On July 1, 2014, the United States Food and Drug Administration granted 'breakthrough therapy' designation to CTL019, the anti-CD19 chimeric antigen receptor T-cell therapy developed at the University of Pennsylvania. This is the first personalized cellular therapy for cancer to be so designated and occurred 25 years after the first publication describing genetic redirection of T cells to a surface antigen of choice. The peer-reviewed literature currently contains the outcomes of more than 100 patients treated on clinical trials of anti-CD19 redirected T cells, and preliminary results on many more patients have been presented. At last count almost 30 clinical trials targeting CD19 were actively recruiting patients in North America, Europe, and Asia. Patients with high-risk B-cell malignancies therefore represent the first beneficiaries of an exciting and potent new treatment modality that harnesses the power of the immune system as never before. A handful of trials are targeting non-CD19 hematological and solid malignancies and represent the vanguard of enormous preclinical efforts to develop CAR T-cell therapy beyond B-cell malignancies. In this review, we explain the concept of chimeric antigen receptor gene-modified T cells, describe the extant results in hematologic malignancies, and share our outlook on where this modality is likely to head in the near future.

2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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DUBLIN--(BUSINESS WIRE)--Research and Markets (http://www.researchandmarkets.com/research/hrgdr7/cell_therapy) has announced the addition of Jain PharmaBiotech's new report "Cell Therapy - Technologies, Markets and Companies" to their offering.

This report describes and evaluates cell therapy technologies and methods, which have already started to play an important role in the practice of medicine. Hematopoietic stem cell transplantation is replacing the old fashioned bone marrow transplants. Role of cells in drug discovery is also described. Cell therapy is bound to become a part of medical practice.

The number of companies involved in cell therapy has increased remarkably during the past few years. More than 500 companies have been identified to be involved in cell therapy and 296 of these are profiled in part II of the report along with tabulation of 280 alliances. Of these companies, 167 are involved in stem cells. Profiles of 72 academic institutions in the US involved in cell therapy are also included in part II along with their commercial collaborations. The text is supplemented with 62 Tables and 17 Figures. The bibliography contains 1,200 selected references, which are cited in the text.

Stem cells are discussed in detail in one chapter. Some light is thrown on the current controversy of embryonic sources of stem cells and comparison with adult sources. Other sources of stem cells such as the placenta, cord blood and fat removed by liposuction are also discussed. Stem cells can also be genetically modified prior to transplantation.

Cell therapy technologies overlap with those of gene therapy, cancer vaccines, drug delivery, tissue engineering and regenerative medicine. Pharmaceutical applications of stem cells including those in drug discovery are also described. Various types of cells used, methods of preparation and culture, encapsulation and genetic engineering of cells are discussed. Sources of cells, both human and animal (xenotransplantation) are discussed. Methods of delivery of cell therapy range from injections to surgical implantation using special devices.

Cell therapy has applications in a large number of disorders. The most important are diseases of the nervous system and cancer which are the topics for separate chapters. Other applications include cardiac disorders (myocardial infarction and heart failure), diabetes mellitus, diseases of bones and joints, genetic disorders, and wounds of the skin and soft tissues.

Regulatory and ethical issues involving cell therapy are important and are discussed. Current political debate on the use of stem cells from embryonic sources (hESCs) is also presented. Safety is an essential consideration of any new therapy and regulations for cell therapy are those for biological preparations.

The cell-based markets was analyzed for 2014, and projected to 2024.The markets are analyzed according to therapeutic categories, technologies and geographical areas. The largest expansion will be in diseases of the central nervous system, cancer and cardiovascular disorders. Skin and soft tissue repair as well as diabetes mellitus will be other major markets.

Key Topics Covered:

Part I: Technologies, Ethics & Regulations

0. Executive Summary

1. Introduction to Cell Therapy

2. Cell Therapy Technologies

3. Stem Cells

4. Clinical Applications of Cell Therapy

5. Cell Therapy for Cancer

6. Cell Therapy for Neurological Disorders

7. Ethical, Legal and Political Aspects of Cell therapy

8. Safety and Regulatory Aspects of Cell Therapy

Part II: Markets, Companies & Academic Institutions

9. Markets and Future Prospects for Cell Therapy

10. Companies Involved in Cell Therapy

11. Academic Institutions

12. References

For more information visit http://www.researchandmarkets.com/research/hrgdr7/cell_therapy

Source: Jain PharmaBiotech

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Formerly Orthopedic Stem Cell Institute We put the power of your own body to work for you.

Our team of board certified, fellowship-trained orthopedic and spine surgeons work with patients from around the world using the newest and most advanced technology to treat orthopedic injuries and bone and joint pain, as well as relieving symptoms and improving the lives of patients with a multitude of illnesses.

The Premier Stem Cell Institute is a leading research and treatment facility in Colorado providing the most innovative and proven techniques and therapies using the bodys natural healing power of stem cells.

A stem cell is a basic cell constantly produced by your body to heal injuries, build new skin, even grow your hair. However, your body wont refix a chronic injury or illness by continuing to attack it with new stem cells unless those cells are extracted and reintroduced into your body via stem cell therapies.

We are a leading research and treatment facility providing the most innovative and proven techniques and therapies using the bodys natural healing power of stem cells. Our services are performed by fellowship-trained surgeons using the most state-of-the-art equipment and technology in the field.All stem cell treatments are not alike. AtPremier Stem Cell Institute, we extract your stem cells from your bone marrow because they are higher quality and result in better outcomes than stem cells from fat (adipose). We treat each patient with the utmost respect and our concierge service makes you feel incredibly well cared for from the first phone call to follow up visits.

They're very personable, they're very helpful..nice people. Bottom line is there's no pain where there was a lot of pain before.

Jon Hoffman, Former NFL Player

I used to dread doing simple things like putting on a coat, a seat belt or reaching for things. I can now do those things without nearly as much difficulty. I want to thank everyone at the clinic for performing the procedure on me. They are making peoples' lives much more enjoyable.

Bob Hyland, Former NFL Player

It's amazing! You're awake the whole time, it's virtually painless, and within an hour you're walking out.

Don Horn, Former NFL Player

of Patients are 70% Better Within 1 Year!

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A patient has become the first in the UK to receive an experimental stem cell treatment that has the potential to save the sight of hundreds of thousands of Britons.

By December, doctors will know whether the woman, who has age-related macular degeneration, has regained her sight after a successful operation at Moorfields Eye Hospital in London last month. Over 18 months, 10 patients will undergo the treatment.

The transplant involves eye cells, called retinal pigment epithelium, derived from stem cells and grown in the lab to form a patch that can be placed behind the retina during surgery.

Related: Stem cell therapy success in treatment of sight loss from macular degeneration

The potential is huge. Although the first patients have the wet form of macular degeneration, the doctors believe it might also eventually work for those who have the dry form, who are the vast majority of the UKs 700,000 sufferers.

The surgery is an exciting moment for the 10-year-old London Project to Cure Blindness, a collaboration between the hospital, the UCL Institute of Ophthalmology and the National Institute for Health Research, which was formed to find a cure for wet age-related macular degeneration, the more serious but less common form of the disease.

Prof Pete Coffey of UCL, one of the founders of the London Project, said he would not be working on the new treatment if he did not believe it would work. He hopes it could become a routine procedure for people afflicted by vision loss, which is as common a problem among older people as dementia.

It does involve an operation, but were trying to make it as straightforward as a cataract operation, he said. It will probably take 45 minutes to an hour. We could treat a substantial number of those patients.

First they have to get approval. The trial is not just about safety, but also efficacy. There will be a regulatory review after the first few transplants to ensure all is going well.

The group of patients chosen have the wet form of the disease and experienced sudden loss of vision within about six weeks. The support cells in the eye, which get rid of daily debris and allow the seeing part to function have died.

There is a possibility of restoring their vision, said Coffey. The aim of the transplant is to restore the support cells so the seeing part of the eye is not affected by what would become an increasingly toxic environment, causing deterioration and serious vision loss. The surgery is being performed by retinal surgeon Prof Lyndon Da Cruz from Moorfields, who is also a co-founder of the London Project.

The team chose people with this dramatic vision loss to see whether the experimental stem cell therapy would reverse the loss of vision. But in those with dry macular degeneration, said Coffey, the process is far slower, which would mean doctors could choose the time to intervene if the treatment works.

Helping people to regain their sight has long been one of the most hopeful prospects for stem cell transplantation. Other research groups have been trialling the use of stem cells in people with Stargardts disease, which destroys the vision at a much earlier age.

Stem cells have moved from the drawing board into human trials with incredible speed, scientists say. The first embryonic stem cell was derived in 1989. Using them in eyes was always going to have a big advantage over other prospects, because it is possible to transplant them without an all-out attack by the immune system, as would happen in other parts of the body. Most people who have any sort of transplant have to take drugs that suppress the immune system for the rest of their lives.

Just like conventional medicines, stem cell therapies will very likely have to be developed and marketed by large commercial concerns. The London Project has the US drug company Pfizer on board.

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An illustration of GRNOPC1, a drug based on human embryonic stem cells, which contains oligodendrocyte progenitor cells.

Geron/UC Irvine

The hope: that one day this treatment may help the paralyzed walk again.

On Friday at the Shepherd Center, a spinal cord and brain injury rehabilitation center in Atlanta, a patient with a recent spinal cord injury made medical history: The paraplegic was injected with two million embryonic stem cells.

The goal: To regenerate spinal cord tissue.

The process, reports CBS Station KPIX correspondent Dr. Kim Mulvihill, involves coaxing the cells into becoming specialized nerve cells, and then injecting them directly into the injured area of the spinal cord.

The embryonic stem cells come from a donated human embryo left over from a fertility treatment, an embryo that would have otherwise been discarded.

Embryonic stem cells have been at the center of funding controversies because the research involves destroying the embryos, which some have argued is akin to abortion. But, many researchers consider embryonic stem cells the most versatile types of stem cells, as they can morph into any type of cell.

While there are some restrictions on federal funding for stem cell lines for research, companies such as Geron do not use federal funding and are therefore free from those restrictions.

The study is approved by the FDA but is privately funded.

The drug - known as GRNOPC1 - contains cells called oligodendrocyte progenitor cells. Those progenitor cells turn into oligodendrocytes, a type of cell that produces myelin, a coating that allows impulses to move along nerves. When those cells are lost because of injury, paralysis can follow.

If GRNOPC1 works, the progenitor cells will produce new oligodendrocytes in the injured area of the patient's spine, potentially allowing for new movement.

The therapy will be injected into the patients' spines one to two weeks after they suffer an injury between their third and 10th thoracic vertebrae, or roughly the middle to upper back. Later trials would include patients with less severe spinal injuries and damage to other parts of the spine.

In lab animals, the results were dramatic - paralyzed rodents moved again.

Dr. Thomas Okarma, President and CEO of Geron, told CBS Station KPIX, "This therapy goes far beyond the reach of pills or scalpels and will achieve a new level of healing with a single injection of healthy replacement cells."

So far, Geron of Menlo Park, Calif., has spent $175 million in developing this treatment.

However, Dr. Arnold Kriegstein, who heads Regeneration Medicine & Stem Cell Research at University of California-San Francisco, told KPIX, "People are just so different from rodents."

Though optimistic, he urged caution. "I think that people looking at the outcome of this trial should really lower their expectations if they're really thinking people will get out of their wheelchairs. It's unlikely to happen."

The drug still faces many years of testing for effectiveness and tolerance if all goes well in the early stage study.

This initial trial is not aimed at a cure for patients, but to establish if the treatment is safe.

Patients must be treated within 14 days of a spinal cord injury and they must undergo short term immune suppression therapy to make sure their bodies don't reject the cells.

If the treatment is deemed safe, the next trial will aim at testing effectiveness and will use a higher quantity of stem cells.

Shepherd Center is one of seven potential sites in the United States for the trial.

The company has said it plans to enroll eight to 10 patients in the study at sites nationwide. The trial will take about two years, with each patient being studied for one year.

