenetic state.[166]

Substrate rigidity is an important biophysical cue influencing neural induction and subtype specification. For example, soft substrates promote neuroepithelial conversion while inhibiting neural crest differentiation of hESCs in a BMP4-dependent manner. Mechanistic studies revealed a multi-targeted mechanotransductive process involving mechanosensitive Smad phosphorylation and nucleocytoplasmic shuttling, regulated by rigidity-dependent Hippo/YAP activities and actomyosin cytoskeleton integrity and contractility.[167]

Mouse embryonic stem cells (mESCs) undergo self-renewal in the presence of the cytokine leukemia inhibitory factor (LIF). Following LIF withdrawal, mESCs differentiate, accompanied by an increase in cellsubstratum adhesion and cell spreading. Restricted cell spreading in the absence of LIF by either culturing mESCs on chemically defined, weakly adhesive biosubstrates, or by manipulating the cytoskeleton allowed the cells to remain in an undifferentiated and pluripotent state. The effect of restricted cell spreading on mESC self-renewal is not mediated by increased intercellular adhesion, as inhibition of mESC adhesion using a function blocking anti E-cadherin antibody or siRNA does not promote differentiation.[168] Possible mechanisms of stem cell fate predetermination by physical interactions with the extracellular matrix have been described.[169][170]

A new method has been developed that turns cells into stem cells faster and more efficiently by 'squeezing' them using 3D microenvironment stiffness and density of the surrounding gel. The technique can be applied to a large number of cells to produce stem cells for medical purposes on an industrial scale.[171][172]

Cells involved in the reprogramming process change morphologically as the process proceeds. This results in physical difference in adhesive forces among cells. Substantial differences in 'adhesive signature' between pluripotent stem cells, partially reprogrammed cells, differentiated progeny and somatic cells allowed to develop separation process for isolation of pluripotent stem cells in microfluidic devices,[173] which is:

Stem cells possess mechanical memory (they remember past physical signals)with the Hippo signaling pathway factors:[174] Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding domain (TAZ) acting as an intracellular mechanical rheostatthat stores information from past physical environments and influences the cells' fate.[175][176]

Stroke and many neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis need cell replacement therapy. The successful use of converted neural cells (cNs) in transplantations open a new avenue to treat such diseases.[177] Nevertheless, induced neurons (iNs), directly converted from fibroblasts are terminally committed and exhibit very limited proliferative ability that may not provide enough autologous donor cells for transplantation.[178] Self-renewing induced neural stem cells (iNSCs) provide additional advantages over iNs for both basic research and clinical applications.[118][119][120][179][180]

For example, under specific growth conditions, mouse fibroblasts can be reprogrammed with a single factor, Sox2, to form iNSCs that self-renew in culture and after transplantation can survive and integrate without forming tumors in mouse brains.[181] INSCs can be derived from adult human fibroblasts by non-viral techniques, thus offering a safe method for autologous transplantation or for the development of cell-based disease models.[180]

Neural chemically induced progenitor cells (ciNPCs) can be generated from mouse tail-tip fibroblasts and human urinary somatic cells without introducing exogenous factors, but - by a chemical cocktail, namely VCR (V, VPA, an inhibitor of HDACs; C, CHIR99021, an inhibitor of GSK-3 kinases and R, RepSox, an inhibitor of TGF beta signaling pathways), under a physiological hypoxic condition.[182] Alternative cocktails with inhibitors of histone deacetylation, glycogen synthase kinase and TGF- pathways (where: sodium butyrate (NaB) or Trichostatin A (TSA) could replace VPA, Lithium chloride (LiCl) or lithium carbonate (Li2CO3) could substitute CHIR99021, or Repsox may be replaced with SB-431542 or Tranilast) show similar efficacies for ciNPC induction.[182] Zhang, et al.,[183] also report highly efficient reprogramming of mouse fibroblasts into induced neural stem cell-like cells (ciNSLCs) using a cocktail of nine components.

Multiple methods of direct transformation of somatic cells into induced neural stem cells have been described.[184]

Proof of principle experiments demonstrate that it is possible to convert transplanted human fibroblasts and human astrocytes directly in the brain that are engineered to express inducible forms of neural reprogramming genes, into neurons, when reprogramming genes (Ascl1, Brn2a and Myt1l) are activated after transplantation using a drug.[185]

Astrocytesthe most common neuroglial brain cells, which contribute to scar formation in response to injurycan be directly reprogrammed in vivo to become functional neurons that formed networks in mice without the need of cell transplantation.[186] The researchers followed the mice for nearly a year to look for signs of tumor formation and reported finding none. The same researchers have turned scar-forming astrocytes into progenitor cells called neuroblasts that regenerated into neurons in the injured adult spinal cord.[187]

Without myelin to insulate neurons, nerve signals quickly lose power. Diseases that attack myelin, such as multiple sclerosis, result in nerve signals that cannot propagate to nerve endings and as a consequence lead to cognitive, motor and sensory problems. Transplantation of oligodendrocyte precursor cells (OPCs), which can successfully create myelin sheaths around nerve cells, is a promising potential therapeutic response. Direct lineage conversion of mouse and rat fibroblasts into oligodendroglial cells provides a potential source of OPCs. Conversion by forced expression of both eight[188] or of the three[189] transcription factors Sox10, Olig2 and Zfp536, may provide such cells.

Cell-based in vivo therapies may provide a transformative approach to augment vascular and muscle growth and to prevent non-contractile scar formation by delivering transcription factors[115] or microRNAs[14] to the heart.[190] Cardiac fibroblasts, which represent 50% of the cells in the mammalian heart, can be reprogrammed into cardiomyocyte-like cells in vivo by local delivery of cardiac core transcription factors ( GATA4, MEF2C, TBX5 and for improved reprogramming plus ESRRG, MESP1, Myocardin and ZFPM2) after coronary ligation.[115][191] These results implicated therapies that can directly remuscularize the heart without cell transplantation. However, the efficiency of such reprogramming turned out to be very low and the phenotype of received cardiomyocyte-like cells does not resemble those of a mature normal cardiomyocyte. Furthermore, transplantation of cardiac transcription factors into injured murine hearts resulted in poor cell survival and minimal expression of cardiac genes.[192]

Meanwhile, advances in the methods of obtaining cardiac myocytes in vitro occurred.[193][194] Efficient cardiac differentiation of human iPS cells gave rise to progenitors that were retained within infarcted rat hearts and reduced remodeling of the heart after ischemic damage.[195]

The team of scientists, who were led by Sheng Ding, used a cocktail of nine chemicals (9C) for transdifferentiation of human skin cells into beating heart cells. With this method, more than 97% of the cells began beating, a characteristic of fully developed, healthy heart cells. The chemically induced cardiomyocyte-like cells (ciCMs) uniformly contracted and resembled human cardiomyocytes in their transcriptome, epigenetic, and electrophysiological properties. When transplanted into infarcted mouse hearts, 9C-treated fibroblasts were efficiently converted to ciCMs and developed into healthy-looking heart muscle cells within the organ.[196] This chemical reprogramming approach, after further optimization, may offer an easy way to provide the cues that induce heart muscle to regenerate locally.[197]

In another study, ischemic cardiomyopathy in the murine infarction model was targeted by iPS cell transplantation. It synchronized failing ventricles, offering a regenerative strategy to achieve resynchronization and protection from decompensation by dint of improved left ventricular conduction and contractility, reduced scarring and reversal of structural remodelling.[198] One protocol generated populations of up to 98% cardiomyocytes from hPSCs simply by modulating the canonical Wnt signaling pathway at defined time points in during differentiation, using readily accessible small molecule compounds.[199]

Discovery of the mechanisms controlling the formation of cardiomyocytes led to the development of the drug ITD-1, which effectively clears the cell surface from TGF- receptor type II and selectively inhibits intracellular TGF- signaling. It thus selectively enhances the differentiation of uncommitted mesoderm to cardiomyocytes, but not to vascular smooth muscle and endothelial cells.[200]

One project seeded decellularized mouse hearts with human iPSC-derived multipotential cardiovascular progenitor cells. The introduced cells migrated, proliferated and differentiated in situ into cardiomyocytes, smooth muscle cells and endothelial cells to reconstruct the hearts. In addition, the heart's extracellular matrix (the substrate of heart scaffold) signalled the human cells into becoming the specialised cells needed for proper heart function. After 20 days of perfusion with growth factors, the engineered heart tissues started to beat again and were responsive to drugs.[201]

Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells (iCMs) in situ represents a promising strategy for cardiac regeneration. Mice exposed in vivo, to three cardiac transcription factors GMT (Gata4, Mef2c, Tbx5) and the small-molecules: SB-431542 (the transforming growth factor (TGF)- inhibitor), and XAV939 (the WNT inhibitor) for 2 weeks after myocardial infarction showed significantly improved reprogramming (reprogramming efficiency increased eight-fold) and cardiac function compared to those exposed to only GMT.[202]

See also: review[203]

The elderly often suffer from progressive muscle weakness and regenerative failure owing in part to elevated activity of the p38 and p38 mitogen-activated kinase pathway in senescent skeletal muscle stem cells. Subjecting such stem cells to transient inhibition of p38 and p38 in conjunction with culture on soft hydrogel substrates rapidly expands and rejuvenates them that result in the return of their strength.[204]

In geriatric mice, resting satellite cells lose reversible quiescence by switching to an irreversible pre-senescence state, caused by derepression of p16INK4a (also called Cdkn2a). On injury, these cells fail to activate and expand, even in a youthful environment. p16INK4a silencing in geriatric satellite cells restores quiescence and muscle regenerative functions.[205]

Myogenic progenitors for potential use in disease modeling or cell-based therapies targeting skeletal muscle could also be generated directly from induced pluripotent stem cells using free-floating spherical culture (EZ spheres) in a culture medium supplemented with high concentrations (100ng/ml) of fibroblast growth factor-2 (FGF-2) and epidermal growth factor.[206]

Unlike current protocols for deriving hepatocytes from human fibroblasts, Saiyong Zhu et al., (2014)[207] did not generate iPSCs but, using small molecules, cut short reprogramming to pluripotency to generate an induced multipotent progenitor cell (iMPC) state from which endoderm progenitor cells and subsequently hepatocytes (iMPC-Heps) were efficiently differentiated. After transplantation into an immune-deficient mouse model of human liver failure, iMPC-Heps proliferated extensively and acquired levels of hepatocyte function similar to those of human primary adult hepatocytes. iMPC-Heps did not form tumours, most probably because they never entered a pluripotent state.

