Category Archives: Embryonic Stem Cells

Diabetes breakthrough: Revolutionary stem cell technique treated ‘severe’ disease in study – Daily Express

The new technique, which was developed at the Washington University School of Medicine in St Louis, was shown to convert human stem cells into cells producing insulin. The natural hormone is produced in the pancreas and allows the body to use glucose (sugar) from food for energy. People who suffer from diabetes struggle to produce enough insulin, which leads to a build-up of sugar in the bloodstream.

The St Louis researchers, however, believe their new technique can be used to effectively control blood sugar levels using converted stem cells.

The technique has so far been successfully tested on mice injected with the converted cells.

According to a report that is due to be published on February 24 in the online edition of the journal Nature Biotechnology, the mice were "functionally cured" for nine months.

Dr Jeffrey R. Millman, the principal investigator and assistant professor of medicine and of biomedical engineering, said: "These mice had very severe diabetes with blood sugar readings of more than 500 milligrams per deciliter of blood levels that could be fatal for a person and when we gave the mice the insulin-secreting cells, within two weeks their blood glucose levels had returned to normal and stayed that way for many months."

The same team of researchers has previously discovered how to convert human stem cells into so-called pancreatic beta cells to make insulin.

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When these cells are injected into the bloodstream, they secret the much-needed hormone.

However, the technique was found to have its limitations and was not proven to effectively control the disease in mice.

Their new research has now proven to be much more efficient and effective.

Embryonic stem cells are a type of cell that can be instructed to develop into all sorts of specialised cells.

These can range from simple tissue and muscle cells, to even brain cells.

Scientists worldwide believe stem cell research could unlock many new therapies for ailments such as Alzheimer's disease and HIV.

Dr Millman said: "A common problem when youre trying to transform a human stem cell into an insulin-producing beta cell or a neuron or a heart cell is that you also produce other cells that you dont want."

"In the case of beta cells, we might get other types of pancreas cells or liver cells."

Pancreas and liver cells do not cause any harm when injected into mice but they do not fight the disease either.

Dr Millman added: "The more off-target cells you get, the less therapeutically relevant cells you have.

"You need about a billion beta cells to cure a person of diabetes.

"But if a quarter of the cells you make are actually liver cells or other pancreas cells, instead of needing a billion cells, youll need 1.25 billion cells.

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"It makes curing the disease 25 percent more difficult."

With their new technique, the researchers found fewer off-target cells were produced and the beta cells that were created had improved.

The technique specifically targets the cell's so-called internal scaffolding or cytoskeleton.

The cytoskeleton is what gives cells their shape and allows them to interact with their environment.

Dr Millman said: "Its a completely different approach, fundamentally different in the way we go about it.

"Previously, we would identify various proteins and factors and sprinkle them on the cells to see what would happen.

"As we have better understood the signals, weve been able to make that process less random."

Although the study's results are promising, the expert added there is a long way to go before the technique can be developed into a treatment for humans.

The converted cells will need to be tested over longer periods of time and in bigger animals.

According to Diabetes UK, some 5.5 million people are estimated to have diabetes in the UK by 2030.

Right now, more than 4.9 million people are affected by the disease and 13.6 million people are at increased risk of type 2 diabetes.

About 90 percent of people with the disease have type 2 diabetes, and only about eight percent have type 1 diabetes.

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Diabetes breakthrough: Revolutionary stem cell technique treated 'severe' disease in study - Daily Express

The Role of Quality and Speed in Custom Model Generation – FierceBiotech

The pressure to produce results quickly during the drug discovery and development process continues to increase as does the role of genetically engineered custom mouse models. However, even the fastest custom mouse model generation projects take about 6 months to reach the stage when a few F1 heterozygous mice are available for experiments or breeding. This timeline increases if more than a few heterozygotes or homozygotes are needed. Taconics ExpressMODEL portfolio of products shifts the deliverable of a model generation project from a few heterozygous F1 mice to a much larger cohort of 10-100 mice while reducing the timeline to obtaining data, adding predictability, all without compromising essential quality control steps. By applying innovative thinking and leveraging our ability to seamlessly integrate custom model generation, embryology and colony management services, the ExpressMODEL portfolio achieves the industry's fastest timelines to study cohort with no compromise in quality for models generated using embryonic stem cell (ESC), CRISPR, or random integration transgenic (RITg) methodology.

Regardless of the methodology used to generate the founder animals, ExpressMODEL is built around the concept that using in vitro fertilization (IVF) rather than conventional breeding to generate the F1 mice from founders has a number of distinct advantages including:

However, ExpressMODEL is more than simply using IVF to produce F1 mice because the quality of the male founders needs to be high in order to fully realize the advantages IVF provides. High quality means that founder males need to have both a high percentage of the desired genetically-modified gene and a high fertility rate. Thus, the candidate founder males need to be well-characterized. Because the different methodologies used for model generation produce founders with different characteristics, we have developed unique founder analysis protocols to fit the three different methodologies. The founders produced from injection of ESCs into blastocyst-stage embryos are called chimeras because they are derived from two different populations of genetically distinct cells that originated from different embryos. The founders produced by introducing CRISPR reagents or transgenic DNA into one-cell embryos (zygotes) are mosaics meaning they are composed of two or more different populations of genetically distinct cells that originated from the same embryo.

ExpressMODEL: Embryonic stem cell (ESC)

ESC-mediated mouse model generation remains the gold standard and best choice for complex projects such as genomic replacement humanizations. Using ExpressMODEL: ESC, the timeline for a typical project that would take 66 weeks to deliver a homozygous study-size cohort is reduced to 54 weeks, saving at least 3 months. The key components of ExpressMODEL: ESC are:

The data from these analyses facilitate the choice of founder male(s) to be utilized for the IVF to produce an F1 heterozygous cohort that is sized to meet the customers downstream goals and timeline requirements. It is important to note that all quality control steps in vector construction and ES cell targeting are preserved.

ExpressMODEL: CRISPR

Two great advantages of CRISPR methodology are the speed at which a genetically engineered model can be generated and the ability to modify a wide range of genetic backgrounds, including existing genetically-engineered models. Using ExpressMODEL: CRISPR, the timeline for a typical project that would take 48 weeks to deliver a homozygous study-size cohort is reduced to 36 weeks, saving at least 3 months. ExpressMODEL: CRISPR combines our ability to produce founders with a low degree of mosaicism and to accurately estimate the degree of mosaicism of each founder male. The key components of ExpressMODEL: CRISPR are:

ExpressMODEL: Random Integration Transgenic (RITg)

More than 30 years since the first RITg model was generated, the method continues to be a favored path to quickly generate gain of function models that express an ectopic gene. However, because genomic integration of the transgene is random in each injected embryo, the resulting founder line are unique and may or may not perform to the desired specifications. Additionally, each founder often has transgene insertions at multiple sites and the configuration and copy number of those insertions differs. Thus, RITg founders can be more genetically complex than CRISPR founders. As a result, common practice is to separately propagate multiple lines to generate offspring for extensive transgene expression studies. These data are then used to determine which founder line(s) to propagate. The cost and time of this downstream breeding and characterization of multiple founder lines greatly exceeds the original cost to generate the lines and takes significant additional time. Because transgene expression is assessed in founder animals, ExpressMODEL: RITg takes the guesswork out of the process and allows one to avoid the cost of breeding and characterizing multiple founder lines. Moreover, it reduces project timeline by at least 12 weeks and potentially up to 24 weeks or more. The key components of ExpressMODEL: RITg are:

Additional customizable options are available including the provision of tissue lysates and fixed tissue for protein expression analyses, and transgene mapping analysis to accurately determine the transgene integration site and configuration.