Geron is among several companies focusing on embryonic stem cell therapy. Advanced Cell Technology Inc. hopes to develop the embryonic stem cell therapy called retinal pigment epithelium, or RPE. That therapy is designed to treat Stargart disease, an inherited condition that affects children and can lead to blindness in adulthood.

Meanwhile, other companies such as StemCells Inc. are focusing on adult stem cells, which can be gathered from a person's skin.

For more info: clinicaltrials.goc - Safety Study of GRNOPC1 in Spinal Cord Injury

2010 CBS Interactive Inc. All Rights Reserved. This material may not be published, broadcast, rewritten, or redistributed. The Associated Press contributed to this report.

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In 2006, Shinya Yamanaka of Kyoto University in Japan was the first to disprove the previous notion that reversible cell differentiation of mammals was impossible. He reprogrammed a fully differentiated mouse cell into a pluripotent stem cell by introducing four genes, Oct-4, SOX2, KLF4, and Myc, into the mouse fibroblast through gene-carrying viruses. With this method, he and his coworkers created induced pluripotent stem cells (iPS cells), the key component in this experiment.[1] Scientists have been able to conduct experiments that show the ability of iPS cells to treat and even cure diseases. In this experiment, tests were run on mice with inherited sickle-cell anemia. Skin cells were turned into cells containing genes that transformed the cells into iPS cells. These replaced the diseased sickled cells, curing the test mice. The reprogramming of the pluripotent stem cells in mice was successfully duplicated with human pluripotent stem cells within about a year of the experiment on the mice.[citation needed]

Sickle-cell anemia is a disease in which the body produces abnormally shaped red blood cells. Red blood cells are flexible and round, moving easily through the blood vessels. Infected cells are shaped like a crescent or sickle (the namesake of the disease). As a result of this disorder the hemoglobin protein in red blood cells is faulty. Normal hemoglobin bonds to oxygen, then releases it into cells that need it. The blood cell retains its original form and is cycled back to the lungs and re-oxygenated.

Sickle cell hemoglobin, however, after giving up oxygen, cling together and make the red blood cell stiff. The sickle shape also makes it difficult for the red blood cell to navigate arteries and causes blockages.[2] This can cause intense pain and organ damage. The sickled red blood cells are fragile and prone to rupture. When the number of red blood cells decreases from rupture (hemolysis), anemia is the result. Sickle cells die in 1020 days as opposed to the traditional 120-day lifespan of a normal red blood cell.

Sickle cell anemia is inherited as an autosomal (meaning that the gene is not linked to a sex chromosome) recessive condition.[2] This means that the gene can be passed on from a carrier to his or her children. In order for sickle cell anemia to affect a person, the gene must be inherited from both the mother and the father, so that the child has two recessive sickle cell genes (a homozygous inheritance). People who inherit one sickle cell gene from one parent and one normal gene from the other parent, i.e. heterozygous patients, have a condition called sickle cell trait. Their bodies make both sickle hemoglobin and normal hemoglobin. They may pass the trait on to their children.

The effects of sickle-cell anemia vary from person to person. People who have the disease suffer from varying degrees of chronic pain and fatigue. With proper care and treatment, the quality of health of most patients will improve. Doctors have learned a great deal about sickle cell anemia since its discovery in 1979. They know its causes, its effects on the body, and possible treatments for complications. Sickle cell anemia has no widely available cure. A bone marrow transplant is the only treatment method currently recognized to be able to cure the disease, though it does not work for every patient. Finding a donor is difficult and the procedure could potentially do more harm than good. Treatments for sickle cell anemia are generally aimed at avoiding crises, relieving symptoms, and preventing complications. Such treatments may include medications, blood transfusions, and supplemental oxygen.

During the first step of the experiment, skin cells (also known as fibroblasts) were collected from infected test mice and put in a culture. The fibroblasts were reprogrammed by infecting them with retroviruses that contained genes common to embryonic stem cells. These genes were the same four used by Yamanaka (Oct-4, SOX2, KLF4, and Myc) in his earlier study. The investigators were trying to produce cells with the potential to differentiate into any type of cell needed (i.e. pluripotent stem cells). As the experiment continued, the fibroblasts multiplied into identical copies of iPS cells. The cells were then treated to form the mutation needed to reverse the anemia in the mice. This was accomplished by restructuring the DNA containing the defective globin gene into DNA with the normal gene through the process of homologous recombination. The iPS cells then differentiated into blood stem cells, or hematopoietic stem cells. The hematopoietic cells were injected back into the infected mice, where they proliferate and differentiate into normal blood cells, curing the mice of the disease.[3][4][verification needed]

To determine whether the mice were cured from the disease, the scientists checked for the usual symptoms of sickle cell disease. They examined the blood for mean corpuscular volume (MCV) and red cell distribution width (RDW) and urine concentration defects. They also checked for sickled red blood cells. They examined the DNA through gel electrophoresis, checking for bands that display an allele that causes sickling. Compared to the untreated mice with the disease, which they used as a control, "the treated animals had marked increases in RBC counts, healthy hemoglobin, and packed cell volume levels".[5]

Researchers examined "the urine concentration defect, which results from RBC sickling in renal tubules and consequent reduction in renal medullary blood flow, and the general deteriorated systemic condition reflected by lower body weight and increased breathing."[5] They were able to see that these parts of the body of the mice had healed or improved. This indicated that "all hematological and systemic parameters of sickle cell anemia improved substantially and were comparable to those in control mice."[5] They cannot say if this will work in humans because a safe way to inject the genes for the induced pluripotent cells is still needed.[citation needed]

The reprogramming of the induced pluripotent stem cells in mice was successfully duplicated in humans within a year of the successful experiment on the mice. This reprogramming was done in several labs and it was shown that the iPS cells in humans were almost identical to original embryonic stem cells (ES cells) that are responsible for the creation of all structures in a fetus.[1] An important feature of iPS cells is that they can be generated with cells taken from an adult, which would circumvent many of the ethical problems associated with working with ES cells. These iPS cells also have potential in creating and examining new disease models and developing more efficient drug treatments.[6] Another feature of these cells is that they provide researchers with a human cell sample, as opposed to simply using an animal with similar DNA, for drug testing.

One major problem with iPS cells is the way in which the cells are reprogrammed. Using gene-carrying viruses has the potential to cause iPS cells to develop into cancerous cells.[1] Also, an implant made using undifferentiated iPS cells, could cause a teratoma to form. Any implant that is generated from using these iPS cells would only be viable for transplant into the original subject that the cells were taken from. In order for these iPS cells to become viable in therapeutic use, there are still many steps that must be taken.[5][7]

In the future, researchers hope that induced pluripotent cells may be used to treat other diseases. Pluripotency is a crucial part of disease treatment because iPS cells are capable of differentiation in a way that is very similar to embryonic stem cells which can grow into fully differentiated tissues. iPS cells also demonstrate high telomerase activity and express human telomerase reverse transcriptase, a necessary component in the telomerase protein complex. Also, iPS cells expressed cell surface antigenic markers expressed on ES cells. Also, doubling time and mitotic activity are cornerstones of ES cells, as stem cells must self-renew as part of their definition. As said, iPS cells are morphologically similar to embryonic stem cells. Each cell has a round shape, a large nucleolus and a small amount of cytoplasm. One day, the process may be used in practical settings to provide a fundamental way of regeneration.

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Information contained on this page is provided by an independent third-party content provider. WorldNow and this Station make no warranties or representations in connection therewith. If you have any questions or comments about this page please contact pressreleases@worldnow.com.

SOURCE Reportlinker

NEW YORK, Oct. 1, 2015 /PRNewswire/ -- Innovative Therapies for treating diseases are being sought after with fresh vigor as new targets, approaches and biology is discovered. Improved health care, nutrition and preventive medicine in the last few decades have all helped in increasing the life expectancy WW. However, this has not translated into any reduction in the incidence or prevalence of chronic or critical illnesses! On the contrary the incidence of chronic diseases like diabetes, obesity, arthritis etc. as well as cancer and the maladies associated with aging (dementia, Alzheimer's etc.) are on the rise!. Consequently the pharma industry continues to grow and is projected to

achieve sales in excess of trillion dollar mark by 2020 By the next decade, one field which is poised to bring a paradigm change in the way diseases are treated is the Stem cell therapy/Regenerative Medicine space. The number of companies and products in the clinic have reached a critical mass warranting a close watch for those interested in keeping pace with the development of new medicines.

Regenerative Medicine and Stem cell based Cell therapies-Drugs of the Future Offering Hope for Cure

EXECUTIVE SUMMARY

- INTRODUCTION

- Tough Choice- "Autologous vs. Allogenic " Therapies

- REGULATORY GUIDELINES

- Marketed Cell based/Stem Cell Products

- Progress and Challenges

- Progress in Specific Therapy Areas

- SELECT UPCOMING MILESTONES IN REGENERATIVE MEDICINE/STEM

CELL FOCUSED COMPANIES (2015-16)

- Appendix

Read the full report: http://www.reportlinker.com/p02629094-summary/view-report.html

About Reportlinker ReportLinker is an award-winning market research solution that finds, filters and organizes the latest industry data so you get all the market research you need - instantly, in one place.

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To view the original version on PR Newswire, visit:http://www.prnewswire.com/news-releases/regenerative-medicine-and-stem-cell-based-cell-therapies-drugs-of-the-future-offering-hope-for-cure-300153074.html

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From the United States Senate to houses of worship, and even to the satirical television show "South Park," stem cells have been in the spotlight -- though not always in the kindest light. Since early research has focused on the use of embryonic stem cells (cells less than a week old), the very act of extracting these cells has raised a raft of ethical questions for researchers and the medical community at large, with federal funding often hanging in the balance.

However, the advances in stem cell research and the subsequent applications to modern medicine can't be ignored. According to the National Institutes of Health (NIH), stem cells are being considered for a wide variety of medical procedures, ranging from cancer treatment to heart disease and cell-based therapies for tissue replacement.

Why? To answer that question, you have to understand what stem cells are. Called "master" cells or "a sort of internal repair system," these remarkable-yet-unspecialized cells are able to divide, seemingly without limits, to help mend or replenish other living cells [sources: Mayo Clinic; NIH]. In short, these cells are the cellular foundation of the entire human body, or literally the body's building blocks.

By studying these cells and how they develop, researchers are closing in on a better understanding of how our bodies grow and mature, and how diseases and other abnormalities take root. The research work that began with mouse embryos in the early 1980s eventually helped scientists devise a way to isolate stem cells from human embryos by the late 1990s.

Embryonic, or pluripotent, stem cells are taken from human embryos that are less than a week old. These cells are wildly versatile, capable of dividing into more stem cells or becoming any type of cell in the human body (roughly 220 types, including muscle, nerve, blood, bone and skin). Researchers have also recently found stem cells in amniotic fluid taken from pregnant women during amniocentesis, a fairly routine procedure used to determine potential complications, such as Down syndrome.

However, recent research has indicated that adult stem cells, once thought to be more limited in their capabilities, are actually much more versatile than originally believed. Though not as "pure" as embryonic stem cells, due to environmental conditions that exist in the real world -- ranging from air pollution to food impurities -- adult stem cells are nonetheless garnering attention, if only because they don't incite the same ethical debate as embryonic stem cells.

So, what are the cutting-edge uses for stem cells?

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As a national pioneer of innovative medicine, Mississippi Stem Cell Treatment Centers motto Excellence with a Human Touch, is at the forefront of what we do. Located in the city of Ocean Springs on the Mississippi Gulf Coast, we provide treatment to promote healing and tissue generation to those suffering from autoimmune, degenerative, inflammatory and ischemic conditions. Our team is highly committed to alleviating your symptoms and enhancing your functionality, quality of life, and wellbeing.

