MENLO PARK, Calif. and NEW YORK, March 11, 2020 (GLOBE NEWSWIRE) -- Forty Seven Inc. (Nasdaq: FTSV) and Rocket Pharmaceuticals Inc. (Nasdaq: RCKT) announced today that they have entered into a research collaboration to pursue clinical proof-of-concept for Forty Sevens novel antibody-based conditioning regimen, FSI-174 (anti-cKIT antibody) plus magrolimab (anti-CD47 antibody), with Rockets ex vivo lentiviral vector hematopoietic stem cell (LVV HSC) gene therapy, RP-L102. The initial collaboration will evaluate this treatment regimen in Fanconi Anemia (FA), a genetic disease that affects patients capacity to produce blood cells and is associated with an increased risk of leukemia and other neoplasms. RP-L102, Rockets gene therapy approach for FA, involves treatment with patients own gene-corrected blood forming stem cells (hematopoietic stem cells, or HSCs).

Gene therapies for monogenic blood disorders have broad potential. One concern associated with these treatments is the toxicity of pre-therapy conditioning regimens that utilize cytotoxic chemotherapy and/or radiation to destroy existing HSCs and facilitate engraftment of gene-corrected HSCs. Forty Sevens all-antibody based conditioning regimen is designed to address the limitations of current pre-treatment conditioning therapies. These regimens are often associated with serious side effects, including severe infection, cognitive impairment, infertility, endocrine dysfunction, secondary malignancies and organ damage. These toxicities are especially difficult for pediatric patients and are particularly severe for patients with FA, who are more sensitive to the DNA-damaging effects of traditional conditioning agents. Preliminary data demonstrate that RP-L102 may confer efficacy without pre-treatment conditioning. The combination of RP-L102 with Forty Sevens all-antibody conditioning regimen may provide patients an alternate treatment option in situations where conditioning may be advantageous.

We are pleased to enter into this collaboration with Forty Seven, said Jonathan Schwartz, M.D., Chief Medical Officer and Senior Vice President of Rocket. RP-L102 Process B is currently being evaluated in a registrational trial without the use of conditioning. In parallel, we are assessing incorporation of a non-genotoxic conditioning regimen as a part of Rockets life-cycle management strategy. Forty Sevens novelall-antibodyconditioning regimen could also beapplied to Rockets other lentiviral programs, in which conditioning is more integral to the gene therapy approach.

We are initiating our first in human healthy volunteer study of FSI-174 in the first quarter this year, and are excited to enter into a partnership with Rocket at this time. Rocket is at the forefront of developing gene therapies for high unmet-need diseases, and this collaboration will provide an opportunity to evaluate the benefit of Forty Sevens novel conditioning regimen with Rockets RP-L102 to help FA patients, says Jens-Peter Volkmer, VP of Research at Forty Seven.

This collaboration is in line with our strategy to study our anti-cKIT and anti-CD47, all-antibody conditioning regimen in combination with several different gene therapies, and to establish clinical proof-of-concept in a broad range of transplant indications, said Mukul Agarwal, VP of Corporate Development at Forty Seven.

Maria Grazia Roncarolo, M.D., Scientific Advisor to Forty Seven, commented, The goal of my lifes work is to bring pediatric patients transformative therapies for currently incurable diseases. We believe Rocket Pharmaceuticals commitment to devastating diseases, such as FA, addresses a critical unmet need and Forty Sevens antibody conditioning creates an alternative avenue to deliver this therapy to those patients. We look forward to seeing how this collaboration may help patients in need.

Under the terms of the agreement, Rocket will provide its ex vivo LVV HSC gene therapy platform and Forty Seven will contribute its innovative antibody-based conditioning regimen for the collaboration.

About FSI-174 and MagrolimabFSI-174 is a humanized monoclonal antibody targeting cKIT, which is a receptor that is highly expressed on hematopoietic stem cells. Magrolimab is a humanized monoclonal antibody targeting CD47, which is a dont eat me signal to macrophages and is expressed on all cells. Magrolimab is currently being investigated in Phase 2 clinical trials to treat cancer and has established clinical efficacy in four indications, including myelodysplastic syndrome, acute myeloid leukemia, diffuse large B cell lymphoma and follicular lymphoma, with a favorable safety profile in over 400 patients treated, including some patients treated continuously for over two years. When combined, FSI-174 sends a positive signal to macrophages to target blood forming stem cells for removal and magrolimab disengages inhibitory signals that block phagocytosis. Combination of these antibodies has shown efficient removal of blood forming stem cells, allowing for transplantation in pre-clinical models.

About Fanconi Anemia Fanconi Anemia (FA) is a rare pediatric disease characterized by bone marrow failure, malformations and cancer predisposition. The primary cause of death among patients with FA is bone marrow failure, which typically occurs during the first decade of life. Allogeneic hematopoietic stem cell transplantation (HSCT), when available, corrects the hematologic component of FA, but requires myeloablative conditioning. Graft-versus-host disease, a known complication of allogeneic HSCT, is associated with an increased risk of solid tumors, mainly squamous cell carcinomas of the head and neck region. Approximately 60-70% of patients with FA have aFANC-Agene mutation, which encodes for a protein essential for DNA repair. Mutation in theFANC-Agene leads to chromosomal breakage and increased sensitivity to oxidative and environmental stress. Chromosome fragility induced by DNA-alkylating agents such as mitomycin-C (MMC) or diepoxybutane (DEB) is the gold standard test for FA diagnosis. Somatic mosaicism occurs when there is a spontaneous correction of the mutated gene that can lead to stabilization or correction of a FA patients blood counts in the absence of any administered therapy. Somatic mosaicism, often referred to as natural gene therapy provides a strong rationale for the development of FA gene therapy because of the selective growth advantage of gene-corrected hematopoietic stem cells over FA cells1.