These results establish the feasibility of significant liver repopulation of mice with human hepatocytes generated in vitro, which removes a long-standing roadblock on the path to autologous liver cell therapy.

Cocktail of small molecules, Y-27632, A-83-01 (a TGF kinase/activin receptor like kinase (ALK5) inhibitor), and CHIR99021 (potent inhibitor of GSK-3), can convert rat and mouse mature hepatocytes in vitro into proliferative bipotent cells - CLiPs (chemically induced liver progenitors). CLiPs can differentiate into both mature hepatocytes and biliary epithelial cells that can form functional ductal structures. In long-term culture CLiPs did not lose their proliferative capacity and their hepatic differentiation ability, and can repopulate chronically injured liver tissue.[208]

Complications of Diabetes mellitus such as cardiovascular diseases, retinopathy, neuropathy, nephropathy and peripheral circulatory diseases depend on sugar dysregulation due to lack of insulin from pancreatic beta cells and can be lethal if they are not treated. One of the promising approaches to understand and cure diabetes is to use pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced PCSs (iPSCs).[209] Unfortunately, human PSC-derived insulin-expressing cells resemble human fetal cells rather than adult cells. In contrast to adult cells, fetal cells seem functionally immature, as indicated by increased basal glucose secretion and lack of glucose stimulation and confirmed by RNA-seq of whose transcripts.[210]

An alternative strategy is the conversion of fibroblasts towards distinct endodermal progenitor cell populations and, using cocktails of signalling factors, successful differentiation of these endodermal progenitor cells into functional beta-like cells both in vitro and in vivo.[211]

Overexpression of the three transcription factors, PDX1 (required for pancreatic bud outgrowth and beta-cell maturation), NGN3 (required for endocrine precursor cell formation) and MAFA (for beta-cell maturation) combination (called PNM) can lead to the transformation of some cell types into a beta cell-like state.[212] An accessible and abundant source of functional insulin-producing cells is intestine. PMN expression in human intestinal "organoids" stimulates the conversion of intestinal epithelial cells into -like cells possibly acceptable for transplantation.[213]

Adult proximal tubule cells were directly transcriptionally reprogrammed to nephron progenitors of the embryonic kidney, using a pool of six genes of instructive transcription factors (SIX1, SIX2, OSR1, Eyes absent homolog 1(EYA1), Homeobox A11 (HOXA11) and Snail homolog 2 (SNAI2)) that activated genes consistent with a cap mesenchyme/nephron progenitor phenotype in the adult proximal tubule cell line.[214] The generation of such cells may lead to cellular therapies for adult renal disease. Embryonic kidney organoids placed into adult rat kidneys can undergo onward development and vascular development.[215]

As blood vessels age, they often become abnormal in structure and function, thereby contributing to numerous age-associated diseases including myocardial infarction, ischemic stroke and atherosclerosis of arteries supplying the heart, brain and lower extremities. So, an important goal is to stimulate vascular growth for the collateral circulation to prevent the exacerbation of these diseases. Induced Vascular Progenitor Cells (iVPCs) are useful for cell-based therapy designed to stimulate coronary collateral growth. They were generated by partially reprogramming endothelial cells.[150] The vascular commitment of iVPCs is related to the epigenetic memory of endothelial cells, which engenders them as cellular components of growing blood vessels. That is why, when iVPCs were implanted into myocardium, they engrafted in blood vessels and increased coronary collateral flow better than iPSCs, mesenchymal stem cells, or native endothelial cells.[216]

Ex vivo genetic modification can be an effective strategy to enhance stem cell function. For example, cellular therapy employing genetic modification with Pim-1 kinase (a downstream effector of Akt, which positively regulates neovasculogenesis) of bone marrowderived cells[217] or human cardiac progenitor cells, isolated from failing myocardium[218] results in durability of repair, together with the improvement of functional parameters of myocardial hemodynamic performance.

Stem cells extracted from fat tissue after liposuction may be coaxed into becoming progenitor smooth muscle cells (iPVSMCs) found in arteries and veins.[219]

The 2D culture system of human iPS cells[220] in conjunction with triple marker selection (CD34 (a surface glycophosphoprotein expressed on developmentally early embryonic fibroblasts), NP1 (receptor - neuropilin 1) and KDR (kinase insert domain-containing receptor)) for the isolation of vasculogenic precursor cells from human iPSC, generated endothelial cells that, after transplantation, formed stable, functional mouse blood vessels in vivo, lasting for 280 days.[221]

To treat infarction, it is important to prevent the formation of fibrotic scar tissue. This can be achieved in vivo by transient application of paracrine factors that redirect native heart progenitor stem cell contributions from scar tissue to cardiovascular tissue. For example, in a mouse myocardial infarction model, a single intramyocardial injection of human vascular endothelial growth factor A mRNA (VEGF-A modRNA), modified to escape the body's normal defense system, results in long-term improvement of heart function due to mobilization and redirection of epicardial progenitor cells toward cardiovascular cell types.[222]

RBC transfusion is necessary for many patients. However, to date the supply of RBCs remains labile. In addition, transfusion risks infectious disease transmission. A large supply of safe RBCs generated in vitro would help to address this issue. Ex vivo erythroid cell generation may provide alternative transfusion products to meet present and future clinical requirements.[223][224] Red blood cells (RBC)s generated in vitro from mobilized CD34 positive cells have normal survival when transfused into an autologous recipient.[225] RBC produced in vitro contained exclusively fetal hemoglobin (HbF), which rescues the functionality of these RBCs. In vivo the switch of fetal to adult hemoglobin was observed after infusion of nucleated erythroid precursors derived from iPSCs.[226] Although RBCs do not have nuclei and therefore can not form a tumor, their immediate erythroblasts precursors have nuclei. The terminal maturation of erythroblasts into functional RBCs requires a complex remodeling process that ends with extrusion of the nucleus and the formation of an enucleated RBC.[227] Cell reprogramming often disrupts enucleation. Transfusion of in vitro-generated RBCs or erythroblasts does not sufficiently protect against tumor formation.

The aryl hydrocarbon receptor (AhR) pathway (which has been shown to be involved in the promotion of cancer cell development) plays an important role in normal blood cell development. AhR activation in human hematopoietic progenitor cells (HPs) drives an unprecedented expansion of HPs, megakaryocyte- and erythroid-lineage cells.[228] See also Concise Review:[229][230] The SH2B3 gene encodes a negative regulator of cytokine signaling and naturally occurring loss-of-function variants in this gene increase RBC counts in vivo. Targeted suppression of SH2B3 in primary human hematopoietic stem and progenitor cells enhanced the maturation and overall yield of in-vitro-derived RBCs. Moreover, inactivation of SH2B3 by CRISPR/Cas9 genome editing in human pluripotent stem cells allowed enhanced erythroid cell expansion with preserved differentiation.[231] (See also overview.[230][232])

Platelets help prevent hemorrhage in thrombocytopenic patients and patients with thrombocythemia. A significant problem for multitransfused patients is refractoriness to platelet transfusions. Thus, the ability to generate platelet products ex vivo and platelet products lacking HLA antigens in serum-free media would have clinical value. An RNA interference-based mechanism used a lentiviral vector to express short-hairpin RNAi targeting 2-microglobulin transcripts in CD34-positive cells. Generated platelets demonstrated an 85% reduction in class I HLA antigens. These platelets appeared to have normal function in vitro[233]

One clinically-applicable strategy for the derivation of functional platelets from human iPSC involves the establishment of stable immortalized megakaryocyte progenitor cell lines (imMKCLs) through doxycycline-dependent overexpression of BMI1 and BCL-XL. The resulting imMKCLs can be expanded in culture over extended periods (45 months), even after cryopreservation. Halting the overexpression (by the removal of doxycycline from the medium) of c-MYC, BMI1 and BCL-XL in growing imMKCLs led to the production of CD42b+ platelets with functionality comparable to that of native platelets on the basis of a range of assays in vitro and in vivo.[234] Thomas et al., describe a forward programming strategy relying on the concurrent exogenous expression of 3 transcription factors: GATA1, FLI1 and TAL1. The forward programmed megakaryocytes proliferate and differentiate in culture for several months with megakaryocyte purity over 90% reaching up to 2x105 mature megakaryocytes per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as one million starting hPSCs.[235] See also overview[236]

A specialised type of white blood cell, known as cytotoxic T lymphocytes (CTLs), are produced by the immune system and are able to recognise specific markers on the surface of various infectious or tumour cells, causing them to launch an attack to kill the harmful cells. Thence, immunotherapy with functional antigen-specific T cells has potential as a therapeutic strategy for combating many cancers and viral infections.[237] However, cell sources are limited, because they are produced in small numbers naturally and have a short lifespan.

A potentially efficient approach for generating antigen-specific CTLs is to revert mature immune T cells into iPSCs, which possess indefinite proliferative capacity in vitro and after their multiplication to coax them to redifferentiate back into T cells.[238][239][240]

Another method combines iPSC and chimeric antigen receptor (CAR)[241] technologies to generate human T cells targeted to CD19, an antigen expressed by malignant B cells, in tissue culture.[242] This approach of generating therapeutic human T cells may be useful for cancer immunotherapy and other medical applications.

Invariant natural killer T (iNKT) cells have great clinical potential as adjuvants for cancer immunotherapy. iNKT cells act as innate T lymphocytes and serve as a bridge between the innate and acquired immune systems. They augment anti-tumor responses by producing interferon-gamma (IFN-).[243] The approach of collection, reprogramming/dedifferentiation, re-differentiation and injection has been proposed for related tumor treatment.[244]

Dendritic cells (DC) are specialized to control T-cell responses. DC with appropriate genetic modifications may survive long enough to stimulate antigen-specific CTL and after that be completely eliminated. DC-like antigen-presenting cells obtained from human induced pluripotent stem cells can serve as a source for vaccination therapy.[245]

CCAAT/enhancer binding protein- (C/EBP) induces transdifferentiation of B cells into macrophages at high efficiencies[246] and enhances reprogramming into iPS cells when co-expressed with transcription factors Oct4, Sox2, Klf4 and Myc.[247] with a 100-fold increase in iPS cell reprogramming efficiency, involving 95% of the population.[248] Furthermore, C/EBPa can convert selected human B cell lymphoma and leukemia cell lines into macrophage-like cells at high efficiencies, impairing the cells' tumor-forming capacity.[249]

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Induced stem cells - Wikipedia

Cardiac Stem Cells Comments Off on Induced stem cells – Wikipedia | November 28th, 2016

BACKGROUND. Low vitamin D status in pregnancy was proposed as a risk factor of preeclampsia.