We employ a method called Stromal Vascular Fraction deployment (SVF). SVF relies on individual patient stem cells and growth factors, and helps accelerate healing and tissue regeneration. The SVF we collect from patients fat tissue is given back to the individual through the deployment process. SVF is an innovative product that can be used to regenerate different types of tissue throughout the body.

Mississippi Stem Cell Treatment Center is an affiliate of the Cell Surgical Network of CA. Our center meets all FDA guidelines for treating patients using their own tissue for therapy. We provide same-day harvesting and treatment in a state-of-the-art environment, which facilitates a faster recovery.

We provide treatment for anyone suffering in the following areas:

At Mississippi Stem Cell Treatment Center, we offer stem cell center treatments for autoimmune disease, as well as stem cell center treatment for people suffering from other degenerative diseases. For more information on our innovative technology, browse our website for a wealth of information on stem cells, or contact us so we can discuss your individual candidate profile.

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Combined NSC transplantation and VPA administration improves functional recovery of hind limbs without CST axon reextension. As VPA has been shown to have effects that are likely to be beneficial to treatment of the injured CNS, such as neuroprotection (2731), induction of neuronal differentiation (26), and promotion of neurite outgrowth (32), we examined the response of SCI model mice to different combinations of VPA administration and NSC transplantation. We prepared NSCs from embryonic forebrains of 3 different Tg mouse lines ubiquitously expressing either GFP (GFP-Tg) (33), GFP and LUC (GFP.LUC-Tg), or GFP, LUC, and the diphtheria toxin (DT) receptor human heparin-binding EGF-like growth factor (TR6) (TR6.GFP.LUC-Tg) (see Methods). The expression of GFP, LUC, and TR6 in NSCs enabled us to distinguish transplanted cells from host cells, to trace the survival of transplanted cells based on LUC activity in a noninvasive fashion, and to specifically ablate transplanted cells (see below), respectively. To obtain a homogeneous population of NSCs, we used adherent monolayer culture (3436). The embryonic forebrains were dissociated and cultured with EGF and basic FGF (bFGF) (36) (Supplemental Figure 1, A and B; supplemental material available online with this article; doi:10.1172/JCI42957DS1). These cells uniformly expressed the stem cell markers Sox2 and nestin but did not express differentiation markers (Supplemental Figure 1, C and D). Under the appropriate conditions for each lineage, these NSCs differentiated into neurons, astrocytes, or oligodendrocytes (Supplemental Figure 1, E and F). NSCs from different Tg mice behaved similarly in these culture conditions (data not shown). NSCs that had been cultured and passaged 510 times in the presence of both EGF and bFGF to maintain the undifferentiated state were used for transplantation studies.

Undifferentiated NSCs were transplanted into the SCI epicenter 7 days after injury. Nontransplanted control and transplanted mice were then intraperitoneally administered VPA or saline daily for 7 days (Figure 1A), whereafter we monitored their hind limb motor function using the open field locomotor scale (BBB score) (79, 37) for 6 weeks. Remarkably, we found that the simultaneous treatment of SCI model mice with NSCs and VPA resulted in a dramatic recovery of hind limb function compared with either treatment alone (Figure 1B and Supplemental Videos 14). There were no significant differences among the data obtained from each SCI model mouse group transplanted with the 3 distinct NSCs. Functional recovery of each treated SCI model mouse reached a plateau at around 6 weeks, the level of which was sustained for more than 3 months. Since mice treated with VPA alone showed no further improvement compared with untreated mice, it is most likely that VPA affected the function of transplanted cells.

A combination of NSC transplantation and VPA administration improves functional recovery of hind limbs without CST axon reextension. (A) Schematic of the NSC transplantation and VPA injection protocol. (B) Time course of functional recovery of hind limbs after SCI. GFP-NSCs, GFP.LUC-NSCs, and TR6.GFP.LUC-NSCs were transplanted into the SCI epicenter 7 days after injury as indicated. Combined treatment with NSC transplantation and VPA administration resulted in the greatest functional recovery. Data represent mean SEM. **P < 0.001 compared with SCI models with no treatment; *P < 0.01 compared with SCI models with no treatment (repeated measures ANOVA). NSC+VPA, total n = 21. (C) Representative pictures of BDA-labeled CST fibers at 5 mm rostral and 5 mm caudal to the lesion site. BDA was injected into the motor cortices 12 weeks after SCI. 2 weeks after the injection, mice were fixed and spinal cord sections were stained. Representative results for a GFP-NSCtransplanted spinal cord are shown. Blue, Hoechst nuclear staining. Scale bar: 20 m. (D) Quantification of the labeled CST fibers in the spinal cords of intact mice, SCI mice receiving no treatment, and SCI mice undergoing combined NSC/VPA treatment. Eight 30-mthick serial parasagittal sections from individual spinal cords were evaluated. The x axis indicates specific locations along the rostrocaudal axis of the spinal cord, and the y axis indicates the ratio of the number of BDA-labeled fibers at the indicated site to that at 6 mm rostral to the lesion site (Th9). **P < 0.001 compared with SCI models without treatment; *P = 0.188 There is no significant difference in the number of BDA-labeled fibers between NSC+VPA-treated mice (blue line) and SCI model mice with no treatment (yellow line) (repeated measures ANOVA). All data shown are from at least 3 experiments in parallel conditions, with error bars representing SEM.

We next sought to determine the basis for this improvement in locomotor function. Since transplanted NSCs have been reported to play a supportive role in the reextension of injured axons (14), we analyzed whether CST axons were regenerated by anterograde labeling using biotinylated dextran amine (BDA) (6, 16, 17). Because BDA was injected into the motor cortex, only the axons of first-order neurons in the CST could be visualized (Figure 1C). In our SCI model mice, the caudal part of the injured site was completely devoid of CST axons (Figure 1, C and D), and the same was true in mice that had undergone combined NSC transplantation and VPA administration (Figure 1, C and D). These data indicated that CST axons did not reextend in mice treated with both NSCs and VPA and therefore that some other mechanism was responsible for the animals dramatic functional locomotor improvement.

Transplanted NSCs encompass the lesion site and extend their processes. Given that host CST axon reextension was not involved in the observed hind limb recovery, we decided to focus on the transplanted cells. We analyzed the migration, morphology, neuronal marker expression, and viability of these cells after coadministration with VPA. Transplant-derived cells migrated to both rostral and caudal areas and displayed processes that extended into the gray matter and dorsal funiculus within 5 weeks of transplantation (Figure 2). Between 20% and 40% of the transplanted cells were found to be surviving in the injured spinal cord after 8 weeks, and 17% still remained viable more than 1 year after transplantation (data not shown). About 20% of the surviving cells had differentiated into microtubule-associated protein 2positive (MAP2-positive) neurons with elongated processes within 5 weeks after transplantation (Figure 2, B and C, and Figure 3, E and F). Survival of the transplanted NSCs was not significantly influenced by VPA administration (Supplemental Figure 8).

Transplanted NSCs migrate from the injection site and encompass the lesion site. Representative results of GFP-NSCtransplanted SCI model mice are shown. (A) A series of immunostaining images of injured spinal cord at 6 weeks after injury. SCI mice received combination treatment with NSC transplantation and VPA administration. Specimens were picked up every 150 m and stained with anti-GFP (green) and MAP2 (not shown) antibodies and Hoechst (blue). The epicenter of the SCI is indicated (*). Scale bar: 1 mm. (B and C) Higher-magnification images of the white boxes in A. GFP-positive transplanted NSCs differentiated into MAP2-positive neurons and extended their processes. Scale bar: 50 m.

VPA promotes neuronal differentiation of transplanted NSCs. Representative results of GFP-NSCtransplanted SCI model mice are shown. (A) Confocal images of NSCs 1 week after transplantation into the injured spinal cords. Spinal cord sections from VPA-treated (+) and untreated () mice were stained with anti-GFP (green), anti-doublecortin (DCX) (immature neuronal marker, red) and anti-GFAP (magenta) antibodies, and Hoechst (blue). VPA administration resulted in an increase in the number of DCX-positive neuronal precursors among transplanted cells (lower panel). Scale bar: 20 m. (BD) The percentages of DCX-, GFAP-, and MBP-positive cells in GFP-positive transplanted cells were quantified. **P < 0.01; *P < 0.05 compared with controls (Students t test). (E) Confocal images of NSCs 5 weeks after transplantation into injured spinal cords. Spinal cord sections from VPA-treated (+) and untreated () mice were stained with anti-GFP (green), anti-MAP2 (neuronal marker, red) and anti-GFAP (magenta) antibodies, and Hoechst (blue). VPA administration increased the numbers of MAP2-positive neurons (lower panel). Scale bar: 20 m. (F and G) The percentages of cells positive for MAP2 or GFAP in GFP-positive transplanted cells in E were quantified. **P < 0.01; *P < 0.05 compared with control (Students t test). All data shown in BD, F, and G are from at least 15 confocal images of 3 individuals in parallel experiments, with error bars representing the SD.

HDAC inhibition promotes neuronal differentiation of NSCs and is critical for transplantation-induced hind limb recovery. In contrast to previous studies, which have indicated that very few transplanted NSCs differentiate into neurons in the injured CNS environment (8, 10, 11, 20), many neurons were observed in the spinal cord after coadministration with VPA. We next examined in more detail the contribution of VPA to differentiation of cultured and transplanted NSCs. To analyze differentiation in vitro, NSCs were treated with either VPA or valpromide (VPM), an amide analog of VPA that is also an antiepileptic but is not an HDAC inhibitor (24), under differentiation culture conditions. VPA enhanced histone acetylation (Supplemental Figure 2A) and promoted neuronal differentiation and neurite outgrowth of the NSCs (Supplemental Figure 3, AC); it also inhibited astrocytic and oligodendrocytic differentiation of NSCs (Supplemental Figure 3, DG). A different HDAC inhibitor, trichostatin A (TSA), also enhanced histone acetylation (Supplemental Figure 2A) and neuronal differentiation of NSCs (not shown) (26). In contrast, VPM neither enhanced histone acetylation nor induced neuronal differentiation, suggesting that HDAC inhibition has an important role in regulating fate determination in NSCs.

We then assessed the histone acetylation status and differentiation profiles of transplanted NSCs. VPA administration enhanced histone acetylation in transplanted cells in the spinal cord (Supplemental Figure 2, B and C). When we examined the differentiation status of transplanted cells 1 week after transplantation, neuronal but not glial differentiation was greatly enhanced by VPA administration (Figure 3, AD, and Supplemental Figure 4A). A similar differentiation tendency of transplanted NSCs to that at 1 week was observed at 5 weeks after transplantation: there was a dramatic increase in the number of cells positive for MAP2 (a relatively late differentiation marker of neurons in comparison with DCX) in VPA-administered mice (Figure 3, EG, and Supplemental Figure 4B). Furthermore, VPM administration to the SCI mice neither promoted neuronal differentiation nor enhanced hind limb motor function, suggesting that HDAC inhibition has an essential role in regulating fate determination of transplanted NSCs and improvement of motor function in vivo (Supplemental Figure 5, AC). In light of the above findings that the percentage of neurons generated from transplanted NSCs increased dramatically with VPA administration, whereas those of astrocytes and oligodendrocytes declined, we anticipated that these neurons would be likely to play a major role in regenerating the disrupted neuronal circuitry of the injured spinal cord.