Taconics ExpressMODEL suite of technologies is designed to reduce the custom model generation timeline from project conception to study cohort without taking any shortcuts that compromise quality. Taconic provides a seamless end-to-end solution incorporating industry leading model generation, embryology, and colony management capabilities that allows your project to travel in the express lane.

Interested in learning more about custom animal model generation? Visit Taconic's website at http://www.taconic.com.

This article was created in collaboration with the sponsoring company and our sales and marketing team. The editorial team does not contribute.

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The Role of Quality and Speed in Custom Model Generation - FierceBiotech

Healthcare Researchers Are Putting HUMAN Immune Systems In Pigs To Study Illnesses-Here’s The Tech Behind It – Tech Times

RJ Pierce, Tech Times 05 October 2021, 09:10 am

Healthcare research has gone a long way from the dark days of old, when today's simplest illnesses can be a death sentence. And now, there's reason to look forward to a brighter future because of this news.

(Photo : Getty Images )

According to BigThink, a team from Iowa State University claimed that they've found a way to integrate human immune systems in pigs, as a way to study illnesses much closer.

In other words, they basically "humanized" the pigs to try and find out how to better treat human diseases in the future.

The implications of their research are quite profound, too. As per the researchers, this breakthrough could theoretically advance healthcare research in areas such as virus and vaccines, cancer, and even stem cell treatments.

Before this, scientists often used mice in their biotech and biomedical experiments. However, the problem is that mice-based results don't translate well to humans.

Aside from mice, primates have also been used in related fields of healthcare research due to their direct biological connections with humans. Nevertheless, a lot of ethical issues popped up, thus leading to the retirement of primates, including chimpanzees, from this type of research eight years ago.

This won't be the first time that healthcare research has produced what's basically human-animal hybrids to study illnesses.

Three years ago, a team of scientists from Rockefeller University in New York managed to create a human-chicken embryo, in an attempt to take a closer look at the intricacies of stem cell therapies.

Read also: Scientists Want To Create Part-Human Part-Animal Chimeras To Find Cure For Diseases

It started when the same scientists from Iowa State University discovered a genetic mutation in pigs that caused an illness called SCID (Severe Combined Immunodeficiency).

Some people may know this from the film "The Boy In The Plastic Bubble" from 1976, which tells the story of a child whose immune system never fully developed. As such, he was forced to literally live inside a sterile bubble because even the slightest cold would kill him.

Upon this discovery, the researchers then developed a pig that's far more immunocompromised compared to a person with SCID, then successfully "humanized" it by injecting human immune stem cells into the livers of piglets.

The researchers were able to do this by using ultrasound imaging as a guide.

Ultrasound imaging, also known as sonography, makes use of high-frequency waves to look inside the body.

(Photo : Getty Images )

The resulting pigs had excellent healthcare research potential, because they were found to have human immune cells in their blood, thymus gland, spleen, and liver.

However, the SCID-afflicted pigs are in constant danger of infections. As such, they have to be housed in so-called bubble biocontainment facilities. These facilities work by maintaining high positive pressure, which keeps dangerous pathogens out. All staff members have to wear sterile protective gear at all times.

They've basically turned into their own versions of the boy in the bubble.

Before this research, pigs have often been used to know more about the human body because of how strikingly similar their anatomy is to humans.

In fact, a few scientists even believe that with how biologically similar pigs are to humans, they might be classified into an animal family occupied by primates, reportedScience.org.au.

But of course, there have been ethical issues involving the use of these human-animal hybrids for healthcare research. Eventually, though, the National Institutes of Health (NIH) relaxed their regulations a bit back in 2016, which made it easier for scientists to transfer human stem cells into animal embryos.

Related: Scientists Grow Sheep Embryos With Human Cells To Revolutionize Organ Transplant

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Healthcare Researchers Are Putting HUMAN Immune Systems In Pigs To Study Illnesses-Here's The Tech Behind It - Tech Times

Stem cells may be the key to saving white rhinos from extinction – Sciworthy

It is too late for conservation efforts to save the northern white rhinoceros, but with recent scientific advancements there may still be hope to bring back this beloved species. In a recently published paper, scientist Marisa Korody and her colleagues at San Diego Zoo Global (USA) and at the Department of Molecular Medicine at Scripps Research (USA) describe their exciting progress on using stem cells to revive the northern white rhino.

The northern white rhino is functionally extinct, meaning there are not enough of these rhinos left to save the species. In fact there are only two northern white rhinos left: a mother and a daughter. But for decades, scientists have preserved cell samples from 15 northern white rhinos containing enough genetic material to potentially bring this species back from the brink. These preserved samples hold fibroblast cells the type of skin cells that secrete collagen from white rhinos. With these scientists newly developed methods, fibroblast cells can be converted into something much more valuable: induced pluripotent stem cells. These stem cells can differentiate into any cell type in the body including heart cells, muscle cells, and reproductive cells.

In theory, by converting fibroblast cells into reproductive cells, scientists could create genetically unique rhino embryos. Alongside other assisted reproduction technologies, scientists could implant a new embryo into a closely-related southern white rhino, where the baby northern white rhino could develop as an otherwise normal pregnancy. By completing this process multiple times, scientists may be able to establish a stable population of northern white rhinos.

In 2011, this research team generated induced pluripotent stem cells from the samples of another endangered species, but unfortunately since this process was found to harm the recipient genomes, this method was largely unsuccessful. Despite this setback, in 2015 the authors met with colleagues worldwide to consider ways to save the northern white rhino, and they concluded that methods involving induced pluripotent stem cells may still be the most promising solution. Over the following years, the scientists worked to improve their methods, and these improvements are documented in their recent paper. These experiments represent the first step in a long-term plan to bring the northern white rhino back through assisted reproduction techniques.

Right from the start, the scientists faced a whole host of challenges. Through trial and error they modified the growth medium for the cells, optimizing it for rhinoceros cells. With their improved growth medium, scientists successfully generated induced pluripotent stem cell lines from 11 rhinoceros individuals. This has never been done before and represents a huge stride forward in the path to recovering this species.

Before trying to make their first rhino, the scientists needed to stress these induced pluripotent stem cells and sequence their genomes to determine if the cell quality is good enough to potentially produce new, viable rhinos. They maintained colonies of these cells in long-term cultures and exposed these colonies to different conditions to give insight into how resilient these cells could be. These tests demonstrated that long-term culture did not affect the potential for these cells to differentiate into cardiac lineage cells, confirming that these cells are stable long-term. The researchers also confirmed that these pluripotent cells could potentially produce gametes, the egg and sperm cells that are used for sexual reproduction. These advancements indicate that with these newly developed protocols, induced pluripotent stem cells are a promising tool that could someday help recover the northern white rhino.

Although this study includes some exciting results, there is still much work to do. For example, scientists must now sequence the genomes of the northern and southern white rhino so other researchers can analyze the stem cells ability to stay the same over time. Despite the work that still needs to be done, these promising advancements could someday help the northern white rhino population recover. This method may also work for saving other endangered or extinct species, as long as the genetic material needed is available. Long-term, these scientists plan to continue a series of experiments that could ultimately bring this beloved rhino, and potentially other endangered species, back from the brink of extinction.

