This new report by Eon Market Research, titled Global Autologous Stem Cell and Non-Stem Cell Based Therapies Market 2020 Research Report, 2015 2025 offers a comprehensive analysis of Autologous Stem Cell and Non-Stem Cell Based Therapies industry at a global as well as regional and country level. Key facts analyzed in this report include the Autologous Stem Cell and Non-Stem Cell Based Therapies market size by players, regions, product types and end industries, history data 2014-2018 and forecast data 2020-2025. This report primarily focuses on the study of the competitive landscape, market drivers and trends, opportunities and challenges, risks and entry barriers, sales channels, distributors in global Autologous Stem Cell and Non-Stem Cell Based Therapies market.

Global Autologous Stem Cell and Non-Stem Cell Based Therapies Market presents market size in terms of volume and value (or whichever applicable) for the entire forecast period and also offers CAGR for the forecast period under consideration.

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Key trends analyzed for the prospective readers of this Autologous Stem Cell and Non-Stem Cell Based Therapies market report include major demand drivers, restraints and key opportunities prevailing in the industry. Certain high-level analysis of Autologous Stem Cell and Non-Stem Cell Based Therapies market such as value chain analysis, Porters five forces analysis, SWOT analysis, and market attractiveness analysis to cover all the circumstances affecting this Autologous Stem Cell and Non-Stem Cell Based Therapies industry is also covered in this report. Portfolio analysis helps to understand the product mix of leading companies in the Autologous Stem Cell and Non-Stem Cell Based Therapies industry.

This report focuses on top manufacturers in the global Autologous Stem Cell and Non-Stem Cell Based Therapies market, with revenue production, sales, gross margin, and market share for each manufacturer, covering

U.S. STEM CELL, INC.Brainstorm Cell TherapeuticsCytoriDendreon CorporationFibrocellLion BiotechnologiesCaladrius BiosciencesOpexa TherapeuticsOrgenesisRegenexxGenzymeAntriaRegeneusMesoblastPluristem Therapeutics IncTigenixMed cell EuropeHolostemMiltenyi Biotec

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Consumption, production, capacity, market share, growth rate, and prices are included for each product type segment of Autologous Stem Cell and Non-Stem Cell Based Therapies market

Embryonic Stem CellResident Cardiac Stem CellsAdult Bone MarrowDerived Stem CellsUmbilical Cord Blood Stem Cells

Consumption, market share and growth rate for each application segment of Autologous Stem Cell and Non-Stem Cell Based Therapies market

Neurodegenerative DisordersAutoimmune DiseasesCancer and TumorsCardiovascular Diseases

The Autologous Stem Cell and Non-Stem Cell Based Therapies market report presents all-inclusive information on raw materials suppliers, equipment suppliers, manufacturing cost, capacity, production, profit margin, capacity utilization rate, etc. The Global Autologous Stem Cell and Non-Stem Cell Based Therapies Market report also covers a systematic geographical analysis.

Key regions analyzed in the global Autologous Stem Cell and Non-Stem Cell Based Therapies market include; North America, Latin America, Europe, Asia Pacific, and Middle East Africa. The country-level analysis included for U.S., UK, France, Germany, Russia, China, Japan, India, and Brazil. The Autologous Stem Cell and Non-Stem Cell Based Therapies industry report provides detailed bifurcation of each segment on global, regional and country level. In a word, the Autologous Stem Cell and Non-Stem Cell Based Therapies market report provides major statistics on the state of the industry and is a valuable source of direction and control for companies and individuals interested in the market.

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Global Autologous Stem Cell and Non-Stem Cell Based Therapies Market Provides An In-Depth Insight Of Sales Analysis-US STEM CELL, INC. - Fashion...

Cardiac Stem Cells Comments Off on Global Autologous Stem Cell and Non-Stem Cell Based Therapies Market Provides An In-Depth Insight Of Sales Analysis-US STEM CELL, INC. – Fashion… | March 8th, 2020

Painless non surgical skin tightening procedures are now available and very popular for people who don't want to go through surgery in order to look better. Call LifeGaines in Boca Raton for more information about "Thread Lifts."

Boca Raton, Florida--(Newsfile Corp. - March 5, 2020) - Thread Lifts are a simple, painless procedure used for skin tightening on the face, neck, or anywhere else on the body. As popular and effective as Botox is, it simply doesn't have the ability to lift the skin. In the past, there hasn't been a great way to produce lifting results without surgery.

Call LifeGaines to inquire about this new method of skin tightening in the South Florida area. Call 561-295-9007.

To view an enhanced version of this image, please visit:https://orders.newsfilecorp.com/files/6848/53186_lifegaines_orig.jpg

Non-surgical aesthetics are in high demand and ThreadLifts, new to the United States, have the ability to produce skin that is instantly lifted and tightened.

This procedure uses no cuts or incisions, only injections. Threads are needles that are pre-loaded with PDO thread. The whole needle is inserted in the tissue at the sub-dermal level, along the surface of the skin and then the needle is pulled out. Threads can be used nearly anywhere on the body, but they are especially effective on the neck and jawline.

What is PDO?

Polydioxanone (PDO) sutures have been used for surgical procedures for many decades. It is one of the safest materials to implant in the body. PDO is completely dissolvable and your skin fully absorbs it within 4 to 6 months, leaving no scar tissue behind. This is especially effective when it's used together with chemical peels, Botox, and fillers to effect a patient's entire facial structure, remove sunspots and other conditions.

It is a great way to rejuvenate and restore youthful contours to brows, cheeks, jowls and the neck area. They are also effective on the breasts, buttocks and upper arms, areas that are prone to sagging due to weight loss, aging, pregnancy and childbirth or poor muscle tone.

Thread Lifts are a great way to rejuvenate and restore youthful contours to brows, cheeks, jowls and the neck area. Results from threads generally last between 12 months to several years depending on the area that has been treated, how many threads are used and what kinds of threads are used in each needle.

Amy Steffey, is a Licensed Nurse Practitioner with LifeGaines Medical and Aesthetics, and with Thread Liftsprocedure, she helps Boca Raton residents regain their confidence with rejuvenating procedures and body contouring.

Amy Steffey works at LifeGaines, which is one of the most highly respected Age Management Medical teams in South Florida. Age Management Medicine pioneer Dr. Richard Gaines is the founder of LifeGaines, and he has years of experience specializing in Hormone Replacement Therapy, Sexual Wellness, Platelet-rich Plasma, Stem Cells, Aesthetics, and Advanced Age Management protocols.

LifeGaines is located at 3785 N Federal Hwy #150, Boca Raton, FL 33431.

Call 561-295-9007 Today to Schedule a Consultation with Amy Steffey at LifeGaines Medical & Aesthetics Center in Boca Raton.

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people-who-dont-want-to-do-surgery.jpg People who don't want to do surgery could consider a non-surgical way of tightening skin. Call LifeGaines to inquire about this new method of skin tightening in the South Florida area. Call 561-295-9007.

To view the source version of this press release, please visit https://www.newsfilecorp.com/release/53186

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Non Surgical Aesthetics Are in High Demand, So Thread Lifts Are a New Way to Achieve Instant Skin Tightening - Yahoo Finance

Skin Stem Cells Comments Off on Non Surgical Aesthetics Are in High Demand, So Thread Lifts Are a New Way to Achieve Instant Skin Tightening – Yahoo Finance | March 6th, 2020

1 Molecular missing link: Research explains why some creams cause a skin rash

Allergic skin reactions can be caused by many different chemical compounds found in skincare creams, cosmetics and other topical consumer products, but how they trigger the reaction has remained somewhat mysteriousuntil now.

New research that suggests the way some chemicals displace natural fat-like molecules known as lipids in skin cells may explain how many common ingredients trigger allergic contact dermatitis.

The breakthrough could help stem soaring cases of rashes, lumps, blisters, itchy eyes and facial swellings. It has been dubbed the molecular missing link because it might have brought a new way to treat the condition.

Currently, the only way to stop allergic contact dermatitis is to identify and avoid coming into contact with the chemical that causes the reaction.

Most allergies are attributed to proteins or synthetically produced peptide antigens that set off the immune system.

A new study from the Hamamatsu University School of Medicine has discovered that docosahexaenoic acid (DHA) could potentially be an important component in creating more efficient lip care products.

The university collaborated with Kose Corporations research laboratories in order to better understand the molecular profile of the lip area.

The study highlighted it was especially important to study the lip in closer detail as it was one of the major targets of cosmetics.

Its important to under the molecular profile specific to human lips to discover the intrinsic ingredients for lip cosmetics.

To gain a better understanding, the team aimed to map out the human lip using imaging mass spectrometry to gather insight into its lipid distribution.

Researchers at Kao Corporation have developed a method to predict odour intensity, a skill previously dependent on the experience of perfumers.

The development of the technology was the result of a project by the firms Sensory Science Research Laboratory, which compiled a database of 314 commonly used fragrance ingredients.

The database of olfaction characteristics allowed scientists to develop a method that can predict the odour intensity based on the concentration of perfumery raw materials (PRMs) present in a gas sample.

Data were obtained from an evaluation testing performed by 18 perfumers and researchers, who scored the intensity of samples emitted from a fragrance diluter with different gas concentrations.

Based on evaluations of those results, the team managed to visualise the relationship between gas concentration and odour intensity.

Singapore biotech start-up Insectta is rearing black soldier flies on its urban farm to produce a more sustainable and purer chitosan for the cosmetics industry.

The company, which claims to have the first insect farm in Singapore, raises black soldier flies that feed on food waste before being converted into viable materials such as chitosan.

It is a useful material that has many functions. For cosmetics, it has antioxidant, antimicrobial and wound-healing properties. Additionally, it can enhance penetration into skin.

Chitosan is conventionally sourced from shrimp and crab. However, the company believes using insects is cleaner and more sustainable.

These flies are not pests, they don't bite. They are native to Singapore and not an invasive species. They feed on food waste which would otherwise go into the incinerator. We're trying to promote a circular economy, said Chua Kai Ning, chief marketing officer of Insectta.

The flies on the farm, which are housed in a small room, can consume around 7.5 tonnes of food waste a month.

Experts in Australia have been urging the public to carry on using sunscreen even though a widely publicised study has shown chemicals can be absorbed from some products into the bloodstream.

US Food and Drug Administration scientists found that sunscreen users might be taking in more of the active ingredients into our blood, far beyond regulatory thresholds.

They tested six of the main active ingredients in sunscreen lotions, sprays and pumps, revealing quantities of sunscreen chemicals in the blood high enough for the products to have to undergo additional FDA safety studies.

This happens when formulations surpass a threshold that requires them to be taken for further testing. The chemicals studied were avobenzone, oxybenzone, octocrylene, ecamsule, homosalate, octisalate and octinoxate.

The researchers stress the findings do not mean that sunscreens are unsafe, merely that more research is needed. The FDA will now conduct more research to determine the maximum levels of sunscreen ingredients that are safe to use.

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Skin science: Top five stories on cosmetics science and formulation - CosmeticsDesign-Asia.com

Skin Stem Cells Comments Off on Skin science: Top five stories on cosmetics science and formulation – CosmeticsDesign-Asia.com | March 6th, 2020

Buyers looking to own a private, well-stocked fishing pond or enjoy the simplicity of a quiet paddle across 18 acres of tranquil water will find all of that and a 5,619-square-foot custom contemporary home at 355 Pond St. in Uxbridge.

Set back from the road on more than 56 scenic acres, the 10-room home, on the market for $1.1 million, offers unique upgrades such as cedar paneled ceilings and 14-karate-gold plated fixtures in the master bath.

The house also features a 2,400-square-foot, wraparound deck that overlooks Lee Pond.

Owner Dr. Charles A. Vacanti said his favorite feature of the property is its pond, which is stocked upstream along Emerson Brook by two fishing clubs.

Both of their ponds drain into our 18-acre pond, he said. It may be the best fishing hole in New England.

Listing agent Gary Smith of Mendon Area Real Estate called the pond and its surrounding property beautiful, while his favorite feature in the home is the spectacular family room.

The sunken combination family and dining room share a wood burning stove with a two-story stone surround. The cathedral ceilings are paneled in cedar and hold skylights and fans. Light from two panels of floor to ceiling glass brightens this room that gives access to the deck.

The modern, eat-in kitchen holds a center island, custom-built cabinetry, granite countertops and large pantry.

The upstairs hallway is an open loft overlooking the family/dining room.

The master suite offers scenic views through a wall of glass with a door that opens to a balcony. The master bath has marble flooring, a cathedral ceiling with skylights, custom built closet and cabinets, a spa hot tub, and large stall shower.

Also upstairs are an office and gym space.

Dr. Vacanti and his wife, Linda, bought the property in 1998.

Since then, the couple has replaced all 27 skylights, all external doors and windows, and the roof. The installation of solar panels, Dr. Vacanti said, has reduced annual utility costs to nearly zero.

Dr. Vacanti is widely known for his research work in stem cell and tissue engineering. While at the University of Massachusetts Medical School in 1996, he grew new cartilage in the shape of a human ear under the skin of a lab mouse. The Vacanti Mouse was part of research on organ generation and the first demonstration of growing new tissue from cells in a lab.

As for the property, Smith called it a beautiful high and dry pasture that just begs for equestrian use, a corporate retreat, Bed and Breakfast, or Rod and Gun Club.

The property is minutes from Route 146.

Original post:
House of the Week: Uxbridge property is a sportsmans paradise - Worcester Telegram

Skin Stem Cells Comments Off on House of the Week: Uxbridge property is a sportsmans paradise – Worcester Telegram | March 6th, 2020

Technology Networksrecently had the pleasureof speaking with Bruce Steel, CEOEquillium, Inc. to learn more about how they are leveraging their comprehensive understanding of immunobiology to develop novel treatments for inflammatory and autoimmune disorders.Bruce provides insight on the various indications the company is currently developing treatments for, elaborates on the clinical progress of their initial product candidate EQ001 (itolizumab), and explains the role CD6 plays in autoimmunity and how it can be targeted therapeutically.

Laura Lansdowne (LL): How can immunobiology be harnessed to develop therapeutics?Bruce Steel (BS): The role of the immune system is to defend the body against foreign organisms and cells, including cancerous cells, and in doing so, it must distinguish accurately between self- and non-self-entities a process called tolerance. Autoimmunity is an immune response directed against the bodys own healthy cells and tissues and is the underlying process in many inflammatory diseases. Autoimmunity results from a loss of tolerance caused in part by an imbalance in the relationship between effector T cells and regulatory T cells. Therefore, developing therapeutics, such as EQ001 (itolizumab), that target these critical regulators of immune activation pathways has the potential dramatically improve the lives of patients with severe autoimmune and inflammatory disorders.LL: What indications are you currently developing treatments for?BS: We select target indications based on three primary criteria: strong scientific rationale for why itolizumab has potential to be a best-in-class therapeutic approach, areas of high unmet medical need where there are little or no treatments available today, and indications where we believe there is an attractive future commercial opportunity. Today we have ongoing clinical trials with itolizumab in acute graft-versus-host disease (GVHD), uncontrolled asthma and lupus/lupus nephritis.

While these three indications are our initial areas of focus, we believe itolizumab has the potential to be developed in other areas such as transplant science, systemic autoimmunity, pulmonary, neurologic, gastrointestinal, renal, vascular, ophthalmic and dermatologic disorders.We licensed itolizumab from our partner Biocon Limited who developed the drug and received regulatory approval in India for the treatment of psoriasis.LL: Can you tell us more about the mechanism of your drug candidate EQ001 (itolizumab)?BS: Itolizumab is a clinical-stage, first-in-class monoclonal antibody that selectively targets the CD6-ALCAM pathway, which plays a central role in modulating the activity and trafficking of effector T cells that drive a number of immune-inflammatory diseases.LL: What role does CD6 play in autoimmunity?BS: CD6 is a novel co-stimulatory receptor that uniquely modulates T cell activity and trafficking. It is a key checkpoint in regulating effector T cells that are central to autoimmune responses. CD6 binds activated leukocyte cell adhesion molecule (ALCAM). ALCAM is expressed on both antigen-presenting cells and tissue including the skin, gut, lung and kidney.In preclinical studies, blockade of CD6 with itolizumab leads to reduction in effector T cell activation and proliferation. Additionally, inhibiting the binding of ALCAM to CD6 with itolizumab modulates lymphocyte trafficking and reduces effector T cell infiltration into inflamed tissues.

Our work with CD6 builds upon the research conducted from researchers at the Dana-Farber Cancer Institute, our partner Biocon, and other leading academic centers. Today there are numerous peer reviewed publications related to the novel CD6 checkpoint receptor and targeting the CD6-ALCAM pathway.LL: Can you elaborate on the clinical progress of itolizumab?BS: Itolizumab is currently being studied in three different indications: acute graft-versus-host disease (aGVHD), uncontrolled moderate to severe asthma and lupus/lupus nephritis. This is an important catalyst year for Equillium as we expect initial data from all three programs in 2H 2020.

Each of these studies will allow us to understand the safety of itolizumab in these different disease areas and understand what potential dose we will carry forward. This is important as we consider making a larger investment in Phase II studies to advance the program.Bruce Steel was speaking with Laura Elizabeth Lansdowne, Senior Science Writer for Technology Networks.

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Exploiting Immunobiology To Treat Severe Autoimmune and Inflammatory Disorders - Technology Networks

Skin Stem Cells Comments Off on Exploiting Immunobiology To Treat Severe Autoimmune and Inflammatory Disorders – Technology Networks | March 6th, 2020

Avoid close contact with sick patients. Stay home if youre feeling unwell. Scrub your hands with soap and water for at least 20 seconds and for goodness sake, stop touching your face.

By now, youve probably heard or seen the advice from the Centers for Disease Control and Prevention (CDC) for staving off COVID-19, the viral epidemic ricocheting across the globe. Most cases of the disease are mild, triggering cold-like symptoms including fever, fatigue, dry cough and shortness of breath. The death rate appears to be lowabout two or three percent, perhaps much less. But the virus responsible, called SARS-CoV-2, is a fearsomely fast spreader, hopping from person to person through the droplets produced by sneezes and coughs. Since COVID-19 was first detected in Chinas Hubei province in December 2019, nearly 100,000 confirmed cases have been reported worldwide, with many more to come.

To curb the virus spread, experts stress the importance of hand hygiene: keeping your hands clean by regularly lathering up with soap and water, or, as a solid second choice, thoroughly rubbing them down with an alcohol-based sanitizer. That might sound like simple, even inconsequential advice. But such commonplace practices can be surprisingly powerful weapons in the war against infectious disease.

[Washing your hands] is one of the most important ways to interrupt transmission of viruses or other pathogens, says Sallie Permar, a physician and infectious disease researcher at Duke University. It can have a major impact on an outbreak.

In the strictest sense of the word, viruses arent technically alive. Unlike most other microbes, which can grow and reproduce on their own, viruses must invade a host such as a human cell to manufacture more of themselves. Without a living organism to hijack, viruses cant cause illness. Yet viral particles are hardy enough to remain active for a while outside of the host, with some staying infectious for hours, days or weeks. For this reason, viruses can easily spread unnoticed, especially when infected individuals dont always exhibit symptomsas appears to be the case with COVID-19.

Researchers are still nailing down the details of exactly how SARS-CoV-2 is transmitted and how resilient it is outside the body. Because the virus seems to hang out in mucus and other airway fluids, it almost certainly spreads when infected individuals cough or sneeze. Released into the air, infectious droplets can land on another person or a frequently touched surface like a doorknob, shopping cart or subway seat. The virus can also transfer through handshakes after someone carrying the virus sneezes or coughs into their hand.

After that, its a short trip for the virus from hand to head. Researchers estimate that, on average, humans touch their faces upwards of 20 times an hour, with about 44 percent of these encounters involving eyes, mouths and nosessome of the quickest entry points into the bodys interior.

Breaking this chain of transmission can help stem the spread of disease, says Chidiebere Akusobi, an infectious disease researcher at Harvards School of Public Health. Sneezing or coughing into your elbow can keep mucus off your mitts; noticing when your hand drifts towards your face can help you reduce the habit.

All this public-health-minded advice boils down to a game of keep away. To actually infect a person, viruses must first get inside the body, where they can infect living cellsso if one lands on your hands, the best next move is to remove or destroy it.

The most important step to curbing infection may be hand-washing, especially before eating food, after using the bathroom and after caring for someone with symptoms. Its simply the best method to limit transmission, says Kellie Jurado, a virologist at the University of Pennsylvanias Perelman School of Medicine. You can prevent yourself from being infected as well as transmitting to others.

According to the CDC, you should wet your handsfront and backwith clean, running water; lather up with soap, paying mind to the easily-forgotten spaces between your fingers and beneath your nails; scrub for at least 20 seconds; then rinse and dry. (Pro tip: If counting bores you or youre sick of the birthday song, try the chorus of these popular songs to keep track.)