1Soulier, J.,et al. (2005) Detection of somatic mosaicism and classification of Fanconi anemia patients by analysis of the FA/BRCA pathway. Blood 105: 1329-1336

About Rocket Pharmaceuticals, Inc. Rocket Pharmaceuticals, Inc. (Nasdaq: RCKT) (Rocket) is advancing an integrated and sustainable pipeline of genetic therapies that correct the root cause of complex and rare childhood disorders. The companys platform-agnostic approach enables it to design the best therapy for each indication, creating potentially transformative options for patients contending with rare genetic diseases. Rocket's clinical programs using lentiviral vector (LVV)-based gene therapy are for the treatment of Fanconi Anemia (FA), a difficult to treat genetic disease that leads to bone marrow failure and potentially cancer, Leukocyte Adhesion Deficiency-I (LAD-I), a severe pediatric genetic disorder that causes recurrent and life-threatening infections which are frequently fatal, and Pyruvate Kinase Deficiency (PKD) a rare, monogenic red blood cell disorder resulting in increased red cell destruction and mild to life-threatening anemia. Rockets first clinical program using adeno-associated virus (AAV)-based gene therapy is for Danon disease, a devastating, pediatric heart failure condition. Rockets pre-clinical pipeline program is for Infantile Malignant Osteopetrosis (IMO), a bone marrow-derived disorder. For more information about Rocket, please visitwww.rocketpharma.com.

For more information, please visit http://www.rocketpharma.com or contact info@rocketpharma.com

About Forty Seven, Inc.Forty Seven, Inc.is a clinical-stage immuno-oncology company that is developing therapies targeting cancer immune evasion pathways based on technology licensed fromStanford University. Forty Sevens lead program, magrolimab, is a monoclonal antibody against the CD47 receptor, a dont eat me signal that cancer cells commandeer to avoid being ingested by macrophages. This antibody is currently being evaluated in multiple clinical studies in patients with myelodysplastic syndrome, acute myeloid leukemia, and non-Hodgkins lymphoma.

For more information, please visitwww.fortyseveninc.comor contactinfo@fortyseveninc.com.

Follow Forty Seven on social media:@FortySevenInc,LinkedIn

Rocket Cautionary Statement Regarding Forward-Looking StatementsVarious statements in this release concerning Rocket's future expectations, plans and prospects, including without limitation, Rocket's expectations regarding the safety, effectiveness and timing of product candidates that Rocket may develop, to treat Fanconi Anemia (FA), Leukocyte Adhesion Deficiency-I (LAD-I), Pyruvate Kinase Deficiency (PKD), Infantile Malignant Osteopetrosis (IMO) and Danon Disease, and the safety, effectiveness and timing of related pre-clinical studies and clinical trials, may constitute forward-looking statements for the purposes of the safe harbor provisions under the Private Securities Litigation Reform Act of 1995 and other federal securities laws and are subject to substantial risks, uncertainties and assumptions. You should not place reliance on these forward-looking statements, which often include words such as "believe," "expect," "anticipate," "intend," "plan," "will give," "estimate," "seek," "will," "may," "suggest" or similar terms, variations of such terms or the negative of those terms. Although Rocket believes that the expectations reflected in the forward-looking statements are reasonable, Rocket cannot guarantee such outcomes. Actual results may differ materially from those indicated by these forward-looking statements as a result of various important factors, including, without limitation, Rocket's ability to successfully demonstrate the efficacy and safety of such products and pre-clinical studies and clinical trials, its gene therapy programs, the preclinical and clinical results for its product candidates, which may not support further development and marketing approval, the potential advantages of Rocket's product candidates, actions of regulatory agencies, which may affect the initiation, timing and progress of pre-clinical studies and clinical trials of its product candidates, Rocket's and its licensors ability to obtain, maintain and protect its and their respective intellectual property, the timing, cost or other aspects of a potential commercial launch of Rocket's product candidates, Rocket's ability to manage operating expenses, Rocket's ability to obtain additional funding to support its business activities and establish and maintain strategic business alliances and new business initiatives, Rocket's dependence on third parties for development, manufacture, marketing, sales and distribution of product candidates, the outcome of litigation, and unexpected expenditures, as well as those risks more fully discussed in the section entitled "Risk Factors" in Rocket's Annual Report on Form 10-K for the year ended December 31, 2019, filed March 6, 2020 with the SEC. Accordingly, you should not place undue reliance on these forward-looking statements. All such statements speak only as of the date made, and Rocket undertakes no obligation to update or revise publicly any forward-looking statements, whether as a result of new information, future events or otherwise.

Forty Seven Cautionary Statement Regarding Forward-Looking StatementsStatements contained in this press release regarding matters that are not historical facts are "forward-looking statements" within the meaning of the Private Securities Litigation Reform Act of 1995. Words such as will, may, assess, could, believe, and similar expressions (as well as other words or expressions referencing future events, conditions, or circumstances) are intended to identify forward-looking statements. These statements include those related to the research and development plans for Rockets and Forty Sevens respective platforms and product candidates, the timing and success of Forty Sevens collaboration with Rocket, Forty Sevens plans to pursue clinical proof-of-concept for FSI-174 plus magrolimab with the LVV HSC gene therapy platform, the focus on diseases that have the potential to be corrected with the combination of RP-L102 and Forty Sevens all-antibody conditioning regimen, the tolerability and efficacy of RP-L102, FSI-174 and magrolimab, the timing and success of any future collaborations between Forty Seven and Rocket, Forty Sevens plans to continue development of FSI-174 plus magrolimab, as well as related timing for clinical trials of the same.

Because such statements are subject to risks and uncertainties, actual results may differ materially from those expressed or implied by such forward-looking statements. The product candidates that Forty Seven develops may not progress through clinical development or receive required regulatory approvals within expected timelines or at all.In addition, clinical trials may not confirm any safety, potency or other product characteristics described or assumed in this press release. Such product candidates may not be beneficial to patients or successfully commercialized. The failure to meet expectations with respect to any of the foregoing matters may have a negative effect on Forty Seven's stock price. Additional information concerning these and other risk factors affecting Forty Seven's business can be found in Forty Seven's periodic filings with theSecurities and Exchange Commissionatwww.sec.gov. These forward-looking statements are not guarantees of future performance and speak only as of the date hereof, and, except as required by law, Forty Seven disclaims any obligation to update these forward-looking statements to reflect future events or circumstances.