Original study: Rewinding Extinction in the Northern White Rhinoceros: Genetically Diverse Induced Pluripotent Stem Cell Bank for Genetic Rescue

Study published on: February 15, 2021

Study author(s): Marisa L. Korody, Sarah M. Ford, Thomas D. Nguyen, Cullen G. Pivaroff, Iigo Valiente-Alandi, Suzanne E. Peterson, Oliver A. Ryder, and Jeanne F. Loring

The study was done at: San Diego Zoo Global (USA), Scripps Research (USA)

The study was funded by: San Diego Zoo Global and San Diego Zoo Wildlife Conservancy donors, including Anne and Christopher Lewis, and the Robert Kleberg and John and Beverly Stauffer Foundations; and Uma Lakshmipathy from Thermo Fisher Scientific for providing supplies

Raw data availability: Contact author for data (all other data are accessible from the article or supplementary materials)

Featured image credit: Hein waschefort, CC BY-SA 3.0, via Wikimedia Commons

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Stem cells may be the key to saving white rhinos from extinction - Sciworthy

Global Allogenic Stem Cell Therapy Market 2021 Size, Share, Growth and Regional Analysis by Segmentation and Country Forecast to 2028 – Digital…

The Report Published by Data Bridge Market Research on Allogenic stem cell therapy Market 2021, This report provides details of Market analysis, size, share and forecast, trends, growth drivers, and challenges, as well as vendor analysis. The company profiles of all the dominating market manufacturers and brands that are making moves such as product launches, joint ventures, mergers, and acquisitions are described in the report. It also becomes easy to analyse the actions of key players and respective effect on the sales, import, export, revenue, and CAGR values. The market data of the Allogenic stem cell therapy report is useful for businesses in characterizing their individual strategies. Allogenic stem cell therapy market report estimates the size of the market with respect to the information on key merchant revenues, development of the industry by upstream and downstream, industry progress, key companies, along with market segments and application.

Allogenic stem cell therapy market is expected to gain market growth in the forecast period of 2020 to 2027. Data Bridge Market Research analyses the market to account to grow at a CAGR of 10.10% in the above-mentioned forecast period.

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Objective Of The Report

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Allogenic Stem Cell TherapyMarket Drivers:

The rapid development in infrastructure for stem cell banking has been directly impacting the growth of allogenic stem cell therapy market.

The high growth of advanced genome-based cell analysis techniques has been driving the market and is acting as a potential driver for the allogenic stem cell therapy market over the forecast period of 2020 to 2027.

The rising awareness correlated to the therapeutic potency of stem cells in effective disease management is also contributing towards the growth of the target market.

The growing public-private investments for the expansion of stem cell therapies, growth in infrastructure related to stem cell banking and processing along with increase in prevalence of chronic diseases such as cardiovascular diseases andcanceras well as increase in funding for stem cell research are also increasing the allogenic stem cell therapy market size.

Moreover, the rising awareness totherapeutic potencyof stem cells in disease organization is actively driving the growth of the target market. In addition, the appearance of IPSCS as an efficient substitute to ESCS and supportive regulations across emerging countrieswill flourish various growth opportunities for the allogenic stem cell therapy market in the above mentioned forecast period.

However, the uncertain regulatory guidelines for product development and commercialization as well as socio-ethical issues coupled with the use of ESCS in disease treatment will hamper the growth of the allogenic stem cell therapy market in the forecast period of 2020 to 2027.

Allogenic Stem Cell TherapyMarket Restraints:

Thetechnical limitations associated with production scale-up will pose as a challenge towards the growth of the market in the above mentioned forecast period.

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Analyze and forecast Allogenic stem cell therapy market on the basis of type, function and application.

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Key points for analysis in the report

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Global Allogenic Stem Cell Therapy Market 2021 Size, Share, Growth and Regional Analysis by Segmentation and Country Forecast to 2028 - Digital...

Human Embryonic Stem Cells Market 2021 Is Booming Across the Globe by Share, Size, Growth, Segments and Forecast to 2027 The Courier – The Courier

Latest research report, titled GlobalHuman Embryonic Stem Cells MarketInsights 2021 and Forecast 2026, This includes overview and deep study of factors which are considered to have greater influence over future course of the market such asmarket size, market share, different dynamics of the industry, Human Embryonic Stem CellsMarket companies, regional analysis of the domestic markets, value chain analysis, consumption, demand, key application areas and more.The study also talks about crucial pockets of the industry such as products or services offered, downstream fields, end using customers, historic data figures regarding revenue and sales, market context and more.

Top Key players profiled in the report include:ESI BIO, Thermo Fisher, BioTime, MilliporeSigma, BD Biosciences, Astellas Institute of Regenerative Medicine, Asterias Biotherapeutics, Cell Cure Neurosciences, PerkinElmer, Takara Bio, Cellular Dynamics International, Reliance Life Sciences, Research & Diagnostics Systems, SABiosciences, STEMCELL Technologies, Stemina Biomarker Discovery, Takara Bio, TATAA Biocenter, UK Stem Cell Bank, ViaCyte, Vitrolifeand More

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On the basis of product, we research the production, revenue, price, market share and growth rate, primarily split into Totipotent Stem Cells, Pluripotent Stem Cells, Unipotent Stem Cells

Market Segment by Applications, can be divided into: Research, Clinical Trials

Global Human Embryonic Stem CellsMarket by Geography:

Asia-Pacific(Vietnam, China, Malaysia, Japan, Philippines, Korea, Thailand, India, Indonesia, and Australia) Europe(Turkey, Germany, Russia UK, Italy, France, etc.) North America(the United States, Mexico, and Canada.) South America(Brazil etc.) The Middle East and Africa(GCC Countries and Egypt.)

Years Considered to Estimate the Market Size: History Year:2018-2021 Base Year:2021 Estimated Year:2021 Forecast Year:2021-2026

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1.Human Embryonic Stem CellsMarket Overview 1.1 Introduction 1.2 Scope 1.3 Assumptions 1.4 Players Covered 1.5 Market Analysis By Type 1.5.1 Global Human Embryonic Stem CellsMarket Size Growth Rate By Type (2021-2026) 1.5.2 1.6 Market By Application 1.6.1 Global Human Embryonic Stem CellsMarket Share By Application (2021-2026) 1.6.2 Application 1

2. Executive Summary

3. Human Embryonic Stem CellsMarket Analysis By Type (Historic 2016-2021) 3.1 Global Human Embryonic Stem CellsMarket Size Analysis (USD Million) 2016-2021 3.1.1 Type 1 3.1.2 3.2 Global Human Embryonic Stem CellsMarket Share Analysis By Type (%) 2016-2021

4. Human Embryonic Stem CellsMarket Analysis By Application (Historic 2016-2021) 4.1 Global Nanoscale Smart Materials Market Size Analysis (USD Million) 2016-2021 4.1.1 Application 1 4.1.2 Application 2 4.1.3 Application 3 4.2 Global Human Embryonic Stem CellsMarket Share Analysis By Application (%) 2016-2021

5. Human Embryonic Stem CellsMarket Analysis By Regions (Historic 2016-2021) 5.1 Global Human Embryonic Stem CellsMarket Size Analysis (USD Million) 2016-2021 5.1.1 Human Embryonic Stem CellsMarket Share By Regions (2016-2021) 5.1.2 United States 5.1.3 Europe 5.1.4 China 5.1.5 Japan 5.1.6 India 5.1.7 Rest Of The World

6. Key Companies Analysis/Company Profile

Continued..

