Category Archives: Somatic Stem Cells

Scientists ‘Reset’ The Age of Stem Cells From a Supercentenarian Who Lived to 114 – ScienceAlert

For the first time, scientists have reprogrammed the stem cells of a 114-year-old woman, the oldest donor to date.

After first transforming cells from her blood sample into induced pluripotent stem cells (iPSCs), the researchers then generated mesenchymal stem cells, which help to maintain and repair tissues like bone, cartilage and fat.

"We set out to answer a big question: Can you reprogram cells this old?" says stem cell biologist Evan Snyder at Sanford Burnham Prebys Medical Discovery Institute in California.

"Now we have shown it can be done, and we have a valuable tool for finding the genes and other factors that slow down the aging process."

Stem cells are sometimes called "cellular Rosetta stones", because they allow us to study disease, cancer, ageing and regeneration like never before.

The most valuable type are embryonic stem cells (ESCs), but their acquisition is linked to some ethical issues, and such cells can be difficult to get a hold of. Thankfully, somatic cells, or adult stem cells, can be found in any human; we have the technology to genetically reprogram these units into induced pluripotent stem cells, which are nearly as potent as ESCs.

Until now, however, we weren't sure just how long an adult's cells remain programmable in this way. While some previous research suggests older stem cells cannot be reprogrammed, in recent years, scientists have been able to generate iPSCs from centenarians, or people who live to be more than 100 years old.

So, what about those who've won an even bigger genetic lottery? What about supercentenarians?

In the whole wide world, currently we know of only 28 people verified to be over the age of 110. This unique population is difficult to study, not only because of its limited sample size but also because of our records, which can be pretty shoddy at times.

Nevertheless, research so far has found supercentenarians not only age slower, they also show a strange immunity in general to chronic age-related diseases, like Alzheimer's and Parkinson's, that doesn't seem to have a lot to do with lifestyle.

"Why do supercentenarians age so slowly?" says Snyder. "We are now set to answer that question in a way no one has been able to before."

To do this, the team reprogrammed lymphoblastsfrom three donors: the supercentenarian woman, a healthy 43-year-old individual, and an 8-year-old child with a condition that causes rapid ageing.

Not only did the supercentenarian cells transform into iPSCs just as easily as the others, the telomeres - sequences of 'protective' DNA that sit at the ends of our chromosomes and shrink as we age - were also reset to more "youthful levels".

Granted, this telomere resetting didn't occur as frequently in older cells, only a third of the time. Nevertheless, the authors say what they were able to achieve on these occasions was equivalent to turning back the cellular clock from age 114 to age zero.

"These data indicate that extreme age is not an absolute barrier to reprogramming with restoration of telomere length," the authors write.

What's more, their technique only required four reprogramming factors - a manageable number that will make it relatively easy for scientists to see how these supercentenarian stem cells compare in the long run.

They claim reprogramming donor cells from extremely or prematurely aged donors is feasible, and that by doing so with retrospective samples, we might be able to expand on this small pool of supercentenarian donors.

The authors of the study hope their research will allow us to better investigate how and why supercentenarians live so long and are so extraordinarily resistant to degenerative disease. There's much we can learn from these remarkable people.

The study was published in Biochemical and Biophysical Research Communications.

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Scientists 'Reset' The Age of Stem Cells From a Supercentenarian Who Lived to 114 - ScienceAlert

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

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

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

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

Researchers ID Protein-Protein Interaction That Promotes Cancer Development – BioSpace

A group of researchers at Purdue University, led by Humaira Gowher, an associate professor in the Department of Biochemistry, identified an epigenetic process that controls the behavior of stem cell enhancers. In other words, Gowher and her team discovered that the OCT4 protein blocks a sequence of events that occurs when stem cells transition to any cells that arent reproductive cells, otherwise called somatic cells.

What does that mean?

If the cellular mechanisms that control the transition from stem cells to somatic cells don't stop correctly, cells can become cancerous. Gowhers research points to a fundamental mechanism behind why some cells become cancerous.

The research was published in the journal Cell Reports.

If you differentiate normal stem cells, they should silence their pluripotency gene enhancers, but these cells are not doing that, Gowher said. Theyre leaving the enhancer program partially open. Weve uncovered the issue that shelves these cells in some kind of intermediate, rather than in terminally differentiated, state.

In earlier research, Gowhers team found that when normal embryonic stem cells differentiate, they control their stemness genes by silencing certain control elements called enhancers. They do this via DNA methylation, which is a form of epigenetic silencing. A methyl group is a carbon atom with three hydrogen atoms attached to it, and when it is added to certain areas of the genome, acts as a switch, turning genes on or off under certain conditions.