Forty SevenInvestors:Hannah Deresiewicz, (212) 362-1200hannah.deresiewicz@sternir.com

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Media:Sarah Plumridge, (312) 506-5218fortyseven@hdmz.com

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Forty Seven and Rocket Pharmaceuticals Announce Research Collaboration for Fanconi Anemia - BioSpace

Bone Marrow Stem Cells Comments Off on Forty Seven and Rocket Pharmaceuticals Announce Research Collaboration for Fanconi Anemia – BioSpace | March 12th, 2020

The College of Engineering for the first time has five faculty members who have been awarded National Science Foundations (NSF) Faculty Early Career Development (CAREER) grants. Four of the recipients of the five-year grants, Lauren B. Andrews,Peter J. Beltramo,Jungwoo Leeand Sarah L. Perry, are assistant proferssors in chemical engineering, while Xian Duis an assistant professor in mechanical and industrial engineering.

Sanjay Raman, dean of the College of Engineering, welcomed news of the grants. These awards are a testimony to the remarkable potential of these early-career UMass engineering faculty, he says. They are also the product of strong faculty development programs at the college and university levels, and outstanding mentorship by colleagues across the college. We look forward to the impactful research and educational innovations of these rising stars in emerging areas such as therapeutics and vaccine development, tissue engineering, biomanufacturing, biosensors and flexible electronics.

Du is the principal investigator on a $571,655 grant that focuses on improvements in roll-to-roll soft lithography. He is establishing a learning-based modeling method that guides the design and control of continuous microcontact printing processes and investigates continuous pattern formation mechanisms.

Andrews, the Marvin and Eva Schlanger Faculty Fellow in chemical engineering, will do researchstudying how communities of bacteria can be engineered to have coordinated behaviors. This will have numerous applications in biomanufacturing, cell-based therapies, and medical diagnostics. Andrewss $589,060 grant will fund research into developing a new approach for effectively programming how cells in a bacterial community work together in a predictive and highly controllable way.

Beltramos $592,332 grant will support his work on understanding the interplay between lipid composition and biomolecule transport in biological membranes. This is fundamental research that could enable the development of such breakthroughs as advanced drug delivery systems, biosensors, and other biomimetic materials.

Lee says his $549,710 grant will fund research that could lead to a greater understanding through which bone remodeling and blood forming processes are functionally coupled in porus, or trabecular bone cavities, by creating tissue engineered stem cell bone marrow models.

Perrys $657,920 grant will fund a study of a groundbreaking new approach to protein stabilization based on nature-inspired strategies. Her research has the ultimate goal of boosting the accessibility of vaccines and other therapeutics, especially in developing countries, and extending the reach of temperature-stable proteins to sensing and catalysis applications.

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Five College of Engineering Faculty Win NSF CAREER Grants - UMass News and Media Relations

Bone Marrow Stem Cells Comments Off on Five College of Engineering Faculty Win NSF CAREER Grants – UMass News and Media Relations | March 12th, 2020

PERTH, Australia Australian stem cell therapy company Mesoblast Ltd. plans to evaluate its allogeneic mesenchymal stem cell (MSC) candidate, remestemcel-L, in patients with acute respiratory distress syndrome (ARDS) caused by coronavirus (COVID-19) in the U.S., Australia, China and Europe.

The company is in active discussions with various governments, regulatory authorities, medical institutions and pharmaceutical companies to implement these activities.

What people are dying of is acute respiratory distress syndrome, which is the bodys immune response to the virus in the lungs, and the immune system goes haywire, and in its battle with the virus it overreacts and causes severe damage to the lungs, Mesoblast CEO Silviu Itescu told BioWorld.

Were going to be evaluating whether an injection of our cells intravenously can tone down the immune system just enough so it gets rid of the virus but doesnt destroy your lungs at the same time.

Recently published results from an investigator-initiated clinical study conducted in China reported that allogeneic MSCs cured or significantly improved functional outcomes in all seven treated patients with severe COVID-19 pneumonia.

We have now looked at our own data in lung disease in adults where half the patients had the same kind of inflammation in the lungs as you get with coronavirus, and our cells significantly reduced the inflammation and significantly improved lung function, Itescu said, noting that he is awaiting emergency use authorization to treat patients under a clinical trial protocol.

In a post-hoc analyses of a 60-patient randomized controlled study in chronic obstructive pulmonary disease (COPD), remestemcel-L infusions were well-tolerated, significantly reduced inflammatory biomarkers, and significantly improved pulmonary function in those patients with elevated inflammatory biomarkers.

Since the same inflammatory biomarkers are also elevated in COVID-19, those data suggest that remestemcel-L could be useful in the treatment of patients with ARDS due to COVID-19. The COPD study results have been submitted for presentation at an international conference, with full results to be submitted for publication shortly.

Mortality in COVID-19-infected patients with the inflammatory lung condition is reported to approach 50% and is associated with older age, co-morbidities such as diabetes, higher disease severity, and elevated markers of inflammation.

Current therapeutic interventions do not appear to be improving in-hospital survival, and remestemcel-L has potential for use in the treatment of ARDS, which is the principal cause of death in COVID-19 infection.

Itescu said he didnt know of any other stem cell companies that were doing this. He said that other companies could try the approach from a research perspective but that Mesoblast has all the patents locked down.

The companys intellectual property portfolio encompasses more than 1,000 patents or patent applications in all major markets and includes the use of MSCs obtained from any source for patients with ARDS, and for inflammatory lung disease due to coronavirus (COVID-19), influenza and other viruses.

Remestemcel-L is being studied in numerous clinical trials across several inflammatory conditions, including in elderly patients with lung disease and adults and children with steroid-refractory acute graft-vs.-host disease (aGVHD).

Mesoblasts stem cell therapy is currently being reviewed by the FDA for potential approval in the treatment of children with steroid-refractory aGVHD. The company submitted the final module of a rolling BLA in January.

Remestemcel-L is being developed for rare pediatric and adult inflammatory conditions. It is an investigational therapy comprising culture-expanded MSCs derived from the bone marrow of an unrelated donor and is administered in a series of intravenous infusions.

The stem cell therapy is believed to have immunomodulatory properties to counteract the inflammatory processes that are implicated in several diseases by down-regulating the production of pro-inflammatory cytokines, increasing production of anti-inflammatory cytokines, and enabling recruitment of naturally occurring anti-inflammatory cells to involved tissues, according to Mesoblast.