Done properly, this process accomplishes several virus-taming tasks. First, the potent trifecta of lathering, scrubbing and rinsing physically removes pathogens from your skin, says Shirlee Wohl, a virologist and epidemiologist at Johns Hopkins University.

In many ways, soap molecules are ideal for the task at hand. Soap can incapacitate SARS-CoV-2 and other viruses that have an outer coating called an envelope, which helps the pathogens latch onto and invade new cells. Viral envelopes and soap molecules both contain fatty substances that tend to interact with each other when placed in close proximity, breaking up the envelopes and incapacitating the pathogen. Basically, the viruses become unable to infect a human cell, Permar says.

Alcohol-based hand sanitizers also target these vulnerable viral envelopes, but in a slightly different way. While soap physically dismantles the envelope using brute force, alcohol changes the envelopes chemical properties, making it less stable and more permeable to the outside world, says Benhur Lee, a microbiologist at the Icahn School of Medicine at Mount Sinai. (Note that alcohol here means a chemical like ethanol or isopropyl alcoholnot a beverage like vodka, which contains only some ethanol.)

Alcohol also can penetrate deep into the pathogens interior, wreaking havoc on proteins throughout the virus. (Importantly, not all viruses come with outer envelopes. Those that dont, like the viruses that cause HPV and polio, wont be susceptible to soap, and to some extent alcohol, in the same way.)

Hand sanitizers made without alcohollike some marketed as baby-safe or naturalwont have the same effect. The CDC recommends searching for a product with at least 60 percent alcohol contentthe minimum concentration found to be effective in past studies. (Some water is necessary to unravel the pathogens proteins, so 100 percent alcohol isnt a good option.)

As with hand-washing, timing matters with sanitizers. After squirting a dollop onto your palm, rub it all over your hands, front and back, until theyre completely drywithout wiping them off on a towel, which could keep the sanitizer from finishing its job, Jurado says.,

But hand sanitizers come with drawbacks. For most people, using these products is less intuitive than hand-washing, and the CDC notes that many people dont follow the instructions for proper application. Hand sanitizers also dont jettison microbes off skin like soap, which is formulated to lift oily schmutz off surfaces, Akusobi says.

Soap emulsifies things like dirt really well, he says. When you have a dirty plate, you dont want to use alcoholthat would help sterilize it, but not clean it.

Similarly, anytime the grit is visible on your hands, dont grab the hand sanitizer; only a full 20 seconds (or more) of scrubbing with soapy water will do. All told, hand sanitizer should not be considered a replacement for soap and water, Lee says. If I have access to soap and water, I will use it.

Technically, it is possible to overdo it with both hand-washing and hand sanitizing, Akusobi says. If your skin is chronically dry and cracking, thats no good. You could be exposing yourself to other infections, he says. But it would take a lot to get to that point.

In recent weeks, hand sanitizers have been flying off the shelves, leading to shortages and even prompting some retailers to ration their supplies. Some people have begun brewing up hand sanitizers at home based on online recipes.

Many caution against this DIY approach, as the end products cant be quality controlled for effectiveness, uniformity or safety, says Eric Rubin, an infectious disease researcher at Harvards School of Public Health. On average, one would imagine that [a homemade sanitizer] would not work as well, so it would be a mistake to rely on it, he says.

As more information on SARS-CoV-2 and COVID-19 emerges, experts stress the importance of awareness. Even as the news changes and evolves, peoples vigilance shouldnt.

Do the small things you need to do to physically and mentally prepare for whats next, Wohl says. But dont panic. That never helps anybody.

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Why Is Washing Your Hands So Important, Anyway? - Smithsonian

Skin Stem Cells Comments Off on Why Is Washing Your Hands So Important, Anyway? – Smithsonian | March 6th, 2020

A new population of stem cells that can generate bone has been revealed by researchers, which they say could have implications in regenerative medicine.

A population of stem cells with the ability to generate new bone has been newly discovered by a group of researchers at the University of Connecticut (UConn) School of Dental Medicine, US.

The researchers present a new population of cells that reside along the vascular channels that stretch across the bone and connect the inner and outer parts of the bone.

This is a new discovery of perivascular cells residing within the bone itself that can generate new bone forming cells, said lead investigator Dr Ivo Kalajzic. These cells likely regulate bone formation or participate in bone mass maintenance and repair.

Stem cells for bone have long been thought to be present within bone marrow and the outer surface of bone, serving as reserve cells that constantly generate new bone or participate in bone repair. Recent studies have described the existence of a network of vascular channels that helped distribute blood cells out of the bone marrow, but no research has proved the existence of cells within these channels that have the ability to form new bones.

In this study, Kalajzic and his team are the first to report the existence of these progenitor cells within cortical bone that can generate new bone-forming cells osteoblasts that can be used to help remodel a bone.

To reach this conclusion, the researchers observed the stem cells within an ex vivo bone transplantation model. These cells migrated out of the transplant and began to reconstruct the marrow cavity and form new bone.

While this study shows there is a population of cells that can help aid formation, more research needs to be done to determine the cells potential to regulate bone formation and resorption, say the scientists.

According to the authors of the study: we have identified and characterised a novel stromal lineagerestricted osteoprogenitor that is associated with transcortical vessels of long bones. Functionally, we have demonstrated that this population can migrate out of cortical bone channels, expand and differentiate into osteoblasts, therefore serving as a source of progenitors contributing to new bone formation.

The results are published inSTEM CELLS.

Original post:
Stem cells that can grow new bone discovered by researchers - Drug Target Review

Bone Marrow Stem Cells Comments Off on Stem cells that can grow new bone discovered by researchers – Drug Target Review | March 6th, 2020

Stem cell research has been the subject of discussion and heated debate for many years. Much of the social and political drama surrounding stem cells is the result of misunderstanding what stem cells are, where they come from, and what they can do for those with injuries and diseases.

Working from a common set of facts is a great way to dispel controversy, however. Whether we fall into the pro-choice or pro-life camp, it is more than evident that supporting stem cell research, including the development of stem cell therapies, is very much a pro-life position to take.

Stem cells function essentially like raw materials for the body. Depending on instructions from the body (or researchers in laboratories), stem cells can become many other types of cells with specialized functions.

The daughters of stem cells either become new stem cells (self-renewal) or they become more specialized cells for use in specific areas of the body (differentiation). These specialized cells include brain cells, heart muscle cells, bone cells, blood cells and others.

There are several reasons why stem cells are the focus of some of the most important medical science research today:

This last avenue of medical research stem cell therapies is the most consequential as well as the most controversial, depending on your point of view. Understanding stem cell therapy and its divisiveness requires understanding where stem cells come from in medical research and why they have considerable palliative potential.

Stem cells come from one of these three sources:

Embryonic stem cells are the most controversial as well as the most important type of stem cells right now. Thanks to a low-information electorate and gross misinformation from within the government, embryonic stem cells remain mired in needless debate.

Despite the rhetoric, these cells arent harvested from slain newborns. Instead, they are carefully gathered from blastocysts. Blastocysts are three-to-five-day-old embryos comprised of around 150 cells. According to some religious-political arguments, blastocysts are potential human beings, and therefore deserve legal protection.

Embryonic stem cells are the most valuable in medical research because they are fully pluripotent, which means they are versatile enough to become any type of cell the body requires to heal or repair itself.

Adults have limited numbers of stem cells in a variety of bodily tissues, including fat and bone marrow. Unlike pluripotent embryonic stem cells, adult stem cells have more limits on the types of cells they can become.

However, medical researchers keep uncovering evidence that adult stem cells may be more pliable than they originally believed. There is reason to believe cells from adult bone marrow may eventually help patients overcome heart disease and neurological problems. However, adult stem cells are more likely than embryonic stem cells to show abnormalities and environment-induced damage, including cell replication errors and toxins.

The newest efforts in stem cell research involve using genetic manipulation to turn adult stem cells into more versatile embryonic variants. This could help side-step the thorny abortion controversy, but its also not clear at present whether these altered stem cells may bring unforeseen side-effects when used in humans.

More research is required to fully understand the medical potential of perinatal stem cells. However, some scientists believe they may in time become a viable replacement for other types of stem cells. Perinatal stem cells come from amniotic fluid and umbilical cord blood.

Using a standard amniocentesis, doctors can extract umbilical cord mesenchymal stem cells, hematopoietic stem cells, amniotic membrane and fluid stem cells, amniotic epithelial cells and others.

Among other things, stem cell therapy is the next step forward for organ transplants. Instead of waiting on a transplant waiting list, patients may soon be able to have new organs grown from their very own stem cells.

Bone marrow transplants are one of the best-known examples of stem cell therapy. This is where doctors take bone marrow cells and induce them to become heart muscle cells.

Stem cell-based therapies hold significant promise across a wide range of medical conditions and diseases. With the right approach, stem cells show the potential to:

As the FDA notes, there is a lot of hype surrounding stem cell therapy. Much of it is warranted, but some of it deserves caution.

According to the FDA, stem cells have the potential to treat diseases or conditions for which few treatments exist. The FDA has a thorough investigational process for new stem cell-based treatments. This includes Investigational New Drug Applications (IND) and conducting animal testing.

However, the FDA notes that not every medical entity submits an IND when they bring a new stem cell therapy to market. It is vital that patients seek out only FDA-reviewed stem cell therapies and learn all they can about the potential risks, which include reactions at the administration site and even the growth of tumors.

The FDA submitted a paper, Clarifying Stem-Cell Therapys Benefits and Risks, to the New England Journal of Medicine in 2017. Its goal is to help patients fully understand what theyre getting themselves into.

For now, a great deal more research is required before we begin deploying stem cell therapies on a larger scale. The only FDA-approved stem cell therapies on the market today involve treating cancer in bone marrow and blood. Some clinics claim their therapy delivers miracle-like cures for everything from sports injuries to muscular dystrophy, but there just isnt enough evidence yet to take them at face value.

Unfortunately, the religious and political climate makes this evidence difficult to achieve. In some parts of the United States, the hostility toward stem cell researchers and medical practitioners has reached dangerous new levels.

Republicans in Ohio and Georgia want to make it illegal for doctors to perform routine procedures on ectopic pregnancies. This condition is life-threatening for the mother and involves the removal of a nonviable embryo from the fallopian tube.

These laws wouldnt just outlaw ectopic pregnancy surgery in the name of potential human life. It would, in fact, require women to undergo a reimplantation procedure after the ectopic pregnancy is corrected by a physician. If this procedure was actually medically possible, it would be dangerous and unnecessary. Thankfully, it doesnt exist outside the nightmarish imaginations of some of the more extreme Christian lawmakers and Planned Parenthood demonstrators.

Acquiring embryonic stem cells from ectopic pregnancies would seem to be the least controversial way to go about it. Unfortunately, even that small step toward medical progress sees itself hampered by reactionary politics.

No matter how theyre acquired, however, the 150 or so cells in blastocysts are packed with medical potential. Its clear that further exploration down this road will unlock unprecedented scientific progress. It will also, almost certainly, save many times more potential life than even the most outlandish estimates of what the achievement will cost us to achieve. Abortions today are rarer and safer than ever, and the vast majority occur within eight weeks of conception.

The medical community is poised for a revolution here, using these and other nonviable embryos and blastocysts. But realizing that potential requires, among other things, that we collectively make peace with modern medicine and family planning.

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Despite Pro-Life Claims, Stem Cell Therapy Has Very Real Benefits and Should Be Accessible - Patheos

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Researchers from UConn School of Dental Medicine have recently discovered a group of stem cells that help in generating a new bone. In regards with this, Dr Ivo Kalajzic, professor of reconstructive sciences, stated that, this newly discovered perivascular stem cells that reside in the bone itself have capability of generating the bone and these cells are highly instrumental in repair & mass maintenance of the bone along with its formation.

Since ages, it has been thought that stem cells only reside in bone marrow and exterior surface of the bone stores the cells that continuously generate new bone or repair the bone. Postdoctoral individuals Dr Sierra Root and Dr Natalie Wee, and collaborators at Harvard, Maine Medical Research Center, and the University of Auckland also were part of this study along with Dr Ivo Kalajzic and confirmed that these new cluster of cells residing in the vascular channels that range across the bone and serve as connection between inner and outer part of the bone is capable of generating a new bone.

This team is also pioneer in bringing forward a study that says existence of these progenitor cells inside cortical bone not only generates a new bone but also help remodeling of the bone. The conclusion was made after these researchers observed that these stem cells within an ex vivo bone transportation model migrated out of the transplant and started manufacturing a new bone marrow cavity along with completely new bone.

In order to establish this, more research needs to done as it will definitely turn out wonderful to the field of medical science and mankind.

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Stem Cells that will aid new bone generation discovered as per latest research - Medical Herald

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Two-year-old Regann Moore lights up as she watches videos on her iPad at home on Thursday, Feb. 20, 2020. Moore has a rare disease known as Krabbe Disease and received a life-saving stem cell donation less than a month after being born.(Photo: Nathan Papes/Springfield News-Leader)

Soon after the News-Leader published a story about 2-year-old Regann Moore,a Springfield child whose life was saved thanks to a newborn screening test, someone tweeted the story toMissouri State Rep. Becky Ruth.

"I bawled my eyes out," Ruth said. "I just cried."

She cried because she knew Regann is alive thanks to the death of Ruth's grandson, Brady.

"I cry and smile when I see these children," Ruth said. "We are always so thankful. For us, we see Brady's death wasn't in vain. His legacy lives on by helping save the lives of other children."

More: Springfield child with rare, deadly disease continues to amaze doctors, family

Regann, who is 2 now, was diagnosed right after she was born withKrabbe Disease, a rare metabolic disorder that must be diagnosed at birth and treated as soon as possible with a stem cell donation.

The newborn screening is important because babies with Krabbe Disease appear healthy at birth. Signs something is wrong usually don't appear until it's too late for treatment to be effective.

That is what happened to Brady in 2009. He wasn't diagnosed with the disease until he was 4.5 months old too late for treatment.

Brady died 10 days before his first birthday.

Brady Cunningham died of Krabbe Disease just before his first birthday.(Photo: Courtesy of the Cunningham family)

That's why Ruth and her family fought to get lawmakers on board with making sure all newborns in Missouri are screened for Krabbe Disease.

TheBrady Alan Cunningham Newborn Screening Act was passed in 2009 and screening began in 2012. Ruthsaid her family was OK with the three-year lag because they realized the lab needed time to become equipped to test for the disease.

Missouri is one of just a few states that do the newborn screening.

Brady's law also includes screening for Pompe, Fabry, Gauche and Niemann-Pick diseases. Since then, SCID, MPS I, MPS II and SMA diseases are screened, as well.

Ruth became a state representative in 2015and said newborn screening is her passion.

Her experience with getting Brady's law passed is what led her to seek office.

"It showed me what just a regular everyday person can do and what a differenceyou can make," Ruth said. "People a lot of times complain about politicians and the legislature, but we also do very good things here."

Ruth said her family knows of another child with Krabbe Disease who was saved thanks to newborn screening and a stem cell transplant.

That child is now 4. Ruth said her family and that child's family have a "strong connection."Ruth said shehopes to someday meet Regann's family.

Brady Cunningham was born in 2008. His family is from Campbell in southeast Missouri.

Bradyappeared healthy at birth and was not tested for Krabbe Disease.

Ruth said he started having health problems after about a month and a half. Brady went through "a myriad of diagnoses," Ruth recalled, including acid reflux and seizures.

"Finally my daughter took him to Children's Hospital in St. Louis," she said. "They promised her he wouldn't leave without a diagnosis."

Missouri State Rep. Becky Ruth was moved to tears after reading about Regann Moore, a Springfield child whose life was saved thanks to newborn screening for Krabbe Disease. Ruth and her family encouraged Missouri lawmakers to make sure all Missouri babies are tested for the deadly disease after her grandson, Brady, died from it.(Photo: Submitted by Becky Ruth)

Three weeks later, Brady was diagnosed with Krabbe Disease, which rapidly destroys the nervous system.

"We were told there was nothing they could do," she said. "It was one of the worst days of all of our lives."

Brady was 4.5 months old when he was diagnosed. In order for a stem cell donation to have any chance of being effective, babies must have the transplant within the first month of their life.

Regann, the Springfield child, was given a stem cell donation thanks to an umbilical cord donation.

Thediseaseaffects about one in every 100,000 people in the United States.

"They are missing an enzyme that helps keep their nervous system intact," said Dr. Shalini Shenoy, Regann's transplant doctor. "Because this is missing, they have degeneration of the brain and nervous system. And if you let it progress, it is fatal very early."

Without the stem cell donation, babies die within the first few months, Shenoy said.

"You can't change someone's genetic makeup," Shenoy said. "But when you put stem cells into their bone marrow from somebody else who is normal, some of these cells migrate into their brain and into their nervous system and supply what they are lacking themselves."

It takes some time for the transplant to begin working for the transplanted cells to "settle down" and begin making the missing enzyme, Shenoy said.

"Because of that, the earlier you transplant a Krabbe patient, the more you will be able to rescue them," she said. "You want to catch them before too much damage is done. Once there's a lot of nerve damage, it's not reversible. If I saw a Krabbe patient two months after they were born or four months after they were born when they already had major problems, it's unlikely I'd be able to rescue them too much."

Since the screening and the stem cell transplant treatment are both relatively recent medical advancements, Shenoy said it's anybody's guess what the future will hold for children who, like Regann, were successfully treated with a stem cell transplant early on.

Ferrell Moore holds his two-year-old daughter Regann Moore at their home on Thursday, Feb. 20, 2020. Regann has a rare disease known as Krabbe Disease and received a life-saving stem cell donation less than a month after being born.(Photo: Nathan Papes/Springfield News-Leader)

Regann can't stand on her own or walk yet. But her family is determined to make that happen. She cannot talk but is learning sign language to communicate.

She has regular visits with speech and occupational therapists.

Regann's dad Ferrell Moore got to take her to the circus recently, something the little girl seemed to enjoy.

"She is the joy of my life," Ferrell Moore said. "When I come home, it couldn't be any better to see her and how happy she is to see me."

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'His legacy lives on': Grandmother who helped create newborn screening law tells history of bill - News-Leader

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INTRODUCTION

In recent years, organoids have emerged as powerful tools for basic research, drug screening, and tissue engineering. The organoids formed in vitro show many features of the structural organization and the functional hallmarks of adult or embryonic anatomical structures (1). In addition, the formation of organoids alleviates the need to perform animal studies and provides an attractive platform for robust quantitative studies on the mechanisms regulating organ homeostasis and tissue repair in vivo (1). The formation of organoids usually starts with populations of stem cells. They are therefore expected to be heterogeneous because pluripotent stem cells [induced pluripotent stem cells (pscs) or embryonic stem cells] have been shown to dynamically and stochastically fluctuate from ground to differentiated state (2). In the same vein, LGR5+ intestinal stem cells are reported to contain several distinct populations (3). As such, the formation of organoids involves the inherent capacity of these heterogeneous populations to self-sort and self-pattern to form an organized three-dimensional (3D) architecture (4). However, the rules underlying organoid formation as well as the contribution of intrinsic population heterogeneity to the organoid self-assembly remain poorly understood (5). Consequently, there is a need for novel quantitative approaches at the single-cell level to reliably understand the mechanisms of spatial tissue patterning in 3D organoids, for which microfluidic and quantitative image analysis methods are well suited.

In this work, we use mesenchymal progenitors, alternatively named mesenchymal stromal cells (MSCs), which constitute a self-renewing population with the ability to differentiate into adipocytes, chondrocytes, and osteoblasts (5). Although human MSCs (HMSCs) express high levels of undifferentiation markers (e.g., CD105, CD44, CD73), they constitute a heterogeneous population of cells that exhibit considerable variation in their biophysical properties and epigenetic status, as well as the basal level of expression of genes related to differentiation, immunoregulation, and angiogenesis (6, 7). Nonetheless, their aggregation leads to the formation of highly cohesive 3D spherical structures [which we designate hereafter as mesenchymal bodies (MBs)] with improved biological activities in comparison to 2D cultures (8). However, little is known on how HMSCs self-organize or whether the intrinsic heterogeneity of the population regulates MB formation and individual cell functions in 3D.

The self-aggregation of HMSCs into MBs can recapitulate the early stages of mesenchymal condensation, and it promotes the secretion of paracrine molecules taking part in the process of ossification (9). During mesenchymal condensation in vivo, mesenchymal progenitors self-aggregate and form dense cell-cell contacts that lead to the initiation of bone organogenesis through endochondral (necessitating a chondrogenic intermediate) and intramembranous (direct osteogenic differentiation) ossification (10). In addition, the formation of these 3D MBs in vivo is associated with the secretion of important paracrine molecules such as prostaglandin E2 (PGE2) and vascular endothelial growth factor (VEGF), which participate in the recruitment of endogenous osteoblasts, osteoclasts, and blood vessels, leading to the initiation/restoration of bone homeostasis (11, 12). In these two ossification processes, the induction of nuclear factor B (NF-B) target genes, such as cyclooxygenase-2 (COX-2), and their downstream products (e.g., PGE2 and VEGF) plays a critical role as developmental regulators of ossification and bone healing (13). However, while mesenchymal condensation is critical for bone organogenesis, there is still a limited understanding on how the cellular spatial organization within 3D MBs regulates the individual cells endocrine functions (14).