METHODS. We assessed the effect of vitamin D supplementation (4,400 vs. 400 IU/day), initiated early in pregnancy (1018 weeks), on the development of preeclampsia. The effects of serum vitamin D (25-hydroxyvitamin D [25OHD]) levels on preeclampsia incidence at trial entry and in the third trimester (3238 weeks) were studied. We also conducted a nested case-control study of 157 women to investigate peripheral blood vitamin Dassociated gene expression profiles at 10 to 18 weeks in 47 participants who developed preeclampsia.

RESULTS. Of 881 women randomized, outcome data were available for 816, with 67 (8.2%) developing preeclampsia. There was no significant difference between treatment (N = 408) or control (N = 408) groups in the incidence of preeclampsia (8.08% vs. 8.33%, respectively; relative risk: 0.97; 95% CI, 0.611.53). However, in a cohort analysis and after adjustment for confounders, a significant effect of sufficient vitamin D status (25OHD 30 ng/ml) was observed in both early and late pregnancy compared with insufficient levels (25OHD <30 ng/ml) (adjusted odds ratio, 0.28; 95% CI, 0.100.96). Differential expression of 348 vitamin Dassociated genes (158 upregulated) was found in peripheral blood of women who developed preeclampsia (FDR <0.05 in the Vitamin D Antenatal Asthma Reduction Trial [VDAART]; P < 0.05 in a replication cohort). Functional enrichment and network analyses of this vitamin Dassociated gene set suggests several highly functional modules related to systematic inflammatory and immune responses, including some nodes with a high degree of connectivity.

CONCLUSIONS. Vitamin D supplementation initiated in weeks 1018 of pregnancy did not reduce preeclampsia incidence in the intention-to-treat paradigm. However, vitamin D levels of 30 ng/ml or higher at trial entry and in late pregnancy were associated with a lower risk of preeclampsia. Differentially expressed vitamin Dassociated transcriptomes implicated the emergence of an early pregnancy, distinctive immune response in women who went on to develop preeclampsia.

TRIAL REGISTRATION. ClinicalTrials.gov NCT00920621.

FUNDING. Quebec Breast Cancer Foundation and Genome Canada Innovation Network. This trial was funded by the National Heart, Lung, and Blood Institute. For details see Acknowledgments.

Hooman Mirzakhani, Augusto A. Litonjua, Thomas F. McElrath, George OConnor, Aviva Lee-Parritz, Ronald Iverson, George Macones, Robert C. Strunk, Leonard B. Bacharier, Robert Zeiger, Bruce W. Hollis, Diane E. Handy, Amitabh Sharma, Nancy Laranjo, Vincent Carey, Weilliang Qiu, Marc Santolini, Shikang Liu, Divya Chhabra, Daniel A. Enquobahrie, Michelle A. Williams, Joseph Loscalzo, Scott T. Weiss

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JCI - Welcome

Bone Marrow Stem Cells Comments Off on JCI – Welcome | November 17th, 2016

Stem cells are undifferentiated biological cells that can differentiate into specialized cells and can divide (through mitosis) to produce more stem cells. They are found in multicellular organisms. In mammals, there are two broad types of stem cells: embryonic stem cells, which are isolated from the inner cell mass of blastocysts, and adult stem cells, which are found in various tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing adult tissues. In a developing embryo, stem cells can differentiate into all the specialized cellsectoderm, endoderm and mesoderm (see induced pluripotent stem cells)but also maintain the normal turnover of regenerative organs, such as blood, skin, or intestinal tissues.

There are three known accessible sources of autologous adult stem cells in humans:

Stem cells can also be taken from umbilical cord blood just after birth. Of all stem cell types, autologous harvesting involves the least risk. By definition, autologous cells are obtained from one's own body, just as one may bank his or her own blood for elective surgical procedures.

Adult stem cells are frequently used in various medical therapies (e.g., bone marrow transplantation). Stem cells can now be artificially grown and transformed (differentiated) into specialized cell types with characteristics consistent with cells of various tissues such as muscles or nerves. Embryonic cell lines and autologous embryonic stem cells generated through somatic cell nuclear transfer or dedifferentiation have also been proposed as promising candidates for future therapies.[1] Research into stem cells grew out of findings by Ernest A. McCulloch and James E. Till at the University of Toronto in the 1960s.[2][3]

The classical definition of a stem cell requires that it possess two properties:

Two mechanisms exist to ensure that a stem cell population is maintained:

Potency specifies the differentiation potential (the potential to differentiate into different cell types) of the stem cell.[4]

In practice, stem cells are identified by whether they can regenerate tissue. For example, the defining test for bone marrow or hematopoietic stem cells (HSCs) is the ability to transplant the cells and save an individual without HSCs. This demonstrates that the cells can produce new blood cells over a long term. It should also be possible to isolate stem cells from the transplanted individual, which can themselves be transplanted into another individual without HSCs, demonstrating that the stem cell was able to self-renew.

Properties of stem cells can be illustrated in vitro, using methods such as clonogenic assays, in which single cells are assessed for their ability to differentiate and self-renew.[7][8] Stem cells can also be isolated by their possession of a distinctive set of cell surface markers. However, in vitro culture conditions can alter the behavior of cells, making it unclear whether the cells shall behave in a similar manner in vivo. There is considerable debate as to whether some proposed adult cell populations are truly stem cells.[citation needed]

Embryonic stem (ES) cells are the cells of the inner cell mass of a blastocyst, an early-stage embryo.[9] Human embryos reach the blastocyst stage 45 days post fertilization, at which time they consist of 50150 cells. ES cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. In other words, they can develop into each of the more than 200 cell types of the adult body when given sufficient and necessary stimulation for a specific cell type. They do not contribute to the extra-embryonic membranes or the placenta.

During embryonic development these inner cell mass cells continuously divide and become more specialized. For example, a portion of the ectoderm in the dorsal part of the embryo specializes as 'neurectoderm', which will become the future central nervous system.[10] Later in development, neurulation causes the neurectoderm to form the neural tube. At the neural tube stage, the anterior portion undergoes encephalization to generate or 'pattern' the basic form of the brain. At this stage of development, the principal cell type of the CNS is considered a neural stem cell. These neural stem cells are pluripotent, as they can generate a large diversity of many different neuron types, each with unique gene expression, morphological, and functional characteristics. The process of generating neurons from stem cells is called neurogenesis. One prominent example of a neural stem cell is the radial glial cell, so named because it has a distinctive bipolar morphology with highly elongated processes spanning the thickness of the neural tube wall, and because historically it shared some glial characteristics, most notably the expression of glial fibrillary acidic protein (GFAP).[11][12] The radial glial cell is the primary neural stem cell of the developing vertebrate CNS, and its cell body resides in the ventricular zone, adjacent to the developing ventricular system. Neural stem cells are committed to the neuronal lineages (neurons, astrocytes, and oligodendrocytes), and thus their potency is restricted.[10]

Nearly all research to date has made use of mouse embryonic stem cells (mES) or human embryonic stem cells (hES) derived from the early inner cell mass. Both have the essential stem cell characteristics, yet they require very different environments in order to maintain an undifferentiated state. Mouse ES cells are grown on a layer of gelatin as an extracellular matrix (for support) and require the presence of leukemia inhibitory factor (LIF). Human ES cells are grown on a feeder layer of mouse embryonic fibroblasts (MEFs) and require the presence of basic fibroblast growth factor (bFGF or FGF-2).[13] Without optimal culture conditions or genetic manipulation,[14] embryonic stem cells will rapidly differentiate.

A human embryonic stem cell is also defined by the expression of several transcription factors and cell surface proteins. The transcription factors Oct-4, Nanog, and Sox2 form the core regulatory network that ensures the suppression of genes that lead to differentiation and the maintenance of pluripotency.[15] The cell surface antigens most commonly used to identify hES cells are the glycolipids stage specific embryonic antigen 3 and 4 and the keratan sulfate antigens Tra-1-60 and Tra-1-81. By using human embryonic stem cells to produce specialized cells like nerve cells or heart cells in the lab, scientists can gain access to adult human cells without taking tissue from patients. They can then study these specialized adult cells in detail to try and catch complications of diseases, or to study cells reactions to potentially new drugs. The molecular definition of a stem cell includes many more proteins and continues to be a topic of research.[16]

There are currently no approved treatments using embryonic stem cells. The first human trial was approved by the US Food and Drug Administration in January 2009.[17] However, the human trial was not initiated until October 13, 2010 in Atlanta for spinal cord injury research. On November 14, 2011 the company conducting the trial (Geron Corporation) announced that it will discontinue further development of its stem cell programs.[18] ES cells, being pluripotent cells, require specific signals for correct differentiationif injected directly into another body, ES cells will differentiate into many different types of cells, causing a teratoma. Differentiating ES cells into usable cells while avoiding transplant rejection are just a few of the hurdles that embryonic stem cell researchers still face.[19] Due to ethical considerations, many nations currently have moratoria or limitations on either human ES cell research or the production of new human ES cell lines. Because of their combined abilities of unlimited expansion and pluripotency, embryonic stem cells remain a theoretically potential source for regenerative medicine and tissue replacement after injury or disease.