In addition to the DNA methylation, Gowher found that an enzyme called Lsd1 aids in the DNA methylation. In some cancers, the stemness genes are partially repressed, which causes only partial differentiation. One possible way to think of this might be popcorn. If the unpopped popcorn is the stem cell and a fully popped popcorn is the normal somatic cell, the partially popped popcorn is the result of this repression and partial differentiation.

If you differentiate normal stem cells, they should silence their pluripotency gene enhancers, but these cells are not doing that, Gowher said. Theyre leaving the enhancer program partially open. Weve uncovered the issue that shelves these cells in some kind of intermediate, rather than in terminally differentiated, state.

When the enhancers are primed, they may potentially reactivate, which leads to more cell multiplication. This mechanism is also related to why cancer stem cells become resistant to differentiation therapy. Differentiation therapy is a type of cancer treatment where malignant cells are treated with drugs that cause them to differentiate into more mature types. Its because malignant cancer cells usually take on a less specialized, stem cell-like dedifferentiated form.

Another way of looking at it, is that that the fully popped popcorn is susceptible to certain types of cancer drugs, while the partially popped popcorn is less so.

What people have seen in some cancers is that enhancers exist in a primed state, Gowher said. It can potentially help the cancer stem cells survive and propagate. Its a pro-survival mechanism.

The activity of Lsd1 is inhibited by stem cell transcription factor OCT4 in these cells.

Because of this aberrant presence of OCT4, cells arent completely differentiating, Gowher said. If you could inhibit the OCT4-Lsd1 interaction, or target degradation of OCT4, that should allow the cells to differentiate. That could be a target for a cancer therapy.

As their research continues, Gowher will try to determine the interface of Lsd1-OCT4 interaction as well as identify other transcription factors that might inhibit Lsd1 activity.

When embryonic stem cells (ESC) differentiate, pluripotency gene (PpG)-specific enhancers are silenced by way of DNA methylation. The authors note that studies by the Cancer Genome Anatomy Project (CGAP) show that one out of three cancers express PpGs. This suggests they play a role in dysregulated cell proliferation during the formation of tumors, i.e., tumorigenesis. There are other factors as well, but it appears that in order to continue to divide and grow, many cancer cells continue to express PpGs, which led to the development of terminal differentiation therapy.

They wrote, To understand the mechanism by which cancer cells retain PpG expression, we investigated the mechanism of enhancer-mediated regulation of PpG expression in ECCs. Our data showed that, in differentiating F9 ECCs, the PpGs are only partially repressed.

This was consistent with deacetylation of H3K27, the 27th amino acid in Histone H3, a type of protein in the DNA molecule. But it also showed that there was an absence of Lsd1-mediated H3K4me1 demethylation at PpGe.

Which is a fairly complicated and detailed way of saying that they identified a mechanism that causes stem cells to head toward becoming regular somatic cells, but get stuck in an in-between state that can become cancer. And by learning more about this mechanism and targeting it, they may be able to halt cancer proliferation in its tracks.

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Researchers ID Protein-Protein Interaction That Promotes Cancer Development - BioSpace

Dental Regenerative Market Size, Share 2020 Regional Trend, Future Growth, Leading Players Updates, Industry Demand, Current and Future Plans by…

Global Dental Regenerative Market 2020 Industry Research report provides a comprehensive exploration of vital market dynamics and their recent trends, along with relevant market segments. The Dental Regenerative report also covers several factors influencing the growth of the Dental Regenerative market, Also, its impact on the individual segments is evaluated in this research. The report highlights the regional market, the leading market players, and several market. In addition, the research evaluated key market aspects, comprising capacity utilization rate, revenue, price, capacity, growth rate, gross, production, consumption, supply, export, market share, cost, import, gross margin, demand, and much more. The study also presents the segmentation of the worldwide Dental Regenerative market on the basis of end-users, applications, geography, and technology.

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The emotional connection of natural teeth and the high nature of natural teeth have drawn attention to the development of bioengineered teeth (tooth regeneration).Tooth regeneration is a stem cell-based regenerative medicine procedure that, in the fields of tissue engineering and stem cell biology, replaces damaged or lost teeth by redrawing from autologous stem cells.As a source of new bioengineered teeth, somatic stem cells are collected and reprogrammed into induced pluripotent stem cells that can be placed directly in the dental plate or in a reabsorbable biopolymer in the shape of a new tooth.Market Analysis and Insights: Global Dental Regenerative MarketIn 2019, the global Dental Regenerative market size was US$ xx million and it is expected to reach US$ xx million by the end of 2026, with a CAGR of xx% during 2021-2026.Global Dental Regenerative Scope and Market SizeDental Regenerative market is segmented by Type, and by Application. Players, stakeholders, and other participants in the global Dental Regenerative market will be able to gain the upper hand as they use the report as a powerful resource. The segmental analysis focuses on revenue and forecast by Type and by Application in terms of revenue and forecast for the period 2015-2026.