In the present work, we interrogate the influence of phenotypic heterogeneity within a population of stem cells on the mechanisms of self-assembly and functional patterning within 3D organoids using HMSCs as a model of heterogeneous progenitor cell population. This is performed using a novel microfluidic platform for high-density formation of mensenchymal bodies, combined with the analysis of individual cells by quantitative image analysis. Our study reveals that the progenitor cell population self-assembles in a developmentally hierarchical manner. We also find that the structural arrangement in mensenchymal bodies is linked with the functional patterning in 3D, through a modulation of the activity of regulatory molecular signaling at a local scale. This study demonstrates the interplay between cell size and differentiation status, which mediates cellular spatial rearrangement in 3D, leading to the regionalized activation of unique biological functions while forming aggregates.

HMSCs are known to constitute a heterogeneous population (6, 7). In this study, fetal HMSCs were derived from the Whartons jelly of the umbilical cord (UC). UC-derived HMSCs are considered to be more primitive than HMSCs derived from adult bone marrow because of their higher proliferative capacity, their ability to form colony-forming unitfibroblast, as well as their lower degree of basal commitment (15). To examine the cellular diversity within the population, HMSCs were first characterized by their expression of membrane markers. Most of the HMSC population consistently expresses CD73, CD90, CD105, and CD146, but not CD31 (an endothelial cell marker), CD34 (a hematopoietic cell marker), CD14 (an immune cell marker), or human leukocyte antigenDR (HLA-DR) (a type of major histocompatibility complex II) (Fig. 1, A to F, and fig. S1, A to C). However, a deeper analysis of the flow cytometric data shows that the HMSC population contains cells of heterogeneous size [coefficient of variation (CV) = 33 to 37%] (Fig. 1, G and I), having a broad distribution in the expression of CD146 (Fig. 1F). Of note, the CD146 level of expression was linked to the size of the cells: The highest levels of CD146 were found for the largest cells (Fig. 1, H and J). Similar correlations with cell size were also observed for CD73, CD90, and CD105 (fig. S1, D to F). In addition, upon specific induction, the HMSC population used in this study successfully adopted an adipogenic (Fig. 1K), an osteogenic (Fig. 1L), or a chondrogenic (Fig. 1M) phenotype, demonstrating their mesenchymal progenitor identity.

(A) Percentage of positive cells for CD31, CD73, CD90, CD105, and CD146 (n = 3). Representative histograms of the distribution of the CD31 (B), CD73 (C), CD90 (D), CD105 (E), and CD146 (F) level of expression are shown. (G) Representative histogram of the forward scatter (FSC) distribution. (H) Correlation between cell size [FSC and side scatter (SSC)] and the level of CD146 expression. (I) Representative histogram of the cell projected area distribution. (J) Representative histogram of the size distribution of the CD146dim, CD146int, and CD146bright (ImageSteam analysis). (K) Representative images of hMSCs differentiated toward adipogenic lineage (Oil Red O staining). (L) Representative images of UC-hMSCs differentiated toward osteogenic lineage in (Alizarin Red S staining). (M) Representative images of UC-hMSCs differentiated toward chondrogenic lineage (Alcian Blue staining in 2D and cryosectioned micromass cultures). Scale bars, 50 m. The images were acquired using a binocular. FITC-A, fluorescein isothiocyanateA; APC-A, allophycocyanin-A.

To interrogate contribution of cellular heterogeneity (i.e., in terms of size and levels of CD marker expression) in the self-organization of HMSCs in 3D, MBs were formed at high density on an integrated microfluidic chip. This was done by encapsulating cells into microfluidic droplets at a density of 380 cells per droplet, with a CV of 24% (fig. S2, A and B). The drops were then immobilized in 250 capillary anchors in a culture chamber, as previously described (Fig. 2, A and B) (16). The loading time for the microfluidic device was about 5 min, after which the typical time for complete formation of MBs was about 4 hours (movie S1), as obtained by measuring the time evolution of the projected area (Fig. 2, C and D) and circularity of individual MBs (Fig. 2E and movie S2). The protocol resulted in the formation of a single MB per anchor (fig. S2C) with an average diameter of 158 m (Fig. 2F), when starting with a seeding concentration of 6 106 cells ml1. The diameter of the aggregates can easily be tuned by modulating the concentration of cells in the seeding solution (fig. S2, A and B). In addition, the complete protocol yielded the reproducible formation of a high-density array of fully viable MBs ready for long-term culture (for the images of the individual fluorescent channel, see Fig. 2G and fig. S2D), as described previously (16). Of interest, the CV of the MB diameter distribution was lower than the CV of the individual cell size and of the cell number in droplets (CV MB diameter = 13.3%, CV cell number per drop = 24%, and CV cell size = 35%), which demonstrates that the production of MBs leads to more homogeneous size conditions, compared with the broad heterogeneity in the cell population.

(A) Chip design. Scale bar, 1 cm. (B) Schematized side view of an anchor through the MB formation and culture protocol. (C) Representative time lapse of an MB formation. Scale bar, 100 m. (D and E) Measurement of the time evolution of the projected area (D) and circularity of each aggregate (E). n = 120 MBs. (F) Distribution of the MB diameter normalized by the mean of each chip (n = 10,072 MBs). (G) Top: Representative images of MBs after agarose gelation and oil-to-medium phase change. Bottom: The same MBs are stained with LIVE/DEAD. Scale bar, 100 m. (H) Representative images of MBs formed in the presence of EDTA, an N-cadherin, or a CD146-conjugated blocking antibody (Ab) (the red color shows the position of the CD146 brightest cells, and the dilution of the antibody was 1/100 and remain in the droplet for the whole experiment). Scale bar, 100 m. The images were acquired using a wide-field microscope.

To gain insight into the cellular components required to initiate the self-organization of HMSCs in 3D, the MB formation was disrupted by altering cell-cell interactions. This was first performed by adding EDTA, a chelating agent of the calcium involved in the formation of cadherin junctions, to the droplet contents. Doing so disrupted the MB formation, as shown in Fig. 2H, where the projected area of the cells increased and the circularity decreased in the presence of EDTA compared with the controls, as previously reported (17). The role of N-cadherins among different types of cadherins was further specified by adding a blocking antibody in the droplets before MB formation. This also led to a disruption of the MB formation, demonstrating that N-cadherin homodimeric interactions are mandatory to initiate the process of HMSC aggregation. CD146 [melanoma cell adhesion molecule (M-CAM)] plays important dual roles: as an adhesion molecule (that binds to Laminin 411) (18) and a marker of the commitment of HMSCs (19). We, thus, interrogate its contribution to MB formation. The addition of a CD146-conjugated blocking antibody also disrupted the formation of the MB (Fig. 2H), demonstrating that cell-cell interactions involving CD146 are also required during MB formation, as reported with other cell types (18). Of note, the brightest signal from the CD146-stained cells was located in the core of the cellular aggregates (Fig. 2H), suggesting that HMSCs self-organize relatively to their degree of commitment.

We found that the population of HMSCs constituted of cells of broad size and expressing different levels of undifferentiated markers [i.e., CD90, CD73, CD105, and CD146 are known to be down-regulated upon differentiation; (20)] and that the cells are capable of self-organizing cohesively in 3D. To better understand how the heterogeneous cells organized within the MBs, we measured how the different cell types composing the population self-assembled spatially in 3D by investigating the role of CD146. For this purpose, the CD146dim and CD146bright cells were separated from the whole HMSC population by flow cytometry (Fig. 3, A and B). The cells were then reseeded on a chip for the MB formation after fluorescently labeling the brighter and/or the dimmer CD146 populations. Image analysis revealed that the CD146bright cells were mostly located in the center of the cellular aggregates, while CD146dim cells were found at the boundaries of the MBs (Fig. 3, C to E, figs. S3A and S5A for confocal images, and movie S1). This organization was stable for a 3-day culture (fig. S3B).

(A) Representative dot plot of the hMSC population separation based on the level of CD146: The CD146dim constitutes 20% of the population expressing the highest levels of CD146; the CD146bright constitutes the 20% of the population expressing the lowest levels of CD146. (B) Fluorescence signal distribution in the CD146dim and CD146bright populations after cell sorting. (C) After cell sorting, the CD146bright or the CD146dim was stained with Vybrant Dil (red) or Vybrant DiO (green), remixed together and allowed to form MBs. Representative images of the CD146bright and CD146dim within the MBs. Scale bar, 100 m (n = 185 MBs). (D) The position of the CD146bright and CD146dim was quantified by correlating the fluorescence signal of the different stained cells as a function of their radial position within the MBs, after the staining of individual population with Vybrant Dil (CD146bright) = 500 and CD146dim (MBs; CD146bright = 85). Error bars show the SD. (E) Schematized representation of the structural organization of MBs. (F and G) RT-qPCR analysis of the relative RUNX-2, CEBP/, and SOX-9 expression to glyceraldehyde-3-phosphate dehydrogenase (GADPH) [Ct (cycle threshold) (D) and relative RNA expression (E)] in the CD146bright and CD146dim populations (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.

As we found that the CD146bright cells were larger than the CD146dim cells, the cells from the HMSC population were also separated on the basis of their relative size (a parameter that also discriminates the CD90, CD105, and CD73bright from the CD90, CD105, and CD73dim cells; fig. S1, D to F). After reseeding on the chip, the MBs were composed of large cells in the core, while the smallest cells were located at the boundaries, as expected from the previous experiments (fig. S3A). Moreover, we found that the speed of self-assembly of each population is not related to the rearrangement of CD146dim and CD146bright cells in 3D, because the mixing of dissociated cells or the fusion of aggregates made each population give rise to the same structural organization (21). It is well established that CD146bright defines the most undifferentiated HMSCs (20). The heterogeneity in level of commitment between the two subpopulations was therefore checked by reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis to quantify differences in the expression of differentiation markers. The analysis showed that the CD146dim cells expressed higher levels of osteogenic differentiation markers (i.e., RUNX-2) than the CD146bright cells (Fig. 3, F and G).

The level of RUNX-2 expression was also quantified at the protein level using immunocytochemistry and image analysis of the MBs on the microfluidic device by developing a layer-by-layer description of the MBs. This mapping was constructed by estimating the boundaries of each cell in the image from a Voronoi diagram, built around the positions of the cell nuclei stained with 4,6-diamidino-2-phenylindole (DAPI) (Fig. 4A) (22). These estimates were then used to associate the fluorescence signal from each cell with one of the concentric layers (Fig. 4B). Such a mapping provides better resolution for discriminating the spatial heterogeneity of protein expression than simply assigning a fluorescence signal to a defined radial coordinate (fig. S4). Moreover, the reliability of the measurements by quantitative image analysis was confirmed by performing several control experiments. In particular, we verified (i) the specificity of the fluorescence labeling, (ii) the absence of limitation for antibody diffusion, and (iii) the absence of the light path alteration in the 3D structure (fig. S5 and Materials and Methods). Consistent with the qPCR data, we found that HMSCs located at the boundaries of the MBs expressed higher levels of the protein RUNX-2 than the cells located in the core (see Fig. 4, C and D, and fig. S7 for individual experiments).

(A and B) The detection of nuclei within MBs enables the construction of a Voronoi diagram (A) that allows the identification of concentric cell layers (B) within the MBs. (C and D) Representative image (C) and quantitative analysis (D) (error bars represent the SD) of RUNX-2 staining within the cell layers of the MB (Nchips = 3 and nMBs = 458). N.S., nonsignificant. (E to H) Quantitative analysis (E) and representative images (F to H) of N-cadherin staining after methanol/acetone (F) (Nchips = 3 and nMBs = 405), after PFA/Triton X-100 fixation and permeabilization (G) (Nchips = 3 and nMBs = 649), and F-actin staining with phalloidin (H) (Nchips = 3 and nMBs = 421). Scale bars, 20 m. The images were acquired using a wide-field microscope. ***P < 0.001. (I) Schematized representation of the structural organization of MBs.

Thus, as CD146 defines the most undifferentiated and clonogenic cells as well as regulates the trilineage differentiation potential of HMSCs, the results indicate that HMSCs self-organize within MBs based on their initial commitment. The most undifferentiated and largest cells are found in the core (r/R < 0.8), while more differentiated cells positioned in the outer layers of the MBs (r/R > 0.8) (Fig. 4, C to E). In addition, these data reveal that HMSCs are conditioned a priori to occupy a specific location within the MBs.

The commitment of HMSCs is known to regulate their level of CD146 expression and the type of cell-cell adhesion molecules (23), which plays a fundamental role in the structural cohesion of the MBs (Fig. 2H). For this reason, we interrogated the organization of cell-cell junctions after the MB formation through measurements of the N-cadherin and F-actin fluorescence signal distribution. Two different protocols were used to discriminate several forms of N-cadherin interactions. First, paraformaldehyde (PFA) fixation and Triton X-100 permeabilization were used, because they were reported to retain in place only the detergent-insoluble forms of N-cadherin. Alternatively, ice-cold methanol/acetone fixation and permeabilization enabled the detection of all forms of N-cadherins (26). The results show a higher density of total N-cadherins in the core of the MBs (Fig. 4, E and F), while a higher density of F-actin was found in the cell layers located near the edge of the MBs (Fig. 4, E and H). The pattern of F-actin distribution was not related to the agarose gel surrounding the MBs (fig. S5B). These results are consistent with the theories of cell sorting in spheroids that postulate that more adhesive cells (i.e., expressing more N-cadherin or CD146) should be located in the core, while more contractile cells (i.e., containing denser F-actin) are located at the edge of the MBs (24). Moreover, our observations are in accordance with recent results demonstrating that HMSCs establishing higher N-cadherin interactions show reduced osteogenic commitment than HMSCs making fewer N-cadherin contacts, potentially through the modulation of Yap/Taz signaling and cell contractility (23).

In contrast, the most triton-insoluble forms of N-cadherins were located at the boundaries of the HMSC aggregates (Fig. 4, E and G), at the same position as the cells containing the denser F-actin. These results demonstrate that different types of cellular interactions were formed between the core and the edges of the MBs, which correlated with the degree of cell commitment that apparently stabilize the adherens junctions (Fig. 4I) (25).

We found above that the degree of commitment was linked with the pattern of HMSC self-organization in MBs (i.e., formation of adherens junctions), which may also regulate their paracrine functions (26). We therefore interrogated the functional consequences of the cellular organization in MBs by investigating the distribution of VEGF- and PGE2-producing cells.

The specific production of COX-2, VEGF, and two other molecules regulating bone homeostasis such as tumor necrosis factorinducible gene 6 (TSG-6) (27) and stanniocalcin 1 (STC-1) (28) was evaluated by RT-qPCR analysis. An increased transcription (20- to 60-fold) of these molecules was measured in 3D in comparison to the monolayer culture (Fig. 5, A and B). Consistent with this observation, while a very limited level of secreted PGE2 and VEGF was measured by enzyme-linked immunosorbent assay (ELISA) in 2D culture, they were significantly increased (by about 15-fold) upon the aggregation of HMSCs in 3D (Fig. 5C). In addition, to interrogate the specific role of COX-2 [the only inducible enzyme catalyzing the conversion of arachidonic acid into prostanoids; (29)] in PGE2 and VEGF production, indomethacin (a pan-COX inhibitor) was added to the culture medium. Indomethacin abrogated the production of PGE2, and it significantly decreased VEGF secretion (Fig. 5C), which suggests an intricate link between COX-2 expression and the secretion of these two molecules (30, 31).

(A and B) RT-qPCR analysis of the relative TSG-6, COX-2, STC-1, and VEGF expression to GADPH (Ct) (A) and relative RNA expression (B) in the 3D and 2D populations (n3D = 3 and n2D = 3). (C) Quantification by ELISA of the PGE-2 and VEGF secreted by hMSCs cultivated in 2D, as MBs or as MBs treated with indomethacin (nchips = 3 and n2D = 3). (D and E) Representative image (D) and quantitative analysis (E) of COX-2 (Nchips = 13 and nMBs = 2936) and (F) VEGF-A (Nchips = 3 and nMBs = 413) staining within the cell layers of the MBs (error bars represent the SD). Scale bars, 50 m. The images were acquired using a wide-field microscope *P < 0.05; ***P < 0.001; a and b: P < 0.05. (G) Schematized representation of the structural organization of MBs.

To further interrogate the link between the COX-2 and the VEGF-producing cells, their location was analyzed by quantitative image analysis at a layer-by-layer resolution. These measurements showed significantly higher levels of COX-2 in the first two layers, compared with the successive layers of the MBs (Fig. 5, D and E), with a continuous decrease of about 40% of the COX-2 signal between the edge and the core. This pattern of COX-2 distribution was not affected by the MB diameter (fig. S7B). Similar observations were made with VEGF (Fig. 5, D and F), demonstrating that cells at the boundaries of the MBs expressed both COX-2 and VEGF (Fig. 5G). Taken with the measurements of Fig. 5C, these results imply that COX-2 acts as an upstream regulator of PGE2 and VEGF secretion. Conversely, oxygen deprivation was unlikely to occur within the center of the MBs because no hypoxic area was detected through the whole MBs (fig. S5). Consequently, it is unlikely that hypoxia-inducible factor1 (HIF-1) signaling mediates the increase in VEGF expression at the boundaries of the MBs. Note that finding the link between these three molecules requires the 3D format, because the molecules are not detected in 2D. Here, the combination of population-scale measurements (Fig. 5C) and cell layer analysis (Fig. 5, E and F) provides strong evidence for this pathway.

Because variations of COX-2 and adherens junction distribution are colocalized within the MBs (Figs. 4, E to G, and 5, D and E), the results point to a link between the quality of cell-cell interactions and the spatial distribution of the COX-2high cells in 3D. The mechanisms leading to the spatial patterning of COX-2 expression in the MBs were therefore explored using inhibitors of the signaling pathways related to anti-inflammatory molecule production and of the molecular pathways regulating the structural organization (table S3): (i) 4-N-[2-(4-phenoxyphenyl)ethyl]quinazoline-4,6-diamine (QNZ) that inhibits NF-B, a critical transcription factor regulating the level of COX-2 expression (32); (ii) N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) that inhibits the canonical Notch pathway, modulating cell-cell interactions and several differentiation pathways; (iii) Y-27632 (Y27) that inhibits ROCK involved in the bundling of F-actin (i.e., formation of stress fibers) to assess the role of actomyosin organization; and (iv) cytochalasin D (CytoD) that inhibits the polymerization of actin monomers.

While the addition of DAPT had virtually no effect on the ability of the cells to form MBs, Y27 led to MBs with more rounded cells, and both QNZ and CytoD strongly interfered with the MB formation process (Fig. 6, A to C). The results indicate that NF-B activation and the promotion of actin polymerization are critical signaling steps initiating the process of MB formation by HMSCs.

(A) Representative images of MBs formed 1 day after the droplet loading. Scale bar, 100 m. Inhibitors are added to the culture medium before the MB formation. (B and C) Quantitative analysis of the aggregates projected area (B) and shape index (C) in the presence of the different inhibitors. Red lines represent the mean value for each condition. (D and E) Representative images (D) (contrast is adjusted individually for a better visualization of the pattern; scale bar, 100 m; the images were acquired using a wide-field microscope) and quantitative analysis (E) of the COX-2 fluorescence signal intensity normalized by the control value with the different inhibitors. For these longer culturing times, QNZ and CytoD are only added during the phase change to allow the MB formation. Small dots represent one MB. Large dots represent the average normalized COX-2 fluorescence signal per chip. Each color corresponds to a specific chip. *P < 0.05. (F) Estimation of inhibitor effect in the cell layers with the COX-2 signal normalized by the control value. Control: Nchips = 11 and nMBs = 2,204; QNZ: Nchips = 6 and nMBs = 1215; DAPT: Nchips = 3 and nMBs = 658; Y27: Nchips = 4 and nMBs = 709; CytoD: Nchips = 3 and nMBs = 459. *P < 0.05; **P < 0.01; ***P < 0.001. (G) Proposed mechanisms regulating the MB formation and the patterning of their biological functions. (i) Regulation of the formation of MBs. (ii and iii) Spatial patterning of hMSC biological properties within MBs.