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Australia's Mesoblast plans to evaluate its stem cell therapy in patients infected with COVID-19 - BioWorld Online

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Graft-versus-host disease can occur any time after a transplant when donor bone marrow or stem cells attack the recipient

CytoDyn Inc (), a late-stage biotechnology company, said Wednesday that it has treated its first patient with its lead drug leronlimab (PRO 140), in its Phase 2 clinical trial for graft-versus-host disease (GvHD) under the modified trial protocol.

Graft-versus-host disease can occur at any time after a transplant. It is a rare condition that typically occurs when donor bone marrow or stem cells attack the recipient.

In a statement, the Vancouver, Washington-based company said 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 after the enrollment of 10 patients under the amended protocol after each patient has been dosed for 30 days, said the company.

CytoDyn CEO Nader Pourhassan pointed out that 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, said Dr Pourhassan.

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, he added.

A preclinical study by Dr Denis R Burger, CytoDyns former chief science officer, and Daniel Lindner, from the Department of Translational Hematology and Oncology Research, at The Cleveland Clinic, was published in the peer-reviewed journal called the Biology of Blood and Marrow Transplantation.

The US Food and Drug Administration earlier granted orphan drug designation to leronlimab for the prevention of GvHD. The designation provides CytoDyn with various incentives and benefits including seven years of US market exclusivity for leronlimab in GvHD, subject to FDA approval for use in this indication.

Leronlimab was earlier granted Fast Track status by the FDA for the treatment of HIV in combination with the cocktail known as highly active antiretroviral therapy (HAART), and for metastatic triple-negative breast cancer, a rare variety which doesnt respond to some treatments.

Leronlimab has completed nine clinical trials and has been given to 800 patients in HIV treatment programs, without a single drug-related serious adverse event. CytoDyn is developing leronlimab to battle multiple diseases. The company has also filed an IND application and a Phase 2 clinical trial protocol with the FDA to treat patients with NASH - damage caused by a build-up of fat in the liver.

Contact the author Uttara Choudhury at[emailprotected]

Follow her onTwitter:@UttaraProactive

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CytoDyn treats first patient with leronlimab in Phase 2 trial for GvHD under modified protocol - Proactive Investors USA & Canada

Bone Marrow Stem Cells Comments Off on CytoDyn treats first patient with leronlimab in Phase 2 trial for GvHD under modified protocol – Proactive Investors USA & Canada | March 10th, 2020

The second person ever to be cleared of HIV has revealed his identity, saying he wants to be an ambassador of hope to others with the condition.

Adam Castillejo, the so-called London patient, was declared free of HIV last year, 18 months after stopping antiretroviral therapy following a stem cell or bone marrow transplant to treat blood cancer.

Castillejo, 40, went public on Monday in an interview with the New York Times and revealed he had been living with HIV since 2003.

In 2012 he was diagnosed with acute myelogenous leukaemia and subsequently underwent a stem cell transplant. Crucially, the medical team picked a donor whose stem cells had two copies of a mutation that meant the white blood cells they developed into were resistant to HIV.

Timothy Brown, known as the Berlin patient and the first person to be cleared of the virus, underwent a similar treatment. However, while Brown and Castillejo had chemotherapy, only Brown had radiotherapy as part of his cancer treatment.

Last year it emerged the procedure had not only successfully treated the cancer, but that Castillejo was in remission for HIV as well. However, he chose to remain anonymous at the time.

I was watching TV and its like, OK, theyre talking about me, he told the New York Times. It was very strange, a very weird place to be.

Now Castillejo has decided to reveal his identity because he wants his case to be a cause for optimism. This is a unique position to be in, a unique and very humbling position, Castillejo said. I want to be an ambassador of hope.

Stem cell transplants are not suitable for most people with HIV because they involve a serious and invasive procedure that carries risks.

However, drug advances mean people who are HIV positive can take a pill every day to reduce their levels of the virus, preventing transmission and helping them to live a long and active life.

Prof Ravindra Gupta, the first author of the new study from Cambridge University, said Castillejos case was important: It is a second case of cure,. It means the first one wasnt an anomaly or a fluke.

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Second person ever to be cleared of HIV reveals identity - The Guardian

Bone Marrow Stem Cells Comments Off on Second person ever to be cleared of HIV reveals identity – The Guardian | March 10th, 2020

Bone remodeling and regeneration are dependent on resident stem/progenitor cells with the capability to replenish mature osteoblasts and repair the skeleton.

Until now, it has been thought that stem cells for bone lie within the bone marrow and the outer surface of the bone. Many studies have described the existence of a network of vascular channels that helped distribute blood cells out of the bone marrow. However, none of the studies had proved the existence of cells within these channels.

A new study by the scientists from the UConn School of Dental Medicine has discovered the population of stem cells that reside along the vascular channels within the cortical bone and have the ability to generate new bone. These stem cells stretch across the bone and connect the inner and outer parts of the bone.

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

This is the first study that reports the existence of these progenitor cells within the cortical bone that can generate new bone-forming cellsosteoblaststhat can be used to help remodel a bone.

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

However, further study is required to determine the cells potential to regulate bone formation and resorption.

The study is presented in the journal Stem Cells.

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These new stem cells have the ability to generate new bone - Tech Explorist

Bone Marrow Stem Cells Comments Off on These new stem cells have the ability to generate new bone – Tech Explorist | March 9th, 2020

A year after the London Patient was introduced to the world as only the second person to be cured of H.I.V., he is stepping out of the shadows to reveal his identity: He is Adam Castillejo.

Six feet tall and sturdy, with long, dark hair and an easy smile, Mr. Castillejo, 40, exudes good health and cheer. But his journey to the cure has been arduous and agonizing, involving nearly a decade of grueling treatments and moments of pure despair. He wrestled with whether and when to go public, given the attention and scrutiny that might follow. Ultimately, he said, he realized that his story carried a powerful message of optimism.

This is a unique position to be in, a unique and very humbling position, he said. I want to be an ambassador of hope.

Last March, scientists announced that Mr. Castillejo, then identified only as the London Patient, had been cured of H.I.V. after receiving a bone-marrow transplant for his lymphoma. The donor carried a mutation that impeded the ability of H.I.V. to enter cells, so the transplant essentially replaced Mr. Castillejos immune system with one resistant to the virus. The approach, though effective in his case, was intended to cure his cancer and is not a practical option for the widespread curing of H.I.V. because of the risks involved.