Transplant-derived neurons reconstruct disrupted neuronal circuits in a relay manner. We next asked how the disrupted neuronal circuits were regenerated following the combined treatment with NSC transplantation and VPA administration. Wheat germ agglutinin (WGA), which can be transsynaptically transported, is one of the best known tracers of neural pathways (38). WGA protein can be transferred across synapses to second- and third-order neurons, permitting functional neuronal circuits to be tracked in the CNS. We injected WGA-expressing adenoviruses into the motor cortex of mouse brain 12 weeks after SCI. In uninjured mice, WGA was detected as intracellular granule-like structures in neurons localized in the ventral horn throughout the spinal cord (Figure 4, A and B). In untreated SCI model mice, WGA granules were almost completely absent from the caudal region below the injured site (Figure 4, A and C). Surprisingly, although we could not observe CST axonal reextension through the lesion site (Figure 1, C and D), WGA granules were clearly present in caudal large neurons located in the spinal cords of mice treated with both NSC and VPA (Figure 4, A and D). Intriguingly, moreover, transplant-derived neurons in or close to the lesion site contained WGA granules (Figure 4E), which were received from more rostral neurons. These data imply that WGA was conveyed through the lesion site to the caudal area via transplant-derived neurons. Considering this finding, together with the fact that WGA could be detected in caudal neurons without CST axonal reextension in mice that had undergone the combined treatment, it seemed conceivable that the transplant-derived neurons reconstructed the disrupted neuronal circuits, thereby acting as relays for transmitting signals between endogenous neurons whose interconnection had been abolished by the injury. In mice that received NSC transplantation alone after SCI, the percentage of WGA-positive cells among MAP2ab-positive cells in the caudal region was higher than that in untreated mice (Figure 4C) but lower than that in mice receiving combined NSC transplantation and VPA administration (Supplemental Figure 6), reflecting the degree of hind limb functional improvement (Figure 1C).

Transplant-derived neurons reconstruct disrupted neuronal circuits in a relay manner. (A) Representative pictures of WGA-labeled neuronal cell bodies located in the ventral horn at 14 weeks after SCI. Spinal cord sections were stained with anti-WGA (red) and -MAP2ab (magenta) antibodies and Hoechst (blue). Scale bar: 20 m. Intense WGA immunoreactivity was observed as intracellular granule-like structures. Left panels show the rostral area (Th4Th7), and right panels show the caudal area (Th11 to lumbar vertebra [L] 1). In uninjured mice, WGA injected into the bilateral motor cortices was transsynaptically transported to neurons in areas rostral and caudal to the injured site (top panels). In the SCI model mice that did not receive treatment, very little WGA was observed in caudal areas (middle panels). However, in spinal cords of animals that underwent dual treatment with NSC and VPA, WGA was clearly observed in neurons in the caudal areas (bottom panels). Representative results of GFP-NSCtransplanted SCI model mice are shown. (BD) The percentages of WGA-positive cells in the neurons localized in the ventral horn were quantified. **P < 0.05 (Students t test). All data shown are from at least 30 images, containing more than 600 cells, from 3 individuals (5 images per area) in parallel experiments, with error bars representing SD. (E) Representative confocal images of WGA-labeled transplant-derived MAP2-positive neurons. Sections were stained with anti-WGA (red), anti-MAP2ab (magenta) and anti-GFP (green) antibodies, and Hoechst (blue). Granule-like WGA structures (yellow arrowheads) could be seen in the GFP and MAP2abdouble-positive transplant-derived neurons. Scale bar: 10 m.

In support of the notion of a relay function for transplant-derived neurons, immunoelectron microscopy revealed that GFP-positive transplant-derived neurons received projections from endogenous neurons (Figure 5, A and B) and that the axon terminals of transplant-derived neurons made synapses with endogenous neurons localized in the ventral horn (Figure 5, CE).

Transplant-derived neurons make synapses with endogenous neurons. (A) Immunoelectron microscopy image of a sagittal section of dual-treated (GFP-NSC and VPA) injured spinal cord (rostral area). A GFP-positive dendrite (Den) made synapses with GFP-negative endogenous axon termini (At) (yellow arrowheads). Scale bar: 1 m. (B) In other rostral regions, a dendrite of a GFP-positive transplant-derived neuron made a synapse (yellow arrowheads) with the axon terminus of a GFP-negative endogenous neuron. Scale bar: 1 m. (C) Sagittal section of dual-treated (NSC and VPA) injured spinal cord (caudal area) stained with anti-GFP antibody (dark brown). The epicenter of the SCI is indicated (*). Scale bar: 500 m. (D) High-magnification image of a large neuron localized in the ventral horn in the white rectangle in C. GFP-positive transplanted neurons extended their processes toward an endogenous neuron (yellow arrowheads). Scale bar: 100 m. (E) Immunoelectron microscopy image of the boxed area in D. GFP-positive axon termini made synapses with the dendrite of a GFP-negative endogenous large neuron (yellow arrowheads). Scale bar: 1 m.

Transplanted cells contribute directly to functional recovery of hind limb movement in SCI mice. To determine whether the transplanted cells made a direct contribution to the functional recovery of hind limbs after SCI, we performed specific ablation of transplanted cells using the toxin receptormediated cell knockout (TRECK) method (Figure 6A and refs. 39, 40). For this purpose, we prepared NSCs from the embryonic forebrains of GFP.LUC Tg and TR6.GFP.LUC Tg mice (Figure 6A and Supplemental Figure 7, A and B). Almost all of the transplanted TR6.GFP.LUC-NSCs were specifically ablated following DT administration (Figure 6, B and C). Furthermore, after ablation of the transplanted cells, the BBB scores of SCI model mice that had undergone combined TR6.GFP.LUC-NSC transplantation and VPA administration declined rapidly to levels similar to those observed in untreated and VPA onlytreated mice. These results were superimposed on the graph in Figure 1B, with the observation period extended to 12 weeks after SCI, as shown in Figure 6D (for clarity, the data for GFP-NSC.VPA and GFP.LUC-NS in Figure 1B were removed). These data indicate that the transplanted cells, in the presence of VPA, made a direct and major contribution to the functional recovery of hind limb movement in SCI model mice.

Ablation of transplanted cells abolishes hind limb motor function recovery. (A) Schematic of the protocols for NSC transplantation and for detection and ablation of transplanted cells. NSCs derived from GFP.LUC- or TR6.GFP.LUC-Tg mice were transplanted into SCI model mice 1 week after injury. VPA was intraperitoneally administered every day for 1 week. Survival of transplanted cells and locomotor function of the mice were monitored weekly for 14 weeks. (B) Survival of transplanted cells was checked every week using a bioluminescence imaging system. 6 weeks after injury (5 weeks after transplantation), each mouse received 2 DT administrations. By the following week, LUC activity had completely disappeared in mice transplanted with TR6.GFP.LUC-NSCs (lower panel). (C) Sagittal sections from SCI model mice transplanted with GFP.LUC- and TR6.GFP.LUC-NSCs 2 weeks after DT injection. All transplanted cells were ablated with DT (lower panel). Scale bar: 1 mm. (D) Time course of the changes in BBB scores in SCI model mice. The hind limb function of mice that had undergone dual treatment with TR6.GFP.LUC-NSCs and VPA dropped drastically after DT administration (black line). *P < 0.0001 compared with GFP.LUC-NSCtransplanted, VPA-administered, and DT-injected SCI model mice (blue line) (repeated measures ANOVA). Data are mean SEM. VPA, n = 8; no treatment, n = 8. (E) Twelve weeks after injury, groups of SCI model mice received NMDA injections, as indicated, into the injury epicenter, to ablate local neurons in the gray matter (blue, black, and yellow lines with triangles). *P < 0.0001 compared with non-NMDAinjected mice in each group (blue, black, and yellow lines with circles) (repeated measures ANOVA). Data represent mean SEM.

Both endogenous and transplant-derived local neurons play an important role in improving hind limb motor function. It has been shown recently that local neurons in the spinal cord play an important role in spontaneous functional recovery after SCI (41, 42). In our SCI model, we also observed slight but significant spontaneous recovery of hind limb function in untreated mice, and similar levels of recovery were sustained after ablation of transplanted cells (Figure 6D). We thus hypothesized that these recoveries were attributable to endogenous local neurons in the spinal cord. Furthermore, it seemed likely that the much higher recovery observed in mice with the combined treatment but without cell ablation (Figure 6D) was effected by transplant-derived local neurons in addition to the endogenous ones. To evaluate the involvement of these local neurons in our treatment regime, we divided each treated mouse group analyzed in Figure 6D into 2 subgroups (except for the TR6.GFP.LUC-NCStransplanted only and VPA-administered only groups). The axon-sparing excitotoxin NMDA was injected at 12 weeks after SCI into the injury epicenter in the injured spinal cords of the mice in 1 subgroup for each treatment to ablate local neurons in the gray matter (4345). In uninjured mice, NMDA injections had no significant effect on hind limb function (data not shown). However, as shown in Figure 6E, NMDA injections completely reversed both spontaneous and treatment-provoked functional recovery of hind limb movement in SCI model mice, indicating that both endogenous and transplant-derived local neurons indeed play an important role in the restoration of hind limb motor function.

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An isolated cardiac muscle cell, beating

Cardiac muscle (heart muscle) is involuntary striated muscle that is found in the walls and histological foundation of the heart, specifically the myocardium. Cardiac muscle is one of three major types of muscle, the others being skeletal and smooth muscle. These three types of muscle all form in the process of myogenesis. The cells that constitute cardiac muscle, called cardiomyocytes or myocardiocytes, contain only three nuclei.[1][2][pageneeded] The myocardium is the muscle tissue of the heart, and forms a thick middle layer between the outer epicardium layer and the inner endocardium layer.

Coordinated contractions of cardiac muscle cells in the heart propel blood out of the atria and ventricles to the blood vessels of the left/body/systemic and right/lungs/pulmonary circulatory systems. This complex mechanism illustrates systole of the heart.

Cardiac muscle cells, unlike most other tissues in the body, rely on an available blood and electrical supply to deliver oxygen and nutrients and remove waste products such as carbon dioxide. The coronary arteries help fulfill this function.

Cardiac muscle has cross striations formed by rotating segments of thick and thin protein filaments. Like skeletal muscle, the primary structural proteins of cardiac muscle are myosin and actin. The actin filaments are thin, causing the lighter appearance of the I bands in striated muscle, whereas the myosin filament is thicker, lending a darker appearance to the alternating A bands as observed with electron microscopy. However, in contrast to skeletal muscle, cardiac muscle cells are typically branch-like instead of linear.

Another histological difference between cardiac muscle and skeletal muscle is that the T-tubules in the cardiac muscle are bigger and wider and track laterally to the Z-discs. There are fewer T-tubules in comparison with skeletal muscle. The diad is a structure in the cardiac myocyte located at the sarcomere Z-line. It is composed of a single T-tubule paired with a terminal cisterna of the sarcoplasmic reticulum. The diad plays an important role in excitation-contraction coupling by juxtaposing an inlet for the action potential near a source of Ca2+ ions. This way, the wave of depolarization can be coupled to calcium-mediated cardiac muscle contraction via the sliding filament mechanism. Cardiac muscle forms these instead of the triads formed between the sarcoplasmic reticulum in skeletal muscle and T-tubules. T-tubules play critical role in excitation-contraction coupling (ECC). Recently, the action potentials of T-tubules were recorded optically by Guixue Bu et al.[3]

The cardiac syncytium is a network of cardiomyocytes connected to each other by intercalated discs that enable the rapid transmission of electrical impulses through the network, enabling the syncytium to act in a coordinated contraction of the myocardium. There is an atrial syncytium and a ventricular syncytium that are connected by cardiac connection fibres.[4] Electrical resistance through intercalated discs is very low, thus allowing free diffusion of ions. The ease of ion movement along cardiac muscle fibers axes is such that action potentials are able to travel from one cardiac muscle cell to the next, facing only slight resistance. Each syncyntium obeys the all or none law.[5]

Intercalated discs are complex adhering structures that connect the single cardiomyocytes to an electrochemical syncytium (in contrast to the skeletal muscle, which becomes a multicellular syncytium during mammalian embryonic development). The discs are responsible mainly for force transmission during muscle contraction. Intercalated discs are described to consist of three different types of cell-cell junctions: the actin filament anchoring adherens junctions, the intermediate filament anchoring desmosomes , and gap junctions. They allow action potentials to spread between cardiac cells by permitting the passage of ions between cells, producing depolarization of the heart muscle. However, novel molecular biological and comprehensive studies unequivocally showed that intercalated discs consist for the most part of mixed-type adhering junctions named area composita (pl. areae compositae) representing an amalgamation of typical desmosomal and fascia adhaerens proteins (in contrast to various epithelia).[6][7][8] The authors discuss the high importance of these findings for the understanding of inherited cardiomyopathies (such as arrhythmogenic right ventricular cardiomyopathy).