Global Dental Regenerative market 2019 research provides a basic overview of the industry including definitions, classifications, applications and industry chain structure. The Global Dental Regenerative market analysis is provided for the international markets including development trends, competitive landscape analysis, and key regions development status. Development policies and plans are discussed as well as manufacturing processes and cost structures are also analyzed. This report also states import/export consumption, supply and demand Figures, cost, price, revenue and gross margins.

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Global Dental Regenerative market competition by top manufacturers, with production, price, revenue (value) and market share for each manufacturer; the TOP PLAYERS including;

The analysis provides an exhaustive investigation of the global Dental Regenerative market together with the future projections to assess the investment feasibility. Furthermore, the report includes both quantitative and qualitative analyses of the Dental Regenerative market throughout the forecast period. The report also comprehends business opportunities and scope for expansion. Besides this, it provides insights into market threats or barriers and the impact of regulatory framework to give an executive-level blueprint the Dental Regenerative market. This is done with an aim of helping companies in strategizing their decisions in a better way and finally attain their business goals.

With tables and figures helping analyze worldwide Global Dental Regenerative market, this research provides key statistics on the state of the industry and is a valuable source of guidance and direction for companies and individuals interested in the market.

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By the product type, the market is primarily split into

By the end users/application, this report covers the following segments

The study objectives of this report are:

In this study, the years considered to estimate the market size of Dental Regenerative are as follows:

Key Stakeholders

Major Points from Table of Contents:

1 Report Overview

1.1 Study Scope

1.2 Key Market Segments

1.3 Players Covered

1.4 Market Analysis by Type

1.4.1 Global Dental Regenerative Market Size Growth Rate by Type (2014-2026)

1.4.2 Major-Type

1.4.3 Independent-Type

1.4.4 Administrator-Type

1.5 Market by Application

1.5.1 Global Dental Regenerative Market Share by Application (2014-2026)

1.5.2 Commercial

1.5.3 Commonweal

1.5.4 Other

1.6 Study Objectives

1.7 Years Considered

2 Global Growth Trends

2.1 Dental Regenerative Market Size

2.2 Dental Regenerative Growth Trends by Regions

2.2.1 Dental Regenerative Market Size by Regions (2014-2026)

2.2.2 Dental Regenerative Market Share by Regions (2014-2019)

2.3 Industry Trends

2.3.1 Market Top Trends

2.3.2 Market Drivers

2.3.3 Market Opportunities

3 Market Share by Key Players

3.1 Dental Regenerative Market Size by Manufacturers

3.1.1 Global Dental Regenerative Revenue by Manufacturers (2014-2019)

3.1.2 Global Dental Regenerative Revenue Market Share by Manufacturers (2014-2019)

3.1.3 Global Dental Regenerative Market Concentration Ratio (CR5 and HHI)

3.2 Dental Regenerative Key Players Head office and Area Served

3.3 Key Players Dental Regenerative Product/Solution/Service

3.4 Date of Enter into Dental Regenerative Market

3.5 Mergers & Acquisitions, Expansion Plans

4 Breakdown Data by Type and Application

4.1 Global Dental Regenerative Market Size by Type (2014-2019)

4.2 Global Dental Regenerative Market Size by Application (2014-2019)

(5, 6, 7, 8, 9, 10, 11) United States, Europe, China, Japan, Southeast Asia, India, Central & South America

Dental Regenerative Market Size (2014-2019)

Key Players

Dental Regenerative Market Size by Type

Dental Regenerative Market Size by Application

12 International Players Profiles

Company Details

Company Description and Business Overview

Dental Regenerative Introduction

Revenue in Dental Regenerative Business (2014-2019)

Recent Development

13 Market Forecast 2019-2026

13.1 Market Size Forecast by Regions

13.2 United States

13.3 Europe

13.4 China

13.5 Japan

13.6 Southeast Asia

13.7 India

13.8 Central & South America

13.9 Market Size Forecast by Product (2019-2026)

13.10 Market Size Forecast by Application (2019-2026)

14 Analysts Viewpoints/Conclusions

15 Appendix

15.1 Research Methodology

15.1.1 Methodology/Research Approach

15.1.1.1 Research Programs/Design

15.1.1.2 Market Size Estimation

12.1.1.3 Market Breakdown and Data Triangulation

15.1.2 Data Source

15.1.2.1 Secondary Sources

15.1.2.2 Primary Sources

15.2 Disclaimer

15.3 Author Details

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Dental Regenerative Market Size, Share 2020 Regional Trend, Future Growth, Leading Players Updates, Industry Demand, Current and Future Plans by...