To assess the role of NF-B and actin polymerization in the pattern and the level of COX-2 expression in the MBs, QNZ and CytoD were added 1 day after the cell seeding, once the MBs were completely formed. In contrast, Y27 and DAPT were included in the initial droplets and maintained in the culture medium for the whole culture period. Typical images showing the COX-2 signal in these different conditions are shown in Fig. 6D (see also fig. S7 for quantification of the individual experiments). Of note, none of the inhibitors had an effect on Casp3 activation, indicating that they do not induce apoptosis at the concentration used in this study (fig. S8). The levels of COX-2 expression in MBs, after 3 days in culture, were significantly reduced with QNZ, also decreasing after the addition of CytoD (Fig. 6E). By contrast, Y27 and DAPT had no effect on the levels of COX-2 expression. As a consequence, the results demonstrate that a sustained NF-B activity after the MB formation is required to promote COX-2 expression. Moreover, the induction of actin polymerization in MBs constitutes a mandatory step to initiate COX-2 production.

To get a deeper understanding on the local regulation of these signaling pathways, we analyzed at the single-cell resolution the distribution of COX-2 within the MBs. The spatial mapping revealed that the COX-2 fluorescence intensity was mostly attenuated at the edge of the MBs treated with CytoD and QNZ, while more limited change in the pattern of its expression was observed in the presence of Y27 and even less so with DAPT (Fig. 6F). Consequently, the results revealed a strong link between cell phenotype, the capability to form functional adherens junctions, and the local regulation of NF-B and actin polymerization leading to the increased expression of PGE2 and VEGF that are mediated by COX-2 in 3D (Fig. 6G). Together, the results indicate that in 3D cell aggregates, the spatial organization has some implications on the specific activation of signaling pathways, resulting in local functional heterogeneity.

Understanding the mechanisms of the formation and the spatial tissue patterning within organoids requires a characterization at single-cell level in 3D. In this study, we used a novel microfluidic and epifluorescence imaging technology to obtain a precise quantitative mapping of the structure, the position, and the link with individual cell functions within MBs. The image analysis provided quantitative data that were resolved on the scale of the individual cells, yielding measurements on 700,000 cells in situ within over 10,000 MBs.

While the microfluidic technology developed here is very efficient for high-density size-controlled MB formation, the method is prone to some limitations. Chief among them, the cultivation in nanoliter-scale drops may subject the cells to nutrient deprivation and by-product accumulation under static culture conditions. This limits the duration of the culture to a few days, depending on the cell type and droplet size. To overcome this limitation, it is possible to continuously perfuse the chip with fresh culture medium after performing the oil-aqueous phase exchange, as we demonstrated previously (16). Alternatively, it is also possible to maintain the cells in liquid droplets (without using a hydrogel) by resupplying culture medium through the fusion of additional drops at later times. This operation requires, however, a new design of the anchors and additional microfluidic steps (21).

The second major drawback of the method emerges from the large distance between the MBs and the microscope objective, which requires the use of very large working distance objectives. This compounds the difficulty of applying different confocal techniques by limiting the fluorescence intensity of the images, which, in turn, reduces the throughput when 3D image stacks are required. Although we have shown above that wide-field imaging can be used to obtain spatial mappings of spheroid structure and cell functions, true single-cell measurements will need to overcome the limitations on imaging in the future.

A Voronoi segmentation was used to categorize the cells into concentric layers, starting from the edge of the MBs and ending with the cells in the central region (22), which allowed us to measure variations in the structural organization and in the protein expressions on a layer-by-layer basis within the 3D cultures. The MBs were found to organize into a core region of undifferentiated cells, surrounded by a shell of committed cells. This hierarchical organization results from the spatial segregation of an initially heterogeneous population, as is generally the case for populations of pluripotent and somatic stem cells (2, 3, 33). The process of aggregation of HMSCs obtained within a few hours takes place through different stages (Fig. 6G): The first steps of the aggregation of MBs are mediated by N-cadherin interactions. In parallel, NF-B signaling is activated, promoting cell survival by preventing anoikis of suspended cells (34, 35). At later stages, the formation of polymerized F-actin and, to a lesser extent, stress fibers mediates the MB compaction, mainly at the edge of the MBs where the cellular commitment helps the stabilization of adherens junctions. The formation of adherens junctions facilitates the cohesion of the 3D structure, probably through the enhanced - and -catenin availability in the CD146dim/RUNX-2+ cells (36, 37, 38), which are recruited in the CCC complexes of the adherens junctions to promote the stable coupling of the F-actin to the N-cadherin (39), which become more insoluble to Triton X-100 than unbounded N-cadherins.

A functional phenotype that correlates with this hierarchical segregation is an increase in endocrine activity of the cells located at the boundaries of the MBs. COX-2 expression is increased in the outer layers of the MBs, which also contain more functional adherens junctions as well as a sustained NF-B activity in this region. The promoter of COX-2 contains RUNX-2 and NF-B cis-acting elements (40). While RUNX-2 is required for COX-2 expression in mesenchymal cells, its level of expression does not regulate the levels of COX-2 (40). The increased COX-2 expression is, in turn, due to the unbundled form of F-actin (i.e., a more relaxed form of actin, in comparison to the dense stress fibers observed in 2D) near the edge of the MBs, which was reported to sustain NF-B activity (41) and to down-regulate COX-2 transcriptional repressors (42). Therefore, NF-B has a high activity in the outer layers of the MB, where it locally promotes COX-2 expression.

These results show that the 3D culture format may provide some insights to understand the mesenchymal cell behavior in vivo, because we found that the expression of key bone regulatory molecules is spatially regulated as a function of the structural organization of the MBs. The 3D structure obtained here recalls some of the conditions found at the initial steps of intramembranous ossification that occurs after mesenchymal condensation (i.e., no chondrogenic intermediate was found in the MBs). In the developing calvaria, the most undifferentiated mesenchymal cells (e.g., Sca-1+/RUNX-2 cells) are located in the intrasutural mesenchyme, which is surrounded by an osteogenic front containing more committed cells (e.g., Sca-1/RUNX-2+ cells) (43, 44). Similarly, we observed that undifferentiated HMSCs (i.e., CD146bright/RUNX-2 HMSCs) were surrounded by osteogenically committed cells (i.e., CD146dim/RUNX-2+ HMSCs), which also coexpressed pro-osteogenic molecules, namely, COX-2 and its downstream targets, PGE2 and VEGF. While the link between COX-2 and PGE2 is well established, there is also evidence that COX-2 can induce the production of VEGF in different cell types, e.g., colon cancer cells (45), prostate cancer cells (46), sarcoma (47), pancreatic cancer cells (48), retinal Mller cells (49), gastric fibroblasts (50), skin or lung fibroblasts (51). In these cases, the mechanism for VEGF production through COX-2 induction is thought to be linked to PGE-2, either in an autocrine/paracrine manner (52) or in an intracrine manner (53).

Beyond HMSCs, spatial organization related to the level of differentiation and cell size has been documented in growing embryoids and organoids, with more committed cells being positioned in the outer layers (54, 55, 56). Our results show that a similar hierarchical structure can also be obtained through the aggregation of a mixed population of adult progenitors. This suggests that cell sorting, based on the size and commitment, plays a dominant role in organizing stem cell aggregates. This data-driven approach of combining high-throughput 3D culture and multiscale cytometry (16) on complex biological models can be applied further for getting a better understanding of the equilibria that determine the structure and the function of cells within multicellular tumor spheroids, embryoid bodies, or organoids.

HMSCs derived from the Whartons jelly of the UC (HMSCs) [American Type Culture Collection (ATCC) PCS-500-010, LGC, Molsheim, France] were obtained at passage 2. Four different lots of HMSCs were used in this study (lot nos. 60971574, 63739206, 63516504, and 63739206). While the lots were not selected a priori, we found consistent results for COX-2 and CD146 distribution within MBs. HMSCs from the different lots were certified for being CD29, CD44, CD73, CD90, CD105, and CD166 positive (more than 98% of the population is positive for these markers) and CD14, CD31, CD34, and CD45 negative (less than 0.6% of the population is positive for these markers) and to differentiate into adipocytes, chondrocytes, and osteocytes (ATCC, certificates of analysis). HMSCs were maintained in T175 cm2 flasks (Corning, France) and cultivated in a standard CO2 incubator (Binder, Tuttlingen, Germany). The culture medium was composed of modified Eagles medium (-MEM) (Gibco, Life Technologies, Saint Aubin, France) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco) and 1% (v/v) penicilin-streptomycin (Gibco). The cells were seeded at 5 103 cells/cm2, subcultivated every week, and the medium was refreshed every 2 days. HMSCs at passage 2 were first expanded until passage 4 [for about five to six population doublings (PDs)], then cryopreserved in 90% (v/v) FBS/10% (v/v) dimethyl sulfoxide (DMSO), and stored in a liquid nitrogen tank. The experiments were carried out with HMSCs at passages 4 to 11 (about 24 to 35 PDs, after passage 2).

HMSCs were harvested by scrapping or trypsinization from T175 cm2 flasks. Then, the cells were incubated in staining buffer [2% FBS in phosphate-buffered saline (PBS)], stained with a mouse anti-human CD146Alexa Fluor 647 (clone P1-H12, BD Biosciences), a mouse anti-human CD31Alexa Fluor 488 (BD Biosciences, San Jose, CA) antibody, a mouse anti-human CD105Alexa Fluor 647 (BD Biosciences, San Jose, CA), a mouse anti-human CD90fluorescein isothiocyanate (FITC) and a mouse anti-human CD73allophycocyanin (APC) (Miltenyi Biotec, Germany), a CD14-APC (Miltenyi Biotec), a CD34-FITC (BioLegend), and an HLA-DRAPC (BD Biosciences).

The percentages of CD73-, CD90-, CD105-, CD146-, CD31-, CD34-, and HLA-DRpositive cells were analyzed using a FACS LSRFortessa (BD Biosciences, San Jose, CA) or an ImageStream (Amnis) flow cytometer. To validate the specificity of the antibody staining, the distributions of fluorescently labeled cells were compared to cells stained with isotype controls: mouse immunoglobulin G1 (IgG1), k-PE-Cy5 (clone MOPC-21, BD Biosciences), and mouse IgG2a K isotype control FITC (BD Biosciences, San Jose, CA). Alternatively, HMSCs were sorted on the basis of their level of expression of CD146 or their size [forward scatter (FSC) and side scatter (SSC)] using a FACSAria III (BD Biosciences, San Jose, CA).

To induce adipogenic differentiation, UC-HMSCs were seeded at 1 104 cells/cm2 in culture medium. The day after, the culture medium was switched to StemPro Adipogenesis Differentiation medium (Life Technologies) supplemented with 10 M rosiglitazone (Sigma-Aldrich) for 2 weeks. To visualize the differentiated adipocytes, the cells were stained with Oil Red O (Sigma-Aldrich). As a control, UC-HMSCs were maintained in culture medium for 2 weeks and stained with Oil Red O, as above.

To induce osteogenic differentiation, UC-HMSCs were seeded at 5 103 cells/cm2 in culture medium. The day after, the culture medium was switched to StemPro Osteogenesis Differentiation medium (Life Technologies) supplemented with 2-nm bone morphogenetic protein 2 (BMP-2) (Sigma-Aldrich) for 2 weeks. To visualize the differentiated osteoblasts, the cells were stained with Alizarin Red S (Sigma-Aldrich). As a control, UC-HMSCs were maintained in culture medium for 2 weeks and stained with Alizarin Red S, as above.

To induce chondrogenic differentiation, UC-HMSCs were seeded at 1 106 cells/ml in a 15-ml conical tube to promote micromass culture. The medium consisted of StemPro Chondrogenic Differentiation medium (Life Technologies). After 3 weeks in culture, the pellets were fixed and cryosectioned and then stained for Alcian Blue 8GX (Sigma-Aldrich). As a control, UC-HMSCs were maintained in 2D using culture medium for 3 weeks and stained with Alcian Blue, as above.

The color images were acquired using a binocular (SMZ18, Nikon) equipped with a camera (D7500, Nikon).

Standard dry-film soft lithography was used for the flow-focusing device (top of the chip) fabrication, while a specific method for the fabrication of the anchors (bottom of the chip) was developed. For the first part, up to five layers of dry-film photoresist consisting of 50-m Eternal Laminar E8020, 33-m Eternal Laminar E8013 (Eternal Materials, Taiwan), and 15-m Alpho NIT215 (Nichigo-Morton, Japan) negative films were successively laminated using an office laminator (PEAK pro PS320) at a temperature of 100C until the desired channel height, either 135, 150, 165, or 200 m, was reached. The photoresist film was then exposed to ultraviolet (Lightningcure, Hamamatsu, Japan) through a photomask of the junction, the channels, and the culture chamber boundaries. The masters were revealed after washing in a 1% (w/w) K2CO3 solution (Sigma-Aldrich). For the anchor fabrication, the molds were designed with RhinoCAM software (MecSoft Corporation, LA) and were fabricated by micromilling a brass plate (CNCMini-Mill/GX, Minitech Machinery, Norcross). The topography of the molds and masters was measured using an optical profilometer (Veeco Wyco NT1100, Veeco, Mannheim, Germany).

For the fabrication of the top of the chip, poly(dimethylsiloxane) [PDMS; SYLGARD 184, Dow Corning, 1:10 (w/w) ratio of curing agent to bulk material] was poured over the master and cured for 2 hours at 70C. For the fabrication of the bottom of the chip, the molds for the anchors were covered with PDMS. Then, a glass slide was immersed into uncured PDMS, above the anchors. The mold was lastly heated on a hot plate at 180C for 15 min. The top and the bottom of the chip were sealed after plasma treatment (Harrick, Ithaca). The chips were filled three times with Novec Surface Modifier (3M, Paris, France), a fluoropolymer coating agent, for 30 min at 110C on a hot plate.

HMSCs were harvested with TrypLE at 60 to 70% confluence, and a solution containing 6 105 cells in 70 l of medium was mixed with 30 l of a 3% (w/v) liquid low-melting agarose solution (i.e., stored at 37C) (Sigma-Aldrich, Saint Quentin Fallavier, France) diluted in culture medium containing gentamicin (50 g/ml; Sigma-Aldrich) (1:3, v/v), resulting in a 100-l solution of 6 106 cells/ml in 0.9% (w/v) agarose.

HMSCs and agarose were loaded into a 100-l glass syringe (SGE, Analytical Science, France), while Fluorinert FC-40 oil (3M, Paris, France) containing 1% (w/w) PEG-di-Krytox surfactant (RAN Biotechnologies, Beverly, USA) was loaded into a 1- and 2.5-ml glass syringes (SGE, Analytical Science). Droplets of cell-liquid agarose were generated in the FC-40 containing PEG-di-Krytox, at the flow-focusing junction, by controlling the flow rates using syringe pumps (neMESYS Low-Pressure Syringe Pump, Cetoni GmbH, Korbussen, Germany) (table S1). After complete loading, the chips were immersed in PBS, and the cells were allowed to settle down and to organize as MBs overnight in the CO2 incubator. Then, the agarose was gelled at 4C for 30 min, after which the PEG-di-Krytox was extensively washed in flushing pure FC-40 in the culture chamber. After washing, cell culture medium was injected to replace the FC-40. All flow rates are indicated in table S1. Further operations were allowed by gelling the agarose in the droplets, such that the resulting beads were retained mechanically in the traps rather than by capillary forces (Fig. 2G). This step allowed the exchange of the oil surrounding the droplets by an aqueous solution, for example, to bring fresh medium for long-term culture, chemical stimuli, or the different solutions required for cell staining.

For the live imaging of the MB formation, the chips were immersed in PBS and then were incubated for 24 hours in a microscope incubator equipped with temperature, CO2, and hygrometry controllers (Okolab, Pozzuoli, Italy). The cells were imaged every 20 min.

2D cultures or MBs were washed in PBS and incubated with a 5 M NucView 488 caspase-3 substrate (Interchim, Montluon, France) diluted in PBS. After washing with PBS, HMSCs were fixed with a 4% (w/v) PFA (Alpha Aesar, Heysham, UK) for 30 min and permeabilized with 0.2 to 0.5% (v/v) Triton X-100 (Sigma-Aldrich) for 5 min. The samples were blocked with 5% (v/v) FBS in PBS for 30 min and incubated with a rabbit polyclonal antiCOX-2 primary antibody (ab15191, Abcam, Cambridge, UK) diluted at 1:100 in 1% (v/v) FBS for 4 hours. After washing with PBS, the samples were incubated with an Alexa Fluor 594conjugated goat polyclonal anti-rabbit IgG secondary antibody (A-11012, Life Technologies, Saint Aubin, France) diluted at 1:100 in 1% (v/v) FBS for 90 min. Last, the cells were counterstained with 0.2 M DAPI for 5 min (Sigma-Aldrich) and then washed with PBS.

The same protocol was used for the staining of VEGF-Aexpressing cells using a rabbit anti-human VEGF-A monoclonal antibody (ab52917, Abcam, Cambridge, UK), which was revealed using the same secondary antibody as above. RUNX-2positive cells were similarly stained using a mouse anti-human RUNX-2 monoclonal antibody (ab76956, Abcam, Cambridge, UK), which was revealed using an Alexa Fluor 488 goat anti-mouse IgG2a secondary antibody (A-21131, Life Technologies, Saint Aubin, France), both diluted at 1:100 in 1% (v/v) FBS.

To measure potential induction of hypoxia within the core of the MBs, the cells were stained with Image-iT Red Hypoxia Reagent (Invitrogen) for 3 hours and then imaged using a fluorescence microscope. As a positive control, the chips containing the MBs were immersed into PBS, incubated overnight in an incubator set at 37C under 3% O2/5% CO2, and lastly imaged as above.

To interrogate the contribution of signaling related to anti-inflammatory molecule production (COX-2 and NF-B) or molecular pathways regulated by the cell structural organization (Notch, ROCK, and F-actin), several small molecules inducing their inhibition were added to the culture medium (table S1). For all the conditions, the final concentration of DMSO was below 0.1% (v/v) in the culture medium.

The cell viability was assessed using LIVE/DEAD staining kit (Molecular Probes, Life Technologies). The MBs were incubated for 30 min in PBS containing 1 M calcein AM and 2 M ethidium homodimer-1, in flushing 100 l of the solution. The samples were then washed with PBS and imaged under a motorized fluorescence microscope (Nikon, France).

For the detection of the functional forms of N-cadherins (i.e., the N-cadherins closely linked to the actin network, which are PFA insoluble), the MBs were fixed with a 4% (w/v) PFA (Alpha Aesar, Heysham, UK) for 30 min and permeabilized with 0.2 to 0.5% (v/v) Triton X-100 (Sigma-Aldrich) for 5 min. Alternatively, the aggregates were incubated for 5 min with 100% cold methanol followed by 1 min with cold acetone, for the detection of total N-cadherins (i.e., the PFA-soluble and PFA-insoluble forms).

Then, the samples were blocked with 5% (v/v) FBS in PBS for 30 min and incubated with a rabbit polyclonal antiN-cadherin primary antibody (ab18203, Abcam, Cambridge, UK) diluted at 1:100 in 1% (v/v) FBS for 4 hours. After washing with PBS, the samples were incubated with an Alexa Fluor 594conjugated goat polyclonal anti-rabbit IgG secondary antibody (A-11012, Life Technologies, Saint Aubin, France) diluted at 1:100 in 1% (v/v) FBS for 90 min. Last, the cells were counterstained with 0.2 M DAPI for 5 min (Sigma-Aldrich) and then washed with PBS.

For the quantification of the polymerized form of actin (F-actin), the MBs were first fixed with a 4% (w/v) PFA (Alpha Aesar, Heysham, UK) for 30 min and permeabilized with 0.2 to 0.5% (v/v) Triton X-100 (Sigma-Aldrich) for 5 min. The samples were then blocked with a 5% (v/v) FBS solution and incubated for 90 min in a 1:100 phalloidinAlexa Fluor 594 (Life Technologies) diluted in a 1% (v/v) FBS solution. The cells were then counterstained with 0.2 M DAPI for 5 min (Sigma-Aldrich) and then washed with PBS.

To ensure the specificity of the antibody to COX-2 and N-cadherin, control UC-HMSCs were permeabilized, fixed, and incubated only with the secondary antibody (Alexa Fluor 594conjugated goat polyclonal anti-rabbit IgG), as above. The absence of fluorescence signal indicated the specific staining for intracellular COX-2 and N-cadherin.

Next, to validate that the distribution of the fluorescence intensity was not related to any antibody diffusion limitation, the MBs were fixed and permeabilized, as above. For this assay, the MBs were not subjected to any blocking buffer. The cells were incubated for 90 min with the Alexa Fluor 594conjugated goat polyclonal anti-rabbit IgG secondary antibody (A-11012, Life Technologies, Saint Aubin, France) diluted at 1:100 in 1% (v/v) FBS. Then, the cells were counterstained for DAPI, as above. Last, the MBs were collected from the chip, deposed on a glass slide, and imaged.

For the analysis of COX-2 expression by flow cytometry, the total MBs were recovered from the chip. The MBs were then trypsinized and triturated to obtain single-cell suspension. UC-HMSCs were stained for COX-2, as above. The percentage of COX-2positive cells was quantified on 5 103 dissociated UC-HMSCs using a Guava easyCyte Flow Cytometer (Merck Millipore, Guyancourt, France). The results were compared to the fluorescence intensity distribution obtained by image analysis.