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Human Embryonic Stem Cells Market 2021 Is Booming Across the Globe by Share, Size, Growth, Segments and Forecast to 2027 The Courier - The Courier

Conversion of mouse embryonic fibroblasts into neural crest cells and functional corneal endothelia by defined small molecules – Science Advances

INTRODUCTION

Corneal disorders are the vital leading cause of blindness, with 12.7 million patients suffering from corneal blindness globally (1). Corneal transplantation, which replaces the damaged cornea with healthy donated corneal tissue, is the primary therapy. However, fewer than 1.5% of patients requiring corneal grafts can receive allotransplants due to a global shortage of donor corneas. Corneal endothelial keratoplasty has grown exponentially and is currently indicated for close to half of all corneal transplantations (2). The corneal endothelium, a monolayer of dedicated cells essential for maintaining the corneas transparency and useful visual function, is naturally nonrenewable in vivo but is lost with age or in various disease states. Therefore, the massive global demand for corneal endothelia allograft cannot be fulfilled at the current rate of cornea donation. Recently, success with surgical grafting of in vitro cultured corneal endothelial cells (CECs) provided a critical proof of principle for a strategy to develop CEC-based cell therapies (3). However, it remains challenging to obtain sufficient CECs from healthy donor tissues.

Direct lineage reprogramming facilitates the generation of functional cell types independent of the donor organ for applications in cell replacement therapy (4). Terminally differentiated cells can be converted to other cell types in vitro by the introduction of lineage-specific transcription factors (TFs), bypassing the pluripotent state (510). Similarly, these conversions can also be induced by the overexpression of specific TFs in vivo (1113). In addition, direct lineage reprogramming mediated by TFs has been used in disease modeling (14), implying its potential for practical applications. Recently, the small moleculebased conversion of fibroblasts to other functional cells represents an attractive reprogramming strategy and has attracted much interest because of its safety and efficiency (1518).

Developmentally, the corneal endothelia originate from neural crest cells (NCCs) (19, 20), which are a population of transient and multipotent cells giving rise to diverse differentiated cell types, including peripheral neurons, glia, melanocytes, and several types of ocular cells (21). During ocular development in vertebrates, NCCs delaminate from the roof plate upon closure of the neural tube and migrate into the eye, where they form the periocular mesenchyme; this tissue further differentiates into diverse cell lineages, including the corneal endothelium, stroma, trabecular meshwork, and others (20, 22, 23).

In this study, we developed a two-step lineage reprogramming strategy to generate chemically induced CECs (ciCECs) from fibroblasts using defined small molecules. We screened a new cocktail of small molecules that could efficiently convert mouse fibroblasts into chemically induced NCCs (ciNCCs) bypassing the pluripotent state. The ciNCCs exhibited typical NCC features and could be further differentiated into ciCECs using another combination of small molecules in vitro. Through lineage tracing in Wnt1-Cre/ROSAtdTomato and Fsp1-Cre/ROSA26tdTomato fibroblasts, we confirmed that the ciNCCs and ciCECs were converted from fibroblast cells. The ciCECs showed similar gene expression profiles and self-renewal capacity to those of primary CECs (pCECs). Transplantation of the ciCECs into an animal model reversed corneal opacity, yielding clear tissue. Our findings provide a new approach to the generation of neural crestlike cells and functional corneal endotheliallike cells, providing a different alternative cell source for regeneration of corneal endothelia and other tissues derived from neural crest.

To identify the chemicals sufficient for ciNCC generation from mouse fibroblasts, we used a lineage tracing strategy to monitor the conversion process and excluded any NCCs from the starting mouse embryonic fibroblasts (MEFs) (Fig. 1A and fig. S1A). Wnt1-Cre transgenic mice have been validated as a lineage-tracing reporter model for NCC development (24, 25). In Wnt1-Cre/ROSA26tdTomato mice, tdTomato protein was faithfully expressed in NCCs. MEFs were isolated from Wnt1-Cre/ROSA26tdTomato mice at embryonic day 13.5 (E13.5). Because the NCC population was marked with tdTomato, we performed fluorescence-activated cell sorting (FACS) to collect the tdTomato population to exclude any NCCs or progenitors (the purified cells are hereinafter referred to as Wnt1-tdTomato MEFs; fig. S1B). We confirmed that the Wnt1-tdTomato MEFs were also negative for other NCC markers, including Sox10, P75, Pax3, HNK1, and AP2 (fig. S1, C and D).

(A) Schematic diagram of the reprogramming of NCCs from MEFs. (B) Effects of individual chemicals on ciNCC generation. Data are means SD; n = 3 independent experiments. (C) Enhancement effects of individual chemicals on the generation of ciNCCs. Data are means SD; n = 3 independent experiments. (D) Schematic illustration of our strategy to convert MEFs into ciNCCs. (E) Generation of Wnt1+ ciNCCs from MEFs using a cocktail of small molecules. DMSO, dimethyl sulfoxide. (F) Quantification of Wnt1+ cells induced by candidate cocktails at day 12. Independent experiments, n = 3. (G) Morphological changes on distinct days during the induction process of ciNCCs. Scale bar, 50 m. (H) Percentages of Wnt1-tdTomato+ cells induced by candidate cocktails on distinct days. Independent experiments, n = 3.

To generate ciNCCs from mouse fibroblasts, we hypothesized that small molecules shown to target NCC lineagespecific signaling would facilitate NCC reprogramming. Therefore, we selected 16 small molecules as candidates based on (i) the epigenetic regulation and signaling modulation of NCC development (2629) and (ii) enhanced neural lineage reprogramming. The small molecules for NCC reprogramming were as follows: valproic acid (VPA), SB431542, RepSox, LDN193189 (LDN), CHIR99021, Y-27632, retinoic acid (RA), Forskolin, A8301, EPZ004777 (EPZ), RG108, 5-azacytidine (5-Aza), SMER28, AM580, Parnate, and BMP4 (table S1). For initial screening, the Wnt1-tdTomato MEFs were cultured in a 24-well plate and treated with small molecules. After testing various small-molecule conditions, we found that a combination of SB431542, CHIR99021, and Forskolin yields consistent tdTomato expression (0.53 0.03%) (Fig. 1B). SB431542 is an inhibitor of transforming growth factor (TGF-) signaling, which is important for NCC differentiation (3032). Similarly, CHIR99021 is an inhibitor of glycogen synthase kinase 3 (GSK3) signaling, which is involved in promoting NCC fate (31, 33, 34). Forskolin, a adenosine 3,5-monophosphate (cAMP) agonist, is crucial in early reprogramming for mesenchymal-to-epithelial transition (MET) (35). For subsequent screening and optimization, we used the combination of SB431542, CHIR99021, and Forskolin as induction basal condition. We found that VPA (a histone deacetylase inhibitor), EPZ004777 [Disruptor of telomeric silencing 1-like (DOPTiL) inhibitor], and 5-Aza (a DNA methylation inhibitor) further enhanced the induction of Wnt1-tdTomato+ cells (Fig. 1C). Therefore, we used a chemically defined medium combined with a cocktail of six small molecules (VPA, CHIR99021, SB431542, Forskolin, 5-Aza, and EPZ004777) (hereafter termed M6) to reprogram mouse fibroblasts into ciNCCs (Fig. 1D). Expression of Wnt1-tdTomato in individual cells was observed as early as day 3 with M6 medium treatment (Fig. 1E). The M6 reprogramming medium effectively induced Wnt1-tdTomato+ cells at 3.67 0.21% (Fig. 1F). On days 5 to 7, inducted Wnt1-tdTomato+ colonies were observed in the M6 reprogramming medium (Fig. 1G and fig. S1E). These Wnt1-tdTomato+ cells and colonies had molecular feature similar to those of primary NCCs (pNCCs). The Wnt1-tdTomato+ cell number increased notably in small colonies at approximately day 12 (Fig. 1H). Although the generation of Wnt1-tdTomato+ NCCs was only as efficient as the conversion of human fibroblasts using TFs (33), merely 2 to 5% of cells were positive for Wnt1-tdTomato in this study. The results were reproducible in different batches of MEFs (n = 8), and MEFs with different genetic backgrounds (C57BL/6, 129C57BL/6, and 129) could also be converted into ciNCCs via M6 conditions (fig. S2). Together, these results indicate that M6 can reprogram MEFs into ciNCCs. Furthermore, M6 could induce neonatal mouse tail-tip fibroblasts (TTFs) into ciNCCs, albeit at a lower reprogramming efficiency (fig. S3).