Human embryonic stem cell colony on mouse embryonic fibroblast feeder layer

The primitive stem cells located in the organs of fetuses are referred to as fetal stem cells.[20] There are two types of fetal stem cells:

Adult stem cells, also called somatic (from Greek , "of the body") stem cells, are stem cells which maintain and repair the tissue in which they are found.[22] They can be found in children, as well as adults.[23]

Pluripotent adult stem cells are rare and generally small in number, but they can be found in umbilical cord blood and other tissues.[24] Bone marrow is a rich source of adult stem cells,[25] which have been used in treating several conditions including liver cirrhosis,[26] chronic limb ischemia [27] and endstage heart failure.[28] The quantity of bone marrow stem cells declines with age and is greater in males than females during reproductive years.[29] Much adult stem cell research to date has aimed to characterize their potency and self-renewal capabilities.[30] DNA damage accumulates with age in both stem cells and the cells that comprise the stem cell environment. This accumulation is considered to be responsible, at least in part, for increasing stem cell dysfunction with aging (see DNA damage theory of aging).[31]

Most adult stem cells are lineage-restricted (multipotent) and are generally referred to by their tissue origin (mesenchymal stem cell, adipose-derived stem cell, endothelial stem cell, dental pulp stem cell, etc.).[32][33]

Adult stem cell treatments have been successfully used for many years to treat leukemia and related bone/blood cancers through bone marrow transplants.[34] Adult stem cells are also used in veterinary medicine to treat tendon and ligament injuries in horses.[35]

The use of adult stem cells in research and therapy is not as controversial as the use of embryonic stem cells, because the production of adult stem cells does not require the destruction of an embryo. Additionally, in instances where adult stem cells are obtained from the intended recipient (an autograft), the risk of rejection is essentially non-existent. Consequently, more US government funding is being provided for adult stem cell research.[36]

Multipotent stem cells are also found in amniotic fluid. These stem cells are very active, expand extensively without feeders and are not tumorigenic. Amniotic stem cells are multipotent and can differentiate in cells of adipogenic, osteogenic, myogenic, endothelial, hepatic and also neuronal lines.[37] Amniotic stem cells are a topic of active research.

Use of stem cells from amniotic fluid overcomes the ethical objections to using human embryos as a source of cells. Roman Catholic teaching forbids the use of embryonic stem cells in experimentation; accordingly, the Vatican newspaper "Osservatore Romano" called amniotic stem cells "the future of medicine".[38]

It is possible to collect amniotic stem cells for donors or for autologuous use: the first US amniotic stem cells bank [39][40] was opened in 2009 in Medford, MA, by Biocell Center Corporation[41][42][43] and collaborates with various hospitals and universities all over the world.[44]

These are not adult stem cells, but rather adult cells (e.g. epithelial cells) reprogrammed to give rise to pluripotent capabilities. Using genetic reprogramming with protein transcription factors, pluripotent stem cells equivalent to embryonic stem cells have been derived from human adult skin tissue.[45][46][47]Shinya Yamanaka and his colleagues at Kyoto University used the transcription factors Oct3/4, Sox2, c-Myc, and Klf4[45] in their experiments on human facial skin cells. Junying Yu, James Thomson, and their colleagues at the University of WisconsinMadison used a different set of factors, Oct4, Sox2, Nanog and Lin28,[45] and carried out their experiments using cells from human foreskin.

As a result of the success of these experiments, Ian Wilmut, who helped create the first cloned animal Dolly the Sheep, has announced that he will abandon somatic cell nuclear transfer as an avenue of research.[48]

Frozen blood samples can be used as a source of induced pluripotent stem cells, opening a new avenue for obtaining the valued cells.[49]

To ensure self-renewal, stem cells undergo two types of cell division (see Stem cell division and differentiation diagram). Symmetric division gives rise to two identical daughter cells both endowed with stem cell properties. Asymmetric division, on the other hand, produces only one stem cell and a progenitor cell with limited self-renewal potential. Progenitors can go through several rounds of cell division before terminally differentiating into a mature cell. It is possible that the molecular distinction between symmetric and asymmetric divisions lies in differential segregation of cell membrane proteins (such as receptors) between the daughter cells.[50]

An alternative theory is that stem cells remain undifferentiated due to environmental cues in their particular niche. Stem cells differentiate when they leave that niche or no longer receive those signals. Studies in Drosophila germarium have identified the signals decapentaplegic and adherens junctions that prevent germarium stem cells from differentiating.[51][52]

Stem cell therapy is the use of stem cells to treat or prevent a disease or condition. Bone marrow transplant is a form of stem cell therapy that has been used for many years without controversy. No stem cell therapies other than bone marrow transplant are widely used.[53][54]

Stem cell treatments may require immunosuppression because of a requirement for radiation before the transplant to remove the person's previous cells, or because the patient's immune system may target the stem cells. One approach to avoid the second possibility is to use stem cells from the same patient who is being treated.

Pluripotency in certain stem cells could also make it difficult to obtain a specific cell type. It is also difficult to obtain the exact cell type needed, because not all cells in a population differentiate uniformly. Undifferentiated cells can create tissues other than desired types.[55]

Some stem cells form tumors after transplantation;[56] pluripotency is linked to tumor formation especially in embryonic stem cells, fetal proper stem cells, induced pluripotent stem cells. Fetal proper stem cells form tumors despite multipotency.[citation needed]

Some of the fundamental patents covering human embryonic stem cells are owned by the Wisconsin Alumni Research Foundation (WARF) they are patents 5,843,780, 6,200,806, and 7,029,913 invented by James A. Thomson. WARF does not enforce these patents against academic scientists, but does enforce them against companies.[57]

In 2006, a request for the US Patent and Trademark Office (USPTO) to re-examine the three patents was filed by the Public Patent Foundation on behalf of its client, the non-profit patent-watchdog group Consumer Watchdog (formerly the Foundation for Taxpayer and Consumer Rights).[57] In the re-examination process, which involves several rounds of discussion between the USTPO and the parties, the USPTO initially agreed with Consumer Watchdog and rejected all the claims in all three patents,[58] however in response, WARF amended the claims of all three patents to make them more narrow, and in 2008 the USPTO found the amended claims in all three patents to be patentable. The decision on one of the patents (7,029,913) was appealable, while the decisions on the other two were not.[59][60] Consumer Watchdog appealed the granting of the '913 patent to the USTPO's Board of Patent Appeals and Interferences (BPAI) which granted the appeal, and in 2010 the BPAI decided that the amended claims of the '913 patent were not patentable.[61] However, WARF was able to re-open prosecution of the case and did so, amending the claims of the '913 patent again to make them more narrow, and in January 2013 the amended claims were allowed.[62]

In July 2013, Consumer Watchdog announced that it would appeal the decision to allow the claims of the '913 patent to the US Court of Appeals for the Federal Circuit (CAFC), the federal appeals court that hears patent cases.[63] At a hearing in December 2013, the CAFC raised the question of whether Consumer Watchdog had legal standing to appeal; the case could not proceed until that issue was resolved.[64]

Diseases and conditions where stem cell treatment is being investigated include:

Research is underway to develop various sources for stem cells, and to apply stem cell treatments for neurodegenerative diseases and conditions, diabetes, heart disease, and other conditions.[80]

In more recent years, with the ability of scientists to isolate and culture embryonic stem cells, and with scientists' growing ability to create stem cells using somatic cell nuclear transfer and techniques to create induced pluripotent stem cells, controversy has crept in, both related to abortion politics and to human cloning.

Hepatotoxicity and drug-induced liver injury account for a substantial number of failures of new drugs in development and market withdrawal, highlighting the need for screening assays such as stem cell-derived hepatocyte-like cells, that are capable of detecting toxicity early in the drug development process.[81]

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Stem cell - Wikipedia

Bone Marrow Stem Cells Comments Off on Stem cell – Wikipedia | November 16th, 2016

1. Introduction

Hematopoietic stem cell transplantation (HSCT) has a half-century history. It is currently an indispensable treatment for not only incurable blood diseases such as aplastic anemia and severe hemolytic anemia, but also malignant hematological diseases such as leukemia and lymphoma. Although allergenic HSCT is also used to treat hereditary diseases, its indications are restricted because of critical complications including regimen-related toxicities involving conditioning, infection, and graft-versus-host disease.

Studies in recent decades have shown that HSCT can have a long-term effect in the treatment of hereditary diseases involving a responsible gene in hematogenous cells. Although the first successful gene therapy using lymphocytes or bone marrow cells for a patient with adenosine deaminase (ADA) deficiency inspired great hope in the future of gene therapy [1-3], subsequent gene therapy using HSCs for patients with X-linked severe combined immunodeficiency (SCID-X1) resulted in tumorigenesis [4]. In addition to the self-renewal and multilineage differentiation capacities of tissue stem cells, HSCs exhibit cell-cycle dormancy, which complicates their use in gene therapy.

However, as technological advances have increased the safety and efficiency of introducing genes into HSCs, gene therapy with HSCs is attracting attention again. In this chapter, advances in the technology of HSC gene therapy, e.g., vector design to avoid genotoxicity and increase transgenic efficiency by taking advantage of the special characteristics of HSCs, are reviewed. In addition, recent studies on HSC gene therapy for various hereditary diseases, such as thalassemia, Fanconi anemia, hemophilia, primary immunodeficiency, mucopolysaccharidosis, Gaucher disease, and X-linked adrenoleukodystrophy (X-ALD) are discussed.

The concept of the HSC was introduced by Till and McCulloch in 1961 [5]. Although a healthy adult produces approximately 1 trillion blood cells each day, they are considered to originate from a single HSC which can potentially be transplanted into a mouse [6, 7]. Generally stem cells are defined as cells capable of self-renewal and multilineage differentiation. In addition to these two characteristics, HSCs have the capability of cell-cycle dormancy, i.e. to enter a state of dormancy (G0 phase) in the cell cycle and can continue blood cell production over a lifetime while protecting themselves from various kinds of stress [8].

Fig. 1 shows HSC surface markers and the typical cytokines regulating HSCs. Stem cell factor (SCF) and thrombopoietin (TPO) are important direct cytokine regulators of HSCs. Although SCF promotes the proliferation and differentiation of hematopoietic progenitor cells, it is thought to not be essential for the initiation of hematopoiesis and HSC self-renewal [9]. TPO and its receptor, c-Mpl, are thought to play important roles in early hematopoiesis from HSCs. In contrast to the CD34+CD38-c-Mpl- population, CD34+CD38-c-Mpl+ cells show significantly better HSC engraftment [10]. Mice lacking either TPO or c-Mpl have deficiencies in progenitor cells of multiple hematopoietic lineages [11]. TPO-mediated signal transduction for the self-renewal of HSCs is negatively regulated by the intracellular scaffold protein Lnk [12, 13]. A signal from angiopoietin-1 via Tie2 regulates HSC dormancy by promoting the adhesion of HSCs to osteoblasts in the bone marrow niche and maintains long-term repopulating activity [14]. Although cytokine-induced lipid raft clustering of the HSC membrane is essential for HSC re-entry into the cell cycle, transforming growth factor- (TGF-) inhibits lipid raft clustering and induces p57Kip2 expression, leading to HSC dormancy [15, 16]. Recently, the hypoxic niche of HSCs has been demonstrated. It, along with the osteoblastic and vascular niches, are important for HSC dormancy [17-19]. They are targets in HSC gene therapy [20].