Global Gene Therapy Market to Cross Around USD 6892 Million By 2027 – Global Newspaper 24

Gene Therapy Market

Facts and Factors Market Researchhas published a new report titled Gene Therapy Market By Type (Germ Line Gene Therapy and Somatic Gene Therapy), By Vector Type (Viral Vectors, Non-Viral Vectors, and Human Artificial Chromosome), and By Therapy Area (Cancer, Neurological Diseases, Infectious Diseases, Genetic Disorders, Rheumatoid Arthritis, and Others): Global Industry Perspective, Comprehensive Analysis, and Forecast, 2018 2027. According to the report, the globalgene therapy marketwas valued at approximately USD 919 million in 2018 and is expected to reach a value of around USD 6,892 million by 2027, at a CAGR of around 25.1% between 2019 and 2027.

Gene therapy is the kind of experimental method that makes use of genes for treating or preventing disease by inserting foreign genetic material like DNA or RNA into the persons cells. Scientists are studying gene therapy for treating various kinds of immuno-deficiencies, Parkinsons disease, HIV, and cancer by using myriad approaches. Today, many of the approaches to gene therapy are undergoing most intensive & rigorously testing. This includes replacing the mutated gene causing disease with the healthy gene copy. Another approach includes knocking out or inactivating a mutated gene operating improperly. Yet another approach includes a new gene into the body to combat the disease.

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New product approval & commercialization to drive the market trends

Between the periods from 2012 to 2018, nearly five single-use gene treatments received approval from the U.S. FDA for treating a rare form of genetic disorders. Moreover, gene treatments that have received approval are being tested by pharmaceutical firms in the market. Apart from this, current approvals of gene therapy products across the U.S., as well as European countries for treating a plethora of life-threatening diseases, are anticipated to steer the growth of gene therapy industry over the forecast timeline. Moreover, gene therapy can also be used for treating neurodegenerative disorders like Alzheimer, amyotrophic lateral sclerosis, and spinal muscular atrophy.

Furthermore, many of the reputed pharma firms like Bristol-Myers Squibb, BioMarin, and Pfizer are investing massively into the research activities pertaining to gene therapy. Apart from this, a rise in the occurrence of cancer is prompting the demand to treat the disease. Gene therapy is one of the key treatment kinds that will propel the market growth over the forecast period. However, inadequate reimbursement policies pertaining to the one-time gene treatments will downgrade market expansion.

In addition to this, conducting of randomized controlled trials can pose a threat to the expansion of the gene therapy industry as a result of the gene therapy features & projected patient population. Nevertheless, the ability of the gene therapy to eliminate the number of ailments with faulty or missing genes like hemophilia A will promote the market growth over the forecast period and thereby nullify the negative impact of hindrances on the business growth.

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Somatic gene therapy to dominate the type segment

The growth of the segment over the forecast timeline is credited to the ability to treat the targeted cells in the patient population. The treatment is not passed to future generations and is restricted to only the patient who receives the somatic gene therapy. Moreover, it is used for treating a huge number of disorders like cystic fibrosis, cancer, and muscular dystrophy.

Cancer to lead the therapy area segment over the forecast period

The segmental expansion is attributed to a large number of pipeline drugs registered over the past few years along with increasing occurrence of cancer as a result of genetic changes.

North America to dominate the overall regional market share during the forecast timespan

North American market, which accrued revenue of USD 380 million in 2018, is set to contribute majorly towards the overall market revenue by 2027. The regional market surge is credited to robust healthcare amenities, high per capita healthcare spending, and improvement in the reimbursement policies.

The key players included in this market are Advanced Cell & Gene Therapy, Audentes Therapeutics, Benitec Biopharma, Biogen, Blubird Bio, Inc., Bristol-Myers Squibb Company, CHIESI Farmaceutici SPA, Eurofins Scientific, Geneta Science, Genzyme Corporation, Gilead, GlaxoSmithKline PLC, Human Stem Cells institute, Novartis AG, Orchard Therapeutics, Pfizer Inc., Sangamo therapeutics, Spark therapeutics, and Voyager Therapeutics.

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This report segments the gene therapy market as follows:

GlobalGene TherapyMarket:By TypeSegment Analysis

GlobalGene TherapyMarket: ByVector TypeSegment Analysis

GlobalGene TherapyMarket: ByTherapy AreaSegment Analysis

Global Gene TherapyMarket: Regional Segment Analysis

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Global Gene Therapy Market to Cross Around USD 6892 Million By 2027 - Global Newspaper 24

Induced Pluripotent Stem Cells Market Predicted to Witness Surge in the Near Future2018 2028 – TechNews.mobi

TMRR, in its recent market report, suggests that the Induced Pluripotent Stem Cells market report is set to exceed US$ xx Mn/Bn by 2029. The report finds that the Induced Pluripotent Stem Cells market registered ~US$ xx Mn/Bn in 2018 and is spectated to grow at a healthy CAGR over the foreseeable period. This Induced Pluripotent Stem Cells market study considers 2018 as the base year, 2019 as the estimated year, and 2019 2029 as the forecast timeframe.