In the reprogramming process of ciNCCs, Wnt1-positive cells appeared as early as day 3. Small cell colonies emerged toward day 5. With the extension of induction time, the Wnt1-positive cells proliferated gradually.

To obtain the ciNCCs, we performed FACS to collect the Wnt1-tdTomato+ cells on days 12 to 16. Established ciNCCs were serially propagated in NCC expansion medium containing N2, B27, basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). Morphologically, M6-induced cells at P3 maintained typical NCC features in monolayer culture (Fig. 2A). After passaging, the ciNCCs became morphologically homogeneous. To further characterize the M6-induced Wnt1-tdTomato+ cells, we sought to examine their gene expression. Our results showed the ciNCCs expressed multiple NCC markers, including P75, HNK1, AP2, and Nestin (Fig. 2B). Furthermore, we tested if the ciNCCs have differentiation potential toward peripheral neurons, Schwann cells, and others. For differentiation of ciNCCs, these cells were cultured in different lineage differentiation media. After cultured for 2 to 4 weeks, the differentiated cells were examined by assessing the expression of the markers by immunostaining. Notably, the ciNCCs could also give rise to cells expressing specific markers for neuron, including Tuj1 and Peripherin (Fig. 2C). For melanocyte differentiation, we observed melanocytes after 2 to 3 weeks of induction (Fig. 2C). Our results of immunostaining showed that the ciNCCs could differentiate to Schwann cells. The induced Schwann cells were GFAP+ and S100+ cells (Fig. 2D). Further differentiation of these ciNCCs in vitro gave rise to mesenchymal lineages, resulting in typical mesenchymal cell morphology. Our results showed that these ciNCC-derived mesenchymal cells could give rise into osteogenic, adipogenic, and chondrogenic cells (Fig. 2E). Collectively, these data indicated that our ciNCCs could be induced to differentiate toward peripheral nervous system lineages and mesenchymal lineages.

(A) Morphology of M6-induced ciNCCs. Scale bar, 400 m. (B) Immunostaining showing that MEF-derived ciNCCs express P75, Sox10, HNK1, AP2, and Nestin. Scale bar, 50 m. (C) Representative images of melanocytes and differentiated ciNCCs stained with peripheral neuron markers. Scale bars, 50 m. (D) Differentiation of ciNCCs into Schwann cells and melanocytes with marker expression. Scale bar, 50 m. (E) ciNCCs were differentiated into mesenchymal lineages and further into adipocytes, chondrocytes, and osteocytes. Scale bars, 100 m.

The mechanisms prompting the differentiation of NCCs to CECs remain unclear (36, 37). The niche surrounding NCCs, for example, lens epithelial cells (LECs), determines their ultimate fate during differentiation (38). To generate mouse CEC-like cells from ciNCCs, we sought small molecules based on their importance in CEC organogenesis and maintenance in vitro. SB431542 (an inhibitor of TGF- signaling) was capable of inducing CEC-like cells from human pluripotent stem cell (PSC) (39, 40). A previous study has reported that the Wnt signaling inhibitor Dkk2 promotes NCC differentiation into CECs (39). We selected the chemical inhibitor CKI-7 as a substitute, which has been shown to block Wnt signaling by inhibiting casein kinase I (41).

We decided to use the medium containing SB431542 and CKI-7 for CEC differentiation from ciNCCs (Fig. 3A). After 7 to 15 days of culture in this differentiation medium, a small population outside or inside the clusters displayed typical tight aggregates with homogeneous and polygonal morphology (Fig. 3B). We also observed that these colonies rapidly expanded, and the small clusters merged into larger ones by days 12 to 15 (Fig. 3B and movie S1). These CEC-like cells grew rapidly and had strong proliferation ability. The ciNCC-derived CEC-like cells formed a monolayer of hexagonal and pentagonal cells. To confirm that the CEC-like cells were derived from ciNCCs, we differentiated tdTomato+ ciNCCs with CEC differentiation medium containing 5 M SB431542 and 5 M CKI-7. These tdTomato+ ciNCCs could also be subsequently induced to differentiate toward polygonal cells (Fig. 3C). We next assessed CEC gene expression including Na+/K+-ATPase (Na+- and K+-dependent adenosine triphosphatase), AQP1, ZO-1, and N-cadherin by immunofluorescence staining. The results showed that these cells expressed CEC-specific markers (Fig. 3D). The TTF-derived ciNCCs also could be induced into CEC-like cells and expressed multiple CEC markers (fig. S3, A and C). In this study, we identified the function of the CEC-like cells by Dil-labeled acetylated low-density lipoprotein (Dil-Ac-LDL) uptake (Fig. 3E). Global gene expression analysis by RNA sequencing (RNA-seq) showed that the CEC-like cells shared a similar gene expression pattern to that of pCECs, but this pattern was distinct from that of the initial MEFs (Fig. 3F). The presence of tight junctions in the CEC-like cells was observed by transmission electron microscopy (TEM) (Fig. 3G). To further monitor the reprogramming process, we confirmed the expression of a panel of NCC and CEC markers at distinct times by using quantitative reverse transcription polymerase chain reaction (qRT-PCR). By day 12, robust expression of NCC genes was detected in cells, including Hnk1, P75, Sox10, Sox9, Pax3, and Ap2 (Fig. 4A). In addition, similar kinetics of gene activation for CEC genes, such as Slc4a1c, Col8a1, Na+/K+-ATPase, Aqp1, and N-cadherin, were also detected by qRT-PCR (Fig. 4B). The genes that are known to be enriched in pCECs were greatly up-regulated in ciCECs. To validate the transition process from fibroblast to CECs, we analyzed the transcriptome by using RNA-seq (Fig. 4C). The expression of CEC signature genes in ciCECs was substantially up-regulated, consistent with that of pCECs, whereas fibroblast signature genes were markedly down-regulated. Notably, a panel of NCC marker genes was initially up-regulated and subsequently down-regulated during the induction process (16, 18, 40, 42, 43). Principal components analysis revealed that M6-treated cells were distinct from the original MEFs, indicating that chemical reprogramming led to marked transcriptional changes (Fig. 4D). These results indicate that the CEC-like cells obtained CEC identity. Collectively, these data suggest that the combination of SB431542 and CKI-7 effectively promoted the generation of CECs within 10 to 15 days in the ciNCC culture. Those fibroblast-originated CEC-like cells are thereafter referred to as ciCECs.