Under light microscopy, intercalated discs appear as thin, typically dark-staining lines dividing adjacent cardiac muscle cells. The intercalated discs run perpendicular to the direction of muscle fibers. Under electron microscopy, an intercalated disc's path appears more complex. At low magnification, this may appear as a convoluted electron dense structure overlying the location of the obscured Z-line. At high magnification, the intercalated disc's path appears even more convoluted, with both longitudinal and transverse areas appearing in longitudinal section.[9]

In contrast to skeletal muscle, cardiac muscle requires extracellular calcium ions for contraction to occur. Like skeletal muscle, the initiation and upshoot of the action potential in ventricular cardiomyocytes is derived from the entry of sodium ions across the sarcolemma in a regenerative process. However, an inward flux of extracellular calcium ions through L-type calcium channels sustains the depolarization of cardiac muscle cells for a longer duration. The reason for the calcium dependence is due to the mechanism of calcium-induced calcium release (CICR) from the sarcoplasmic reticulum that must occur during normal excitation-contraction (EC) coupling to cause contraction. Once the intracellular concentration of calcium increases, calcium ions bind to the protein troponin, which allows myosin to bind to actin and contraction to occur.

Until recently, it was commonly believed that cardiac muscle cells could not be regenerated. However, a study reported in the April 3, 2009 issue of Science contradicts that belief.[10] Olaf Bergmann and his colleagues at the Karolinska Institute in Stockholm tested samples of heart muscle from people born before 1955 who had very little cardiac muscle around their heart, many showing with disabilities from this abnormality. By using DNA samples from many hearts, the researchers estimated that a 20-year-old renews about 1% of heart muscle cells per year, and about 45 percent of the heart muscle cells of a 50-year-old were generated after he or she was born.

One way that cardiomyocyte regeneration occurs is through the division of pre-existing cardiomyocytes during the normal aging process.[11] The division process of pre-existing cardiomyocytes has also been shown to increase in areas adjacent to sites of myocardial injury. In addition, certain growth factors promote the self-renewal of endogenous cardiomyocytes and cardiac stem cells. For example, insulin-like growth factor 1, hepatocyte growth factor, and high-mobility group protein B1 increase cardiac stem cell migration to the affected area, as well as the proliferation and survival of these cells.[12] Some members of the fibroblast growth factor family also induce cell-cycle re-entry of small cardiomyocytes. Vascular endothelial growth factor also plays an important role in the recruitment of native cardiac cells to an infarct site in addition to its angiogenic effect.

Based on the natural role of stem cells in cardiomyocyte regeneration, researchers and clinicians are increasingly interested in using these cells to induce regeneration of damaged tissue. Various stem cell lineages have been shown to be able to differentiate into cardiomyocytes, including bone marrow stem cells. For example, in one study, researchers transplanted bone marrow cells, which included a population of stem cells, adjacent to an infarct site in a mouse model. Nine days after surgery, the researchers found a new band of regenerating myocardium.[13] However, this regeneration was not observed when the injected population of cells was devoid of stem cells, which strongly suggests that it was the stem cell population that contributed to the myocardium regeneration. Other clinical trials have shown that autologous bone marrow cell transplants delivered via the infarct-related artery decreases the infarct area compared to patients not given the cell therapy.[14]

Occlusion (blockage) of the coronary arteries by atherosclerosis and/or thrombosis can lead to myocardial infarction (heart attack), where part of the myocardium is injured due to ischemia (not receiving enough oxygen). This occurs because coronary arteries are functional end arteries - i.e. there is almost no overlap in the areas supplied by different arteries (anastomoses) so that if one fails, others cannot adequately perfuse the region, unlike in other tissues.

Certain viruses lead to myocarditis (inflammation of the myocardium). Cardiomyopathies are inherent diseases of the myocardium, many of which are caused by genetic mutations.

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Cardiac muscle - Wikipedia, the free encyclopedia

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WHAT ARE STEM CELLS?

Stem cells are super unique in that they have the ability to go through numerous cycles and cell divisions while maintaining the undifferentiated state. Primarily, stem cells are capable of self-renewal and can transform themselves into other cell types of the same tissue. Their crucial role is to replenish dying cells and regenerate damaged tissue. Stem cells have a limited life expectation due to environmental and intrinsic stress factors. Because their life is endangered by internal and external stresses, stem cells have to be protected and supported to delay preliminary aging. In aged bodies, the number and activity of stem cells in reduced.

Until several years ago, the tart, unappealing breed of the Swiss-grown Uttwiler Sptlauber apples, did not seem to offer anything of value. That was until Swiss scientists discovered the unusual longevity of the stem cells that kept these apples alive months after other apples shriveled and fell off their trees. In the rural region of Switzerland, home of these magical apples, it was discovered that when the unpicked apples or tree bark was punctured, Swiss Apple trees have the ability to heal themselves and last longer than other varieties. What was the secret to these apples prolonged lives?

These scientists got to work to find out. What they revealed was that apple stem cells work just like human stem cells, they work to maintain and repair skin tissue. The main difference is that unlike apple stem cells, skin stem cells do not have a long lifespan, and once they begin depleting, the signs of aging start kicking in (in the forms of loose skin, wrinkles, the works). Time to harness these apple stem cells into anti aging skin care! Not so fast. As mentioned, Uttwiler Sptlauber apples are now very rare to the point that the extract can no longer be made in a traditional fashion. The great news is that scientists developed a plant cell culture technology, which involves breeding the apple stem cells in the laboratory.

Human stem cells on the skins epidermis are crucial to replenish the skin cells that are lost due to continual shedding. When epidermal stem cells are depleted, the number of lost or dying skin cells outpaces the production of new cells, threatening the skins health and appearance.

Like humans, plants also have stem cells. Enter the stem cells of the Uttwiler Sptlauber apple tree, whose fruit demonstrates an exceptionally long shelf-life. How can these promising stem cells help our skin?

Studies show that apple stem cells boosts production of human stem cells, protect the cell from stress, and decreases wrinkles. How does it work? The internal fluid of these plant cells contains components that help to protect and maintain human stem cells. Apple stem cells contain metabolites to ensure longevity as the tree is known for the fact that its fruit keep well over long periods of time.

When tested in vitro, the apple stem cell extract was applied to human stem cells from umbilical cords and was found to increase the number of the stem cells in culture. Furthermore, the addition of the ingredient to umbilical cord stem cells appeared to protect the cells from environmental stress such as UV light.

Apple stem cells do not have to be fed through the umbilical cord to benefit our skin! The extract derived from the plant cell culture technology is being harnessed as an active ingredient in anti aging skincare products. When delivered into the skin nanotechnology, the apple stem cells provide more dramatic results in decreasing lines, wrinkles, and environmental damage.

Currently referred to as The Fountain of Youth, intense research has proved that with just a concentration level of 0.1 % of the PhytoCellTec (apple stem cell extract) could proliferate a wealth of human stem cells by an astounding 80%! These wonder cells work super efficiently and are completely safe. Of the numerous benefits of apple stems cells, the most predominant include:

Skin Layers

Skin Cell Activity Before

Skin Cell Activity After 1 Hour

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explore

Stem cells play many important roles in our bodies from embryonic development through adulthood.

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Stem cells can now be created from differentiated cells.

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Learn about some different types of stem cells and their potential for treating diseases.

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Send activating signals to stem cells and watch them get to work!

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Stem cell therapies have been curing diseases for decades.

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Researchers are working on new ways to use stem cells in medicine.

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New developments in research are changing the conversation about stem cells.

Supported by a Science Education Partnership Award (SEPA) Grant No. R25RR023288 from the National Center for Research Resources, a component of the NIH. The contents provided here are solely the responsibility of the authors and do not necessarily represent the official views of NIH.

APA format: Genetic Science Learning Center (2014, June 22) Stem Cells. Learn.Genetics. Retrieved September 26, 2015, from http://learn.genetics.utah.edu/content/stemcells/ MLA format: Genetic Science Learning Center. "Stem Cells." Learn.Genetics 26 September 2015 <http://learn.genetics.utah.edu/content/stemcells/> Chicago format: Genetic Science Learning Center, "Stem Cells," Learn.Genetics, 22 June 2014, <http://learn.genetics.utah.edu/content/stemcells/> (26 September 2015)

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This is a sixth post of the series Not Lost in Translation.

If youre trying to develop a cellular product and just entering the field of cell therapy, you should be aware of existent standards. Why is it important? Knowing standards in your field allows to:

Even though, cell therapy filed relatively new, there are numerous related standards. Unfortunately, many professionals are unaware about organizations and standards in cell therapy field. The purpose of this post is to indicate few leadig organizations, providing standards and types of standards in cell products development. Significant part of this topic was summarized from the recent public FDA workshop Synergizing Efforts in Standards Development for Cellular Therapies and Regenerative Medicine Products.

Type of standards in cell therapy:

Standards-developing organizations and examples: ISO International Organization for Standardization Developing and providing international standards, including medical devices, laboratory testing and some, related to cell therapy and tissue engineered products. Examples: ISO/TC 194/SC 1 Tissue product safety ISO/TC 150/SC 7 Tissue-engineered medical products

ASTM International American Society for Testing and Materials ASTM leading international standards organization. ASTM has Subcommittee F04.43 for developing standards in cell therapy and tissue engineering. Examples: ASTM F2210 Standard Guide for Processing Cells, Tissues, and Organs for Use in Tissue Engineered Medical Products ASTM F2739 Standard Guide for Quantitating Cell Viability Within Biomaterial Scaffolds ASTM F2315 Standard Guide for Immobilization or Encapsulation of Living Cells or Tissue in Alginate Gels ASTM F2944 Standard Test Method for Automated Colony Forming Unit (CFU) Assays

USP U.S. Pharmacopeial Convention Provides standards for use ancillary and raw materials for cellular and tissue products. Examples: Chapter 1046 Cell and Gene Therapies Products Chapter 1047 Gene Therapy Products Chapter 1043 Ancillary Materials for Cell, Gene and Tissue-Engineered Products Chapter 92 Growth Factors and Cytokines Used in Cell Therapy Manufacturing Chapter 90 Fetal Bovine SerumQuality Attributes and Functionality Tests

GBSI Global Biological Standard Institute Developing standards for life sciences, including biomedical research.

ATCC American Type Culture Collection Manufactures and provides reference material (including cells), developing biological standards for basic and translational research. Examples: ATCC Certified reference material ATCC Standards Development Organization

BSI British Standards Institution Has a project for developing regenerative medicine definitions and guidelines for clinical cell products characterization. Examples: PAS 93:2011 Characterization of human cells for clinical applications. Guide PAS 84:2012 Cell therapy and regenerative medicine. Glossary

FACT Foundation for the Accreditation of Cellular Therapy Provides standards for collection and processing cellular products. Accredits clinical stem cell labs, cord blood banks and more than minimal manipulation cell therapy facilities. Examples: FACT-JACIE International Standards for Cellular Therapy Product Collection, Processing and Administration FACT-JACIE Cellular Therapy Accreditation Manual

AABB American Association of Blood Banks Center for Cellular Therapies In cell therapy field, AABB has very similar functions with FACT. Examples: Standards for Cellular Therapy Services

ICCBBA International Council for Commonality in Blood Bank Automation Management of the ISBT-128 Standard the terminology, identification, coding and labeling of medical products of human origin (including blood, cell, tissue, and organ products).