Hematopoietic stem cell (HSC) surface markers and typical cytokines that regulate HSCs. Stem cell factor (SCF) promotes the proliferation and differentiation of HSCs. Thrombopoietin (TPO) and its receptor, c-Mpl, play important roles in early hematopoiesis, especially self-renewal. Signals from angiotensin-1 via Tie2 and transforming growth factor - via its receptors regulate HSC dormancy. (This figure is based on the illustration by BioLegend, Inc. San Diego, CA, U.S.A. http://www.biolegend.com/cell_markers)

While making a HSC with few opportunities for cell division into a transgenic target, it is important to design a safe and efficient vector for inserting a gene into the host chromosome. Furthermore, since a hematogenous cell also has many cells which exhibit its function in the specialization process to a mature effector cell, it is also important to select differentiation-specific or non-specific promoters or enhancers during the vector design process.

Vectors derived from the Retroviridae family, RNA viruses with reverse transcriptase activity, are widely used for inserting genes in host chromosomes. Although adeno-associated virus (AAV) vectors can also insert genes into host chromosomes, this process is inefficient and partial. Gammaretroviruses and lentiviruses are members of the Retroviridae family that are commonly used as vectors in HSC gene therapy. Generally, the former is called simply a retroviral vector and the latter is called a lentiviral vector. When a gene is inserted in the chromosome of an HSC with a Retroviridae vector, genotoxicity can occur.

Retroviral vectors are commonly constructed from the Moloney murine leukemia virus (MoMLV) genome. Retroviral genomes have a gag/pol gene that codes for viral structure proteins, protease and reverse transcriptase, an env gene that codes for the envelope glycoprotein and the packaging signal. These genes are flanked by long terminal repeats (LTR) which contain enhancers and promoters. A retroviral vector consists of a packaging plasmid that does not have the packaging signal but does include the gag/pol gene, a transfer vector with the packaging signal, and the target gene cDNA. After transfection of these plasmids into producer cells (e.g., 297T cells, NIH3T3 cell, etc.), a target vector is obtained by collecting the culture solution.

Expression of a target gene can be inhibited by mechanisms such as methylation of CpG islands in the promoter region, insertion of a negative control region (NCR) into the LTR, and the presence of a repressor binding site (RBS) downstream of the 5 LTR. Other vectors, such as the murine stem cell virus (MSCV) vector [21], the myeloproliferative sarcoma virus vector, the negative control region deleted (MND) vector [22], and the MFG-S vector [23] were developed to improve the efficiency of transgene expression; they are widely used in clinical applications of gene therapy involving HSCs.

Since the retroviral viral genome cannot cross the nuclear membrane, it can be incorporated into a chromosome only during the phase of mitosis when the nuclear membrane has disassembled. Since many HSCs are thought to exist in a dormant phase, insertions into the HSC genome with a retroviral vector require a proliferation stimulus by cytokines. Although various combinations of cytokines to suppress the decrease in HSC self-renewal have been studied, stem cell factor (SCF), fms-related tyrosine kinase-3 (Flt-3) ligand, interleukin-3 (IL-3), TPO, among others, are commonly used [24, 25].

Human immunodeficiency virus type 1 (HIV-1), the representative lentivirus, differs from gammaretroviruses in that it can be incorporated during a non-mitotic phase. This is one advantage of lentiviral vectors in HSC gene therapy.

Both lentiviruses and gammaretroviruses have gag, pol, and env genes sandwiched between LTRs with promoter activity at both ends. In addition, lentiviruses have accessory genes (vif, vpr, vpu, nef) and regulatory genes (tat, rev). Double-stranded cDNA produced from the viral genome enters the cell, and a pre-integration complex is formed with a host protein. This complex can pass through the pores of the nuclear membrane during non-mitotic phases, allowing the viral genome to be inserted into the host cell chromosome.

HIV provirus (A) and the four plasmids of a third-generation lentiviral vector (B). The viral long terminal repeats (LTRs), reading frames of the viral genes, splice donor site (SD), splicing acceptor site (SA), packaging signal (), and rev-responsive element (RRE) are indicated. The packaging plasmid contains the gag and pol genes under the influence of the CMV promoter, intervening sequences, and the polyadenylation site (polyA) of the human -globin gene. As the transcripts of the gag and pol genes contain cis-repressive sequences, they are expressed only if rev promotes their nuclear export by binding to the RRE. All tat and rev exons have been deleted, and the viral sequences upstream of the gag gene have been replaced. The rev plasmid expresses rev cDNA. The SIN vector plasmid contains HIV-1 cis-acting sequences and an expression cassette for the transgene. It is the only portion transferred to the target cells and does not contain wild-type copies of the HIV LTR. The 5 LTR is chimeric, with the RSV enhancer and promoter replacing the U3 region to rescue transcriptional dependence on tat. The 3 LTR has an almost completely deleted U3 region, which includes the TATA box. As the latter is the template used to generate both copies of the LTR in the integrated provirus, transduction of this vector results in transcriptional inactivation of both LTRs; thus, it is a self-inactivating (SIN) vector. The envelope plasmid encodes a heterologous envelope to pseudotype the vector, here shown coding for vesicular stomatitis virus (VSV)-G. Only the relevant parts of the constructs are shown (Reproduced with modifications from [26]).

Although first-generation lentiviral vectors included modification genes, they were removed in the second generation because it was discovered that the modification genes are not required for infection during non-mitotic phases. In the third generation, further modifications included the deletion of tat, use of multiple vector plasmids, and introduction of self-inactivating (SIN) vectors. The structure of HIV-1 and a typical third-generation lentiviral vector system are shown in Fig. 2 [26]. Approximately one-third of the HIV-1 genome has been deleted, and the vector system has been divided into four plasmids, namely, the packaging plasmid, rev plasmid, SIN vector plasmid and envelope plasmid. To prevent production of wild type HIV-1, tat, a regulatory gene indispensable to viral reproduction was deleted, and the rev gene was moved to a separate plasmid. Moreover, since the HIV-1 LTR promoter is weak in the absence of tat, it was replaced with the cytomegalovirus (CMV) promoter in the packaging plasmid. Since an envelope plasmid can only infect CD4 positive cells with a HIV-1 envelope, the envelope gene was replaced with the vesicular stomatitis virus G glycoprotein (VSV-G) envelope. The SIN vector further improved safety by replacing the enhancer / promoter portion of the LTR, suppressing the activation of unnecessary genes with the integrated gene (Fig. 3) [27].

Mechanism of gene activation induced by vector insertion. The genomic integration site of an MLV-based retroviral vector is depicted. With this MLV vector design, the enhancer and promoter within the U3 region (blue rectangle) of the long terminal repeat (LTR) drive transcription of the transgene (indicated by the parallel arrow arising from the blue rectangle). Vector integration near Gene X is shown in the top panel. The enhancer elements located in the U3 region (blue rectangle) of the vector can interact with the regulatory elements upstream of Gene X to increase its basal transcription rate to inappropriately high levels, potentially altering the growth of the cell. Two alternatives for eliminating the use of the powerful enhancer in the U3 region include (1) middle panel: use of a self-inactivating (SIN) MLV-based vector in which the U3 region has been deleted. An internal cellular promoter is used to drive transgene expression and (2) bottom panel: use of a SIN lentiviral vector in which U3 (yellow rectangle) has been eliminated. This system also uses an internal cellular promoter to drive transgene expression (Reproduced with modification from [27]).

To improve the gene transfer into HSCs, Verhoeyen and colleagues designed lentiviral vectors displaying early-acting cytokines such as TPO and SCF. This vector can promote survival of CD34 positive HSCs and achieve selective transduction of long-term repopulating cells in a humanized mouse model (Fig. 4) [28, 29].

Lentiviral vector particles (HIV-1) display recombinant membrane envelope proteins such as stem cell factor (SCF), thrombopoietin (TPO), and vesicular stomatitis virus G glycoprotein (VSV-G). This vector can specifically target vector particles to hematopoietic stem cells (HSCs) expressing c-kit and c-mpl receptors for SCF and TPO, respectively. VSV-G envelope protein can bind to phospholipids in the HSC cell membrane. (Karlsson S, Gene therapy: efficient targeting of hematopoietic stem cells. Blood. 2005;106(10):3333)

The most serious problem with using viral vectors to incorporate a gene into a chromosome is the potential development of clonal proliferative diseases such as leukemia, which was observed in clinical trials involving gene therapy for SCID-X1 and chronic granulomatous disease (CGD). Although this problem of genotoxicity represents a great hurdle in the development of clinical applications for gene therapy, there is promising ongoing research on the mechanisms underlying genotoxicity and how to avoid it.

The mechanisms of retrovirus-induced oncogenesis are shown in Fig. 5 [30]. In oncogene capture, an acute transforming replication-competent retrovirus captures a cellular proto-oncogene and mediates transformation. This mechanism does not occur in replication-incompetent vectors. Second, the provirus 3 LTR can trigger increased transcription of a cellular proto-oncogene. Third, enhancers in the provirus LTRs can activate transcription from nearby cellular proto-oncogene promoters. Fourth, a novel isoform can be expressed when transcription from the provirus 5 LTR creates a novel truncated isoform of a cellular proto-oncogene via splicing. Fifth, an inserted provirus can disrupt transcription by causing premature polyadenylation. The same mechanisms can occur in cellular oncogenesis when a gene is inserted by a retroviral vector [30].

Retroviral mechanisms of oncogenesis. The detailed mechanisms are shown in the text. The integrated provirus is indicated by two LTRs. Cellular proto-oncogene promoter and exons are indicated by black and grey boxes respectively (Reproduced from [30]).