To interrogate the influence of the MB opacity in the COX-2 and N-cadherin fluorescence signals, the samples were treated by the Clear(T2) method after immunostaining (57). Briefly, the MBs were incubated for 10 min in 25% (v/v) formamide/10% (w/v) polyethylene glycol (PEG) (Sigma-Aldrich), then for 5 min in 50% (v/v) formamide/20% (w/v) PEG, and lastly for 60 min in 50% (v/v) formamide/20% (w/v) PEG, before their imaging. The fluorescence signal distribution was compared with the noncleared samples.

The MBs were collected from the chip and then fixed using PFA, as above. The MBs were incubated overnight in a 30% sucrose solution at 4C. Then, the sucrose solution was exchanged to O.C.T. medium (optimal cutting temperature; Tissue-Tek) in inclusion molds, which were slowly cooled down using dry ice in ethanol. The molds were then placed at 80C. On the day of the experiments, the O.C.T. blocks were cut at 7 m using a cryostat (CM3050 S, Leica). The cryosections were placed on glass slides (SuperFrost Plus Adhesion, Thermo Fisher Scientific), dried at 37C, and rehydrated using PBS. The cryosections were permeabilized and stained for COX-2, as above. The slides were lastly mounted in mounting medium containing DAPI (Fluoromount-G, Invitrogen).

All the images used for the quantitative analysis were taken using a motorized wide-field microscope (Ti, Eclipse, Nikon), equipped with a CMOS (complementary metal-oxide semiconductor) camera (ORCA-Flash4.0, Hamamatsu) and a fluorescence light-emitting diode source (Spectra X, Lumencor). The images were taken with a 10 objective with a 4-mm working distance (extra-long working distance) and a 0.45 numerical aperture (NA) (Plan Apo , Nikon).

For control experiments, images were taken using a motorized (Ti2, Nikon) confocal spinning disc microscope equipped with lasers (W1, Yokogawa) and the same camera and objective as above. Alternatively, the samples were imaged with a multiphoton microscope (TCS SP8 NLO, MP, Leica). The objective was an HCX PL APO CS 10, 0.40 NA, working distance of 2.2 mm (Leica).

All immunostained samples were counterstained with DAPI, and most of the images (i.e., for N-cadherin, COX-2, VEGF-A, and F-actin) were taken using red light excitation that is known to penetrate deeper into the 3D objects than dyes emitting at lower weight length (e.g., DAPI, FITC). For wide-field microscopy, the focal plane was defined as the area containing the maximal number of DAPI-stained nuclei covering the focal area, while z stacks were taken for the whole in-focus planes containing DAPI-stained nuclei using spinning discs and two-photon confocal microscopy.

Wide-field imaging is sensitive for the emission of fluorescence from inside and outside the focal plane (i.e., from the out-of-focus upper and bottom planes of the spheroids) (58). Consistently, more DAPI signal from nuclei is emitted from the core than in the edges of MBs using epifluorescence microscopy (fig. S5I). We confirmed that our interpretation of the signal distribution from epifluorescence images was consistent with confocal and two-photon microscopy by comparing with images taken from the median z plane and the maximal z projection (fig. S5, N to P).

Consequently, the results unambiguously demonstrate that even if there are more cells in the z plane of the middle area of the MBs, the contribution of the out-of-focus signal from N-cadherin, COX-2, VEGF-A, and F-actin staining in this area of the MBs is minimal using wide-field imaging. Because of the higher throughput of wide-field microscopy, this method was chosen to quantitatively analyze the distribution of these immunolabeled proteins within MBs.

The culture supernatants of six-well plates were collected, while the total medium content of the chip was recovered by flushing the culture chamber with pure oil. A PGE2 human ELISA kit (ab133055, Abcam, Cambridge, UK) was used for the quantification of PGE2 concentration in the culture supernatant, following the manufacturers instructions. Briefly, a polynomial standard curve of PGE2 concentration derived from the serial dilution of a PGE2 standard solution was generated (r2 > 0.9). The absorbance was measured using a plate reader (Chameleon, Hidex, Finland).

A VEGF-A human ELISA kit (Ab119566, Abcam, Cambridge, UK) was used for the quantification of VEGF-A concentration in the culture supernatant of 2D cultures or from the chips. A linear standard curve of VEGF-A concentration derived from the serial dilution of a VEGF-A standard solution was generated (r2 > 0.9). The absorbance was measured using a plate reader (Chameleon, Hidex, Finland).

The total MBs of a 3-day culture period were harvested from the chips, as described above. Alternatively, cells cultured on regular six-well plates were recovered using trypsin after the same cultivation time; CD146dim and CD146bright isolated cells were immediately treated for RNA extraction after sorting. The total RNA of 1 104 cells were extracted and converted to complementary DNA (cDNA) using SuperScript III CellsDirect cDNA Synthesis System (18080200, Invitrogen, Life Technologies), following the manufacturers instructions. After cell lysis, a comparable quality of the extracted RNA was observed using a bleach agarose gel, and similar RNA purity was obtained by measurement of the optical density at 260 and 280 nm using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE) between total RNA preparations from 2D and on-chip cultures.

The cDNA was amplified using a GoTaq qPCR Master Mix (Promega, Charbonnieres, France) or a FastStart Universal SYBR Green Master Mix (containing Rox) (Roche) and primers (Life Technologies, Saint Aubin, France or Eurofins Scientific, France) at the specified melting temperature (Tm) (table S2) using a MiniOpticon (Bio-Rad) or a QuantStudio 3 (Thermo Fisher Scientific) thermocycler. As a negative control, water and total RNA served as template for PCR. To validate the specificity of the PCR, the amplicons were analyzed by dissociation curve and subsequent loading on a 2.5% (w/v) agarose gel and migration at 100 V for 40 min. The PCR products were revealed by ethidium bromide (Sigma-Aldrich) staining, and the gels were imaged using a transilluminator. The analysis of the samples not subjected to reverse transcription (RT) indicated negligible genomic DNA contamination (i.e., <0.1%), while no amplification signal was observed for the water template (no template control). The amount of TSG-6, COX-2, STC-1, VEGF-A, RUNX-2, CEBP-, and SOX-9 transcripts was normalized to the endogenous reference [glyceraldehyde-3-phosphate dehydrogenase (GADPH)], and the relative expression to a calibrator (2D cultures) was given by 2Ct calculation. At least five biological replicates of 2D and on-chip cultures were analyzed by at least duplicate measurements. The standard curves for GADPH, TSG-6, COX-2, and STC-1 were performed using a five serial dilution of the cDNA templates and indicated almost 100% PCR efficiency.

The image analysis allowed us to perform a multiscale analysis (16) of the MBs. For each chip, single images of the anchors were acquired automatically with the motorized stage of the microscope. The analysis was conducted on a montage of the detected anchors using a custom MATLAB code (r2016a, MathWorks, Natick, MA). Two distinct routines were used: one with bright-field detection and one for the fluorescence experiments.

For the bright field-detection described previously (16), the cells were detected in each anchor as pixels with high values of the intensity gradient. This allowed for each cell aggregate to compute morphological parameters such as the projected area A and the shape index SI that quantifies the circularity of an objectSI=4APwhere P is the perimeter. Shape index values range from 0 to 1, with 1 being assigned for perfect disk.

The MB detection with fluorescence staining (DAPI/Casp3/COX-2, DAPI/phalloidin, DAPI/N-cadherin, or LIVE/DEAD) was performed as described previously (16). First, morphological data were extracted at the MB level, such as the equivalent diameter of the MBs or the shape index. Also, the mean fluorescence signal of each MB was defined as the subtraction of the local background from the mean raw intensity.

At the cellular level, two different methods were used, both relying on the detection of the nuclei centers with the DAPI fluorescence signal. On the one hand, each cell location could be assigned to a normalized distance from the MB center (r/R) to correlate a nuclear fluorescence signal with a position in the MB, as previously described (16). On the other hand, the cell shapes inside the MBs were approximated by constructing Voronoi diagrams on the detected nuclei centers. Basically, the edges of the Voronoi cells are formed by the perpendicular bisectors of the segments between the neighboring cell centers. These Voronoi cells were used to quantify the cellular cytoplasmic signal (COX-2, F-actin and N-cadherin, VEGF and RUNX-2). In detail, to account for the variability of the cytoplasmic signal across the entire cell (nucleus included), the fluorescence signal of a single cell was defined as the mean signal of the 10% highest pixels of the corresponding Voronoi cell.

Image processing was also used to get quantitative data on 2D cultures, as previously described (16). Last, different normalization procedures were chosen in this paper. When an effect was quantified compared with a control condition, the test values were divided by the mean control value, and the significance was tested against 1. For some other data, the values were simply normalized by the corresponding mean at the chip level to discard the interchip variation from the analysis.

*P < 0.05; **P < 0.01; ***P < 0.001; NS, nonsignificant. Details of each statistical test and P values can be found in table S4.

Acknowledgments: C. Frot is gratefully acknowledged for the help with the microfabrication, and F. Soares da Silva is gratefully acknowledged for the help in flow cytometry. The group of Biomaterials and Microfluidics (BMCF) of the Center for Innovation and Technological Research as well as the Center for Translational Science (CRT)Cytometry and Biomarkers Unit of Technology and Service (CB UTechS is also acknowledged for the access to the microfabrication and flow cytometry platform at the Institut Pasteur). Funding: The research leading to these results received funding from the European Research Council (ERC) grant agreement 278248 Multicell. Author contributions: S.S., C.N.B., and A.C. conceived the experiments. S.S. performed the experiments. R.F.-X.T. wrote the image processing code and performed the image analysis. R.F.-X.T., S.S., G.A., and A.B. performed the image and data analyses. S.S., C.N.B., and A.C. discussed the results and wrote the manuscript. All authors discussed the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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Mapping the structure and biological functions within mesenchymal bodies using microfluidics - Science Advances

Bone Marrow Stem Cells Comments Off on Mapping the structure and biological functions within mesenchymal bodies using microfluidics – Science Advances | March 6th, 2020

Article In Brief

A unique molecular structureevident in induced pluripotent stem cells taken from people with young-onset Parkinson's diseasesuggests that the defects may be present throughout patients' lives, and that they could therefore be used as diagnostic markers.

Induced pluripotent stem cells (iPSCs) taken from patients with young-onset Parkinson's disease (YOPD) and grown into dopamine-producing neurons displayed a molecular signature that was corrected in vitro, as well as in the mice striatum, by a drug already approved by the US Food and Drug Administration (FDA), a study published in the January 27 online edition of Nature Medicine found.

Although the patients had no known genetic mutations associated with PD, the neurons grown from their iPSCs nonetheless displayed abnormally high levels of soluble alpha-synucleina classic phenotype of the disease, but one never before seen in iPSCs from patients whose disease developed later in life. Surprisingly, for reasons not yet understood, the cells also had high levels of phosphorylated protein kinase C-alpha (PKC).

In addition, the cells also had another well-known hallmark of PD: abnormally low levels of lysosomal membrane proteins, such as LAMP1. Because lysosomes break down excess proteins like alpha-synuclein, their reduced levels in PD have long been regarded as a key pathogenic mechanism.

When the study team tested agents known to activate lysosomal function, they found that a drug previously approved by the FDA as an ointment for treating precancerous lesions, PEP005, corrected all the observed abnormalities in vitro: it reduced alpha-synuclein and PKC levels while increasing LAMP1 abundance. It also decreased alpha-synuclein production when delivered to the mouse striatum.

Unexpectedly, however, PEP005 did not work by activating lysosomal function; rather, it caused another key protein-clearing cellular structure, the proteasome, to break down alpha-synuclein more readily.

The findings suggest that the defects seen in the iPSCs are present throughout patients' lives, and that they could therefore be used as diagnostic markers. Moreover, the drug PEP005 should be considered a potentially promising therapeutic candidate for YOPD and perhaps even for the 90 percent of PD patients in whom the disease develops after the age of 50, according to the study's senior author, Clive Svendsen, PhD, director of the Cedars-Sinai Board of Governors Regenerative Medicine Institute and professor of biomedical sciences and medicine at Cedars-Sinai.

These findings suggest that one day we may be able to detect and take early action to prevent this disease in at-risk individuals, said study coauthor Michele Tagliati, MD, FAAN, director of the movement disorders program and professor of neurology at Cedars-Sinai Medical Center.

But the study still raises questions regarding the biological mechanisms, and certainly does not warrant off-label prescribing of PEP005 at this time, said Marco Baptista, PhD, vice president of research programs at the Michael J. Fox Foundation, who was not involved with the study.

Repurposing PEP005 is a long way away, Dr. Baptista said. This is not something that neurologists should be thinking about prescribing or recommending to their patients.

Accumulation of alpha-synuclein has been seen in iPSC-derived dopaminergic cultures taken from patients with known genetic defects, but such defects account for only about 10 percent of the PD population. In those without known mutations, on the other hand, no defects in iPSC-derived dopamine-producing neurons have been seen. Until now, however, such studies had been conducted only in patients who had developed PD after age 50.

My idea was why to look in young-onset patients, said Dr. Svendsen.

The idea paid off more richly than he expected. We were shocked to find a very, very prominent phenotype, a buildup of alpha-synuclein, in the neurons of these patients who are genetically normal, Dr. Svendsen said. None of the controls had a buildup of synuclein, and all but one of the early PD patients had a twofold increase in it.

The signature is so consistent, he said, that it offers a natural model that can be interrogated to further understand its workings.

Because high levels of PKC were also seen, Dr. Svendsen said, We picked a bunch of drugs known to reduce PKC. We found one, PEP005, which is actually extracted from the milkweed plant, and it completely reduced synuclein levels almost to normal in dopaminergic neurons. And it also increased dopamine levels in those cells, so we got two for one.

After observing the effects of PEP005 in vitro, We put it into the mouse brain and found it reduced synuclein in vivo, Dr. Svendsen said. But we had to infuse it right into the brain. We're now trying to work out how to get it across the blood-brain barrier more efficiently.

To determine how PEP005 lowers cellular levels of alpha-synuclein, his group tested whether it was activating the lysosome, but found to their surprise that it did not do this until after the synuclein had already been degraded.

Then we asked whether it could be the proteosome, which also breaks down proteins but normally doesn't break down synuclein, Dr. Svendsen said. But when we applied PEP005, it did activate the proteasome. So we think that might be the mechanism.

Because the drug is currently applied externally, Dr. Svendsen said, the next step will be to see if it crosses the blood-brain barrier when applied to the skin of mice, and whether that results in a lowering of synuclein levels in dopaminergic neurons.

Justin Ichida, PhD, the Richard N. Merkin assistant professor of stem cell biology and regenerative medicine at the USC Keck School of Medicine, said the findings are quite important in the field. The potential diagnostic tools they made could be important in clinical care. And identifying a drug that may very effectively reverse the disease in neurons is a very important discovery.

He wondered, however, whether the increase in alpha-synuclein is truly specific to Parkinson's neurons or if it would also be seen in iPSC neurons from patients with Alzheimer's disease or amyotrophic lateral sclerosis.

I wonder if alpha-synuclein accumulating is a sign of PD in a dish or is a consequence of neurodegeneration or impaired protein degradation in general, Dr. Ichida said. That's a key question if you want to use this molecular signature as a diagnostic tool.

He also questioned if proteins other than alpha-synuclein, such as tau, would also be seen to accumulate in the iPSCs of YOPD patients.

If one of the protein-clearance mechanisms in the cell is working poorly, you would imagine that other things would also accumulate, Dr. Ichida said.

In response, Dr. Svendsen said that while some proteins other than alpha-synuclein were reported in the paper at increased levels, We did not look at tau specifically, but are in the process of looking right now. It could be that synuclein and some other proteins are somehow altered to evade them from being degraded by the lysosome, or that there is a general lysosomal problem.

Patrik Brundin, MD, PhD, director of the Center for Neurodegenerative Science and Jay Van Andel Endowed Chair at Van Andel Research Institute in Grand Rapids, MI, called the paper very interesting and thought-provoking. If these findings hold up, they could shift our understanding of young-onset PD. They imply that there is a strong genetic component that has not been picked up in prior genetic studies.

Dr. Brundin said he would like to see the results replicated in another lab using different sets of reagents. It is so intriguing and rather unexpected that one wonders if the observations really apply, as the study states, to 95 percent of all YOPD.

He also questioned whether all the young-onset PD patients are similar. Clearly the iPSCs studied here are not monogenetic PD, so they must be very diverse genetically and still all have the same alpha-synuclein change.

Dr. Brundin also asked why the abnormalities seen in YOPD neurons have not previously been seen in older cases of PD. Is there a specific cutoff regarding age-of-onset when these purposed genetic differences apply? he asked.

Dr. Svendsen responded: We don't know why the YO have this phenotype or exactly what the cut off is. We have, however, looked at one adult-onset case that did not show this phenotype. Also, one of our YO cases did not show this phenotype. Thus some patients even with early onset may not have it. We are currently testing many more cases from older-onset patients.

Dr. Brundin also wanted to know whether non-dopaminergic neurons have the same deficits described in the study.

We don't know which neurons specifically have the protein deficit as we cannot do single-cell proteomics, Dr. Svendsen answered. It could be a little in all cells or a lot in a small set. Immunocytochemistry is not quantitative but showed that it is more likely a general increase in synuclein and not specific to dopaminergic neurons.

While the findings in iPSCs suggest that the abnormal levels of alpha-synuclein must be present at birth, Dr. Brundin said, I do not know how to reconcile the present findings with genetic data.

The absence of previously described mutations in the YOPD patients means only that more work must be done to uncover the genetic underpinnings, Dr. Svendsen said.

We're just at the tip of the iceberg with understanding the genome, he said. It's such a bizarrely complex beast. Perhaps there are a thousand different proteins interacting to stop the synuclein from being degraded. In 10 years, we probably will be clever enough to see it. We know it must be there. Now the genome guys will go after it.

Dr. Baptista from the Michael J. Fox Foundation said he agreed with the view that there must be genetic alterations underpinning the defects seen in the iPSCs.

Just because we call something non-genetic could simply reflect the current ignorance of the field, he said. I think the discoveries are simply difficult to make.

He added that he wished that the main comparator in the study was not healthy controls, and that there were more older-onset iPSCs to compare against YOPD patients' samples.

Dr. Svendsen said it could be that the iPSCs from older-onset patients might yet be found with additional study to display abnormalities similar to those seen in YOPD.

Right now we only see it in young onset, he said. We may need to leave the cultures longer to see in the older-onset patients. We are doing those experiments now.

Drs. Tagliati and Svendsen disclosed that an intellectual patent is pending for diagnostic and drug screening for molecular signatures of early-onset Parkinson's disease. Dr. Ikeda is a co-founder of AcuraStem Inc. Dr. Brundin has received commercial support as a consultant from Renovo Neural, Inc., Lundbeck A/S, AbbVie, Fujifilm-Cellular Dynamics International, Axial Biotherapeutics, and Living Cell Technologies. He has also received commercial support for research from Lundbeck A/S and Roche and has ownership interests in Acousort AB and Axial Biotherapeutics. Dr. Baptista had no disclosures.

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Molecular Signature of Young-Onset Parkinson's Disease Is... : Neurology Today - LWW Journals

Skin Stem Cells Comments Off on Molecular Signature of Young-Onset Parkinson’s Disease Is… : Neurology Today – LWW Journals | March 5th, 2020

Abstract

Mesenchymal stem cells (MSCs) encapsulation by three-dimensionally (3D) printed matrices were believed to provide a biomimetic microenvironment to drive differentiation into tissue-specific progeny, which made them a great therapeutic potential for regenerative medicine. Despite this potential, the underlying mechanisms of controlling cell fate in 3D microenvironments remained relatively unexplored. Here, we bioprinted a sweat gland (SG)like matrix to direct the conversion of MSC into functional SGs and facilitated SGs recovery in mice. By extracellular matrix differential protein expression analysis, we identified that CTHRC1 was a critical biochemical regulator for SG specification. Our findings showed that Hmox1 could respond to the 3D structure activation and also be involved in MSC differentiation. Using inhibition and activation assay, CTHRC1 and Hmox1 synergistically boosted SG gene expression profile. Together, these findings indicated that biochemical and structural cues served as two critical impacts of 3D-printed matrix on MSC fate decision into the glandular lineage and functional SG recovery.

Mesenchymal stem cells (MSCs) hold great promise for therapeutic tissue engineering and regenerative medicine, largely because of their capacity for self-renewal and multipotent properties (1). However, their uncertain fate has a major impact on their envisioned therapeutic use. Cell fate regulation requires specific transcription programs in response to environmental cues (2, 3). Once stem cells are removed from their microenvironment, their response to environmental cues, phenotype, and functionality could often be altered (4, 5). In contrast to growing information concerning transcriptional regulation, guidance from the extracellular matrix (ECM) governing MSC identity and fate determination is not well understood. It remains an active area of investigation and may provide previously unidentified avenues for MSC-based therapy.