ISCT International Society for Cellular Therapy ISCT leverages expertise of cell therapy professionals to develop guidelines and recommendations for cellular products development, characterization, and quality. Examples: Minimal criteria for defining multipotent mesenchymal stromal cells Potency assay development for cellular therapy products Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells IFATS/ISCT statement

Coordination and harmonization As you can see, there are many organizations, involved in different aspects of cell therapy standardization. How can we make sure that there are no overlaps between them? How to coordinate and harmonize their activities? There are some good existent examples of such coordination:

*********************** This post is a part of Not Lost in Translation online community project. In this series we will try to bridge the translational gaps between scientific discovery in research labs and clinical cell applications for therapies. We will look at challenges in translation of cell product development and manufacturing in academic and industry settings. If you would like to contribute to this community project, please contact us!

Tagged as: cell therapy, reference material, standard, translation

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Standards in Cell Therapy

IPS Cell Therapy Comments Off on Standards in Cell Therapy | September 25th, 2015

Every one of us completely regenerates our own skin every 7 days. A cut heals itself and disappears in a week or two. Every single cell in our skeleton is replaced every 7 years.

The future of medicine lies in understanding how the body creates itself out of a single cell and the mechanisms by which it renews itself throughout life.

When we achieve this goal, we will be able to replace damaged tissues and help the body regenerate itself, potentially curing or easing the suffering of those afflicted by disorders like heart disease, Alzheimers, Parkinsons, diabetes, spinal cord injury and cancer.

Research at the institute leverages Stanfords many strengths in a way that promotes that goal. The institute brings together experts from a wide range of scientific and medical fields to create a fertile, multidisciplinary research environment.

There are four major research areas of emphasis at the institute:

Theres no way to know, beforehand, which particular avenue of stem cell research will most expediently yield a successful treatment or cure. Therefore, we need to vigorously pursue a broad number of promising leads concurrently.

--Philip A. Pizzo, MD Carl and Elizabeth Naumann Professor Dean, Stanford University School of Medicine

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Research - Stem Cell Biology and Regenerative Medicine ...

Uncategorized Comments Off on Research – Stem Cell Biology and Regenerative Medicine … | September 25th, 2015

Induced pluripotent stem cells (iPSCs) are adult cells that have been genetically reprogrammed to an embryonic stem celllike state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells. Although these cells meet the defining criteria for pluripotent stem cells, it is not known if iPSCs and embryonic stem cells differ in clinically significant ways. Mouse iPSCs were first reported in 2006, and human iPSCs were first reported in late 2007. Mouse iPSCs demonstrate important characteristics of pluripotent stem cells, including expressing stem cell markers, forming tumors containing cells from all three germ layers, and being able to contribute to many different tissues when injected into mouse embryos at a very early stage in development. Human iPSCs also express stem cell markers and are capable of generating cells characteristic of all three germ layers.

Although additional research is needed, iPSCs are already useful tools for drug development and modeling of diseases, and scientists hope to use them in transplantation medicine. Viruses are currently used to introduce the reprogramming factors into adult cells, and this process must be carefully controlled and tested before the technique can lead to useful treatment for humans. In animal studies, the virus used to introduce the stem cell factors sometimes causes cancers. Researchers are currently investigating non-viral delivery strategies. In any case, this breakthrough discovery has created a powerful new way to "de-differentiate" cells whose developmental fates had been previously assumed to be determined. In addition, tissues derived from iPSCs will be a nearly identical match to the cell donor and thus probably avoid rejection by the immune system. The iPSC strategy creates pluripotent stem cells that, together with studies of other types of pluripotent stem cells, will help researchers learn how to reprogram cells to repair damaged tissues in the human body.

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What are induced pluripotent stem cells? [Stem Cell ...

IPS Cell Therapy Comments Off on What are induced pluripotent stem cells? [Stem Cell … | September 18th, 2015

Last year, Patricia Beals was told she'd need a double knee replacement to repair her severely arthritic knees or she'd probably spend the rest of her life in a wheelchair.

Hoping to avoid surgery, Beals, 72, opted instead for an experimental treatment that involved harvesting bone marrow stem cells from her hip, concentrating the cells in a centrifuge and injecting them back into her damaged joints.

"Almost from the moment I got up from the table, I was able to throw away my cane," Beals says. "Now I'm biking and hiking like a 30-year-old."

A handful of doctors around the country are administering treatments like the one Beals received to stop or even reverse the ravages of osteoarthritis. Stem cells are the only cells in the body able to morph into other types of specialized cells. When the patient's own stem cells are injected into a damaged joint, they appear to transform into chondrocytes, the cells that go on to produce fresh cartilage. They also seem to amplify the body's own natural repair efforts by accelerating healing, reducing inflammation, and preventing scarring and loss of function.

Christopher J. Centeno, M.D., the rehab medicine specialist who performed Beals' procedure, says the results he sees from stem cell therapy are remarkable. Of the more-than-200 patients his Bloomfield, Colo., clinic treated over a two-year period, he says, "two thirds of them reported greater than 50 percent relief and about 40 percent reported more than 75 percent relief one to two years afterward."

According to Centeno, knees respond better to the treatment than hips. Only eight percent of his knee patients opted for a total knee replacement two years after receiving a stem cell injection. The complete results from his clinical observations will be published in a major orthopedic journal later this year.

The Pros and Cons

The biggest advantage stem cell injections seem to offer over more invasive arthritis remedies is a quicker, easier recovery. The procedure is done on an outpatient basis and the majority of patients are up and moving within 24 hours. Most wear a brace for several weeks but still can get around. Many are even able to do some gentle stationary cycling by the end of the first week.

There are also fewer complications. A friend who had knee replacement surgery the same day Beals had her treatment developed life-threatening blood clots and couldn't walk for weeks afterwards. Six months out, she still hasn't made a full recovery.

Most surgeries don't go so awry, but still: Beals just returned from a week-long cycling trip where she covered 20 to 40 miles per day without so much as a tweak of pain.

As for risks, Centeno maintains they are virtually nonexistent.

"Because the stem cells come from your own body, there's little chance of infection or rejection," he says.

Not all medical experts are quite so enthusiastic, however. Dr. Tom Einhorn, chairman of the department of orthopedic surgery at Boston University, conducts research with stem cells but does not use them to treat arthritic patients. He thinks the idea is interesting but the science is not there yet.

"We need to have animal studies and analyze what's really happening under the microscope. Then, and only then, can you start doing this with patients," he says.

The few studies completed to date have examined how stem cells heal traumatic injuries rather than degenerative conditions such as arthritis. Results have been promising but, as Einhorn points out, the required repair mechanisms in each circumstance are very different.

Another downside is cost: The injections aren't approved by the FDA, which means they aren't covered by insurance. At $4,000 a pop -- all out of pocket -- they certainly aren't cheap, and many patients require more than one shot.

Ironically, one thing driving up the price is FDA involvement. Two years ago, the agency stepped in and stopped physicians from intensifying stem cells in the lab for several days before putting them back into the patient. This means all procedures must be done on the same day, no stem cells may be preserved and many of the more expensive aspects of the treatment must be repeated each time.

Centeno says same day treatments often aren't as effective, either.

But despite the sky-high price tag and lack of evidence, patients like Beals believe the treatment is nothing short of a miracle. She advises anyone who is a candidate for joint replacement to consider stem cells first.

"Open your mind up and step into it," she says. "Do it. It's so effective. It's the future and it works."

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Stem Cell Treatment May Help Ease Osteoarthritis Pain ...

Uncategorized Comments Off on Stem Cell Treatment May Help Ease Osteoarthritis Pain … | September 17th, 2015

George Reed's heart wasn't doing so well: He's 71, and after suffering a heart attack years earlier, Reed had undergone open heart surgery and was put on multiple medications. But nothing seemed to help the dizziness and chest pain he experienced daily.

"I'd get dizzy and just fall over -- sometimes twice a day. I would run my head into the concrete. I was a bloody mess," the Perry, Ohio, native says. Despite his doctor's best efforts, Reed continued to experience angina, a type of chest pain that occurs when the heart doesn't get enough oxygen-rich blood; it can be accompanied by dizziness. So when he was recommended for an experimental study that would inject his own stem cells into his damaged heart, Perry signed on. "I needed something to change," he says.

Researchers gave Reed a drug commonly used in bone marrow transplants that stimulates the marrow to make more stem cells. Then they removed some of Reed's blood, isolated the stem cells and injected them into and around the damaged areas of his heart.

"The goal was to grow new blood vessels with stem cells from the patient's own body," says Dr. Tim Henry, a co-author of the study and director of research at the Minneapolis Heart Institute Foundation.

Within a few months, Reed, along with many of the other 100 or so patients at 26 hospital centers who'd received this stem cell treatment, reported feeling better than he had in years.

"When it started kicking in, I felt like a kid. I felt good," Reed says. He wasn't passing out and falling down anymore.

For Jay Homstad, 49, who was part of the Minnesota branch of the study, he felt the changes most in his ability to walk and be active.

"My activity level increased tenfold. Before, I struggled with chest pain every day. My activity level was about as close to zero as you could get. Now I can participate ... just in life. It may sound silly, but the best part is that in the wintertime I could go out and walk with my dog along the Red River. When you're walking through snow that is waist deep, you can tell there's a difference," Homstad says.

Homstad had had about a dozen surgeries and nine stents put in before he enrolled in the study, but he still struggled with angina daily. Within a few months of the stem cell shots, he could walk farther, and his chest pain subsided and was kept at bay for nearly four years.

"These are people for whom other treatment hasn't worked. They're debilitated by their chest pain, but their other options are really limited, that's why we picked them," says Henry. If the positive results seen in this study hold up in the next phase of the study, which is set to begin enrollment in the fall, this type of cardiac stem cell injection could be added to the arsenal of weapons against angina. The upcoming phase three trial has already been approved by the Food and Drug Administration.

Shot to the Heart, Before It's too Late

While several smaller studies have suggested that injecting stem cells into damaged heart tissue might be effective, this study, in its scope and rigor, was the first of its kind. A total of 167 patients were recruited and randomly assigned to receive a lower dose of stem cells, a higher dose or a placebo. The patients didn't know who got what treatment, and neither did the doctors treating them.

When tracked for a year after the injection, patients who received the lower dose of stem cells could last longer during a treadmill exercise than those who had received the placebo, and they averaged seven fewer episodes of chest pain in a week. To put this in perspective, a popular drug to treat angina, Ranolazine, reduced chest pain by fewer than two episodes a week in clinical trials.

Although the goal of the stem cell shots was to grow new blood vessels, it's impossible to tell if these stem cells were actually growing into blood vessels or if they were just triggering some other kind of healing process in the body, Henry says. Tests in animal models, however, do suggest that new blood vessels are forming, says Dr. Marco Costa, a co-author of the study and George Reed's doctor at UH Case Medical Center in Cleveland.

For now, the only gauge of the injections is improvement in symptoms.

Despite the positive results of the study, cardiologists remain "cautiously optimistic" about stem cells as a treatment for angina.

"The number of patients is relatively small, so this trial would probably not carry much scientific weight," says Dr. Jeff Brinker, a professor of cardiology at Johns Hopkins University. The results did justify the next, larger trial, he says, which would offer more answers as to whether this treatment is actually working the way researchers suspect.

The fact that lower doses of stem cells were puzzlingly more effective than larger ones is cause for caution, says Dr. Steve Nissen, chairman of the department of cardiovascular medicine at the Cleveland Clinic.

"The jury is still out for stem cell therapies to treat heart disease," says Dr. Cam Paterson, a cardiologist at the University of North Carolina at Chapel Hill.

But the results so far provide cautious hope for heart patients like George Reed and Jay Homstad.

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Injecting the Heart With Stem Cells Helps Chest Pain - ABC ...

Cardiac Stem Cells Comments Off on Injecting the Heart With Stem Cells Helps Chest Pain – ABC … | September 13th, 2015

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Its a shame that National Geographic has become part of a corporate empire that is not always consistent, to put it nicely, with data-based reality. Can NatGeo maintain its credibility and impact, when it is owned by a climate change denier (quoted for example as dissing folks as extreme greenies) who also has other verynon-scientificpriorities?