Only one other individual with H.I.V. Timothy Ray Brown, the so-called Berlin Patient, in 2008 has been successfully cured, and there have been many failed attempts. In fact, Mr. Castillejos doctors could not be sure last spring that he was truly rid of H.I.V., and they tiptoed around the word cure, instead referring to it as a remission.

Still, the news grabbed the worlds attention, even that of President Trump.

And by confirming that a cure is possible, it galvanized researchers.

Its really important that it wasnt a one-off, it wasnt a fluke, said Richard Jefferys, a director at Treatment Action Group, an advocacy organization. Thats been an important step for the field.

For Mr. Castillejo, the experience was surreal. He watched as millions of people reacted to the news of his cure and speculated about his identity. I was watching TV, and its, like, OK, theyre talking about me, he said. It was very strange, a very weird place to be. But he remained resolute in his decision to remain private until a few weeks ago.

For one, his doctors are more certain now that he is virus-free. We think this is a cure now, because its been another year and weve done a few more tests, said his virologist, Dr. Ravindra Gupta of the University of Cambridge.

Mr. Castillejo also tested his own readiness in small ways. He set up a separate email address and telephone number for his life as LP, as he refers to himself, and opened a Twitter account. He began talking weekly with Mr. Brown, the only other person who could truly understand what he had been through. In December, Mr. Castillejo prepared a statement to be read aloud by a producer on BBC Radio 4.

After talking through his decision with his doctors, friends and mother, he decided the time was right to tell his story.

I dont want people to think, Oh, youve been chosen, he said. No, it just happened. I was in the right place, probably at the right time, when it happened.

Mr. Castillejo grew up in Caracas, Venezuela. His father was of Spanish and Dutch descent which later turned out to be crucial and served as a pilot for an ecotourism company. Mr. Castillejo speaks reverently of his father, who died 20 years ago, and bears a strong resemblance to him. But his parents divorced when he was young, so he was primarily raised by his industrious mother, who now lives in London with him. She taught me to be the best I could be, no matter what, he said.

As a young man, Mr. Castillejo made his way first to Copenhagen and then to London in 2002. He was found to have H.I.V., the virus that causes AIDS, in 2003.

I do recall when the person told me and the panic set in, he said. At the time, an H.I.V. diagnosis was often seen as a death sentence, and Mr. Castillejo was only 23. It was a very terrifying and traumatic experience to go through.

With the support of his partner at the time, Mr. Castillejo persevered. He turned the passion for cooking he had inherited from his grandmother into a job as a sous chef at a fashionable fusion restaurant. He adopted an unfailingly healthy lifestyle: He ate well, exercised often, went cycling, running and swimming.

Then, in 2011, came the second blow. Mr. Castillejo was in New York City, visiting friends and brunching on the Upper East Side, when a nurse from the clinic where he went for regular checkups called him. Where are you? she asked. When Mr. Castillejo told her, she would say only that they had some concerns about his health and that he should come in for more tests when he returned to London.

He had been experiencing fevers, and the tests showed that they were the result of a Stage 4 lymphoma. I will never forget my reaction as once again my world changed forever, he said. Once again, another death sentence.

Years of harsh chemotherapy followed. Mr. Castillejos H.I.V. status complicated matters. Each time his oncologists adjusted his cancer treatment, the infectious-disease doctors had to recalibrate his H.I.V. medications, said Dr. Simon Edwards, who acted as a liaison between the two teams.

There is little information about how to treat people with both diseases, and H.I.V.-positive people are not allowed to enter clinical trials. So with each new chemotherapy combination, Mr. Castillejos doctors were venturing further into unchartered territory, Dr. Edwards said.

With each treatment that seemed to work and then didnt, Mr. Castillejo fell into a deeper low. He saw fellow patients at the clinic die and others get better, while he kept returning, his body weakening with each round.

I was struggling mentally, he said. I try to look at the bright side, but the brightness was fading.

In late 2014, the extreme physical and emotional toll of the past few years caught up to Mr. Castillejo, and two weeks before that Christmas he disappeared. His friends and family imagined the worst, and filed a missing persons report. Mr. Castillejo turned up four days later outside London, with no memory of how he had ended up there or what he had done in the interim. He described it as switching off from his life.

Around that same time, he said, he felt so defeated that he also contemplated going to Dignitas, the Swiss company that helps terminally ill people take their own lives: I felt powerless. I needed control, to end my life on my own terms. He made it through that dark period, and emerged with a determination to spend whatever was left of his life fighting.

Still, in the spring of 2015, his doctors told him he would not live to see Christmas. A bone-marrow transplant from a donor is sometimes offered to people with lymphoma who have exhausted their other options, but Mr. Castillejos doctors did not have the expertise to try that, especially for someone with H.I.V.

His close friend, Peter, was not ready to give up, and together they searched online for alternatives. (Peter declined to reveal his last name because of privacy concerns.) They discovered that at a hospital in London was Dr. Ian Gabriel, an expert in bone-marrow transplants for treating cancer, including in people with H.I.V. Because of their last-ditch effort, Mr. Castillejo said, Were here today. You never, never know.

Within a week, he met with Dr. Gabriel, who tried a third and final time to tap Mr. Castillejos own stem cells for a transplant. When that failed, Dr. Gabriel explained that Mr. Castillejos Latin background might complicate the search for a bone-marrow donor who matched the genetic profile of his immune system. To everyones surprise, however, Mr. Castillejo quickly matched with several donors, including a German one perhaps a legacy from his half-Dutch father who carried a crucial mutation called delta 32 that hinders H.I.V. infection. A transplant from this donor offered the tantalizing possibility of curing both Mr. Castillejos cancer and the H.I.V.

When Dr. Gabriel called with the news in the fall of 2015, Mr. Castillejo was on the top deck of one of Londons iconic red buses, on his way to see his general practitioner for a checkup. His thoughts raced alongside the scenery: He had only recently been told he was going to die, and now he was being told he might be cured of both cancer and H.I.V.

I was trying to digest what just happened, he recalled. But after that call, I had a big smile on my face. Thats where the journey began as LP.