Even if a gene is inserted into a HSC similarly, it is also known that there are diseases which may develop a tumor, and diseases a tumor is not accepted to be. Each type of virus has a unique integration profile, and the following observations have been made [30]: (a) Different retroviral vectors have distinct integration profiles. (b) The route of entry does not appear to strongly affect distribution of integration sites. VSV-Gpseudotyped HIV vectors have an integration profile similar to HIV virions with the native HIV envelope despite differences in the route of entry. (c) The integration profile is largely independent of the target cell type, although the transcriptional program and epigenetic status of the target cell can influence integration site selection. (d) For lentiviruses, which can integrate independently of mitosis, the cell-cycle status of the target cell has only a modest effect on the distribution of integration sites.

In order to avoid genotoxicity, various SIN vectors have been developed and improved. In general, lentiviral vectors are considered to have a lower risk of oncogenesis than retroviral vectors [31]. However, when a HSC is the target cell, more attention should be required because tumorigenesis can occur when the cell with the inserted gene undergoes differentiation.

Diseases in which gene therapy using HSCs are being studied are shown in Table 1. They are roughly divided into hematological disorders, immunodeficiencies, and metabolic diseases. Most are congenital or hereditary diseases. The characteristic clinical features and recent basic science or clinical studies on HSC gene therapy for each disease are discussed below.

Clinical applications of hematopoietic stem cell gene therapy.

Hemoglobin A (HbA), comprising 98% of adult human hemoglobin, is a tetramer with two -globin and two -globin chains combined with a heme group. -thalassemia is an autosomal hemoglobin disorder caused by decreased -globin chain synthesis. Although individuals with -thalassemia minor (heterozygote) may be asymptomatic or have mild to moderate microcytic anemia, -thalassemia major (homozygote) progresses to serious anemia by one or two years of age, and hemosiderosis, iron overload caused by transfusion or increased iron absorption, develops. Since most patients develop life-threatening complications such as heart failure by adolescence, HSCT has been performed in patients with advanced disease [32]. In recent years, gene therapy using a lentiviral vector containing a functional -globin gene has been performed in an HbE/ -thalassemia (E/ 0) transfusion-dependent adult male, who subsequently did not require transfusions for over 21 months [33].

The human -globin locus is located in a large 70kb area which also contains some -like globulin genes (, G, A, , ). Gene switching takes place according to the development stage, and the -globin gene is transcribed and expressed specifically after birth. A powerful enhancer called the LCR (locus control region) exists on the 5 side of the promoter. The LCR contains five DNase I hypersensitive sites, referred to as HS5 to HS1 starting from the 5 side. Furthermore, HS5 contains CCCTC-binding factor (CTCF)-dependent insulator.

The structure of the lentiviral SIN vector used in gene therapy for -thalassemia is shown in Fig. 6. To improve safety, two stop codons were inserted into the packaging signal () of GAG, the HS5 portion with insulator activity was deleted, and two copies of the 250 base pair (bp) core of the cHS4 chromatin insulators (chicken -globin insulators) were inserted in the U3 region of the HIV 3 LTR. Furthermore, the amino acid at the 87th position of -globin was changed from threonine to glutamine. This altered -globin can be distinguished from normal adult -globin by high performance liquid chromatography (HPLC) analysis in individuals receiving red blood cell transfusion and +-thalassemia patients [33].

Diagram of the human -globin gene in a lentiviral vector. HIV LTR, human immunodeficiency type-1 virus long terminal repeat; +, packaging signal; cPPT/flap, central polypurine tract/DNA flap; RRE, rev-responsive element; p, human -globin promoter; ppt, polypurine tract; HS, DNase I Hypersensitive Sites (Reproduced with color modification from [33])

A clinical study using this vector was performed in two -thalassemia patients. As with autologous bone marrow transplantation, some of the patients marrow cells were cryopreserved as a backup. The lentiviral vector particles containing a functional -globin were introduced into the remaining cells. After the transfected cells were cultured for one week ex vivo, some were also cryopreserved. The patients were conditioned with intravenous busulfan (3.2 mg/kg/day for four days) without the addition of cyclophosphamide, before transplantation using the autologous gene-modified cryopreserved cells (Fig. 7) [34].

The first patient failed to engraft because the HSCs had been compromised by how they were handled, not because of any issues with the gene therapy vector, and ultimately used backup bone marrow. The second patient, as described previously, achieved long-term -globin production; one-third of the patients hemoglobin was produced by the genetically modified cells [33].

Furthermore, the detailed examination of the transgenic cells showed significantly increased expression of high mobility group AT-hook 2 (HMGA2), which interacts with transcription factors to regulate gene expression, in the clones where gene insertion occurred in the HMGA2 gene. The proportion of the HMGA2 overexpressing clones increased with time, to over 50% of transgenic cells at 20 months after gene therapy. In this patient, the HMGA2 overexpressing cells were only 5% of all circulating hematopoietic cells and there was no evidence of malignant transformation. However, researchers point out that there was expressive production of a truncated form of the HMGA2 protein. Since truncated or overexpressed HMGA2 is observed with some blood cancers and non-malignant expansions of blood cells, caution is recommended with this therapy [34].

Gene-therapy procedure for patient with b-thalassemia. a. Hematopoietic stem cells (HSCs) are collected from the bone marrow of a patient with -thalassemia and maintained them in culture. b, Lentiviral-vector particles containing a functional -globin gene were then introduced into the cells and allowed them to expand further in culture. c. To eradicate the patients remaining HSCs and make room for the geneticaaly modified cells, the patient underwent chemotherapy. d. The genetically modified HSCs were then transplanted into the patient (Reproduced from [34]).

Recently, researchers generated a LCR-free SIN lentiviral vector that combines two hereditary persistence of fetal hemoglobin (HPFH)-activating elements, resulting in therapeutic levels of A-globin protein produced by erythroid progenitors derived from thalassemic HSCs [35]. Both lentiviral-mediated -globin gene addition and genetic reactivation of endogenous -globin genes are considered potentially capable of providing therapeutic levels of hemoglobin F to patients with -globin deficiency [36]. In addition, a trial of -globin induction with -globin production using mithramycin, an inducer of -globin expression, to remove excess -globin proteins in -thalassemic erythroid progenitor cells was reported [37].

Fanconi anemia is a hereditary disease characterized by cellular hypersensitivity to DNA crosslinking agents. It leads to bone marrow failure, such as aplastic anemia, by approximately eight years of age. Since there is a high risk of developing malignancy, HSCT has been performed as a curative treatment for bone marrow insufficiency. Although the ten-year probability of survival after transplant from an Human leukocyte antigen (HLA) -identical donor is over 80%, results with other donors are not satisfactory. HSC gene therapy is considered an alternative in cases where there is no HLA-identical donor available [38-40].

There are currently 13 discovered Fanconi anemia complement groups and 13 distinct genes (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, FANCN) have been cloned. Mutations in FANCB are associated with an X-linked form of Fanconi anemia; mutations in the other genes are associated with autosomal recessive transmission. Although frequencies vary by geographical region, FANCA gene abnormalities are found in more than half of all Fanconi anemia patients [41]. Although one of the major hurdles in the development of gene therapy for Fanconi anemia is the increased sensitivity of Fanconi anemia stem cells to free radical-induced DNA damage during ex vivo culture and manipulation, retroviral and lentiviral vectors have been successfully employed to deliver complementing Fanconi anemia cDNA to HSCs with targeted disruptions of the FANCA and FANCC genes [20, 42-44]. In a phase I trial of FANCA gene therapy, gene transfer was performed with patient bone marrow-derived CD34+ cells and the MSCV retroviral vector [38]. Whether sufficient HSCs can be obtained is a potential problem in Fanconi anemia patients due to possible bone marrow insufficiency, but in this study, sufficient target CD34+ cells were obtained from most patients. Two patients had FANCA-transduced cells successfully infused. The procedure was safe, well tolerated, and resulted in transient improvements in hemoglobin and platelet counts [39]. However, transduced cell products were not obtained in one patient who required cryopreserved bone marrow. The first clinical study of FANCC gene therapy using a retroviral vector involved four patients. Although functional FANCC gene expression was observed in peripheral blood and bone marrow cells, the results were transient [43].

Engraftment efficiency of FANCA-modified cells using a lentiviral vector was studied in a mouse model. Rapid transduction with four hours of culture using only SCF and megakaryocyte growth and development factor and minimal differentiation of gene-induced cells is better than standard 96-hour culture using a variety of cytokines, including SCF, interleukin-11, Flt-3 ligand, and IL-3 [44]. Moreover, a recent trial demonstrated enhanced viability and engraftment of gene-corrected cells in patients with FANCA abnormalities with short transduction (overnight), low oxidative stress (5% oxygen), and the anti-oxidant N-acetyl-L-cysteine [20]. Lentiviral transduction of unselected Fanconi anemia bone marrow cells mediates efficient phenotypic correction of hematopoietic progenitor cells and CD34- mesenchymal stromal cells, with increased efficacy in hematopoietic engraftment [45]. In Fancg -/- mice, the wild-type mesenchymal stem and progenitor cells play important roles in the reconstitution of exogenous HSCs in vitro [46]. Recently, a new approach that directly injects lentiviral vector particles into the femur for FANCC gene transfer in mice was able to successfully introduce the FANCC gene to HSCs. This result provides evidence that targeting the HSCs directly in their native environment enables efficient and long-term correction of bone marrow defects in Fanconi anemia [47].

In recent years, the design of lentiviral vectors used for gene therapy in Fanconi anemia has improved. Although the vav and phosphoglycerate kinase (PGK) promoters are relatively weak, physiological levels of FANCA gene expression can be obtained in lymphoblastoid cells. CMV and spleen focus-forming virus (SFFV) promoters result in overexpression of FANCA. The PGK-FANCA lentiviral vectors with either a wild-type woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or a mutated WPRE in the 3 region have higher levels of FANCA gene expression. In conclusion, lentiviral vectors with a mutated WPRE and a PGK promoter are considered the most suitable with respect to safety and efficiency for Fanconi anemia gene therapy [48].

There was a recent interesting report on the use of induced pluripotent stem cells (iPS cell). Instead of introducing a repaired gene into the HSCs of a patient with a FANCA gene abnormality, the modified gene was introduced into more stable somatic cells, e.g. fibroblasts, and iPS cells were derived from the genetically modified somatic cells. If HSCs can be produced from genetically modified iPS cells, hematological function can be efficiently reconstructed in patients with hematologic disorders [49].