The Induced Pluripotent Stem Cells market research focuses on the market structure and various factors (positive and negative) affecting the growth of the market. The study encloses a precise evaluation of the Induced Pluripotent Stem Cells market, including growth rate, current scenario, and volume inflation prospects, on the basis of DROT and Porters Five Forces analyses. In addition, the Induced Pluripotent Stem Cells market study provides reliable and authentic projections regarding the technical jargon.

Important regions covered in the Induced Pluripotent Stem Cells market research include Region 1 (Country 1, country 2), Region 2 (Country 1, country 2), Region 3 (Country 1, country 2) and Region 4 (Country 1, country 2).

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The Induced Pluripotent Stem Cells market study answers critical questions including:

The content of the Induced Pluripotent Stem Cells market report includes the following insights:

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On the basis of component, the global Induced Pluripotent Stem Cells market report covers the following segments:

Notable Development

Over the past few years, fast emerging markets in the global induced pluripotent stem cells are seeing the advent of patents that unveil new techniques for reprogramming of adult cells to reach embryonic stage. Particularly, the idea that these pluripotent stem cells can be made to form any cells in the body has galvanized companies to test their potential in human cell lines. Also, a few biotech companies have intensified their research efforts to improve the safety of and reduce the risk of genetic aberrations in their approved human cell lines. Recently, this has seen the form of collaborative efforts among them.

Lineage Cell Therapeutics and AgeX Therapeutics have in December 2019 announced that they have applied for a patent for a new method for generating iPSCs. These are based on NIH-approved human cell lines, and have been undergoing clinical-stage programs in the treatment of dry macular degeneration and spinal cord injuries. The companies claim to include multiple techniques for reprogramming of animal somatic cells.

Such initiatives by biotech companies are expected to impart a solid push to the evolution of the induced pluripotent stem cells.

North America is one of the regions attracting colossal research funding and industry investments in induced pluripotent stem cells technologies. Continuous efforts of players to generate immune-matched supply of pluripotent cells to be used in disease modelling has been a key accelerator for growth. Meanwhile, Asia Pacific has also been showing a promising potential in the expansion of the prospects of the market. The rising number of programs for expanding stem cell-based therapy is opening new avenues in the market.

All the players running in the global Induced Pluripotent Stem Cells market are elaborated thoroughly in the Induced Pluripotent Stem Cells market report on the basis of R&D developments, distribution channels, industrial penetration, manufacturing processes, and revenue. In addition, the report examines, legal policies, and comparative analysis between the leading and emerging Induced Pluripotent Stem Cells market players.

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Induced Pluripotent Stem Cells Market Predicted to Witness Surge in the Near Future2018 2028 - TechNews.mobi

Global Gene Therapy Market Worth Reach USD 6892 Million By 2027 – TheInfobiz

Facts and Factors Market Researchhas published a new report titled Gene Therapy Market By Type (Germ Line Gene Therapy and Somatic Gene Therapy), By Vector Type (Viral Vectors, Non-Viral Vectors, and Human Artificial Chromosome), and By Therapy Area (Cancer, Neurological Diseases, Infectious Diseases, Genetic Disorders, Rheumatoid Arthritis, and Others): Global Industry Perspective, Comprehensive Analysis, and Forecast, 2018 2027.

According to the report, the globalgene therapy market was valued at approximately USD 919 million in 2018 and is expected to reach a value of around USD 6,892 million by 2027, at a CAGR of around 25.1% between 2019 and 2027.

Gene therapy is the kind of experimental method that makes use of genes for treating or preventing disease by inserting foreign genetic material like DNA or RNA into the persons cells. Scientists are studying gene therapy for treating various kinds of immuno-deficiencies, Parkinsons disease, HIV, and cancer by using myriad approaches. Today, many of the approaches to gene therapy are undergoing most intensive & rigorously testing. This includes replacing the mutated gene causing disease with the healthy gene copy. Another approach includes knocking out or inactivating a mutated gene operating improperly. Yet another approach includes a new gene into the body to combat the disease.

Request for Free Sample Report @ https://www.fnfresearch.com/sample/gene-therapy-market-by-type-germ-line-gene

(The sample of this report is readily available on request).

This Free report sample includes:

New product approval & commercialization to drive the market trends

Between the periods from 2012 to 2018, nearly five single-use gene treatments received approval from the U.S. FDA for treating a rare form of genetic disorders. Moreover, gene treatments that have received approval are being tested by pharmaceutical firms in the market. Apart from this, current approvals of gene therapy products across the U.S., as well as European countries for treating a plethora of life-threatening diseases, are anticipated to steer the growth of gene therapy industry over the forecast timeline. Moreover, gene therapy can also be used for treating neurodegenerative disorders like Alzheimer, amyotrophic lateral sclerosis, and spinal muscular atrophy.