With the possibility of an H.I.V. cure, the case immediately took on intense importance for everyone involved. Dr. Edwards, who had cared for Mr. Castillejo since 2012, had, as a young doctor in the early 1990s, seen many men his age die of H.I.V. What a privilege it would be to go from no therapy to a complete cure in my lifetime, he recalled telling Mr. Castillejo. So you have to get better no pressure.

Dr. Edwards involved Dr. Gupta, his former colleague and one of the few virologists in London he knew to be doing H.I.V. research. Dr. Gupta initially was skeptical; the approach had worked only once, 12 years earlier, with Mr. Brown. But Dr. Gupta also knew that the payoff could be huge. Antiretroviral drugs can suppress the virus to undetectable levels, but any interruption in the treatment can bring the virus roaring back, so a cure for H.I.V. is still the ultimate goal.

Dr. Gupta began carefully monitoring Mr. Castillejos H.I.V. status. In late 2015, Mr. Castillejo was preparing to receive the transplant when another major setback arose. His viral load shot back up with H.I.V. that appeared to be resistant to the drugs he had been taking.

This gave Dr. Gupta a rare glimpse at the typically suppressed virus, and allowed him to confirm that the viral strain was one that would be cleared by the transplant. But it also delayed the transplant by several months while the doctors adjusted Mr. Castillejos medications. He eventually received the transplant on May 13, 2016.

The next year was punishing. Mr. Castillejo spent months in the hospital. He lost nearly 70 pounds, contracted multiple infections and underwent several more operations. He had some hearing loss and began wearing a hearing aid. His doctors fretted over how to get his H.I.V. pills into his ulcer-filled mouth by crushing and dissolving them, or by feeding them to him through a tube. One of the doctors came to me and said to me, You must be very special, because I have more than 40 doctors and clinicians discussing your medication, Mr. Castillejo recalled.

Even after he left the hospital, the only exercise he initially was allowed to do was walking, so he walked for hours around the trendy Shoreditch neighborhood. He went to the flower market there every Sunday, treated himself to salted beef beigels to celebrate small successes and admired the colorful murals and vintage clothes.

A year on, as he became stronger, he slowly began thinking about forgoing the H.I.V. medications to see if he was rid of the virus. He took his last set of antiretroviral drugs in October 2017. Seventeen months later, in March 2019, Dr. Gupta announced the news of his cure.

Neither he nor Mr. Castillejo was prepared for what came next. Dr. Gupta found himself presenting the single case to a standing-room-only crowd at a conference, and shaking hands afterward with dozens of people. Mr. Castillejo was overwhelmed by the nearly 150 media requests to reveal his identity, and began to see a role he might play in raising awareness of cancer, bone-marrow transplants and H.I.V.

He has enrolled in several studies to help Dr. Gupta and others understand both diseases. So far, his body has shown no evidence of the virus apart from fragments the doctors call fossils and what seems to be a long-term biological memory of having once been infected.

Others in the H.I.V. community are reassured by this news, but expressed concern for Mr. Castillejos privacy and mental health.

It can be very important for people to have these kinds of beacons of hope, Mr. Jefferys, the Treatment Action Group director, said. At the same time, thats a lot of weight for someone to carry.

Mr. Castillejos friends have similar worries. But he is as ready as he will ever be, he said. He sees LP as his work identity and is determined to live his private life to its fullest. Having lost his lustrous dark hair several times over, he has now grown it to shoulder length. He has always enjoyed adventures, and with careful preparation he has begun traveling again, describing himself to fellow travelers only as a cancer survivor. He celebrated his 40th birthday with a trip to Machu Picchu, in Peru.

But in conversations about his status as the second person ever to be cured of H.I.V., Mr. Castillejo still adamantly refers to himself as LP, not Adam. When you call me LP, it calms me down, he said. LP to my name, that is kind of a big step.

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The London Patient, Cured of H.I.V., Reveals His Identity - The New York Times

Bone Marrow Stem Cells Comments Off on The London Patient, Cured of H.I.V., Reveals His Identity – The New York Times | March 9th, 2020

Steven Hodi Jr., the i3 Centers other PI, and director of Melanoma Center and the Center for Immuno-Oncology at Dana-Farber, and professor of medicine at Harvard Medical School (HMS), is leading the clinical cancer vaccine trial. He has been at the forefront of developing cancer immunotherapies using immune checkpoint inhibitors, a class of drugs able to re-activate tumor-destroying T cells that are muted in the tumor microenvironment. The funding for this center provides a unique opportunity to unite key investigators for translating fundamental advancements in immunology and biomedical engineering into highly synergistic approaches to improve the treatments for cancer patients, said Hod

Using both in vivo and ex vivo biomaterials-based approaches, the i3 Center aims to boost tumor-specific activities of cytotoxic T cells, by boosting different stages of the normal process by which T cells develop, and acquire anti-cancer activity. T cells normal development starts in the bone marrow where hematopoietic stem cells generate T cell progenitor cells. These migrate to the thymus to differentiate into nave T cells, which then travel further to lymph nodes. There, they encounter cancer-derived antigens presented to them by specialized antigen-presenting cells (APCs) that can activate T cells to recognize and eliminate cancer cells.

In relation to adoptive T cell therapies in which T cells are given to patients to fight their cancers, one team at the i3 Center will be led by Dana-Farber researchers Catherine J. Wu and Jerome Ritz, who along with Mooney, will develop and test biomaterials that can better mimic normal APCs in activating and directing the function of patient-derived T cells outside the human body, prior to their transplantation. Wu is chief of the Division of Stem Cell Transplantation and Cellular Therapies, and Ritz is executive director of the Connell and OReilly Families Cell Manipulation Core Facility at Dana-Farber.

We need to make efforts to enhance the ability of theimmune systemto recognizetumor cells. One directionmylaboratoryis taking makes use of innovative biomaterialsto help us to efficiently expandpolyclonaltumor-specificfunctionally-effectiveT cellsex vivoin a way that can be readily translated to theclinical setting. In our studies, we are currently focusing on melanoma and acute myeloid leukemia, said Wu, whose research interests include understanding the basis of effective human anti-tumor responses, including the identification and targeting of the tumor-specific antigens.