Hemophilia is a common congenital coagulopathy caused by coagulation factor VIII (hemophilia A) or IX (hemophilia B) deficiency. Although the genes encoding both factor VIII (Xq28) and factor IX (Xq27) are located on the X chromosome and most cases are X-linked, many sporadic variations have been reported. Factor substitution therapies have been used to treat hemophilia for many years. However, there is great hope for gene therapy with hemophilia because coagulation factors have short half-lives (factor VIII, 8 to 12 hours; factor IX, 18 to 24 hours), and an inhibitor is produced in many cases. Furthermore, it is possible for gene therapy to suppress immunogenicity by introducing a mutant protein that lacks the domain with which the inhibitor interacts. Since both coagulation factors are usually produced in the liver, there are few studies involving HSCs. In addition to hepatocytes, trials introducing the modified gene directly into splenic cells, endothelial cells, myoblasts, fibroblasts, etc. have been reported [50-52]. Since the factor IX gene (34 kb) is smaller than the factor VIII gene (186 kb), hemophilia B gene therapy can be possible with an adenovirus vector or an AAV vector. Therefore, hemophilia B is progressing more as a field of gene therapy research even through there are five times more patients with hemophilia A [51-53].

Recently, human factor VIII variant genes were successfully introduced into the HSCs of a mouse with hemophilia A resulting in therapeutic levels of factor VIII variant protein expression. This variant factor VIII has changes in the B and A2 domain in addition to the A1 domain for improved secretion and reduced immunogenicity (wild-type factor VIII has six domains, A1, A2, B, A3, C1, and C2) [54]. To ameliorate the symptoms of hemophilia A, partial replacement of the mutated liver cells by healthy cells in hemophilia A mice was challenged with allogeneic bone marrow progenitor cell transplantation. In this study, the bone marrow progenitor cell-derived hepatocytes and sinusoidal endothelial cells synthesized factor VIII, showing that autologous gene-modified bone marrow progenitor cells have the potential to treat hemophilia [55].

Although HSCT has been widely performed as curative treatment for primary immunodeficiencies, gene therapy has been considered when there is no HLA-identical donor available. As previously shown, the first successful gene therapy was performed in a patient with ADA deficiency in the U.S. in 1990. Since the gene was introduced into T lymphocytes, frequent treatment was required. However, this treatment was associated with an unacceptable level of toxicity. Since transfected vector and normal ADA gene expression in T lymphocytes continued for two years after the cessation of treatment [1], gene therapy attracted attention. With advances in HSC gene-transfer technology, gene therapy for many primary immunodeficiencies can now be considered [56].

SCID-X1 is an X-linked disease caused by deficiency of the common (c) chain in the IL-2 receptor. Because the c chain is common to the IL-4, IL-7, IL-9, IL-15, and IL-21 receptors, in SCID-X1 patients, there are defects in T and natural killer (NK) cells, and B cell dysfunction are usually observed [57]. Patients begin suffering from various infections starting several weeks after birth. Without curative treatment, such as HSCT, patients die in infancy.

In SCID-X1, since T cells are lacking, engraftment of the gene-transduced cells can be achieved without pre-conditioning therapy. In the clinical studies of SCID-X1 patients in France and the U.K., the MFG retroviral vector was used with HSCs obtained from the patient. After gene therapy, many patients had improvements in immune function. However, since the genes regulating lymphocyte proliferation, such as LIM domain only 2 (LMO2), Bmi1, cyclin D2 (CCND2) are near the gene insertion region, there was a high frequency of T-cell leukemia after treatment. Furthermore, in the patients who developed leukemia, additional chromosomal changes, including activating mutations of Notch1, changes in the T cell receptor region, and deletion of tumor suppressor genes, e.g. cyclin-dependent kinase-2A (CDKN2A) were observed [58]. Almost gene integration sites by the retroviral vector were inside or near genes that are highly expressed in CD34 positive stem cells. Furthermore, the activity of protein kinases or transferases coded by these activated genes was stronger in CD3 positive T cells than CD34 positive cells [59]. Thus, gene integration mediated by a retrovirus influences the target cells dormant capacity for survival, engraftment, and proliferation.

Although continuous T cell production was founded in many cases, there was little reconstruction of myeloid cells and B cells, and some patients required continuous immunoglobulin substitution therapy. The use of conditioning therapy is also related to immunological reconstruction after c chain gene therapy. There is decreased NK cell reconstruction without conditioning therapy, so conditioning chemotherapy is required for the engraftment of undifferentiated stem cells [58]. A trial of SCID-X1 gene therapy in the U.S. involved three patients ranging from 10 to 14 years of age. They had poor immunological recovery after allergenic HSCT and T cell recovery was only observed in the youngest patient, suggesting there is a limit to the recovery of the function of the thymus in older children [60].

To study whether activation of genes near the region of gene insertion or inserted c chain gene expression itself induces oncogenicity during SCID-X1 gene therapy, a study of the human c chain gene being expressed under the control of the human CD2 promoter and LTR (CD2- c chain gene) was performed in mice. When the CD2- c chain gene was expressed in transgenic mice, a few abnormalities involving T cells were observed, but tumorigenesis was not observed and T and B cell functions were recovered in c chain-gene deficient mice. This study demonstrated that when the c c chain gene is expressed externally, SCID-X1 may be treated safely [61].

Although SIN vectors were developed from earlier retroviral [62] or lentiviral vectors [63] to reduce the risk of oncogenicity in SCID-X1 gene therapy, genotoxicity unrelated to mutations in gene insertion regions or c chain gene overexpression have been reported with lentiviral vectors in recent years, and it seems that more sophisticated vector development is required [64].

ADA is an enzyme that catalyzes the conversion of purine metabolism products adenosine and deoxyadenosine into inosine or deoxyinosine. ADA-SCID is an autosomal recessive disease that results in the accumulation of adenosine, deoxyadenosine, and deoxyadenosinetriphosphate (dATP). Accumulated phosphorylated purine metabolism products act on the thymus and cause the maturational or functional disorder of lymphocytes. Because ADA-SCID patients have both T and B cell production fail, patients have a severe combined immunodeficiency disease with a clinical presentation similar to SCID-X1 results, but unlike SCID-X1, many patients have a low level of T cells. Although enzyme replacement therapy with polyethylene glycolmodified bovine ADA (PEG-ADA) was developed to treat ADA-SCID, it is limited by the development of neutralizing antibodies and the cost of lifelong treatment.

In ADA-SCID, since T cell counts are increased by PEG-ADA, gene therapy to increase peripheral T cell counts was attempted during the early stages of gene therapy. Although adverse events were not observed and continuous expression of ADA was achieved in many patients, reconstruction of immune function was not obtained and substitution therapy with PEG-ADA remained necessary. Therefore, HSCs were no longer the target of gene therapy for ADA-SCID. Since ADA-SCID patients have T cells, nonmyeloablative conditioning was performed to achieve gene-transduced HSC engraftment [25, 65].

In a joint Italian-Israeli study started in 2000, ten ADA-SCID children were infused with CD34 positive cells transduced with a MoMLV retroviral vector containing the ADA gene after nonmyeloablative conditioning with busulfan (2mg/kg/day for two days). T cell counts or function were improved in nine out of the ten patients, and PEG-ADA was discontinued in eight. Many patients also had improvements in B or NK cell function, and immunoglobulin substitution therapy was discontinued in five patients. Although some patients had serious adverse events including prolonged neutropenia, hypertension, Epstein-Barr virus infection, and autoimmune hepatitis, there were no cases of treatment-induced leukemia [25].

As with SCID-X1, the retroviral vector gene insertion region is also near genes that control cell proliferation or self-duplication, such as LMO2, or proto-oncogenes [66]. In clinical studies performed in France, the U.S., and the U.K., none of the ADA-SCID patients had adverse events related to insertional mutagenesis, such as leukemia [67, 68]. Thus, HSC gene therapy for ADA-SCID using a lentiviral vector [69] is expected to become the alternative therapy in cases without a suitable donor for HSCT [70]. As an alternative to HSC-based gene therapy, a study using an AAV vector has reported ADA gene expression in various tissues, including heart, skeletal muscle, and kidney [71].

CGD is a disease caused by an abnormality in nicotinamide dinucleotide phosphate (NADPH) oxidase expressed in phagocytes, resulting in failure to produce reactive oxygen species and decreased ability to kill bacteria or fungi after phagocytosis. NADPH oxidase consists of gp91phox (Nox2) and p22 phox which together constitute the membrane-spanning component flavocytochrome b558 (CYBB), and the cytosolic components p47phox, p67phox, p40phox, and Rac. CGD is caused by a functional abnormality in any of these components. Mutations in gp91phox on the X chromosome account for approximately 70% of CGD cases. CGD patients are afflicted with recurrent opportunistic bacterial and fungal infections, leading to the formation of chronic granulomas. Although lifelong antibiotic prophylaxis reduces the incidence of infections, the overall annual mortality rate remains high (2%5%) and the success rate of HSCT is limited by graft-versus-host-disease and inflammatory flare-ups at infected sites [56].

In the initial trials of CGD gene therapy without any conditioning therapy, p47phox or gp91phox gene was inserted using a retroviral vector. The inserted gene was expressed in peripheral blood granulocytes three to six weeks after re-infusion and mobilization by granulocyte colony-stimulating factor (G-CSF), but there was no clinical effect within six months [72-74].

In a German study where gp91phox was inserted with busulfan conditioning (8mg/kg), there were fewer infections after gene therapy. Gene expression was observed in 20% of leukocytes in the first month, rising to 80% at one year. However, in the gene insertion region there are genes related to myeloid cell proliferation, such as myelodysplastic syndrome 1-ecotropic virus integration site 1 (MDS1/EVI1), PR domain containing protein 16 (PRDM16), SET binding protein 1 (SETBP1). Two patients developed myelodysplasia [75]. These two patients had monosomy 7, considered to be related to EVI1 activation. One died of severe sepsis 27 months after gene therapy. Although the gene-inserted cells remained expressed in this patient, methylation of the CpG site in the LTR of the viral vector was observed and the expression of the inserted gp91phox gene was decreased. Interestingly, methylation was restricted to the promoter region of the LTR; the enhancer region was not methylated. Therefore, although gp91phox gene expression was decreased, the activation of EVI1 near the inserted region occurred, leading to clonal proliferation [76]. Since there is a possibility that the transcription activity of genes related to myeloid cell proliferation near the gene insertion site will be increased, there remains a concern about tumorigenesis with peripheral stem cells mobilization by G-CSF in CGD patients, as with X-SCID [74].