(A) Schematic diagram for the chemical reprogramming of ciCECs from MEFs. (B) Bright-field images of initial MEFs, reprogrammed ciNCC colonies, and ciCECs. Scale bar, 400 m. (C) Morphological changes on different days during the induction process of ciCECs from Wnt1-tdTomato+ ciNCCs. Scale bar, 400 m. (D) Immunofluorescence staining of ciCECs for the corneal endothelium markers Na+/K+-ATPase, AQP1, vimentin, N-cadherin, laminin, and ZO-1. Scale bars, 50 m. (E) LDL uptake function in ciCECs at P2 and pCECs at P3. Scale bar, 50 m. (F) Heatmap of differentially expressed genes among the samples at the indicated time points. These numbers below the heatmap indicate independent biological replicates. Red and blue indicate up-regulated and down-regulated genes, respectively. (G) TEM of ciCECs showing the tight junctions. Scale bar, 5 m.

(A) qRT-PCR analysis showing the expression of NCC genes at the indicated time points. Gene expression (log2) was normalized to that in MEFs. (B) qRT-PCR analysis of the expression of the indicated CEC genes in MEF-derived ciCECs at different passages, MEFs, and pCECs. (C) Heatmap of differentially expressed genes among samples at the indicated time points. The number below the heatmap indicates independent biological replicates (n = 12). Red and blue indicate up-regulated and down-regulated genes, respectively. (D) Principal components analysis of samples from day 0 (D0), day 7 (D7), and day 12 (D12) of reprogramming, ciCECs, and the control pCECs.

To confirm the origin of the initial fibroblasts for the small moleculebased reprogramming, we sought a genetic lineage-tracing strategy to purify fibroblast-specific protein 1 (Fsp1)tdTomato+ fibroblasts (Fig. 5A). Fsp1-Cre has been validated as a specific fibroblast marker for lineage tracing (44); thus, Fsp1-Cre mice were crossed with ROSA26tdTomato mice. MEFs were isolated from transgenic mice (Fsp1-Cre/ROSA26tdTomato) at E13.5, and the fibroblasts expressed tdTomato specifically; these cells are hereafter named tdMEFs (fig. S4A). To avoid possible contamination of the MEFs with NCC progenitor cells, we carried out FACS to collect the tdTomato+/p75 population (Fig. 5B and fig. S5G). These tdMEFs were negative for all NCC markers, including P75, HNK1, Sox10, and AP2 (Fig. 5C).

(A) Schematic diagram illustrating the genetic lineage-tracing strategy. MEFs were obtained by sorting for p75/tdTomato+ cells from MEFs derived from E13.5 mouse embryos of the Fsp1-Cre/ROSA26tdTomato background. (B) FACS result showing the p75/tdTomato+ cells from MEFs with a genetic background of Fsp1-Cre/ROSA26 tdTomato. (C) Immunostaining analysis showing negative results for Sox10, P75, HNK1, AP2, Sox2, and Pax6 in the p75/tdTomato+ cells. Scale bar, 100 m. (D) p75/tdTomato+ cell differentiation toward ciNCCs and ciCECs. Scale bars, 400 m. (E) Immunostaining analysis showing positive results for ZO-1, laminin, Na+/K+-ATPase, and AQP1 in the Fsp1-tdTomato-MEFderived ciCECs. Scale bar, 50 m.

Then, these tdMEFs were induced with the abovementioned M6 medium. Epithelial clusters expressing tdTomato were observed during ciNCC induction (Fig. 5D and fig. S4B). We passaged the Fsp1-tdTomato+ ciNCCs and cultured them in NCC expansion medium for further experiments (after 2 weeks of induction). Immunofluorescence analysis confirmed that these Fsp1-tdTomato+ ciNCCs were positive for the NC markers P75, HNK1, Sox10, and AP2, indicating that the colonies of Fsp1-tdTomato+ ciNCCs had the differentiation potential toward CEC-like cells (fig. S4C). Furthermore, the Fsp1-tdTomato+ ciNCCs could differentiate into tdTomato+ ciCECs (fig. S4D). By immunostaining, we found that differentiated CEC-like cells coexpressed Na+/K+-ATPase, AQP1, laminin, ZO-1, Na+/K+-ATPase, and tdTomato (Fig. 5E). Notably, all these ciCECs also expressed tdTomato, demonstrating a conversion from fibroblasts. The results confirmed that the ciNCCs and ciCECs were converted by two-step lineage reprogramming from fibroblasts.

Because the mechanism of our lineage reprogramming might be similar to that of chemically reprogrammed induced PSCs (iPSCs) (9, 16), we sought to assess whether the ciCECs went through an iPSC stage. We performed a comparison between chemical iPSC reprogramming and ciCEC induction from MEFs derived from mice harboring an Oct4 promoterdriven green fluorescent protein (GFP) (OG2) reporter (35, 45). The M6-treated MEFs morphologically underwent a characteristic MET, and small cell colonies gradually emerged toward day 6 (fig. S5A). These cell colonies expressed the NCC marker Sox10 (fig. S5B). In contrast, we did not observe any Oct4-GFPpositive cells during the entire process from MEFs to ciCECs based on our method (fig. S5, C and D). Furthermore, the ciCECs maintained a normal karyotype during 10 continuous passages in vitro (fig. S5E). To evaluate the potential risk of tumorigenesis, a total of 5 106 ciCECs and 2 106 mouse embryonic stem cells (mESCs) were transplanted subcutaneously into nonobese diabetic mice (NOD)/severe combined immunodeficient (SCID) mice. Notably, no tumors formed over 6 months after transplantation with ciCECs, whereas large teratomas developed in the mice transplanted with mESCs after 4 to 8 weeks (fig. S5F). This result suggests that the ciCECs have no tumorigenic potential. To better understand their differentiation in vivo, we transplanted ciCECs into the anterior chambers of the eyes of NOD/SCID mice. After 4 to 8 weeks, the transplanted ciCECs did not form tumors over 6 months after transplantation. These results demonstrated that our approach could directly reprogram MEFs to ciNCCs and eventually ciCECs while bypassing the iPSC stage.

The maintenance of cultured CECs with morphology and normal physiological function in vitro has proven challenging (46). We aimed to test whether large numbers of functional ciCECs could be generated from fibroblasts to enable the large-scale application of ciCECs. On the basis of our observations that ciCECs cultured in medium with SB431542 (5 M) and CKI-7 (5 M) were small, polygonally shaped cells (fig. S6A), we hypothesized that SB431542 and CKI-7 would promote ciCEC growth in vitro. We evaluated the long-term expansion capacity of ciCECs in vitro by continuously passaging ciCECs at a 1:6 ratio and found that the phenotype was similar between P3 and P30 (Fig. 6, A and B). This result showed that SB431542 and CKI-7 strongly promoted ciCEC expansion. These ciCECs sustained themselves as a homogeneous cell population for at least 30 passages (P30) with hexagonal morphology in small moleculebased medium. In addition, we successfully clonogenically cultured these ciCECs to 10 passages and demonstrated consistent morphologies. Our results of immunostaining showed that the rate of Ki67-positive cells was higher in ciCECs at P3 than in pCECs at P3 (fig. S6B). ciCECs were highly proliferative, as 24.6, 37.8, and 48.1% of these cells showed incorporation of ethynyl deoxyuridine (EdU) at P1, P3, and P6 (fig. S6C). The results of FACS analysis with propidium iodide (PI) staining showed that the cell cycle distribution (G0-G1, S, and G2-M phases) was 46.30, 45.11, and 8.59% for ciCECs at P3 and 67.30, 21.50, and 11.20% for pCECs at P3 (fig. S6D). They rapidly expanded into large, homogeneous colonies with population doubling times of 22.3 3.7 hours (Fig. 6C). Large vacuole-like structures were found on the surface of the ciCECs at P2 to P10 (Fig. 6D). These vacuole-like structures disappeared at P20 when they were serially propagated. Notably, the ciCECs at P30 also expressed typical CEC markers, including Na+/K+-ATPase, AQP1, and ZO-1 (Fig. 6E). When assayed by imaging for the ability to migrate into the space created by a scratch wound, ciCECs cultured in medium with SB431542 and CKI-7 at different passages showed a stronger capability of proliferation and migration as compared to that of pCECs (fig. S6, E and F). Collectively, these results demonstrated that SB431542 and CKI-7 had a robust and general effect on long-term expansion of ciCECs in vitro.