A second project explores the use of DNA origami, biocompatible nanostructures composed of DNA, to create cancer vaccines. DNA origami could provide significant advantages in presenting tumor-specific antigens and immune-enhancing adjuvants to APCs because the concentrations, ratios, and geometries of all components can be modulated with nano-scale precision to determine configurations that are more effective than other vaccination strategies. The project will be run by Wyss Institute Core Faculty member William Shih, Derin Keskin, lead immunologist at Dana-Farbers Translational Immunogenomics Lab, and Mooney.

In a third project, David Scadden, professor at Harvards Department of Stem Cell and Regenerative Biology, will collaborate with Mooney to build on their previous work. They will engineer biomaterials that recreate key features of the normal hematopoietic stem cell niche in the bone marrow. Such implantable biomaterials could help rapidly amplify T cell progenitor cells, and enhance T cell-mediated anti-cancer immunity. Scadden also is the Gerald and Darlene Jordan Professor of Medicine at Harvard University, and co-director of the Harvard Stem Cell Institute.

The i3 Centers investigators anticipate that it will stimulate additional cross-disciplinary concepts and research, due to the culture of continuous interactions, sharing of findings, data and samples between all investigators, as well strong biostatistical expertise provided by Donna Neuberg, a senior biostatistician broadly involved with exploring immune-modulating cancer interventions at the Dana-Farber.

This new i3 Center for cancer immunotherapy innovation really embodies how the Wyss Institute with its unparalleled capabilities in bioengineering and serving as a site for multidisciplinary collaboration, and can liaise with clinicians and researchers at our collaborating institutions to confront major medical problems and bring about transformative change, said Wyss Founding Director Donald Ingber. He is also theJudah Folkman Professor of Vascular Biologyat HMS and the Vascular Biology Program at Boston Childrens Hospital, and Professor of Bioengineering at SEAS.

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NIH-funded i3 Center formed to advance cancer immunotherapy - Harvard Gazette

Bone Marrow Stem Cells Comments Off on NIH-funded i3 Center formed to advance cancer immunotherapy – Harvard Gazette | March 8th, 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.

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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

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

Bone Marrow Stem Cells Comments Off on Stem Cells that will aid new bone generation discovered as per latest research – Medical Herald | 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

Bone Marrow Stem Cells Comments Off on Despite Pro-Life Claims, Stem Cell Therapy Has Very Real Benefits and Should Be Accessible – Patheos | March 6th, 2020

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

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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."

Read or Share this story: https://www.news-leader.com/story/news/local/ozarks/2020/03/05/grandma-who-helped-pass-newborn-screening-law-tells-story-bill/4954655002/

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

Bone Marrow Stem Cells Comments Off on ‘His legacy lives on’: Grandmother who helped create newborn screening law tells history of bill – News-Leader | March 6th, 2020

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

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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The Insight Partners is a one stop industry research provider of actionable intelligence. We help our clients in getting solutions to their research requirements through our syndicated and consulting research services. We are committed to provide highest quality research and consulting services to our customers. We help our clients understand the key market trends, identify opportunities, and make informed decisions with our market research offerings at an affordable cost.

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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

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

Why is it that when a woman runs for an office or does something remarkable so very often the first thing people say is look at her hair or WHAT is she wearing! Well, there is a woman whose photos adorn the halls of the volunteer organization where I volunteer. And because she is the founder of the organization there are lots of photos in the office and in books about the history of the organization. The organization is Hadassah, The Womens Zionist Organization of America. The woman, the founder, is Henrietta Szold (1860-1945).

When I look at her photos, I dont focus on her hair, which changed little over the decades of her work. I dont look at the black pocketbook she often carried which seems not to have changed over the decades of her work. I dont think about her shoes, which were sturdy and usually black. Nope when I look at Miss Henrietta Szolds photo the first thing I think of look at how strong she is, look at the determination and purpose in her eyes, look at what those eyes have seen and those hands have touched. The rest unimportant and anecdotal.

As we are commemorating Womens History and Womens Empowerment Month in March, its very appropriate to tell you that even before Henrietta started Hadassah in a New York City synagogue in 1912 with a study group, she had, in her young life, already broken gender barriers and established institutions. Henrietta started the first night school for immigrants in the US and she studied at the Jewish Theological Seminary in an era when the idea of a female rabbi was unthinkable. An empowered woman look no further absolutely.

When Henriettas eyes first saw the disease and living conditions of the Jews in pre-state Israel on a trip with her mother in 1911, her mission of practical Zionism and her purpose were born. When you read her letters looking for funding or in subsequent years, letters between her and the nurses she sent over to provide pasteurized milk to babies and new moms, and to set up public health stations in Jerusalem to fight off the flies on the eyes of children suffering from trachoma, her words are full of determination and, excuse the old fashioned word gumption. This woman had moxie, this woman had chutzpah, this woman had guts. And thank G-d she did. Because she and her womens organization built the State of Israel. Hadassah created the medical infrastructure of Palestine and continues to do so today in Israel, a mere 108 years later. When there is purpose to what you are committed to and that purpose is accompanied by action there can be longevity. How about an organization that looks forward to the next 100 years? How about volunteers that are active for decades?

They say the proof is in the pudding, that you can judge something only once you have used or experienced it. So: Been there. Done that. Doing it.

Ive been a life member of Hadassah for 54 years, worked in just about every capacity and position at every possible level of the organization. I have gone to Israel so many times but when I juxtapose the memories of my first visit in 1966 with my most recent visit in 2018, WOW, the differences are staggering. Close to the top of the list are the ongoing changes at Hadassah Hospitals and Youth Villages. In the early years, beginning in 1913 when Henrietta sent over the first two nurses, Hadassah Hospitals and Clinics covered the map and over the years. Hadassah and the Hadassah Medical Organization (HMO) created many firsts the first medical school, dental school, nursing school, cancer institute, childrens hospice, ambulatory surgery center, ER unit for premature babies, and trauma treatment center in Israel.

Today the two hills of healing stand at opposite ends of Jerusalem Mt. Scopus (opened in 1939, closed in 1948, reopened in 1975) and the Ein Kerem campus, a tertiary care facility, built in 1961 as Ben Gurion told Hadassah to build in the southwest outskirts of Jerusalem and the city would grow out to it. Which is just what happened. Today, a light rail and bus bring people from all over the area to the hospital. In 2012, the Sarah Wetsman Davidson Hospital Tower opened adding 500 beds and 20 operating theaters. In 2020, Hadassah is re-imagining and re-energizing the campus with its 360 Degrees of Healing Campaign.