Furthermore, many of the reputed pharma firms like Bristol-Myers Squibb, BioMarin, and Pfizer are investing massively into the research activities pertaining to gene therapy. Apart from this, a rise in the occurrence of cancer is prompting the demand to treat the disease. Gene therapy is one of the key treatment kinds that will propel the market growth over the forecast period. However, inadequate reimbursement policies pertaining to the one-time gene treatments will downgrade market expansion.

Inquire more about this report before purchase @ https://www.fnfresearch.com/inquiry/gene-therapy-market-by-type-germ-line-gene(You may enquire a report quote OR available discount offers to our sales team before purchase).

In addition to this, conducting of randomized controlled trials can pose a threat to the expansion of the gene therapy industry as a result of the gene therapy features & projected patient population. Nevertheless, the ability of the gene therapy to eliminate the number of ailments with faulty or missing genes like hemophilia A will promote the market growth over the forecast period and thereby nullify the negative impact of hindrances on the business growth.

Somatic gene therapy to dominate the type segment

The growth of the segment over the forecast timeline is credited to the ability to treat the targeted cells in the patient population. The treatment is not passed to future generations and is restricted to only the patient who receives the somatic gene therapy. Moreover, it is used for treating a huge number of disorders like cystic fibrosis, cancer, and muscular dystrophy.

Request Customized Copy of Report @ https://www.fnfresearch.com/customization/gene-therapy-market-by-type-germ-line-gene(We customize your report according to your research need. Ask our sales team for report customization).

Cancer to lead the therapy area segment over the forecast period

The segmental expansion is attributed to a large number of pipeline drugs registered over the past few years along with increasing occurrence of cancer as a result of genetic changes.

North America to dominate the overall regional market share during the forecast timespan

North American market, which accrued revenue of USD 380 million in 2018, is set to contribute majorly towards the overall market revenue by 2027. The regional market surge is credited to robust healthcare amenities, high per capita healthcare spending, and improvement in the reimbursement policies.

The key players included in this market are Advanced Cell & Gene Therapy, Audentes Therapeutics, Benitec Biopharma, Biogen, Blubird Bio, Inc., Bristol-Myers Squibb Company, CHIESI Farmaceutici SPA, Eurofins Scientific, Geneta Science, Genzyme Corporation, Gilead, GlaxoSmithKline PLC, Human Stem Cells institute, Novartis AG, Orchard Therapeutics, Pfizer Inc., Sangamo therapeutics, Spark therapeutics, and Voyager Therapeutics.

Browse the fullGene Therapy Market By Type (Germ Line Gene Therapy and Somatic Gene Therapy), By Vector Type (Viral Vectors, Non-Viral Vectors, and Human Artificial Chromosome), and By Therapy Area (Cancer, Neurological Diseases, Infectious Diseases, Genetic Disorders, Rheumatoid Arthritis, and Others): Global Industry Perspective, Comprehensive Analysis, and Forecast, 2018 2027Report athttps://www.fnfresearch.com/gene-therapy-market-by-type-germ-line-gene

This report segments the gene therapy market as follows:

Global Gene Therapy Market: By Type Segment Analysis

Global Gene Therapy Market: By Vector Type Segment Analysis

Global Gene Therapy Market: By Therapy Area Segment Analysis

Global Gene Therapy Market: Regional Segment Analysis

About Us:

Facts & Factors is a leading market research organization offering industry expertise and scrupulous consulting services to clients for their business development. The reports and services offered by Facts and Factors are used by prestigious academic institutions, start-ups, and companies globally to measure and understand the changing international and regional business backgrounds. Our clients/customers conviction on our solutions and services has pushed us in delivering always the best. Our advanced research solutions have helped them in appropriate decision-making and guidance for strategies to expand their business.

Contact Us:

Facts & Factors

Global Headquarters

Level 8, International Finance Center, Tower 2,8 Century Avenue, Shanghai,Postal 200120, ChinaTel: +86 21 80360450

Email:sales@fnfresearch.com

Web:https://www.fnfresearch.com

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Global Gene Therapy Market Worth Reach USD 6892 Million By 2027 - TheInfobiz

New research shows what happens to your lung cells once you quit smoking – Daily Gaming Worlld

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We know that smoking cessation is an excellent way to lower your risk of lung cancer. So far, the experts were not sure why this was the case. Our latest research has shown that in people who quit smoking, the body actually fills the airways with normal, non-cancerous cells that help protect the lungs and reduce their risk of cancer.

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Cancer develops when a single villain cell takes on genetic changes called mutations that instruct that cell to ignore all normal growth restrictions, causing it to rapidly multiply out of control. Throughout our lives, all of our cells accept mutations at a constant rate about 20-50 mutations per cell and year. Fortunately, the vast majority of these mutations are completely harmless and do not affect our cells in any measurable way.

But occasionally a mutation in the wrong gene ends up in the wrong cell, driving the cell to cancer. We call these genetic changes driver mutations. For the cell to become a full-blown cancer cell, five to ten or more of these driver mutations are likely to be needed.