Recently, next-generation gene therapy for CGD using lineage- and stage-restricted lentiviral vectors to avoid tumorigenesis [77] and novel approaches involving iPSs derived from CGD patients using zinc finger nuclease (ZFN)-mediated gene targeting were studied [78]. Specific gene targeting can be performed in human iPSs using ZFNs to induce sequence-specific double-strand DNA breaks that enhance site-specific homologous recombination. A single-copy of gp91phox was targeted into one allele of the "safe harbor" AAVS1 locus in iPSs [79].

WAS is a severe X-linked immunodeficiency caused by mutations in the gene encoding the WAS protein (WASP), a key regulator of signaling and cytoskeletal reorganization in hematopoietic cells. Mutations in WAS gene result in a wide spectrum of clinical manifestations ranging from relatively mild X-linked thrombocytopenia to the classic WAS phenotype characterized by thrombocytopenia, immunodeficiency, eczema, high susceptibility to developing tumors, and autoimmune manifestations [80]. Preclinical and clinical evidence suggest that WASP-expressing cells have a proliferative or survival advantage over WASP-deficient cells, supporting the development of gene therapy [56]. Furthermore, up to 11% of WAS patients have somatic mosaicism due to spontaneous in vivo reversion to the normal genotype, and in WAS patients, accumulation of normal T-cell precursors are sometimes seen [81].

In one preclinical study introducing the WAS gene into human T and B cells or mouse HSCs using a retroviral vector, recovery of T cell function and immune reactions to infection were observed [82, 83]. The first clinical study of WAS using HSCs involved two young boys in Germany. The WASP-expressing retroviral vector was transfected into CD34 positive cells obtained by apheresis of peripheral blood. Busulfan was used for conditioning therapy (4mg/kg/day for two days). Over two years, WASP gene expression by HSCs, lymphoid and myeloid cells, and platelets was sustained, and the number and function of monocytes, T, B, and NK cells normalized. Clinically, hemorrhagic diathesis, eczema, autoimmunity, and the predisposition to severe infections were diminished. Since comprehensive insertion-site analysis showed vector integration near multiple genes controlling growth and immunologic responses in a persistently polyclonal hematopoiesis, careful monitoring for tumorigenesis is necessary, as with SCID-X1 and CGD [84, 85].

SIN lentiviral vectors using the minimal domain of the WAS promoter or other ubiquitous promoters, such as the PGK promoter, are currently being developed for WAS gene therapy. Preclinical studies using the HSCs obtained from mice or human patients have yield good results in terms of gene expression and genotoxicity [86-90].

Since a study using human embryonic stem cells (hESCs) and WAS-promoterdriven lentiviral vectors labeled by green fluorescent protein (GFP) showed highly specific gene expression in hESCs-derived HSCs, the WAS promoter will be used specifically in the generation of hESC-derived HSCs [91].

JAK3 deficiency is characterized by the absence of T and NK cells and impaired function of B cells, similar to SCID-X1. Treatment consists of HSCT with an HLA-identical or HLA-haplo-identical donor, often the parents of the patient, with T cell depletion. Engraftment is successful in most cases.

Although the recovery of T cell function is usually observed after HSCT, there are usually no improvements in B or NK cell function [92]. One case report involved introduction of JAK3 into the patients bone marrow CD34 positive cells using the MSCV retroviral vector. In this study, immunological recovery was not achieved although gene expression was observed for seven months [93]. Since JAK activation can cause T-cell lymphoma, tumorigenesis remains a concern with JAK gene therapy [92].

PNP metabolizes adenosine into adenine, inosine into hypoxanthine, and guanosine into guanine. PNP deficiency is an autosomal recessive metabolic disorder characterized by lethal T cell defects resulting from the accumulation of products from purine metabolism.

In PNP-deficient mice, transplantation of bone marrow cells transduced with a lentiviral vector containing human PNP resulted in human PNP expression, improved thymocyte maturation, increased weight gain, and extended survival. However, 12 weeks after transplant, the benefit of PNP-transduced cells and the percentage of engrafted cells decreased [94].

LAD-1 is a primary immunodeficiency disease caused by abnormalities in the leukocyte integrin CD11/CD18 heterodimer due to mutations in the CD18 gene. It is similar to canine leukocyte adhesion deficiency (CLAD). LAD-1 patients begin experiencing repeated serious bacterial infections immediately after birth.

In order to suppress gene activation near the gene insertion region in CLAD and to obtain the sufficient expression of the CD18 gene, researches have used various promoters with a lentiviral vector or foamy virus, a retroviral vector. In vivo animal experiments using a PGK or an elongation factor 1 promoter did not lead to symptom improvement [95-97], but improvement was seen with CD11b and CD18 promoters, respectively, with a SIN lentiviral vector in one animal study [98].

MPS is a general term for diseases characterized by glycosaminoglycan (GAG) accumulation into lysosomes as a result of deficiencies in lysosomal enzymes that degrade GAG. Although there are more than ten enzymes that are known to degrade GAG, MPS is divided into seven types: type I (-L-iduronidase deficiency, Hurler syndrome, Sheie syndrome, Hurler-Sheie syndrome), type II (iduronate sulfatase deficiency, Hunter syndrome), type III (heparan N-sulfatase deficiency, -N-acetylglucosaminidase deficiency, -glucosaminidase acetyltransferase deficiency, N-acetylglucosamine 6-sulfatase deficiency, Sanfilippo syndrome), type IV (galactose 6-sulfatase deficiency, Morquio syndrome), type VI (N-acetylgalactosamine 4-sulfatase deficiency, Maroteaux-Lamy syndrome), type VII (-glucuronidase deficiency, Sly syndrome), and type IX (hyaluronidase deficiency). Type II is X-linked; the other types are autosomal recessive. Although lysosomes are found in almost all cells, MPS mainly affects internal organs such as the brain, heart, bones, joints, eyes, liver, and spleen. The extent of disease, including mental retardation, varies with MPS type.

In types I, II, and VI, enzyme replacement therapy is performed. HSCT is performed in types I, II, IV, and VII. Gene therapy for types I, II, III, and VII type have been investigated. There are trials using an AAV or adenovirus vector to insert the modified gene into various cell types, including hepatocytes, muscle cells, myoblasts, and fibroblasts [99].

The first study of HSC gene therapy for MPS using a retroviral vector was performed on type VII mice in 1992, resulting in decreased accumulation of GAG in the liver and spleen but not in the brain and eyes [100]. Subsequent studies in type I and III animal models showed decreases in GAG accumulation in the kidneys and brain. Introductory efficiency and immunological reactions are considered challenges in HSC gene therapy for MPS [99].

Restoring or preserving central nervous system (CNS) function is one of the major challenges in the treatment of MPS. Since replaced enzymes easily cannot pass the blood-brain barrier (BBB), a high dose of enzyme is needed to improve CNS function. Gene therapy faces the same challenge. Even with high expression of enzyme by, for example, hepatocytes, the BBB prevents efficient delivery into the CNS. When a lentiviral vector is directly injected into the body, gene expression in brain tissue is observed, although the underlying mechanism is unknown. There are also trials where AAV vectors are directly injected into the CNS of mice or dogs and gene expression was observed in brain tissue [99].

Recently, a lentiviral vector using an ankyrin-1-based erythroid-specific hybrid promoter/enhancer (IHK) was used with HSCs to obtain gene expression only in erythroblasts for type I MPS. This approach resulted in decreased accumulation of GAG in the liver, spleen, heart, and CNS via enzyme expression in erythroblasts [101].

Gaucher disease is the most common lysosomal storage disorder. It is caused by deficiency of glucocerebroside-cleaving enzyme (-glucocerebrosidase), resulting in the accumulation of glucocerebroside in the reticuloendothelial system [102]. This autosomal recessive disease presents with hepatosplenomegaly, anemia, thrombocytopenia, and convulsions with or without mental retardation. It is classified into three types based on the clinical course or existence of neurological symptoms: type I (non-neuropathic, adult type), type II (acute neuropathic, infantile type), and type III (chronic neuropathic, juvenile type). Enzyme replacement therapy has been established in type I. As with MPS, since it is difficult to improve CNS symptoms with enzyme replacement therapy, HSCT is used, especially with type III. Gene therapy is considered in cases with little improvement with enzyme replacement therapy [103].

For Gaucher disease without CNS symptoms, a animal model using an AAV vector to produce enzyme in hepatocytes yielded good results [103]. HSC gene therapy using a retroviral vector was attempted in type I mice. The treated cells had higher -glucocerebrosidase activity than the HSCs from wild-type mice. Glucocerebroside levels normalized five to six months after treatment and no infiltration of Gaucher cells could be observed in the bone marrow, spleen, and liver [104]. In recent years, development of lentiviral vectors including the human glucocerebrosidase gene [105] and low-risk HSCT with nonmyeloablative doses of busulfan (25mg/kg) and no radiation therapy have been attempted in mice [106].

X-ALD is a peroxisomal disease in which a lipid metabolism abnormality causes demyelination of CNS tissues and dysfunction of the adrenal gland. It results from mutations in the ATP-binding cassette sub-family D (ABCD1) gene that codes for the adrenoleukodystrophy (ALD) protein. Behavioral disorders, mental retardation, or both occur by the age of five or six. Once symptoms appear, they progress to gait disorder and visual impairment within several months and the prognosis is poor. Increased levels of very long chain fatty acids (VLCFA), such as C25:0 or C26:0, are observed in the CNS, plasma, erythrocytes, leucocytes, etc. If the neurological defects are not severe, arrest of or improvement in symptoms can be obtained with HSCT [107].

One study has reported the introduction of wild-type ABCD1 using a lentiviral vector into peripheral blood CD34 positive cells of two patients with no HLA-identical donor. The patients received a transfusion of autologous gene-modified cells after myeloablative conditioning therapy. At three years of follow-up, ALD proteins were expressed in approximately 714% of neutrophils, monocytes, and T cells. Clinically, cerebral demyelination stopped 14 and 16 months after gene therapy, respectively, similar to results with allergenic HSCT [108, 109].

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Recent Advances in Hematopoietic Stem Cell Gene Therapy ...

IPS Cell Therapy Comments Off on Recent Advances in Hematopoietic Stem Cell Gene Therapy … | November 13th, 2016