(A) ciCECs were expanded for 3 days in serum-free control medium. Scale bar, 200 m. (B) Serial expansion of ciCECs in the serum-free medium with addition of SB431542 and CKI-7. Scale bar, 200 m. (C) Average population doubling times (means SD, n = 3; ***P < 0.001) of the ciCECs cultured in medium with and without SB431542 and CKI-7. (D) Bright-field image of ciCECs at P5, which were expanded for 3 days in culture condition with addition of SB431542 and CKI-7. Arrows indicate the vacuole-like structures. Scale bar, 50 m. (E) These ciCECs at P30 were fixed and stained for Na+/K+-ATPase, AQP1, and ZO-1. Scale bar, 50 m.

To evaluate whether the ciCECs had the capacity to regenerate the corneal endothelia in vivo, we transplanted them into a well-established rabbit model with bullous keratopathy by mechanically scraping corneal endothelia from Descemets membrane (47, 48). A previous study found that injection of human pCECs supplemented with a ROCK inhibitor restored endothelial function (49). Corneal edema decreased much earlier after ciCEC transplantation in the grafted eyes than in the untreated eyes. Compared to the untreated eyes, which showed that the clarity of the cornea was unchanged, we noted that the clarity of the cornea of the grafted eyes increased gradually after transplantation (Fig. 7A). After 7 days, the corneas of the grafted eyes became transparent, while corneal opacity and stromal oedema remained poor in the untreated eyes. The results of slit-lamp examination showed that the clarity of the cornea of the grafted eyes was also notably improved after injection, and the pupil and iris texture could be clearly observed (Fig. 7B and fig. S7A).

(A) Diagram depicting the transplantation of ciCECs with the ROCK inhibitor into model rabbits. (B) Corneal transparency in the grafted eyes was notably improved after transplantation, while corneal opacity and stromal oedema were still serious in the untreated controls. (C) Slit-lamp microscopic images showing that the clarity of the grafted corneas was substantially improved after transplantation, while corneal opacity and stromal oedema remained in the untreated controls. (D) Immunohistochemistry showing that surviving tdTomato+ ciCECs were attached to Descemets membrane. Scale bar, 100 m. (E) Visante OCT showing ameliorated corneal oedema (reduced corneal thickness) in a grafted eye. (F and G) Trend in corneal thickness and corneal clarity after transplantation. There were significant differences in corneal thickness and transparency between the untreated controls and the grafted groups. The results are means and SEM for biological replicates (n = 9). (H) Live confocal imaging of corneal endothelia confirmed full coverage of polygonal cells on Descemets membrane in the grafted eyes. Photo credit: Zi-Bing Jin, Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology & Visual Science Key Laboratory, Beijing, 100730 China.

Next, we investigated ciCEC survival in the grafted eyes. Eyeballs were enucleated at postsurgical day 28 to evaluate the transplanted ciCECs. Fluorescence microscopy examination confirmed the existence of tdTomato-tagged grafted cells. Visante optical coherence tomography (OCT) of the anterior segment also showed that the corneal thickness decreased after ciCEC injection (Fig. 7C and fig. S7C). Confocal microscopy confirmed full coverage of polygonal endothelial cells on Descemets membrane in the ciCEC-grafted disease models, while this could not be detected in the untreated models due to their severe corneal opacity (Fig. 7D). Under magnification, the grafted ciCECs tightly adhered to the posterior surface of the cornea in a monolayer, whereas the Descemets membranes in the untreated model were bare and had no detectable CECs on day 28. Immunohistochemistry demonstrated ZO-1 expression, indicating the adhesion of injected cells onto the corneal tissue (Fig. 7E and fig. S7D).

There was a rapid decrease in corneal thickness within 4 weeks after ciCEC injection, followed by a more gradual decrease over the next 2 weeks (Fig. 7F and fig. S7E). In the untreated group, mean corneal thickness was approximately 1200 m throughout the 42 days of observation. In contrast, it decreased rapidly in the grafted group, being significantly less than that in the untreated group. We observed that the mean corneal thickness at postoperative days 14 (P < 0.01), 21, 28, 35, and 42 (P < 0.001) in the ciCEC-grafted group was significantly less than that in the control group, indicating that corneal edema was markedly reversed. Meanwhile, corneal clarity gradually increased after ciCEC transplantation, and grafted corneas had higher corneal transparency compared to untreated controls (Fig. 7G). These results strongly suggest that ciCEC transplantation repopulated and self-organized on the posterior surface of the cornea and has the capacity to regenerate the corneal endothelium. In this study, the ciCECs were injected combined with a ROCK inhibitor (Y-27632) into the anterior chambers of the eyes (Fig. 7H and movie S2). Each recipient received 1 106 ciCECs. Fellow eyes (normal) and untreated eyes [phosphate-buffered saline (PBS) injection] were used as experimental controls.

Previous studies have reported that NCCs can generate from fibroblasts by introducing NCC-specific TFs (33, 42). In this study, we demonstrated that ciNCCs could be generated from MEFs via a small-molecule reprogramming method in vitro. The generated ciNCCs resemble NCCs in the expression profile of signature genes, capacity for self-renewal, and the differentiation potential into derivative neurons, Schwann cells, and mesenchymal lineages. These finding demonstrated that direct lineage-specific conversion to NCCs from MEFs could be achieved by the manipulation of signaling pathways with small molecules. In addition, given the broad spectrum of NCC derivatives, our approach for developing ciNCCs holds great potential as a different source to generate other NCC-derivative cell types (50).

Furthermore, we successfully generated functional ciCECs from ciNCCs. We explored defined differentiation conditions including SB431542 and CKI-7 to achieve this process in vitro. ciCECs showed an expression profile and function close to those of mature CEC. These ciCECs were found to expand further and maintain contact-inhibited hexagonal phenotype in the defined serum-free chemical medium. The maintenance of pCECs in vitro has been proven challenging. When grown under normal culture conditions in vitro, pCECs often show morphological fibroblastic changes and lose their physiological function (43, 51). A TGF- inhibitor maintains the CEC phenotype during the process of ciCEC generation. A previous study showed that SB431542 assisted in the maintenance of cultivated CECs with a normal polygonal and functional phenotype (52). We found that the ciCECs could maintain normal polygonal morphology and function in the medium with SB431542 and CKI-7 over the long term. Notably, these cells could be serially expanded up to 30 passages and maintained contact-inhibited hexagonal phenotype. The contact-inhibited plays important roles in keeping the monolayer structure of ciCECs. Thus, it does not allow ciCECs to overproliferate in vitro and in vivo. During the passage, main genes related to maturation were increased, indicating that they were mature ciCECs.