Thanks to advances in DNA sequencing technology, we are now able to examine all 3 billion DNA bases that make up the genetic blueprint of a cell (called the genome). By sequencing lung cancer cell DNA in smokers and non-smokers, we know that smoking increases the number of mutations.

The binding of tobacco carcinogens to DNA is affected by their chemical properties, which means that certain types of mutations occur more often than others. For tobacco, this leads to a clear signature of mutations that occur in the genome, which differs from other causes of DNA damage.

Our team was interested in the earliest stages of developing lung cancer. In particular, we try to understand what happens to normal cells when they are exposed to tobacco smoke.

To investigate this, we developed methods to isolate individual normal cells from small biopsies of a patients airways, and then grown these cells in an incubator to obtain enough DNA for sequencing. We then analyzed the genome of 632 cells from 16 study participants, including four non-smokers, six ex-smokers and three current smokers (all in middle age or older) and three children.

Among the never smokers, we found that the number of cell mutations increased with age. So when someone is 60 years old, every normal lung cell contains about 1,000 to 1,500 mutations. These mutations are caused by the normal wear and tear of life, the same type of mutation that we see in other organs in the body. Only about 5% of the cells of never-smokers were found to have driver mutations.

Also read: Early breast cancer treatment in India costs 10 years of average annual wages: WHO

With the current smokers, however, the picture was very different. We found that each lung cell had an average of 5,000 additional mutations that exceeded the expectations of a never-smoking age. It was even more striking that the variation from cell to cell also increased dramatically in smokers.

Some individual cells had 10,000-15,000 mutations ten times more mutations than we would have expected if the person had not smoked. These additional mutations had the signature that we would expect from the chemicals in tobacco smoke, which confirms that they can be traced directly to cigarettes.

In addition to an increase in the total number of mutations, there is also a significant increase in driver mutations. More than a quarter of the lung cells of all smokers currently examined had at least one drive mutation. Some even had two or three. Given that five to ten of these mutations can cause cancer, it is clear that many normal lung cells are likely to become cancerous in middle-aged or older smokers.

Our most exciting result was the people who quit smoking. We found that ex-smokers had two groups of cells. One group had the thousands of additional mutations seen in current smokers, but the other group was essentially normal. The normal cell group had the same number of mutations as we would expect in the cells from someone who had never smoked.

This nearly normal group of cells was four times larger in former smokers than in current smokers. This suggests that these cells increase to refill the airway lining after someone quits smoking. We have seen this expansion of nearly normal cells in former smokers who have smoked a pack of cigarettes daily for more than 40 years.

The reason why this finding is so exciting is that this almost normal group of cells protects against cancer. When we examine a former smokers lung cancer cell, it always comes from the badly damaged group of cells not from the almost normal group.

Now we know why our risk of cancer decreases so much because the body fills the airways with cells that are essentially normal. The next step will be to find out how this group of cells manages to avoid damage from cigarette smoke and how we can encourage them to recover even more.

One possible explanation which emerges from previous work on mouse models is that a group of stem cells is buried deep in the glands that produce the mucus secreted from the airways. This place would of course be better protected from tobacco smoke than the surface of the airways.

Our research is currently repeating that quitting smoking at any age not only slows the accumulation of further damage, but also wakes up cells that were not damaged by previous lifestyle choices.

Sam Janes, Professor of Respiratory Medicine, UCL and Peter Campbell, Head of Cancer, Aging and Somatic Mutation, Wellcome Trust Sanger Institute

This article was republished in The Conversation under a Creative Commons license. Read the original article.

Also read: One in 15 Indians will die from cancer, the WHO report says

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New research shows what happens to your lung cells once you quit smoking - Daily Gaming Worlld

Global Gene Therapy Market to Cross USD 6892 Million By 2027 – TheInfobiz

Facts and Factors Market Researchhas published a new report titled Gene Therapy Market By Type (Germ Line Gene Therapy and Somatic Gene Therapy), By Vector Type (Viral Vectors, Non-Viral Vectors, and Human Artificial Chromosome), and By Therapy Area (Cancer, Neurological Diseases, Infectious Diseases, Genetic Disorders, Rheumatoid Arthritis, and Others): Global Industry Perspective, Comprehensive Analysis, and Forecast, 2018 2027.

According to the report, the globalgene therapy marketwas valued at approximately USD 919 million in 2018 and is expected to reach a value of around USD 6,892 million by 2027, at a CAGR of around 25.1% between 2019 and 2027.