Hadassah Hospitals were first in Israel with heart, liver, lung and bone marrow transplants, computer-guided hip replacement (first in the world), macular degeneration clinical trial using embryonic stem cells to repair vision (second in the world) and bone marrow registry for Arabs (only one in the world). Hadassah Medical Organization (HMO) triage procedures and surgical techniques developed by Hadassah doctors were used following the Boston Marathon bombing. HMO doctors and nurses have been first responders in the Philippines, Haiti, Indonesia and Thailand in the wake of natural disasters. Hadassah doctors recently brought humanitarian spinal surgery to Ethiopia.

No rest for this woman she found more purpose and then, more purpose, as time went on.

As a 70-year-old woman, Henrietta was a member of the Palestine Zionist Executive, the Jewish Agency Executive and the Vaad Leumi. Photos show her as often being the only woman in the room. In these capacities she directed the health/educational development and social services of the population. And then, Henrietta took over the daily operations of Youth Aliyah in Palestine. Youth Aliyah was created to bring Jewish children out of Nazi Germany and bring them to Palestine.

In 1943, Henrietta waited in the cold at the Atlit Detention Camp as 750 children from Iran disembarked a train (120 followed a few months later), saved from the atrocities of Nazi Germany. Hadassah joined the life-saving work of Youth Aliyah and continues to be a major supporter to this day. Today, Hadassah-supported youth villages, Meir Shfeyah, Ramat Hadassah Szold and Hadassah Neurim, set at-risk children in Israel on the road to success and since its beginning, more than 300,000 young people from 80 lands have graduated from Youth Aliyah.

Always with an eye to the future, Henrietta Szolds connection to Young Judaea, began in 1909, when she prompted the Federation of American Zionists to call for a junior Zionist convention of delegates from Zionist youth societies. Young Judaea was formally established as a national Zionist youth organization at that New York convention. And then, under the leadership of Henrietta Szold, the department of education was formed by the Zionist Organization of America (ZOA), which briefly sponsored Young Judaea from 1918 to 1921. Over the years, there were many different connections between Henrietta, Hadassah and Young Judaea. Today, Hadassah and Young Judaea continue their connection through their shared mission to forge a strong commitment to Jewish life, instill a love of Israel and Zionism, connect American kids to Israel through education and programs, develop leaders for the Jewish community, and advocacy. Henrietta Szold and Hadassah in the room!

Today Hadassah has 300,000 members, Associates and supporters. It is the largest Jewish womens membership organization in the United States. With members in every Congressional district, Hadassahs advocacy work in spear-heading important legislation, most recently, the Never Again Education Act working to ensure Holocaust education in public schools, is a direct modern-day application of Henriettas legacy and an illustration of purpose with action. Hadassah women are in the room!

So, I think you can see that Henrietta started a run a run of practical Zionism that stretches across decades and centuries. A run I am proud to be part of since it has enabled me to work for Israel while living here in New York. It has allowed me to make differences around the world through medical research and protocols that are shared. Four generations of life members in my family. Three generations of Hadassah Presidents in my family. Once I make our new grandson an Associate, five generations of men affiliated with Hadassah. For me, personally, Hadassah is a family affair.

Today Hadassah strives to empower women of all ages to make a difference and to become leaders in the Jewish community by continuing Henriettas legacy of Practical Zionism through our work in Israel, our advocacy here in the US, on issues that affect Israel, the Jewishcommunity and health. Henrietta asked the artist of her sculpture to make my eyes look to the future. A most meaningful and purposeful statement.

Boy, I would love to know what Henrietta carried in that black bag of hers, or better yet, what her bag would hold today. I can only imagine that shewasthe one with the tissues to wipe the eyes of the young children as they disembarked the train. Shewould bethe one with the cell phone to reach out to anyone who would listen to her pleas for assistance and for funding to facilitate medical research and care of youth. Shewould bethe one with the small flashlight to bring a big light unto the nations. Over the years I have implored our members to release their inner Henrietta. A woman of purpose and perseverance to emulate for sure.

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A Woman of Purpose and Perseverance - Thrive Global

Bone Marrow Stem Cells Comments Off on A Woman of Purpose and Perseverance – Thrive Global | March 5th, 2020

What happened

Shares of Aimmune Therapeutics (NASDAQ: AIMT) fell 23.3% in February, according to data provided by S&P Global Market Intelligence, as the launch of its new peanut allergy treatment took time to unfold.

Aimmune rose 40% last year as investors anticipated Palforzia, the firsttreatment approved by the Food and Drug Administration for peanut allergy in children. The regulatory agency approved the treatment on Jan. 31, and now investors are waiting to see how the first weeks of sales progress. The process is taking time because of the risk management procedure involved in the launch.

Image source: Getty Images.

Palforzia, an orally administered powder made of peanut protein, works by desensitizing patients to the allergen. In order to lower risk in case a new patient suffers a reaction to the therapy, the FDA has set up the Risk Evaluation and Mitigation Strategy (REMS) program. Physicians and patients must enroll in REMS and follow guidelines before treatment can begin. This process, along with the FDA's standard procedure of examining and releasing the first batches of biologic product, lengthened the timeline from approval to sales. Aimmune said during its recent earnings call that it expects to record the first Palforzia sales this month.

Getting physicians and patients on board in the next few months will be crucial for Aimmune. DBVTechnologies (NASDAQ: DBVT) is close behind, with an FDA decision on its peanut allergy drug expected in August. Aimmune now has the advantage of being first to market, before rivals enter with competing products. Aimmune is also expecting a decision on Palforzia from the Europeanregulatory agency in the fourth quarter, and said a decision in Switzerland could come in mid-2021.

All of these elements represent catalysts for the shares over the next year or so. If Aimmune can assure its position as market leader, the shares of this biotech company will benefit in the long term.

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The views and opinions expressed herein are the views and opinions of the author and do not necessarily reflect those of Nasdaq, Inc.

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Why Aimmune Therapeutics Shares Fell 23.3% in February - Nasdaq

Bone Marrow Stem Cells Comments Off on Why Aimmune Therapeutics Shares Fell 23.3% in February – Nasdaq | March 5th, 2020