Over the past decade, engineering three-dimensional (3D) ECM to direct MSC differentiation has demonstrated great potential of MSCs in regenerative medicine (6). 3D ECM has been found to be useful in providing both biochemical and biophysical cues and to stabilize newly formed tissues (7). Culturing cells in 3D ECM radically alters the interfacial interactions with the ECM as compared with 2D ECM, where cells are flattened and may lose their differentiated phenotype (8). However, one limitation of 3D materials as compared to 2D approaches was the lack of spatial control over chemistry with 3D materials. One possible solution to this limitation is 3D bioprinting, which could be used to design the custom scaffolds and tissues (9).

In contrast to traditional engineering techniques, 3D cell printing technology is especially advantageous because it can integrate multiple biophysical and biochemical cues spatially for cellular regulation and ensure complex structures with precise control and high reproducibility. In particular, for our final goal of clinical practice, extrusion-based bioprinting may be more appropriate for translational application. In addition, as a widely used bioink for extrusion bioprinting, alginate-based hydrogel could maintain stemness of MSC due to the bioinert property and improve biological activity and printability by combining gelatin (10).

Sweat glands (SGs) play a vital role in thermal regulation, and absent or malfunctioning SGs in a hot environment can lead to hyperthermia, stroke, and even death in mammals (11, 12). Each SG is a single tube consisting of a functionally distinctive duct and secretory portions. It has low regenerative potential in response to deep dermal injury, which poses a challenge for restitution of lost cells after wound (13). A major obstacle in SG regeneration, similar to the regeneration of most other glandular tissues, is the paucity of viable cells capable of regenerating multiple tissue phenotypes (12). Several reports have described SG regeneration in vitro; however, dynamic morphogenesis was not identified nor was the overall function of the formed tissues explored (1416). Recent advances in bioprinting and tissue engineering led to the complexities in the matrix design and fabrication with appropriate biochemical cues and biophysical guidance for SG regeneration (1719).

Here, we adopted 3D bioprinting technique to mimic the regenerative microenvironment that directed the specific SG differentiation of MSCs and ultimately guided the formation and function of glandular tissue. We used alginate/gelatin hydrogel as bioinks in this present study due to its good cytocompatibility, printability, and structural maintenance in long-time culture. Although the profound effects of ECM on cell differentiation was well recognized, the importance of biochemical and structural cues of 3D-printed matrix that determined the cell fate of MSCs remained unknown; thus, the present study demonstrated the role of 3D-printed matrix cues on cellular behavior and tissue morphogenesis and might help in developing strategies for MSC-based tissue regeneration or directing stem cell lineage specification by 3D bioprinting.

The procedure for printing the 3D MSC-loaded construct incorporating a specific SG ECM (mouse plantar region dermis, PD) was shown schematically in Fig. 1A. A 3D cellular construct with cross section 30 mm 30 mm and height of 3 mm was fabricated by using the optimized process parameter (20). The 3D construct demonstrated a macroporous grid structure with hydrogel fibers evenly distributed according to the computer design. Both the width of the fibers and the gap between the fibers were homogeneous, and MSCs were embedded uniformly in the hydrogel matrix fibers to result in a specific 3D microenvironment. (Fig. 1B).

(A) Schematic description of the approach. (B) Full view of the cellular construct and representative microscopic and fluorescent images and the quantitative parameters of 3D-printed construct (scale bars, 200 m). Photo credit: Bin Yao, Wound Healing and Cell Biology Laboratory, Institute of Basic Medical Sciences, General Hospital of PLA. (C) Representative microscopy images of cell aggregates and tissue morphology at 3, 7, and 14 days of culture (scale bars, 50 m) and scanning electron microscopy (sem) images of 3D structure (scale bars, 20 m). PD+/PD, 3D construct with and without PD. (D) DNA contents, collagen, and GAGs of native tissue and PD. (E) Proliferating cells were detected through Ki67 stain at 3, 7, and 14 days of culture. (F) Live/dead assay show cell viability at days 3, 7, and 14. *P < 0.05.

During the maintenance of constructs for stem cell expansion, MSCs proliferated to form aggregates of cells but self-assembled to an SG-like structure only with PD administration (Fig. 1C and fig. S1, A to C). We carried out DNA quantification assay to evaluate the cellular content in PD and found the cellular matrix with up to 90% reduction, only 3.4 0.7 ng of DNA per milligram tissue remaining in the ECM. We also estimated the proportions of collagen and glycosaminoglycans (GAGs) in ECM through hydroxyproline assay and dimethylmethylene blue assay, the collagen contents could increase to 112.6 11.3%, and GAGs were well retained to 81 9.6% (Fig. 1D). Encapsulated cells were viable, with negligible cell death apparent during extrusion and ink gelation by ionic cross-linking, persisting through extended culture in excess of 14 days. The fluorescence intensity of Ki67 of MSCs cultured in 2D condition decreased from days 3 (152.7 13.4) to 14 (29.4 12.9), while maintaining higher intensity of MSCs in 3D construct (such as 211.8 19.4 of PD+3D group and 209.1 22.1 of PD3D group at day 14). And the cell viability in 3D construct was found to be sufficiently high (>80%) when examined on days 3, 7, and 14. The phenomenon of cell aggregate formation and increased cell proliferation implied the excellent cell compatibility of the hydrogel-based construct and promotion of tissue development of 3D architectural guides, which did not depend on the presence or absence of PD (Fig. 1, E and F).

The capability of 3D-printed construct with PD directing MSC to SGs in vitro was investigated. The 3D construct was dissolved, and cells were isolated at days 3, 7, and 14 for transcriptional analysis. Expression of the SG markers K8 and K18 was higher from the 3D construct with (3D/PD+) than without PD (3D/PD); K8 and K18 expression in the 3D/PD construct was similar to with control that MSCs cultured in 2D condition, which implied the key role of PD in SG specification. As compared with the 2D culture condition, 3D administration (PD+) up-regulated SG markers, which indicated that the 3D structure synergistically boosted the MSC differentiation (Fig. 2A).

(A) Transcriptional expression of K8, K18, Fxyd2, Aqp5, and ATP1a1 in 3D-bioprinted cells with and without PD in days 3, 7, and 14 culture by quantitative real-time polymerase chain reaction (qRT-PCR). Data are means SEM. (B) Comparison of SG-specific markers K8 and K18 in 3D-bioprinted cells with and without PD (K8 and K18, red; DAPI, blue; scale bars, 50 m). (C and D) Comparison of SG secretion-related markers ATP1a1 (C) and Ca2+ (D) in 3D-bioprinted cells with and without PD [ATP1a1 and Ca2+, red; 4,6-diamidino-2-phenylindole (DAPI), blue; scale bars, 50 m].

In addition, we tested secretion-related genes to evaluate the function of induced SG cells (iSGCs). Although levels of the ion channel factors of Fxyd2 and ATP1a1 were increased notably in 2D culture with PD and ATP1a1 up-regulated in the 3D/PD construct, all the secretory genes of Fxyd2, ATP1a1, and water transporter Aqp5 showed the highest expression level in the 3D/PD+ construct (Fig. 2A). Considering the remarkable impact, further analysis focused on 3D constructs.

Immunofluorescence staining confirmed the progression of MSC differentiation. At day 7, cells in the 3D/PD+ construct began to express K8 and K18, which was increased at day 14, whereas cells in the 3D/PD construct did not express K8 and K18 all the time (Fig. 2B and fig. S2A). However, the expression of ATP1a1 (ATPase Na+/K+ transporting subunit alpha 1) and free Ca2+ concentration did not differ between cells in the 3D/PD+ and 3D/PD constructs (Fig. 2, C and D). By placing MSCs in such a 3D environment, secretion might be stimulated by rapid cell aggregation without the need for SG lineage differentiation. Cell aggregationimproved secretion might be due to the benefit of cell-cell contact (fig. S2B) (21, 22).

To map the cell fate changes during the differentiation between MSCs and SG cells, we monitored the mRNA levels of epithelial markers such as E-cadherin, occludin, Id2, and Mgat3 and mesenchymal markers N-cadherin, vimentin, Twist1, and Zeb2. The cells transitioned from a mesenchymal status to a typical epithelial-like status accompanied by mesenchymal-epithelial transition (MET), then epithelial-mesenchymal transition (EMT) occurred during the further differentiation of epithelial lineages to SG cells (fig. S3A). In addition, MET-related genes were dynamically regulated during the SG differentiation of MSCs. For example, the mesenchymal markers N-cadherin and vimentin were down-regulated from days 1 to 7, which suggested cells losing their mesenchymal phenotype, then were gradually up-regulated from days 7 to 10 in their response to the SG phenotype and decreased at day 14. The epithelial markers E-cadherin and occludin showed an opposite expression pattern: up-regulated from days 1 to 5, then down-regulated from days 7 to 10 and up-regulated again at day 14. The mesenchymal transcriptional factors ZEB2 and Twist1 and epithelial transcriptional factors Id2 and Mgat3 were also dynamically regulated.

We further analyzed the expression of these genes at the protein level by immunofluorescence staining (figs. S3B and S4). N-cadherin was down-regulated from days 3 to 7 and reestablished at day 14, whereas E-cadherin level was increased from days 3 to 7 and down-regulated at day 14. Together, these results indicated that a sequential and dynamic MET-EMT process underlie the differentiation of MSCs to an SG phenotype, perhaps driving differentiation more efficiently (23). However, the occurrence of the MET-EMT process did not depend on the presence of PD. Thus, a 3D structural factor might also participate in the MSC-specific differentiation (fig. S3C).

To investigate the underlying mechanism of biochemical cues in lineage-specific cell fate, we used quantitative proteomics analysis to screen the ECM factors differentially expressed between PD and dorsal region dermis (DD) because mice had eccrine SGs exclusively present in the pads of their paws, and the trunk skin lacks SGs. In total, quantitative proteomics analyses showed higher expression levels of 291 proteins in PD than DD. Overall, 66 were ECM factors: 23 were significantly up-regulated (>2-fold change in expression). We initially determined the level of proteins with the most significant difference after removing keratins and fibrin: collagen triple helix repeat containing 1 (CTHRC1) and thrombospondin 1 (TSP1) (fig. S5). Western blotting was performed to further confirm the expression level of CTHRC1 and TSP1, and we then confirmed that immunofluorescence staining at different developmental stages in mice revealed increased expression of CTHRC1 in PD with SG development but only slight expression in DD at postnatal day 28, while TSP1 was continuously expressed in DD and PD during development (Fig. 3, A to C). Therefore, TSP1 was required for the lineage-specific function during the differentiation in mice but was not dispensable for SG development.

(A and B) Differential expression of CTHRC1 and TSP1in PD and back dermis (DD) ECM of mice by proteomics analysis (A) and Western blotting (B). (C) CTHRC1 and TSP1 expression in back and plantar skin of mice at different developmental times. (Cthrc1/TSP1, red; DAPI, blue; scale bars, 50 m).

According to previous results of the changes of SG markers, 3D structure and PD were both critical to SG fate. Then, we focused on elucidating the mechanisms that underlie the significant differences observed in 2D and 3D conditions with or without PD treatment. To this end, we performed transcriptomics analysis of MSCs, MSCs treated with PD, MSCs cultured in 3D construct, and MSC cultured in 3D construct with PD after 3-day treatment. We noted that the expression profiles of MSCs treated with 3D, PD, or 3D/PD were distinct from the profiles of MSCs (Fig. 4A). Through Gene Ontology (GO) enrichment analysis of differentially expressed genes, it was shown that PD treatment in 2D condition induced up-regulation of ECM and inflammatory response term, and the top GO term for MSCs in 3D construct was ECM organization and extracellular structure organization. However, for the MSCs with 3D/PD treatment, we found very significant overrepresentation of GO term related to branching morphogenesis of an epithelial tube and morphogenesis of a branching structure, which suggested that 3D structure cues and biochemical cues synergistically initiate the branching of gland lineage (fig S6). Heat maps of differentially expressed ECM organization, cell division, gland morphogenesis, and branch morphogenesis-associated genes were shown in fig. S7. To find the specific genes response to 3D structure cues facilitating MSC reprogramming, we analyzed the differentially expressed genes of four groups of cells (Fig. 4B). The expression of Vwa1, Vsig1, and Hmox1 were only up-regulated with 3D structure stimulation, especially the expression of Hmox1 showed a most significant increase and even showed a higher expression addition with PD, which implied that Hmox1 might be the transcriptional driver of MSC differentiation response to 3D structure cues. Differential expression of several genes was confirmed by quantitative polymerase chain reaction (qPCR): Mmp9, Ptges, and Il10 were up-regulated in all the treated groups. Likewise, genes involving gland morphogenesis and branch morphogenesis such as Bmp2, Tgm2, and Sox9 showed higher expression in 3D/PD-treated group. Bmp2 was up-regulated only in 3D/PD-treated group, combined with the results of GO analysis, we assumed that Bmp2 initiated SG fate through inducing branch morphogenesis and gland differentiation (Fig. 4C).

(A) Gene expression file of four groups of cells (R2DC, MSCs; R2DT, MSC with PD treatment; R3DC, MSC cultured in 3D construct; and R3DT, MSC treated with 3D/PD). (B) Up-regulated genes after treatment (2DC, MSCs; 2DT, MSC with PD treatment; 3DC, MSC cultured in 3D construct; and 3DT, MSC treated with 3D/PD). (C) Differentially expressed genes were further validated by RT-PCR analysis. [For all RT-PCR analyses, gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with 40 cycles, data are represented as the means SEM, and n = 3].

To validate the role of HMOX1 and CTHRC1 in the differentiation of MSCs to SG lineages, we analyzed the gene expression of Bmp2 by regulating the expression of Hmox1 and CTHRC1 based on the 3D/PD-treated MSCs. The effects of caffeic acid phenethyl ester (CAPE) and tin protoporphyrin IX dichloride (Snpp) on the expression of Hmox1 were evaluated by quantitative real-time (qRT)PCR. Hmox1 expression was significantly activated by CAPE and reduced by Snpp. Concentration of CTHRC1 was increased with recombinant CTHRC1 and decreased with CTHRC1 antibody. That is, it was negligible of the effects of activator and inhibitor of Hmox1 and CTHRC1 on cell proliferation (fig. S8, A and B). Hmox1 inhibition or CTHRC1 neutralization could significantly reduce the expression of Bmp2, while Hmox1 activation or increased CTHRC1 both activated Bmp2 expression. Furthermore, Bmp2 showed highest expression by up-regulation of Hmox1 and CTHRC1 simultaneously and sharply decreased with down-regulation of Hmox1 and CTHRC1 at the same time (Fig. 5A). Immunofluorescent staining revealed that the expression of bone morphogenetic protein 2 (BMP2) at the translational level with CTHRC1 and Hmox1 regulation showed a similar trend with transcriptional changes (Fig. 5B). Likewise, the expression of K8 and K18 at transcriptional and translational level changed similarly with CTHRC1 and Hmox1 regulation (fig. S9, A and B). These results suggested that CTHRC1 and Hmox1 played an essential role in SG fate separately, and they synergistically induced SG direction from MSCs (Fig. 5C).

(A and B) Transcriptional analysis (A) and translational analysis (PD, MSCs; PD+, MSCs with 3D/PD treatment; CAPE, MSCs treated with 3D/PD and Hmox1 activator; Snpp, MSCs treated with 3D/PD and Hmox1 inhibitor; Cthrc1, MSCs treated with 3D/PD and recombinant CTHRC1; anti, MSCs treated with 3D/PD and CTHRC1 antibody: +/+, MSCs treated with 3D/PD and Hmox1 activator and recombinant CTHRC1; and /, MSCs treated with 3D/PD and Hmox1 inhibitor and CTHRC1 antibody. Data are represented as the means SEM and n = 3) (B) of bmp2 with regulation of CTHRC1 and Hmox1. (C) The graphic illustration of 3D-bioprinted matrix directed MSC differentiation. CTHRC1 is the main biochemical cues during SG development, and structural cues up-regulated the expression of Hmox1 synergistically initiated branching morphogenesis of SG. *P < 0.05.

Next, we sought to assess the repair capacity of iSGCs for in vivo implications, the 3D-printed construct with green fluorescent protein (GFP)labeled MSCs was transplanted in burned paws of mice (Fig. 6A). We measured the SG repair effects by iodine/starch-based sweat test at day 14. Only mice with 3D/PD treatment showed black dots on foot pads (representing sweating), and the number increased within 10 min; however, no black dots were observed on untreated and single MSC-transplanted mouse foot pads even after 15 min (Fig. 6B). Likewise, hematoxylin and eosin staining analysis revealed SG regeneration in 3D/PD-treated mice (Fig. 6C). GFP-positive cells were characterized as secretory lumen expressing K8, K18, and K19. Of note, the GFP-positive cells were highly distributed in K14-positive myoepithelial cells of SGs but were absent in K14-positive repaired epidermal wounds (Fig. 6, D and E). Thus, differentiated MSCs enabled directed restitution of damaged SG tissues both at the morphological and functional level.

(A) Schematic illustration of approaches for engineering iSGCs and transplantation. (B) Sweat test of mice treated with different cells. Photo credit: Bin Yao, Wound Healing and Cell Biology Laboratory, Institute of Basic Medical Sciences, General Hospital of PLA. (C) Histology of plantar region without treatment and transplantation of MSCs and iSGCs (scale bars, 200 m). (D) Involvement of GFP-labeled iSGCs in directed regeneration of SG tissue in thermal-injured mouse model (K14, red; GFP, green; DAPI, blue; scale bar, 200 m). (E) SG-specific markers K14, K19, K8, and K18 detected in regenerated SG tissue (arrows). (K14, K19, K8, and K18, red; GFP, green; scale bars, 50 m).

A potential gap in MSC-based therapy still exists between current understandings of MSC performance in vivo in their microenvironment and their intractability outside of that microenvironment (24). To regulate MSCs differentiation into the right phenotype, an appropriate microenvironment should be created in a precisely controlled spatial and temporal manner (25). Recent advances in innovative technologies such as bioprinting have enabled the complexities in the matrix design and fabrication of regenerative microenvironments (26). Our findings demonstrated that directed differentiation of MSCs into SGs in a 3D-printed matrix both in vitro and in vivo was feasible. In contrast to conventional tissue-engineering strategies of SG regeneration, the present 3D-printing approach for SG regeneration with overall morphology and function offered a rapid and accurate approach that may represent a ready-to-use therapeutic tool.

Furthermore, bioprinting MSCs successfully repaired the damaged SG in vivo, suggesting that it can improve the regenerative potential of exogenous differentiated MSCs, thereby leading to translational applications. Notably, the GFP-labeled MSC-derived glandular cells were highly distributed in K14-positive myoepithelial cells of newly formed SGs but were absent in K14-positive repaired epidermal wounds. Compared with no black dots were observed on single MSC-transplanted mouse foot pads, the black dots (representing sweating function) can be observed throughout the entire examination period, and the number increased within 10 min on MSC-bioprinted mouse foot pads. Thus, differentiated MSCs by 3D bioprinting enabled exclusive restitution of damaged SG tissues morphologically and functionally.

Although several studies indicated that engineering 3D microenvironments enabled better control of stem cell fates and effective regeneration of functional tissues (2730), there were no studies concerning the establishment of 3D-bioprinted microenvironments that can preferentially induce MSCs differentiating into glandular cells with multiple tissue phenotypes and overall functional tissue. To find an optimal microenvironment for promoting MSC differentiation into specialized progeny, biochemical properties are considered as the first parameter to ensure SG specification. In this study, we used mouse PD as the main composition of a tissue-specific ECM. As expected, this 3D-printed PD+ microenvironment drove the MSC fate decision to enhance the SG phenotypic profile of the differentiated cells. By ECM differential protein expression analysis, we identified that CTHRC1 was a critical biochemical regulator of 3D-printed matrix for SG specification. TSP1 was required for the lineage-specific function during the differentiation in mice but was not dispensable for SG development. Thus, we identified CTHRC1 as a specific factor during SG development. To our knowledge, this is the first demonstration of CTHRC1 involvement in dictating MSC differentiation to SG, highlighting a potential therapeutic tool for SG injury.

The 3D-printed matrix also provided architectural guides for further SG morphogenesis. Our results clearly show that the 3D spatial dimensionality allows for better cell proliferation and aggregation and affect the characteristics of phenotypic marker expression. Notably, the importance of 3D structural cues on MSC differentiation was further proved by MET-EMT process during differentiation, where the influences did not depend on the presence of biochemical cues. To fully elucidate the underlying mechanisms, we first examined how 3D structure regulating stem cell fate choices. According to our data, Hmox1 is highly up-regulated in 3D construct, which were supposed to response to hypoxia, with a previously documented role in MSC differentiation (31, 32). It is suggested that 3D microenvironment induced rapid cell aggregation leading to hypoxia and then activated the expression of Hmox1.