Gene therapy is the kind of experimental method that makes use of genes for treating or preventing disease by inserting foreign genetic material like DNA or RNA into the persons cells. Scientists are studying gene therapy for treating various kinds of immuno-deficiencies, Parkinsons disease, HIV, and cancer by using myriad approaches. Today, many of the approaches to gene therapy are undergoing most intensive & rigorously testing. This includes replacing the mutated gene causing disease with the healthy gene copy. Another approach includes knocking out or inactivating a mutated gene operating improperly. Yet another approach includes a new gene into the body to combat the disease.

Request Free Sample Copy of Research Report @ https://www.fnfresearch.com/sample/gene-therapy-market-by-type-germ-line-gene

(The sample of this report is readily available on request).

This Free report sample includes:

New product approval & commercialization to drive the market trends

Between the periods from 2012 to 2018, nearly five single-use gene treatments received approval from the U.S. FDA for treating a rare form of genetic disorders. Moreover, gene treatments that have received approval are being tested by pharmaceutical firms in the market. Apart from this, current approvals of gene therapy products across the U.S., as well as European countries for treating a plethora of life-threatening diseases, are anticipated to steer the growth of gene therapy industry over the forecast timeline. Moreover, gene therapy can also be used for treating neurodegenerative disorders like Alzheimer, amyotrophic lateral sclerosis, and spinal muscular atrophy.

Furthermore, many of the reputed pharma firms like Bristol-Myers Squibb, BioMarin, and Pfizer are investing massively into the research activities pertaining to gene therapy. Apart from this, a rise in the occurrence of cancer is prompting the demand to treat the disease. Gene therapy is one of the key treatment kinds that will propel the market growth over the forecast period. However, inadequate reimbursement policies pertaining to the one-time gene treatments will downgrade market expansion.

In addition to this, conducting of randomized controlled trials can pose a threat to the expansion of the gene therapy industry as a result of the gene therapy features & projected patient population. Nevertheless, the ability of the gene therapy to eliminate the number of ailments with faulty or missing genes like hemophilia A will promote the market growth over the forecast period and thereby nullify the negative impact of hindrances on the business growth.

Inquire more about this report before purchase @ https://www.fnfresearch.com/inquiry/gene-therapy-market-by-type-germ-line-gene

(You may enquire a report quote OR available discount offers to our sales team before purchase).

Somatic gene therapy to dominate the type segment

The growth of the segment over the forecast timeline is credited to the ability to treat the targeted cells in the patient population. The treatment is not passed to future generations and is restricted to only the patient who receives the somatic gene therapy. Moreover, it is used for treating a huge number of disorders like cystic fibrosis, cancer, and muscular dystrophy.

Cancer to lead the therapy area segment over the forecast period

The segmental expansion is attributed to a large number of pipeline drugs registered over the past few years along with increasing occurrence of cancer as a result of genetic changes.

Request Customized Copy of Report @ https://www.fnfresearch.com/customization/gene-therapy-market-by-type-germ-line-gene

(We customize your report according to your research need. Ask our sales team for report customization).

North America to dominate the overall regional market share during the forecast timespan

North American market, which accrued revenue of USD 380 million in 2018, is set to contribute majorly towards the overall market revenue by 2027. The regional market surge is credited to robust healthcare amenities, high per capita healthcare spending, and improvement in the reimbursement policies.

The key players included in this market are Advanced Cell & Gene Therapy, Audentes Therapeutics, Benitec Biopharma, Biogen, Blubird Bio, Inc., Bristol-Myers Squibb Company, CHIESI Farmaceutici SPA, Eurofins Scientific, Geneta Science, Genzyme Corporation, Gilead, GlaxoSmithKline PLC, Human Stem Cells institute, Novartis AG, Orchard Therapeutics, Pfizer Inc., Sangamo therapeutics, Spark therapeutics, and Voyager Therapeutics.

Browse detail report with in-depth TOC @ https://www.fnfresearch.com/gene-therapy-market-by-type-germ-line-gene

This report segments the gene therapy market as follows:

GlobalGene TherapyMarket:By TypeSegment Analysis

GlobalGene TherapyMarket: ByVector TypeSegment Analysis

GlobalGene TherapyMarket: ByTherapy AreaSegment Analysis

Global Gene TherapyMarket: Regional Segment Analysis

About Us:

Facts & Factors is a leading market research organization offering industry expertise and scrupulous consulting services to clients for their business development. The reports and services offered by Facts and Factors are used by prestigious academic institutions, start-ups, and companies globally to measure and understand the changing international and regional business backgrounds. Our clients/customers conviction on our solutions and services has pushed us in delivering always the best. Our advanced research solutions have helped them in appropriate decision-making and guidance for strategies to expand their business.

Contact Us:

Facts & Factors

Global Headquarters

Level 8, International Finance Center, Tower 2,8 Century Avenue, Shanghai,Postal 200120, ChinaTel: +86 21 80360450

Email:sales@fnfresearch.com

Web:https://www.fnfresearch.com

See the original post here:
Global Gene Therapy Market to Cross USD 6892 Million By 2027 - TheInfobiz