Theres been an increasing amount of discussion of the technology that could produce GM humans. This dialogue includes the new Hinxton Statement (my take on that here) and George Churchs quoted that Hinxton (which BTW did not call for a moratorium of any kind) was being too cautious nonetheless. Church is quoted:

seems weak on addressing why we should single out genome editing relative to other medicines that are potentially dangerous

Should we push pause, stop, or fast-forward on human genetic modification? asks Lisa Ikemoto.Is there a rewind or edit button too?

The NEJM published a new piece on stem cell clinics run amok and the lack of an effective FDA response. Sounds awfully familiar including the use of Wild West in the title, right? My gripe with these authors is that they didnt give credit where credit is due to those of us on the front lines of this battle and in particular to social media-based efforts to promote evidence-based medicine in the stem cell arena. Still, their message was on target.

Are men more likely to commit large-scale scientific fraud? Check out RetractionWatchs leaderboard.Of course the sheer number of retractions does not take into account the impact of any one or two given retractions that had a disproportionate toxic effect like the STAP pubs. Maybe another calculation to do is the # of citations to a retracted paper.

DrugMonkey talks about perceived scientific backstabbing.

The international stem cell policy and ethics think tank, the Hinxton Group, weighed in yesterday on heritable human genetic modification with a new policy statement.

The Hinxton statement is in many ways in agreement with the Baltimore, et al. Nature paper proposing a prudent path forward for human germline genetic modification, which came out of the Napa Meeting earlier this year.

However, while several of the Napa authors have now thrown their public support behind a clinical pause or moratorium on heritable human modification (e.g. Jennifer Doudnaas well asDavid Baltimore and Paul Berg in a later piece in the WSJ), Hinxton didnt explicitlyaddress either positively or negatively the question of a moratorium.

My initial reading of the Hinxton statement is that I mostly agree with it. In my own proposed ABCD planon human germline modification from earlier this year, however, I included at least a temporary clinical moratorium.

I also would have appreciated a more detailed risk-benefit analysis in the Hinxton statement. For instance, I didnt see a discussion of specific possible risks in their statement. Via myown risk-benefit analysis, I come to the conclusion that on the whole a temporary clinical moratorium has the potential for far more benefit than harm.

What would be the specific, possible benefits of a moratorium?

If the scientific community has united behind a moratorium on clinical use not only will that discourage rogue or potentially ill-advised stabs at clinical use, but also if a few such dangerous efforts proceed anyway (which is fairly likely) and come to public light, these unfortunate events will be placed in the appropriate context of the scientific community having a moratorium in place. Therefore, a moratorium both discourages premature and dangerous clinical use as well as putting potential future human gene editing clinical mishaps into the proper context for the pubic.

Another potential benefit of a moratorium is that it could discourage lawmakers from passing reactionary, overly restrictive legislation that bans both clinical applications and important in vitro research. It would give the politicians and the public the right sense that the scientific community is handling this situation with appropriate caution. If you dont think that a law on human germline modification is likely in the US, consider that conservative lawmakers have already proposed such a law be included as part of the pending appropriations bill and Congress a few months ago held a hearing on germline human modification.

Other benefits of a moratorium include that it would a) demonstrate to the public that the research community is capable of reaching consensus aboutimportant ethical issues and b) increase accountability within the research community. Any rogue researchers or clinicians who would violate the moratorium, even if it were not illegal for them to do so, would at least be subject to the disapproval and possible sanction of their professional peers or institutions. Without a moratorium in place, it is far less likely there would be these kinds of consequences.

What about risks to a clinical moratorium?The primary possible risk of a clinical moratorium is that it could, should human heritable genetic modification someday down the road be viewed as a wise course to pursue directly, impede clinical translation. This warrants discussion, but in my view the risk here is somewhat reduced by the possibility that continuing basic research develops a compelling case that a blanket clinical moratorium might no longer be needed.

The other risk here is that amoratorium on clinical use also might in theory discourage some potentially valuable pre-clinical research as well. In other words, some researchers may adopt the mindset that if they cannot get to their ultimate goal of making clinical impact, why do the preclinical studies? I expect that many researchers would instead go ahead and do the preclinical work with the expectation that a clinical moratorium could be lifted and in fact their own preclinical work might help build a case for moving beyond a moratorium.

I agreestrongly with Hinxton on the need for continuation of basic science on CRISPR and other gene editing technologies limited to the lab. In my view, we should have a nuanced policy though, whereby we support continuation of gene editing research in human cells and even in some cases human embryos in the lab under specific conditions (see again my ABCD plan for details), but in whichwe also put an unambiguous hold onclinical applications at this time.

In the absence of a framework that includes a clinical moratorium, we probably do not have the luxury of a reasonably long time frame (e.g. measured in a few years) for open discussionto sort things out carefully. To be clear, open and diverse discussion is crucial, but we just do not have a whole lot of time to do it as things stand today. Why? In the mean time absent a moratorium, I believe that some will go ahead and do clinical experiments on human germline editing. This would not only put individual research subjects at risk, but also pose dangers in terms of public trust and support to the wider scientific community. In a relatively permissive environment lacking a clinical moratorium, one or two instances of rogue researchers clinically using gene editing in a heritable manner could end up leading to a backlash in which even in vitro gene editing research is stymied.

Stemcentrx scientists working with targeted molecules that can kill some types of lung cancer. MIT Tech Review Image.

A stem cell biotech in the news this week was one thathad mostly flown under the radar previously.

Stemcentrx hasa focus on killing cancer stem cells as a novel approach to treating cancer. Antonio Regalado had a nice articleyesterday on the company. He reports that Stemcentrx has around a half a billion in funding. It is valued in the billions. These are very unusual figures for a stem cell biotech.

Stemcentrx isdeveloping novel cancer therapeutics such as antibodies that target cancer stem cells. Their development pipeline at least in part uses a model of serial xenograft tumor transplantation in mice.Cancer stem cells are also sometimes called tumor initiating cells (TIC). As a cancer stem cell researcher myself, I find Stemcentrx intriguing.

The company published an encouraging bit of preclinical data recently in Science Translational Medicinewith a team of authors including leading company scientist, Scott Dylla. In this paper the team presented evidence that they have a product in the form of a loaded antibody (conjugated to a toxin) against a molecule called DLL3 important to TIC biological function and survival. DLL3 is part of the Notch signaling pathway. Stay tuned tomorrow for my interview with Dr. Dylla.

They showed that this anti-DLL3 antibody,SC16LD6.5, exhibited anti-tumor activities in xenograft models of pulmonary neuroendocrine tumors such as small cell lung cancer. The company also has a clinical trial ongoing but not currently recruiting using this drug, and they have another trial for ovarian cancer based on antibody targeting as well.

SC16LD6.5 also exhibited some degree of toxicity in rats and a non-human primate model so thats a possible issue moving forward, but the toxic effects were at least partially reversible and when youre dealing with a deadly disease some toxicity for treatment is kind of to be expected.

Can Stemcentrx survive and hopefully even thrive as a company selling products that kill cancer stem cells? Well have a clearer picture on this in a few years, but in general biotechs of this type in this arena have a high failure rate. We need to keep in mind the long, sobering path ahead between these kinds of preclinical result and getting an approved drug to patients.

At the same time, this team has the money and talent to potentially succeed, and again, theres that half a billion in funding, which all by itself makes this stem cell biotech noordinary company. Theres another unique thing going on here: famed tech investor Peter Thiel is one of the major funders of the company.

Those of us in the cancer stem cell field have long been engaged in the debate overwhether these special cells exist in specific solid tumors and their functions in tumorigenesis. I believe they are present and important in some, but not all of such tumors. The controversial nature of TICs in lung cancer specifically makes SC16LD6.5 a high-risk, high reward kind ofproduct.

More weapons against lung cancer will be of coursea good thing and targeting cancer stem cells is an innovative approach. The company isrecruiting for many positions including scientists so if you are interested take a look.

I hope Stemcentrx succeeds and I look forward to reading more of their work as the years go by.

The winner of the inaugural Ogawa-Yamanaka Prize is Dr. Masayo Takahashi, MD, PhD.

According to the Gladstone Institutepress release, Dr. Takahashi was awarded the prize for her trailblazing work leading the first clinical trial to use induced pluripotent stem (iPS) cells in humans.

The prize, including a $150,000 cash award, will be given at a ceremony next week at the Gladstone on September 16. If you are interested in listening in, you can register for the webcast here.

Dr. Takahashi started the first ever human clinical study using iPS cells, which is focused on treating of macular degeneration using retinal pigmented epithelial cells derived from human iPS cells.

Congratulations to Dr. Takahashi for the great and well-deserved honor of the Ogawa-Yamanaka Prize.

As readers of this blog likely recall, Dr. Takahashi received our blogsStem Cell Person of the Year Award last year in honor of her pioneering work and that included a $2,000 prize.

Otherpast winners of our Stem Cell Person of the Year Award have gone on to get additional awards too.

The 2013 Stem Cell Person of the Year, Dr. Elena Cattaneo, went on to win the ISSCR Public Service Award in 2014 along with colleagues.

And our 2012 Stem Cell Person of the Year Award winner, stellar patient advocateRoman Reed, went on in 2013 to receive the GPI Stem Cell Inspiration Award.

The more we can recognize the pioneers and outside-the-box thinkers in the stem cell field, the better.

I recently ran a poll on my blog about how the FDA is doing on handling stem cell clinics.

There is substantial debate in the stem cell arena about how the FDA handles stem cell clinics ranging from the view that the agency is far too strict to excessively lenient.

The results of the poll reflect a great deal of dissatisfaction with the job that the FDA is doing on stem cell clinics.

Only 9% of respondents felt that the FDA is currently do things just about right.

While the top 2 answers were polar extremes, by a large margin the top answer was that the FDA was much too lenient.

Although Internet polls of this kind are not scientific, they can reflect sentiments of a community.

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Knoepfler Lab Stem Cell Blog | Building innovative ...

Uncategorized Comments Off on Knoepfler Lab Stem Cell Blog | Building innovative … | September 12th, 2015

Cell culture is the process by which cells are grown under controlled conditions, generally outside of their natural environment. In practice, the term "cell culture" now refers to the culturing of cells derived from multicellular eukaryotes, especially animal cells, in contrast with other types of culture that also grow cells, such as plant tissue culture, fungal culture, and microbiological culture (of microbes). The historical development and methods of cell culture are closely interrelated to those of tissue culture and organ culture. Viral culture is also related, with cells as hosts for the viruses.

The laboratory technique of maintaining live cell lines (a population of cells descended from a single cell and containing the same genetic makeup) separated from their original tissue source became more robust in the middle 20th century.[1][2]

The 19th-century English physiologist Sydney Ringer developed salt solutions containing the chlorides of sodium, potassium, calcium and magnesium suitable for maintaining the beating of an isolated animal heart outside of the body.[3] In 1885, Wilhelm Roux removed a portion of the medullary plate of an embryonic chicken and maintained it in a warm saline solution for several days, establishing the principle of tissue culture.[4]Ross Granville Harrison, working at Johns Hopkins Medical School and then at Yale University, published results of his experiments from 1907 to 1910, establishing the methodology of tissue culture.[5]

Cell culture techniques were advanced significantly in the 1940s and 1950s to support research in virology. Growing viruses in cell cultures allowed preparation of purified viruses for the manufacture of vaccines. The injectable polio vaccine developed by Jonas Salk was one of the first products mass-produced using cell culture techniques. This vaccine was made possible by the cell culture research of John Franklin Enders, Thomas Huckle Weller, and Frederick Chapman Robbins, who were awarded a Nobel Prize for their discovery of a method of growing the virus in monkey kidney cell cultures.

Cells can be isolated from tissues for ex vivo culture in several ways. Cells can be easily purified from blood; however, only the white cells are capable of growth in culture. Mononuclear cells can be released from soft tissues by enzymatic digestion with enzymes such as collagenase, trypsin, or pronase, which break down the extracellular matrix. Alternatively, pieces of tissue can be placed in growth media, and the cells that grow out are available for culture. This method is known as explant culture.