Through regulation of the expression of Hmox1 and addition or of CTHRC1 in the matrix, we confirmed that each of them is critical for SG reprogramming, respectively. Thus, biochemical and structural cues of 3D-printed matrix synergistically creating a microenvironment could enhance the accuracy and efficiency of MSC differentiation, thereby leading to resulting SG formation. Although we further need a more extensive study examining the role of other multiple cues and their possible overlap function in regulating MSC differentiation, our findings suggest that CTHRC1 and Hmox1 provide important signals that cooperatively modulate MSC lineage specification toward sweat glandular lineage. The 3D structure combined with PD stimulated the GO functional item of branch morphogenesis and gland formation, which might be induce by up-regulation of Bmp2 based on the verification of qPCR results. Although our results could not rule out the involvement of other factors and their possible overlapping role in regulating MSC lineage specification toward SGs, our findings together with several literatures suggested that BMP2 plays a critical role in inducing branch morphogenesis and gland formation (3335).

In summary, our findings represented a novel strategy of directing MSC differentiation for functional SG regeneration by using 3D bioprinting and pave the way for a potential therapeutic tool for other complex glandular tissues as well as further investigation into directed differentiation in 3D conditions. Specifically, we showed that biochemical and structural cues of 3D-printed matrix synergistically direct MSC differentiation, and our results highlighted the importance of 3D-printed matrix cues as regulators of MSC fate decisions. This avenue opens up the intriguing possibility of shifting from genetic to microenvironmental manipulations of cell fate, which would be of particular interest for clinical applications of MSC-based therapies.

The main aim and design of the study was first to determine whether by using 3D-printed microenvironments, MSCs can be directed to differentiate and regenerate SGs both morphologically and functionally. Then, to investigate the underlying molecular mechanism of biochemical and structural cues of 3D-printed matrix involved in MSCs reprogramming. The primary aims of the study design were as follows: (i) cell aggregation and proliferation in a 3D-bioprinted construct; (ii) differentiation of MSCs at the cellular phenotype and functional levels in the 3D-bioprinted construct; (iii) the MET-EMT process during differentiation; (iv) differential protein expression of the SG niche in mice; (v) differential genes expression of MSCs in 3D-bioprinted construct; (vi) the key role of CTHRC1 and HMOX1 in MSCs reprogramming to SGCs; and (vii) functional properties of regenerated SG in vivo.

Gelatin (Sigma-Aldrich, USA) and sodium alginate (Sigma-Aldrich, USA) were dissolved in phosphate-buffered saline (PBS) at 15 and 1% (w/v), respectively. Both solutions were sterilized under 70C for 30 min three times at an interval of 30 min. The sterilized solutions were packed into 50-ml centrifuge tubes, stored at 4C, and incubated at 37C before use.

From wild-type C57/B16 mice (Huafukang Co., Beijing) aged 5 days old, dermal homogenates were prepared by homogenizing freshly collected hairless mouse PD with isotonic phosphate buffer (pH 7.4) for 20 min in an ice bath to obtain 25% (w/v) tissue suspension. The supernatant was obtained after centrifugation at 4C for 20 min at 10,000g. The DNA content was determined using Hoechst 33258 assay (Beyotime, Beijing). The fluorescence intensity was measured to assess the amount of remaining DNA within the decellularized ECMs and the native tissue using a fluorescence spectrophotometer (Thermo Scientific, Evolution 260 Bio, USA). The GAGs content was estimated via 1,9-dimethylmethylene blue solution staining. The absorbance was measured with microplate reader at wavelength of 492 nm. The standard curve was made using chondroitin sulfate A. The total COL (Collagen) content was determined via hydroxyproline assay. The absorbance of the samples was measured at 550 nm and quantified by referring to a standard curve made with hydroxyproline.

MSCs were bioprinted with matrix materials by using an extrusion-based 3D bioprinter (Regenovo Co., Bio-Architect PRO, Hangzhou). Briefly, 10 ml of gelatin solution (10% w/v) and 5 ml of alginate solution (2% w/v) were warmed under 37C for 20 min, gently mixed as bioink and used within 30 min. MSCs were collected from 100-mm dishes, dispersed into single cells, and 200 l of cell suspension was gently mixed with matrix material under room temperature with cell density 1 million ml1. PD (58 g/ml) was then gently mixed with bioink. Petri dishes at 60 mm were used as collecting plates in the 3D bioprinting process. Within a temperature-controlled chamber of the bioprinter, with temperature set within the gelation region of gelatin, the mixture of MSCs and matrix materials was bioprinted into a cylindrical construct layer by layer. The nozzle-insulation temperature and printing chamber temperature were set at 18 and 10C, respectively; nozzles with an inner diameter of 260 m were chosen for printing. The diameter of the cylindrical construct was 30 mm, with six layers in height. After the temperature-controlled bioprinting process, the printed 3D constructs were immersed in 100-mM calcium chloride (Sigma-Aldrich, USA) for 3 min for cross-linking, then washed with Dulbeccos modified Eagle medium (DMEM) (Gibco, USA) medium for three times. The whole printing process was finished in 10 min. The 3D cross-linked construct was cultured in DMEM in an atmosphere of 5% CO2 at 37C. The culture medium was changed to SG medium [contains 50% DMEM (Gibco, New York, NY) and 50% F12 (Gibco) supplemented with 5% fetal calf serum (Gibco), 1 ml/100 ml penicillin-streptomycin solution, 2 ng/ml liothyronine sodium (Gibco), 0.4 g/ml hydrocortisone succinate (Gibco), 10 ng/ml epidermal growth factor (PeproTech, Rocky Hill, NJ), and 1 ml/100 ml insulin-transferrin-selenium (Gibco)] 2 days later. The cell morphology was examined and recorded under an optical microscope (Olympus, CX40, Japan).

Fluorescent live/dead staining was used to determine cell viability in the 3D cell-loaded constructs according to the manufacturers instructions (Sigma-Aldrich, USA). Briefly, samples were gently washed in PBS three times. An amount of 1 M calcein acetoxymethyl (calcein AM) ester (Sigma-Aldrich, USA) and 2 M propidium iodide (Sigma-Aldrich, USA) was used to stain live cells (green) and dead cells (red) for 15 min while avoiding light. A laser scanning confocal microscopy system (Leica, TCSSP8, Germany) was used for image acquisition.

The cell-printed structure was harvested and fixed with a solution of 4% paraformaldehyde. The structure was embedded in optimal cutting temperature (OCT) compound (Sigma-Aldrich, USA) and sectioned 10-mm thick by using a cryotome (Leica, CM1950, Germany). The sliced samples were washed repeatedly with PBS solution to remove OCT compound and then permeabilized with a solution of 0.1% Triton X-100 (Sigma-Aldrich, USA) in PBS for 5 min. To reduce nonspecific background, sections were treated with 0.2% bovine serum albumin (Sigma-Aldrich, USA) solution in PBS for 20 min. To visualize iSGCs, sections were incubated with primary antibody overnight at 4C for anti-K8 (1:300), anti-K14 (1:300), anti-K18 (1:300), anti-K19 (1:300), anti-ATP1a1 (1:300), anti-Ki67 (1:300), antiN-cadherin (1:300), antiE-cadherin (1:300), anti-CTHRC1 (1:300), or anti-TSP1 (1:300; all Abcam, UK) and then incubated with secondary antibody for 2 hours at room temperature: Alexa Fluor 594 goat anti-rabbit (1:300), fluorescein isothiocyanate (FITC) goat anti-rabbit (1:300), FITC goat anti-mouse (1:300), or Alexa Fluor 594 goat anti-mouse (1:300; all Invitrogen, CA). Sections were also stained with 4,6-diamidino-2-phenylindole (Beyotime, Beijing) for 15 min. Stained samples were visualized, and images were captured under a confocal microscope.

To harvest the cells in the construct, the 3D constructs were dissolved by adding 55 mM sodium citrate and 20 mM EDTA (Sigma-Aldrich, USA) in 150 mM sodium chloride (Sigma-Aldrich, USA) for 5 min while gently shaking the petri dish for better dissolving. After transfer to 15-ml centrifuge tubes, the cell suspensions were centrifuged at 200 rpm for 3 min, and the supernatant liquid was removed to harvest cells for further analysis.

Total RNA was isolated from cells by using TRIzol reagent (Invitrogen, USA) following the manufacturers protocol. RNA concentration was measured by using a NanoPhotometer (Implen GmbH, P-330-31, Germany). Reverse transcription involved use of a complementary DNA synthesis kit (Takara, China). Gene expression was analyzed quantitatively by using SYBR green with the 7500 Real-Time PCR System (Takara, China). The primers and probes for genes were designed on the basis of published gene sequences (table S1) (National Center for Biotechnology Information and PubMed). The expression of each gene was normalized to that for glyceraldehyde-3-phosphate dehydrogenase and analyzed by the 2-CT method. Each sample was assessed in triplicate.

The culture medium was changed to SG medium with 2 mM CaCl2 for at least 24 hours, and cells were loaded with fluo-3/AM (Invitrogen, CA) at a final concentration of 5 M for 30 min at room temperature. After three washes with calcium-free PBS, 10 M acetylcholine (Sigma-Aldrich, USA) was added to cells. The change in the Fluo 3 fluorescent signal was recorded under a laser scanning confocal microscopy.

Cell proliferation was evaluated through CCK-8 (Cell counting kit-8) assay. Briefly, cells were seeded in 96-well plates at the appropriate concentration and cultured at 37C in an incubator for 4 hours. When cells were adhered, 10 l of CCK-8 working buffer was added into the 96-well plates and incubated at 37C for 1 hour. Absorbance at 450 nm was measured with a microplate reader (Tecan, SPARK 10M, Austria).

Proteomics of mouse PD and DD involved use of isobaric tags for relative and absolute quantification (iTRAQ) in BGI Company, with differentially expressed proteins detected in PD versus DD. Twofold greater difference in expression was considered significant for further study.

Tissues were grinded and lysed in radioimmunoprecipitation assay buffer (Beyotime, Nanjing). Proteins were separated by 12% SDSpolyacrylamide gel electrophoresis and transferred to a methanol-activated polyvinylidene difluoride membrane (GE Healthcare, USA). The membrane was blocked for 1 hour in PBS with Tween 20 containing 5% bovine serum albumin (Sigma-Aldrich, USA) and probed with the antibodies anti-CTHRC1 (1:1000) and anti-TSP1 (1:1000; both Abcam, UK) overnight at 4C. After 2 hours of incubation with goat anti-rabbit horseradish peroxidaseconjugated secondary antibody (Santa Cruz Biotechnology, CA), the protein bands were detected by using luminal reagent (GE Healthcare, ImageQuant LAS 4000, USA).

Total RNA was prepared with TRIzol (Invitrogen), and RNA sequencing was performed using HiSeq 2500 (Illumina). Genes with false discovery rate < 0.05, fold difference > 2.0, and mean log intensity > 2.0 were considered to be significant.

CAPE or Snpp was gently mixed with bioink at a concentration of 10 M. Physiological concentration of CTHRC1 was measured by enzyme linked immunosorbent assay (ELISA) (80 ng/ml), and then recombinant CTHRC1 or CTHRC1 antibody was added into the bioink at a concentration of 0.4 g/ml. The effect of inhibitor and activator was estimated by qRT-PCR or ELISA.

Mice were anesthetized with pentobarbital (100 mg/kg) and received subcutaneous buprenorphine (0.1 mg/kg) preoperatively. Full-thickness scald injuries were created on paw pads with soldering station (Weller, WSD81, Germany). Mice recovered in clean cages with paper bedding to prevent irritation or infection. Mice were monitored daily and euthanized at 30 days after wounding. Mice were maintained in an Association for Assessment and Accreditation of Laboratory Animal Careaccredited animal facility, and procedures were performed with Institutional Animal Care and Use Committeeapproved protocols.

MSCs in 3D-printed constructs with PD were cultured with DMEM for 2 days and then replaced with SG medium. The SG medium was changed every 2 days, and cells were harvested on day 12. The K18+ iSGCs were sorting through flow cytometry and injected into the paw pads (1 106 cells/50 l) of the mouse burn model by using Microliter syringes (Hamilton, 7655-01, USA). Then, mice were euthanized after 14 days; feet were excised and fixed with 10% formalin (Sigma-Aldrich, USA) overnight for paraffin sections and immunohistological analysis.

The foot pads of anesthetized treated mice were first painted with 2% (w/v) iodine/ethanol solution then with starch/castor oil solution (1 g/ml) (Sigma-Aldrich, USA). After drying, 50 l of 100 M acetylcholine (Sigma-Aldrich, USA) was injected subcutaneously into paws of mice. Pictures of the mouse foot pads were taken after 5, 10, and 15 min.

All data were presented as means SEM. Statistical analyses were performed using GraphPad Prism7 statistical software (GraphPad, USA). Significant differences were calculated by analysis of variance (ANOVA), followed by the Bonferroni test when performing multiple comparisons between groups. P < 0.05 was considered as a statistically significant difference.

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/10/eaaz1094/DC1

Fig. S1. Biocompatibility of 3D-bioprinted construct and cellular morphology in 2D monolayer culture.

Fig. S2. Expression of SG-specific and secretion-related markers in MSCs and SG cells in vitro.

Fig. S3. Transcriptional and translational expression of epithelial and mesenchymal markers in 3D-bioprinted cells with and without PD.

Fig. S4. Expression of N- and E-cadherin in MSCs and SG cells in 2D monolayer culture.

Fig. S5. Proteomic microarray assay of differential gene expression between PD and DD ECM in postnatal mice.

Fig. S6. GO term analysis of differentially expressed pathways.

Fig. S7. Heat maps illustrating differential expression of genes implicated in ECM organization, cell division, and gland and branch morphogenesis.

Fig. S8. The expression of Hmox1 and the concentration of CTHRC1 on treatment and the related effects on cell proliferation.

Fig. S9. The expression of K8 and K18 with Hmox1 and CTHRC1 regulation.

Table S1. Primers for qRT-PCR of all the genes.

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

Acknowledgments: Funding: This study was supported in part by the National Nature Science Foundation of China (81571909, 81701906, 81830064, and 81721092), the National Key Research Development Plan (2017YFC1103300), Military Logistics Research Key Project (AWS17J005), and Fostering Funds of Chinese PLA General Hospital for National Distinguished Young Scholar Science Fund (2017-JQPY-002). Author contributions: B.Y. and S.H. were responsible for the design and primary technical process, conducted the experiments, collected and analyzed data, and wrote the manuscript. Y.W. and R.W. helped perform the main experiments. Y.Z. and T.H. participated in the 3D printing. W.S. and Z.L. participated in cell experiments and postexamination. S.H. and X.F. collectively oversaw the collection of data and data interpretation and revised the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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Biochemical and structural cues of 3D-printed matrix synergistically direct MSC differentiation for functional sweat gland regeneration - Science...

Skin Stem Cells Comments Off on Biochemical and structural cues of 3D-printed matrix synergistically direct MSC differentiation for functional sweat gland regeneration – Science… | March 5th, 2020

I consider my nighttime skincare routine as me time. I always double cleanse; first with Bioderma Sensibio H20 Micellar Water, 10.80, and then I use Rodial Pink Diamond Cleansing Balm, 55, and massage it into my skin. Its a really light and gentle balm that has enough slip for me to be able to easily move it around my face, and it helps dissolve any left over SPF and make-up while also getting rid of the days dirt and grime.

After a good 10-15 minute cleanse, I sweep Pestle and Mortar NMF Lactic Acid Toner, 28, around my face. Lactic acid works underneath the skin, helping to get rid of dead skin cells without dehydrating my skin. Then I press SkinCeuticals HA Intensifer, 90, into my skin to help increase hydration levels.

I use my final product once Im in bed; Decleors Bigarade Neroli Night Balm, 45, which I do a deep pressure massage with and then I finish off using a gua sha to help drain any fluids and relax the muscles.

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3 skin experts share their morning and evening skincare routines - Stylist Magazine

Skin Stem Cells Comments Off on 3 skin experts share their morning and evening skincare routines – Stylist Magazine | March 5th, 2020

New Delhi: The usage of stem cells to cure or treat a disease or repair the injured tissue is defined as stem cell therapy. The best example of the stem cell treatment is seen in patients suffering from restoring the vision of the damaged eyes, grafting of the skin in severe burnt conditions.

Stem cell treatments for brain or neural diseases like Parkinsons and Alzheimers disease, multiple sclerosis, preventing heart strokes, curing diabetes, kidney disorders, autism, and spinal cord injuries are progressively making their way.

Undifferentiated cells that are able to differentiate and transform into any type of cells of the body when and where needed. They have an enormous potential to repair, heal and regenerate. Stem cells come from blood, bone marrow, umbilical cord blood and adipose tissue.

Autologous stem cell therapy: Patient receives stem cells from his/her own body

Allogeneic stem cell therapy: Patient receives the stem cells donated by another individual

Autologous stem cell therapy is better than allogeneic stem cell therapy as chances of mismatching are not there and they pose the minimum risk of immune rejection. Also, no side effects or adverse effects are seen as a persons own blood cells are used. They start the healing process immediately in a natural way.

The usage of stem cells to cure or treat a disease or repair the injured tissue is defined as stem cell therapy. Stem cells can be obtained from the bone marrow, adipose tissues etc. Due to their tremendous potential to prevent and to treat various health conditions and to repair the injured tissues global research investigation is continuously being done as to explore the maximum advantage of these cell lines.

The best example of the stem cell treatment is seen in patients suffering from restoring the vision of the damaged eyes, grafting of the skin in severe burnt conditions. Stem cell treatments for brain or neural diseases like Parkinsons and Alzheimers disease, multiple sclerosis, preventing heart strokes, curing diabetes, kidney disorders, autism, and spinal cord injuries are progressively making their way.

Depending upon the disease, different stem cell source can be used in a specific condition. The procedure may involve the extraction of stem cells from adipose tissue-derived stem cells with the combination of PRP (Platelet-rich plasma) or can be obtained from bone marrow that can differentiate into progenitor cells that differentiate into various other tissues which can help in the therapy.

The stem cells are isolated from the bone marrow or adipose tissues followed by their processing and enrichment under sterile conditions. These activated stem cells are placed back into the patients body at the target site for repairing the damaged tissue. It is necessary that the stem cells are injected in the specific area of injury as only then the desired results will be achieved.

Adipose stem cells are preferred over bone marrow stem cells as they are easy to isolate and contain a higher number of stem cells.

The stem cells injections are gaining much interest because it is devoid of the painful procedure, takes less time in comparison to surgery, there are no host and recipient rejections as stem cells are harvested from the patients body itself and a targeted delivery system is available.

The stem cells obtained are processed in a sophisticated stem cell lab and after activation is inserted back into the host with the help of intravenous, intramuscular, intraarterial, intradermal and intrathecal injections as per the requirement of the treatment process.

What is the use of anaesthetics and why? Usually, local anaesthetics are used during a stem cell procedure to numb the area but sometimes general anaesthesia is also given while extracting the stem cells from bone marrow. But it is necessary to find out what anaesthetic your doctor uses during orthopaedic stem cell treatments.

A number of anaesthetics have been found to kill the stem cells thus; the treatments end result will greatly depend on the use of anaesthetics. Some anaesthetics very well sync with the stem cell and hence, aid in the treatment.

Stem cells are to be extracted and processed in a clean room, under aseptic conditions maintaining a controlled environment. The doctor should explain the entire process and the number of viable stem cells infused into the patient during the process. Also, the precision of the injections to provide good quality of stem cells at the site of injury will help in better and faster recovery of the patients damaged area.

Cost of the treatment and its duration varies from one patient to another. The disease which needs to be cured, the severity, age factor, health condition, etc, define the duration of the therapy. One may respond during the treatment phase itself while the other may show results after a few sessions or weeks. Depending upon the disease diagnosed, the stem cells extracted, duration of the therapy, other adjuvants used in the process, the cost of the stem cell therapy can vary.

It is essential that after the stem cell therapy the patient should visit the stem cell doctor for recuperation therapies. The primary goals of such therapy is the prevention of secondary complications, analysis of the recovery of motor, sensory and all the bodily functioning, psychological support/counselling for depression, mood swings or anxiety etc. and reintegration into the community.

There can be different sets of precautions which need to be followed at various steps for the recovery of the damaged tissues. The treatment and post-treatment conditions may vary from person to person depending upon the disease and the severity.

Stem cell therapy has shown results in treating serious ailments like leukaemia, grafting tissues, autism, orthopaedic conditions and skin problems etc. Stem Cell Therapy has been successfully used in the treatment of around 80 serious disorders.

Survival rates among patients who received stem cell treatment are significantly high, whether cell donors are related or unrelated to them. With the ongoing research around the world, scientists are exploring new possibilities in which a number of life-threatening diseases can be prevented and cured hence, the stem cells have proved to be promising in the near future as many aspects are yet to be revealed.