Cells that are cultured directly from a subject are known as primary cells. With the exception of some derived from tumors, most primary cell cultures have limited lifespan.

An established or immortalized cell line has acquired the ability to proliferate indefinitely either through random mutation or deliberate modification, such as artificial expression of the telomerase gene. Numerous cell lines are well established as representative of particular cell types.

For the majority of isolated primary cells, they undergo the process of senescence and stop dividing after a certain number of population doublings while generally retaining their viability (described as the Hayflick limit).

Cells are grown and maintained at an appropriate temperature and gas mixture (typically, 37C, 5% CO2 for mammalian cells) in a cell incubator. Culture conditions vary widely for each cell type, and variation of conditions for a particular cell type can result in different phenotypes.

Aside from temperature and gas mixture, the most commonly varied factor in culture systems is the cell growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrients. The growth factors used to supplement media are often derived from the serum of animal blood, such as fetal bovine serum (FBS), bovine calf serum, equine serum, and porcine serum. One complication of these blood-derived ingredients is the potential for contamination of the culture with viruses or prions, particularly in medical biotechnology applications. Current practice is to minimize or eliminate the use of these ingredients wherever possible and use human platelet lysate (hPL). This eliminates the worry of cross-species contamination when using FBS with human cells. hPL has emerged as a safe and reliable alternative as a direct replacement for FBS or other animal serum. In addition, chemically defined media can be used to eliminate any serum trace (human or animal), but this cannot always be accomplished with different cell types. Alternative strategies involve sourcing the animal blood from countries with minimum BSE/TSE risk, such as The United States, Australia and New Zealand,[6] and using purified nutrient concentrates derived from serum in place of whole animal serum for cell culture.[7]

Plating density (number of cells per volume of culture medium) plays a critical role for some cell types. For example, a lower plating density makes granulosa cells exhibit estrogen production, while a higher plating density makes them appear as progesterone-producing theca lutein cells.[8]

Cells can be grown either in suspension or adherent cultures. Some cells naturally live in suspension, without being attached to a surface, such as cells that exist in the bloodstream. There are also cell lines that have been modified to be able to survive in suspension cultures so they can be grown to a higher density than adherent conditions would allow. Adherent cells require a surface, such as tissue culture plastic or microcarrier, which may be coated with extracellular matrix (such as collagen and laminin) components to increase adhesion properties and provide other signals needed for growth and differentiation. Most cells derived from solid tissues are adherent. Another type of adherent culture is organotypic culture, which involves growing cells in a three-dimensional (3-D) environment as opposed to two-dimensional culture dishes. This 3D culture system is biochemically and physiologically more similar to in vivo tissue, but is technically challenging to maintain because of many factors (e.g. diffusion).

Cell line cross-contamination can be a problem for scientists working with cultured cells.[9] Studies suggest anywhere from 1520% of the time, cells used in experiments have been misidentified or contaminated with another cell line.[10][11][12] Problems with cell line cross-contamination have even been detected in lines from the NCI-60 panel, which are used routinely for drug-screening studies.[13][14] Major cell line repositories, including the American Type Culture Collection (ATCC), the European Collection of Cell Cultures (ECACC) and the German Collection of Microorganisms and Cell Cultures (DSMZ), have received cell line submissions from researchers that were misidentified by them.[13][15] Such contamination poses a problem for the quality of research produced using cell culture lines, and the major repositories are now authenticating all cell line submissions.[16] ATCC uses short tandem repeat (STR) DNA fingerprinting to authenticate its cell lines.[17]

To address this problem of cell line cross-contamination, researchers are encouraged to authenticate their cell lines at an early passage to establish the identity of the cell line. Authentication should be repeated before freezing cell line stocks, every two months during active culturing and before any publication of research data generated using the cell lines. Many methods are used to identify cell lines, including isoenzyme analysis, human lymphocyte antigen (HLA) typing, chromosomal analysis, karyotyping, morphology and STR analysis.[17]

One significant cell-line cross contaminant is the immortal HeLa cell line.

As cells generally continue to divide in culture, they generally grow to fill the available area or volume. This can generate several issues:

Among the common manipulations carried out on culture cells are media changes, passaging cells, and transfecting cells. These are generally performed using tissue culture methods that rely on aseptic technique. Aseptic technique aims to avoid contamination with bacteria, yeast, or other cell lines. Manipulations are typically carried out in a biosafety hood or laminar flow cabinet to exclude contaminating micro-organisms. Antibiotics (e.g. penicillin and streptomycin) and antifungals (e.g.amphotericin B) can also be added to the growth media.

As cells undergo metabolic processes, acid is produced and the pH decreases. Often, a pH indicator is added to the medium to measure nutrient depletion.

In the case of adherent cultures, the media can be removed directly by aspiration, and then is replaced. Media changes in non-adherent cultures involve centrifuging the culture and resuspending the cells in fresh media.

Passaging (also known as subculture or splitting cells) involves transferring a small number of cells into a new vessel. Cells can be cultured for a longer time if they are split regularly, as it avoids the senescence associated with prolonged high cell density. Suspension cultures are easily passaged with a small amount of culture containing a few cells diluted in a larger volume of fresh media. For adherent cultures, cells first need to be detached; this is commonly done with a mixture of trypsin-EDTA; however, other enzyme mixes are now available for this purpose. A small number of detached cells can then be used to seed a new culture. Some cell cultures, such as RAW cells are mechanically scraped from the surface of their vessel with rubber scrapers.

Another common method for manipulating cells involves the introduction of foreign DNA by transfection. This is often performed to cause cells to express a protein of interest. More recently, the transfection of RNAi constructs have been realized as a convenient mechanism for suppressing the expression of a particular gene/protein. DNA can also be inserted into cells using viruses, in methods referred to as transduction, infection or transformation. Viruses, as parasitic agents, are well suited to introducing DNA into cells, as this is a part of their normal course of reproduction.

Cell lines that originate with humans have been somewhat controversial in bioethics, as they may outlive their parent organism and later be used in the discovery of lucrative medical treatments. In the pioneering decision in this area, the Supreme Court of California held in Moore v. Regents of the University of California that human patients have no property rights in cell lines derived from organs removed with their consent.[19]

It is possible to fuse normal cells with an immortalised cell line. This method is used to produce monoclonal antibodies. In brief, lymphocytes isolated from the spleen (or possibly blood) of an immunised animal are combined with an immortal myeloma cell line (B cell lineage) to produce a hybridoma which has the antibody specificity of the primary lymphocyte and the immortality of the myeloma. Selective growth medium (HA or HAT) is used to select against unfused myeloma cells; primary lymphoctyes die quickly in culture and only the fused cells survive. These are screened for production of the required antibody, generally in pools to start with and then after single cloning.

A cell strain is derived either from a primary culture or a cell line by the selection or cloning of cells having specific properties or characteristics which must be defined. Cell strains are cells that have been adapted to culture but, unlike cell lines, have a finite division potential. Non-immortalized cells stop dividing after 40 to 60 population doublings[20] and, after this, they lose their ability to proliferate (a genetically determined event known as senescence).[21]

Mass culture of animal cell lines is fundamental to the manufacture of viral vaccines and other products of biotechnology.

Biological products produced by recombinant DNA (rDNA) technology in animal cell cultures include enzymes, synthetic hormones, immunobiologicals (monoclonal antibodies, interleukins, lymphokines), and anticancer agents. Although many simpler proteins can be produced using rDNA in bacterial cultures, more complex proteins that are glycosylated (carbohydrate-modified) currently must be made in animal cells. An important example of such a complex protein is the hormone erythropoietin. The cost of growing mammalian cell cultures is high, so research is underway to produce such complex proteins in insect cells or in higher plants, use of single embryonic cell and somatic embryos as a source for direct gene transfer via particle bombardment, transit gene expression and confocal microscopy observation is one of its applications. It also offers to confirm single cell origin of somatic embryos and the asymmetry of the first cell division, which starts the process.

Research in tissue engineering, stem cells and molecular biology primarily involves cultures of cells on flat plastic dishes. This technique is known as two-dimensional (2D) cell culture, and was first developed by Wilhelm Roux who, in 1885, removed a portion of the medullary plate of an embryonic chicken and maintained it in warm saline for several days on a flat glass plate. From the advance of polymer technology arose today's standard plastic dish for 2D cell culture, commonly known as the Petri dish. Julius Richard Petri, a German bacteriologist, is generally credited with this invention while working as an assistant to Robert Koch. Various researchers today also utilize culturing laboratory flasks, conicals, and even disposable bags like those used in single-use bioreactors.

Aside from Petri dishes, scientists have long been growing cells within biologically derived matrices such as collagen or fibrin, and more recently, on synthetic hydrogels such as polyacrylamide or PEG. They do this in order to elicit phenotypes that are not expressed on conventionally rigid substrates. There is growing interest in controlling matrix stiffness,[22] a concept that has led to discoveries in fields such as:

Cell culture in three dimensions has been touted as "Biology's New Dimension".[37] Nevertheless, the practice of cell culture remains based on varying combinations of single or multiple cell structures in 2D.[38] That being said, there is an increase in use of 3D cell cultures in research areas including drug discovery, cancer biology, regenerative medicine and basic life science research.[39] There are a variety of platforms used to facilitate the growth of three-dimensional cellular structures such as nanoparticle facilitated magnetic levitation,[40] gel matrices scaffolds, and hanging drop plates.[41]

3D Cell Culturing by Magnetic Levitation method (MLM) is the application of growing 3D tissue by inducing cells treated with magnetic nanoparticle assemblies in spatially varying magnetic fields using neodymium magnetic drivers and promoting cell to cell interactions by levitating the cells up to the air/liquid interface of a standard petri dish. The magnetic nanoparticle assemblies consist of magnetic iron oxide nanoparticles, gold nanoparticles, and the polymer polylysine. 3D cell culturing is scalable, with the capability for culturing 500 cells to millions of cells or from single dish to high-throughput low volume systems.

Cell culture is a fundamental component of tissue culture and tissue engineering, as it establishes the basics of growing and maintaining cells in vitro. The major application of human cell culture is in stem cell industry, where mesenchymal stem cells can be cultured and cryopreserved for future use. Tissue engineering potentially offers dramatic improvements in low cost medical care for hundreds of thousands of patients annually.

Vaccines for polio, measles, mumps, rubella, and chickenpox are currently made in cell cultures. Due to the H5N1 pandemic threat, research into using cell culture for influenza vaccines is being funded by the United States government. Novel ideas in the field include recombinant DNA-based vaccines, such as one made using human adenovirus (a common cold virus) as a vector,[42][43] and novel adjuvants.[44]

Plant cell cultures are typically grown as cell suspension cultures in a liquid medium or as callus cultures on a solid medium. The culturing of undifferentiated plant cells and calli requires the proper balance of the plant growth hormones auxin and cytokinin.

Cells derived from Drosophila melanogaster (most prominently, Schneider 2 cells) can be used for experiments which may be hard to do on live flies or larvae, such as biochemical studies or studies using siRNA. Cell lines derived from the army worm Spodoptera frugiperda, including Sf9 and Sf21, and from the cabbage looper Trichoplusia ni, High Five cells, are commonly used for expression of recombinant proteins using baculovirus.

For bacteria and yeasts, small quantities of cells are usually grown on a solid support that contains nutrients embedded in it, usually a gel such as agar, while large-scale cultures are grown with the cells suspended in a nutrient broth.

The culture of viruses requires the culture of cells of mammalian, plant, fungal or bacterial origin as hosts for the growth and replication of the virus. Whole wild type viruses, recombinant viruses or viral products may be generated in cell types other than their natural hosts under the right conditions. Depending on the species of the virus, infection and viral replication may result in host cell lysis and formation of a viral plaque.

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Cell culture - Wikipedia, the free encyclopedia

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