Read: Egg stem cells do not exist, says researchers

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10 things to know about stem cell therapy - Newsd.in

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A population of stem cells with the ability to generate new bone has been newly discovered by a group of researchers at the UConn School of Dental Medicine.

In the journal STEM CELLS, lead investigator Dr. Ivo Kalajzic, professor of reconstructive sciences, postdoctoral fellows Dr. Sierra Root and Dr. Natalie Wee, and collaborators at Harvard, Maine Medical Research Center, and the University of Auckland present a new population of cells that reside along the vascular channels that stretch across the bone and connect the inner and outer parts of the bone.

This is a new discovery of perivascular cells residing within the bone itself that can generate new bone forming cells, said Kalajzic. These cells likely regulate bone formation or participate in bone mass maintenance and repair.

Stem cells for bone have long been thought to be present within bone marrow and the outer surface of bone, serving as reserve cells that constantly generate new bone or participate in bone repair. Recent studies have described the existence of a network of vascular channels that helped distribute blood cells out of the bone marrow, but no research has proved the existence of cells within these channels that have the ability to form new bones.

In this study, Kalajzic and his team are the first to report the existence of these progenitor cells within cortical bone that can generate new bone-forming cells osteoblasts that can be used to help remodel a bone.

To reach this conclusion, the researchers observed the stem cells within an ex vivo bone transplantation model. These cells migrated out of the transplant, and began to reconstruct the bone marrow cavity and form new bone.

While this study shows there is a population of cells that can help aid bone formation, more research needs to be done to determine the cells potential to regulate bone formation and resorption.

This study was funded by the Regenerative Medicine Research Fund (RMRF; 16-RMB-UCHC-10) by CT Innovations and by National Institute of Arthritis and Musculoskeletal and Skin.

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UConn Researchers Discover New Stem Cells That Can Generate New Bone - UConn Today

Bone Marrow Stem Cells Comments Off on UConn Researchers Discover New Stem Cells That Can Generate New Bone – UConn Today | March 5th, 2020

The Global Stem Cells Market is expected to grow from USD 115.46 Million in 2018 to USD 325.84 Million by the end of 2025 at a Compound Annual Growth Rate (CAGR) of 15.97%.

The Stem Cells Market research presents a study by combining primary as well as secondary research. The report gives insights on the key factors concerned with generating and limiting Stem Cells market growth.

Additionally, the report also studies competitive developments, such as mergers and acquisitions, new partnerships, new contracts, and new product developments in the global Stem Cells market. The past trends and future prospects included in this report makes it highly comprehensible for the analysis of the market. Moreover, the latest trends, product portfolio, demographics, geographical segmentation, and regulatory framework of the Stem Cells market have also been included in the study.

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Stem Cells Market Segment by Manufacturers includes: The report deeply explores the recent significant developments by the leading vendors and innovation profiles in the Global Stem Cells Market including are Anterogen Co., Ltd., Holostem Terapie Avanzate Srl, Medipost Co., Ltd., Osiris Therapeutics, Inc., Pharmicell Co., Ltd., Allosource, JCR Pharmaceuticals Co., Ltd., Nuvasive, Inc., and RTI Surgical, Inc.. On the basis of Cell Source, the Global Stem Cells Market is studied across Adipose Tissue-Derived Mesenchymal Stem Cells, Bone Marrow-Derived Mesenchymal Stem Cells, and Cord Blood/Embryonic Stem Cells.On the basis of Type, the Global Stem Cells Market is studied across Allogeneic Stem Cell Therapy and Autologous.On the basis of Therapeutic Application , the Global Stem Cells Market is studied across Cardiovascular Diseases, Gastrointestinal Diseases, Musculoskeletal Disorders, Surgeries, and Wounds and Injuries.

Global Stem Cells market report covers all the major participants and the retailers will be in conscious of the development factors, market barriers & threats, and the opportunities that the market will offer in the near future. The report also features the historical revenue of the market; industry trends, market volume, and consumption in order to gain perceptions about the political and technical environment of the Stem Cells market share.

This report focuses on the Stem Cells in Global market, especially in

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The report gives detailed analysis in terms of qualitative and quantitative data pertaining to the projected potential opportunities that influence markets growth for the forecast period. With a major focus on the key elements and segments of the global Stem Cells market that might affect the growth prospects of the market, making it a highly informative document.

Major Points covered in this Report:

Market Segmentation:

Regional market analysis

The content of the study subjects includes a total of 15 chapters:

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About Us:We, Regal Intelligence, aim to change the dynamics of market research backed by quality data. Our analysts validate data with exclusive qualitative and analytics driven intelligence. We meticulously plan our research process and execute in order to explore the potential market for getting insightful details. Our prime focus is to provide reliable data based on public surveys using data analytics techniques. If you have come here, you might be interested in highly reliable data driven market insights for your product/service,reach us here 24/7.

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Stem Cells Market Top Impacting Factors to Growth of the Industry by 2025 - Bandera County Courier

Bone Marrow Stem Cells Comments Off on Stem Cells Market Top Impacting Factors to Growth of the Industry by 2025 – Bandera County Courier | March 5th, 2020

VANCOUVER, Washington, March 04, 2020 (GLOBE NEWSWIRE) -- CytoDyn Inc. (CYDY), (CytoDyn or the Company"), a late-stage biotechnology company developing leronlimab (PRO 140), a CCR5 antagonist with the potential for multiple therapeutic indications, announced today the treatment of the first patient in its Phase 2 clinical trial for graft-versus-host disease (GvHD) under the modified trial protocol.

The modified protocol now includes reduced intensity conditioning (RIC) patients and an open-label design under which all enrollees receive leronlimab. The modified protocol also provides for a 50% increase in the dose of leronlimab to more closely mimic preclinical dosing. The next review of data by the independent data monitoring committee (iDMC) will occur following enrollment of 10 patients under the amended protocol after each patient has been dosed for 30 days.

Nader Pourhassan, Ph.D., president and chief executive officer of CytoDyn, added, GvHD is a life-threatening complication following bone marrow transplantation in patients with leukemia who have compromised immune systems due to treatment with aggressive cancer therapies. We selected GvHD as one of our immunology indications for leronlimab, as it targets and masks the CCR5 receptor on T cells. This receptor on T cells is an important mediator of inflammatory diseases including GvHD, especially in organ damage that is the most frequent cause of death in these patients. Dr. Pourhassan concluded that, Based upon the compelling results in our preclinical studies, we are optimistic about the opportunities for leronlimab to provide a therapy for transplant patients to mitigate GvHD.

The Companys preclinical study by Denis R. Burger, Ph.D., CytoDyns former Chief Science Officer, and Daniel Lindner, M.D., Ph.D. of the Department of Translational Hematology and Oncology Research, The Cleveland Clinic, was published online in the peer-reviewed journal Biology of Blood and Marrow Transplantation.

The Company previously reported that the U.S. Food and Drug Administration (FDA) granted orphan drug designation to leronlimab (PRO 140) for the prevention of GvHD. Orphan drug designation is granted to development-stage drugs that have shown promise in addressing serious medical needs for patients living with rare conditions. This designation provides CytoDyn with various incentives and benefits including seven years of U.S. market exclusivity for leronlimab (PRO 140) in GvHD, subject to FDA approval for use in this indication.

About Graft-versus-Host Disease (GvHD)Graft-versus-host disease is a risk when patients receive the transplant of bone marrow stem cells donated from another person. GvHD occurs when the donors immune cells attack the patients normal cells. GvHD can be acute or chronic. Its severity depends on the differences in tissue type between patient and donor. The older the patient, the more frequent and serious the reaction may be. Acute GvHD can occur soon after the transplanted cells begin to appear in the recipient and can range from mild, moderate or severe, and be life-threatening if its effects are not controlled. Certain approved drugs exist that can help prevent or lessen GvHD. However, GvHD does not always respond to these treatments, and it can still result in fatal outcomes. Furthermore, many deaths related to GvHD occur because of infections that develop in patients whose immune systems are suppressed by such drugs.

About Leronlimab (PRO 140)The U.S. Food and Drug Administration (FDA) has granted a Fast Track designation to CytoDyn for two potential indications of leronlimab for deadly diseases. The first as a combination therapy with HAART for HIV-infected patients and the second is for metastatic triple-negative breast cancer. Leronlimab is an investigational humanized IgG4 mAb that blocks CCR5, a cellular receptor that is important in HIV infection, tumor metastases, and other diseases including NASH. Leronlimab has successfully completed nine clinical trials in over 800 people, including meeting its primary endpoints in a pivotal Phase 3 trial (leronlimab in combination with standard antiretroviral therapies in HIV-infected treatment-experienced patients).

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In the setting of HIV/AIDS, leronlimab is a viral-entry inhibitor; it masks CCR5, thus protecting healthy T cells from viral infection by blocking the predominant HIV (R5) subtype from entering those cells. Leronlimab has been the subject of nine clinical trials, each of which demonstrated that leronlimab can significantly reduce or control HIV viral load in humans. The leronlimab antibody appears to be a powerful antiviral agent leading to potentially fewer side effects and less frequent dosing requirements compared with daily drug therapies currently in use.

In the setting of cancer, research has shown that CCR5 plays an important role in tumor invasion and metastasis. Increased CCR5 expression is an indicator of disease status in several cancers. Published studies have shown that blocking CCR5 can reduce tumor metastases in laboratory and animal models of aggressive breast and prostate cancer. Leronlimab reduced human breast cancer metastasis by more than 98% in a murine xenograft model. CytoDyn is therefore conducting aPhase 1b/2 human clinical trial in metastatic triple-negative breast cancer and was granted Fast Track designation in May 2019. Additional research is being conducted with leronlimab in the setting of cancer and NASH with plans to conduct additionalclinical studies when appropriate.

The CCR5 receptor appears to play a central role in modulating immune cell trafficking to sites of inflammation and may be important in the development of acute GvHD and other inflammatory conditions. Clinical studies by others further support the concept that blocking CCR5 using a chemical inhibitor can reduce the clinical impact of acute GvHD without significantly affecting the engraftment of transplanted bone marrow stem cells. CytoDyn is currently conducting a Phase 2 clinical study with leronlimab to further support the concept that the CCR5 receptor on engrafted cells is critical for the development of acute GvHD and that blocking this receptor from recognizing certain immune signaling molecules is a viable approach to mitigating acute GvHD. The FDA has granted orphan drug designation to leronlimab for the prevention of GvHD.

About CytoDynCytoDyn is a biotechnology company developing innovative treatments for multiple therapeutic indications based on leronlimab, a novel humanized monoclonal antibody targeting the CCR5 receptor. CCR5 appears to play a key role in the ability of HIV to enter and infect healthy T-cells. The CCR5 receptor also appears to be implicated in tumor metastasis and in immune-mediated illnesses, such as GvHD and NASH. CytoDyn has successfully completed a Phase 3 pivotal trial with leronlimab in combination with standard antiretroviral therapies in HIV-infected treatment-experienced patients. CytoDyn plans to seek FDA approval for leronlimab in combination therapy and plans to complete the filing of a Biologics License Application (BLA) in the first quarter of 2020 for that indication. CytoDyn is also conducting a Phase 3 investigative trial with leronlimab as a once-weekly monotherapy for HIV-infected patients and plans to initiate a registration-directed study of leronlimab monotherapy indication, which if successful, could support a label extension. Clinical results to date from multiple trials have shown that leronlimab can significantly reduce viral burden in people infected with HIV with no reported drug-related serious adverse events (SAEs). Moreover, results from a Phase 2b clinical trial demonstrated that leronlimab monotherapy can prevent viral escape in HIV-infected patients, with some patients on leronlimab monotherapy remaining virally suppressed for more than five years. CytoDyn is also conducting a Phase 2 trial to evaluate leronlimab for the prevention of GvHD and a Phase 1b/2 clinical trial with leronlimab in metastatic triple-negative breast cancer. More information is atwww.cytodyn.com.

Forward-Looking StatementsThis press releasecontains certain forward-looking statements that involve risks, uncertainties and assumptions that are difficult to predict. Words and expressions reflecting optimism, satisfaction or disappointment with current prospects, as well as words such as believes, hopes, intends, estimates, expects, projects, plans, anticipates and variations thereof, or the use of future tense, identify forward-looking statements, but their absence does not mean that a statement is not forward-looking. The Companys forward-looking statements are not guarantees of performance, and actual results could vary materially from those contained in or expressed by such statements due to risks and uncertainties including: (i)the sufficiency of the Companys cash position, (ii)the Companys ability to raise additional capital to fund its operations, (iii) the Companys ability to meet its debt obligations, if any, (iv)the Companys ability to enter into partnership or licensing arrangements with third parties, (v)the Companys ability to identify patients to enroll in its clinical trials in a timely fashion, (vi)the Companys ability to achieve approval of a marketable product, (vii)the design, implementation and conduct of the Companys clinical trials, (viii)the results of the Companys clinical trials, including the possibility of unfavorable clinical trial results, (ix)the market for, and marketability of, any product that is approved, (x)the existence or development of vaccines, drugs, or other treatments that are viewed by medical professionals or patients as superior to the Companys products, (xi)regulatory initiatives, compliance with governmental regulations and the regulatory approval process, (xii)general economic and business conditions, (xiii)changes in foreign, political, and social conditions, and (xiv)various other matters, many of which are beyond the Companys control. The Company urges investors to consider specifically the various risk factors identified in its most recent Form10-K, and any risk factors or cautionary statements included in any subsequent Form10-Q or Form8-K, filed with the Securities and Exchange Commission. Except as required by law, the Company does not undertake any responsibility to update any forward-looking statements to take into account events or circumstances that occur after the date of this press release.

CYTODYN CONTACTSInvestors: Dave Gentry, CEORedChip CompaniesOffice: 1.800.RED.CHIP (733.2447)Cell: 407.491.4498dave@redchip.com

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CytoDyn Treats First Patient with Leronlimab in Phase 2 Trial for GvHD under Modified Trial Protocol - Yahoo Finance

Bone Marrow Stem Cells Comments Off on CytoDyn Treats First Patient with Leronlimab in Phase 2 Trial for GvHD under Modified Trial Protocol – Yahoo Finance | March 5th, 2020

After performing well on the stock market last year -- with shares climbing by 37.3% -- Incyte's (NASDAQ:INCY) stock is down by 11% year to date. And while it would be easy to attribute this poor performance to the COVID-19 epidemic -- which has now spread to more than 50 countries and is hurting the stock market -- the fact is, Incyte's struggles predate these developments.In early January, before most of us had even heard of the coronavirus, Incyte's shares dropped by about 12% after the company reported disappointing results from a pivotal phase 3 clinical trial.

The clinical trial investigated the efficacy of itacitinib and corticosteroids as a combination treatment for treatment-naive acute graft-versus-host disease (GVHD), a condition that can develop in a patient following a stem cell transplant. The treatment failed to meet its primary or secondary endpoints. Despite this setback, Incyte has a plan to get back on the right track, and here are two things that could help the company do just that.

Image source: Getty Images.

Incyte's top selling-product is Jakafi, which treats several conditions, including a rare bone marrow cancer called myelofibrosis. Jakafi also treats patients with polycythemia vera, a condition that leads to an abnormal increase in the production of red blood cells.Lastly, in May 2019, the U.S. Food and Drug Administration (FDA) approved Jakafi for the treatment of steroid-refractory acute GVHD, a condition that occurs when a patient receives a stem cell transplant and the donor's cells trigger an immune response and attack the recipient's organs.Incidences of this condition number about 5,700 cases a year.

Also, steroid-refractory acute GVHD has a one-year mortality rate of about 70%.Jakafi is the first and only FDA-approved treatment for steroid-refractory acute GVHD. Thanks to this relatively new indication, sales of Jakafi could continue growing, as they have been doing for the past few years. During the fourth quarter, Jakafi's net product revenue was $466.5 million, 23% higher than the year-ago period. For the full year, Jakafi's net product revenue was $1.7 billion, a 21% increase compared to 2018.According to Incyte's executive vice president, Barry P. Flannelly, "Patient demand continued to drive the uptake of Jakafi and growth was strong across all three indications."

Furthermore, Incyte collects royalty revenues from Novartis (NYSE:NVS), which holds the rights to Jakafi outside the U.S. Incyte's royalty revenue for Jakafi for the fourth quarter and the full year were $65 million and $225.9 million, respectively, which represented an increase of 17% for the fourth quarter and 16% for the full year.

According to Incyte, Jakafi has been growing its revenue at a compound annual growth rate of 29% since 2016. And the company hopes its crown jewel will continue performing well in the future. Incyte's CEO Herve Hoppenot said, "On the commercial side, we will work to drive continued Jakafi growth in all three indications."

While Jakafi is performing well, Incyte does rely heavily on this product. During the fourth quarter, Jakafi's net product revenue accounted for about 80.5% of the company's total revenue. Fortunately, Incyte is trying to decrease its top-line exposure to its top-selling drug. In February, Incyte submitted capmatinib to the FDA as a potential treatment for an aggressive type of non-small cell lung cancer (NSCLC) called metastatic MET exon 14 skipping (METex14) mutated NSCLC.

There are currently no approved therapies that specifically target this type of NSCLC, which occurs in 3% to 4% of advanced NSCLC cases. Lung cancer is the most common type of cancer in the world, and NSCLC is the most common form of lung cancer. The FDA granted capmatinib a priority review designation, which means the review process for this drug will go faster than usual.

Also, in November 2019, Incyte submitted a New Drug Application to the FDA for pemigatinib as a potential treatment for cholangiocarcinoma, a rare cancer that affects about 0.3 to 3.4 per 100,000 people in North America and Europe. The FDA also granted pemigatinib priority review.In addition to those products that are currently being reviewed by regulatory authorities, Incyte boasts several more pipeline candidates for a variety of other conditions.This could help the company become less reliant on Jakafi in the future.

Incyte's heavy reliance on Jakafi remains a concern, and for that reason, Incyte probably isn't a strong buy. However, Jakafi's revenue should continue climbing, and unless Incyte runs into regulatory roadblocks, it should have several more products to drive its sales even higher. In short, investors should keep an eye on this biotech company.

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These 2 Things Will Help Incytes Stock Rebound - Motley Fool

Bone Marrow Stem Cells Comments Off on These 2 Things Will Help Incytes Stock Rebound – Motley Fool | March 5th, 2020

The latest Stem Cell Therapy market study offers an all-inclusive analysis of the major strategies, corporate models, and market shares of the most noticeable players in this market. The study offers a thorough analysis of the key persuading factors, market figures in terms of revenues, segmental data, regional data, and country-wise data. This study can be described as most wide-ranging documentation that comprises all the aspects of the evolving Stem Cell Therapy market.

The research report provides deep insights into the global market revenue, parent market trends, macro-economic indicators, and governing factors, along with market attractiveness per market segment. The report provides an overview of the growth rate of Stem Cell Therapy market during the forecast period, i.e., 20202027. Most importantly, the report further identifies the qualitative impact of various market factors on market segments and geographies. The research segments the market on the basis of product type, application, technology, and region. To offer more clarity regarding the industry, the report takes a closer look at the current status of various factors including but not limited to supply chain management, niche markets, distribution channel, trade, supply, and demand and production capability across different countries.

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Stem cell therapy is a technique which uses stem cells for the treatment of various disorders. Stem cell therapy is capable of curing broad spectrum of disorders ranging from simple to life threatening. These stem cells are obtained from different sources, such as, adipose tissue, bone marrow, embryonic stem cell and cord blood among others. Stem cell therapy is enables to treat more than 70 disorders, including degenerative as well as neuromuscular disorders. The ability of a stem cell to renew itself helps in replacing the damaged areas in the human body.

MARKET DYNAMICSIncrease in the number of stem cell banking facilities and rising awareness on the benefits of stem cell for curing various disorders are expected to drive the market during the forecast period. Rise in number of regulations to promote stem cell therapy and increase in number of funds for research in developing countries are expected to offer growth opportunities to the market during the coming years.

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The study conducts SWOT analysis to evaluate strengths and weaknesses of the key players in the Stem Cell Therapy market. Further, the report conducts an intricate examination of drivers and restraints operating in the market. The report also evaluates the trends observed in the parent market, along with the macro-economic indicators, prevailing factors, and market appeal according to different segments. The report also predicts the influence of different industry aspects on the Stem Cell Therapy market segments and regions.

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Stem Cell Therapy Market Segmented by Region/Country: North America, Europe, Asia Pacific, Middle East & Africa, and Central & South America

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Stem Cell Therapy Market 2020 To 2027-Expanding Worldwide with Top Players Future Business Scope and Investment Analysis Report - Monroe Scoop

Bone Marrow Stem Cells Comments Off on Stem Cell Therapy Market 2020 To 2027-Expanding Worldwide with Top Players Future Business Scope and Investment Analysis Report – Monroe Scoop | March 5th, 2020