Corneal endothelium is derived from periocular NCCs (53). Previous reports have shown that PSCs can differentiate into CECs with a conditioned medium (36, 37, 39). Defined medium containing small molecules promoted PSC to differentiate into CEC-like cells (39). Most approaches for the generation of CECs from stem cells in vitro were stepwise procedures according to the developmental process. Consistent with previous studies, the lineage conversion from mouse fibroblasts is also a stepwise procedure. The induction strategy for ciCECs is composed of two major steps, including an initial chemical conversion of ciNCCs from mouse fibroblasts, followed by lineage-specific differentiation into ciCECs. The ciCECs have advantages over the PSC-derived CECs in certain aspects. The small moleculebased conversion from fibroblasts makes the generation of ciCECs safe, which have no tumorigenic potential. Moreover, ciCECs can be produced from individual patients, thus developing individualized cell therapy (40).

The generation of large numbers of functional CECs for cell-based approaches to corneal endothelial dysfunction is an important goal. Direct reprogramming of fibroblasts to CECs could offer a solution to this problem. The ciCECs exhibited a monolayer of hexagonal shaped cells in vitro. In vivo engraftment of ciCECs substantially reversed the corneal opacity in the rabbit corneal endothelial dysfunction model, indicating their therapeutic effect for corneal endothelial deficiency. A previous study has shown that cultivated rabbit CECs injected with Y-27632 were successful in recovering complete transparency of the corneas (47). However, further commenting is needed on the immunologic rejection of xenotransplantation that occurs in this study.

It has been known that MEFs contain a heterogeneous population of nonfibroblast precursor cells (16). To avoid possible contamination of NCCs in the starting MEFs, we carried out a lineage-tracing experiment to track the origin of the ciNCCs and ciCECs. We verified that the Wnt1+ ciNCCs were generated from non-NCC fibroblasts. To further confirm the origin of the ciNCCs, we confirmed that the tdTomato-positive ciNCCs generated from fibroblasts and engrafted Fsp1-tdTomato ciCECs in the animal model by using a fibroblast-specific Fsp1-Cre lineage-tracing reporter in MEFs.

In conclusion, our study presents a new strategy to generate functional CECs through induction of NCCs with chemically defined small-molecule cocktails, providing a new source of neural crest derivatives for the purpose of corneal engineering and regeneration.

The Wnt1-Cre [Tg(Wnt1-Cre)11Rth], Fsp1-Cre [BALB/c-Tg (S100a4-cre)1Egn/YunkJ], and ROSA26-tdTomato [Gt(ROSA)26Sortm14(CAG-tdTomato)Hze] mice were obtained from The Jackson Laboratory; the Oct4-GFP transgenic allelecarrying mice (CBA/CaJC57BL/6J) were also obtained from The Jackson Laboratory; and the 129Sv/Jae and C57BL/6 mice were obtained from Shanghai Vital River Laboratory. The rabbits used in this study were of the New Zealand white strain and were obtained from JOINN Laboratories (Suzhou) Inc., Suzhou, China. All the animals were housed under stable conditions (21 2C) with a 12-hour dark/light cycle. All animal experiments were approved by the Animal Ethics Committee of Wenzhou Medical University, Wenzhou, China.

MEFs were isolated from E13.5 embryos as previously described (54). Briefly, the neural tissues (including head, spinal cord, and tail), limbs, gonads, and visceral tissues of the E13.5 mouse embryos were carefully removed and discarded before MEF isolation. The remaining tissues were sliced into small pieces, trypsinized, and plated onto 10-cm dishes in fibroblast medium. Mouse NCCs were isolated from the neural tube of E8.5 embryos under a dissection microscope as previously described (55). The mouse meninges and vessels were removed and discarded. The remaining brain tissues were sliced into small pieces, dissociated with 0.25% trypsin (Gibco) for 15 min at 37C, washed with Dulbeccos modified Eagles medium (DMEM)/F12 twice, and then plated in a T25 flask bottle in NCC medium, which is composed of DMEM/F12 (Gibco) supplemented with 1 N2 (Gibco), 1 B27 (Gibco), bFGF (20 ng/ml) (PeproTech), and EGF (10 ng/ml) (PeproTech). Mouse pCECs were isolated from postnatal day 30 (P1)2 pups following a published protocol (56). They were cultured in DMEM containing 10% fetal bovine serum (FBS) (Gibco), 0.1 mM nonessential amino acids (NEAAs) (Sigma-Aldrich), 2 mM GlutaMAX (Gibco), and 2 mM penicillin-streptomycin (Gibco). The mESCs were maintained in ESC medium, which is composed of DMEM with 1 N2 (Gibco), 1 B27 (Gibco), leukemia inhibitory factor (LIF), 0.1 mM nonessential amino acids (Sigma-Aldrich), 2 mM GlutaMAX (Gibco), 2 mM penicillin-streptomycin (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), CHIR99021 (3 mM), and PD0325901 (1 mM).

Primary MEFs were isolated from E13.5 mouse embryos with a genetic background of Wnt1-Cre/ROSA26tdTomato (Wnt1-Cre mice ROSA26tdTomato mice) and Fsp1-Cre/ROSA26tdTomato (Fsp1-Cre mice ROSA26tdTomato mice). The MEFs at P2 were dissociated with 0.25% trypsin at 37C for 5 min and neutralized with MEF medium. To prepare the Fsp1-MEFs, the resulting fibroblasts were sorted to obtain tdTomato+/p75 cells by FACS. These MEFs were stained with a specific antibody against p75 and subjected to FACS for tdTomato+/p75 cells. During FACS, a Matrigel-coated 24-well plate was prewarmed at 37C for at least 30 min before seeding the tdMEFs. The tdMEFs were planted immediately after FACS into the prewarmed Matrigel-coated 24-well plate at 1.5 104 cells per well in MEF medium in 5% CO2 and 20% O2 at 37C for 5 hours to allow the cells to attach to the plate.

Small molecules, including the GSK3 inhibitor CHIR99021, the TGF- inhibitor SB431542, the cAMP inducer Forskolin, and CKI-7, were acquired from Sigma-Aldrich. bFGF was acquired from PeproTech. All chemical components are described in table S1.

M6 reprogramming medium preparation. The basal medium contained knockout DMEM (Gibco), 10% KSR (KnockOut serum replacement) (Gibco), 10% FBS (Gibco), 1% NEAA (Gibco), and 0.1 mM 2-mercaptoethanol (Gibco) supplemented with the small molecules CHIR99021 (3 M), SB431542 (5 M), Forskolin (10 M), VPA (500 mM), EPZ004777 (5 M), and 5-Aza (0.5 M). The medium was shaken for 30 min to ensure that all components were fully dissolved.

CEC differentiation and medium preparation. DMEM/F12/GlutaMAX (Gibco), 10% KSR (Gibco), 1% NEAA (Gibco), and 0.1 mM 2-mercaptoethanol (Gibco) were supplemented with SB431542 (5 M) and CKI-7 (5 M).

Chemical conversion of NCCs from mouse fibroblasts. The MEFs and TTFs were plated at 5 104 cells per well on six-well tissue culture plates in fibroblast medium. The culture plates were precoated with fibronectin or laminin for more than 2 hours. After overnight culture, the medium was exchanged with M6 chemical medium, which was refreshed every 2 days. NCC-like cells appeared and increased at days 3 to 5. After 7 to 10 days of induction, FACS was performed to collect Wnt1+ cells.

Generation of ciCECs from mouse ciNCCs. On days 12 to 16, the M6 chemical medium was replaced with CEC differentiation medium, which was refreshed every 2 days. Endothelial-like cell clusters appeared as early as day 20. During days 30 to 35, these CEC-like cell colonies were counted or further detected.

Cells were washed once with 1 PBS and then fixed with 4% paraformaldehyde at room temperature for 10 to 15 min, followed by permeabilization with 0.2% Triton X-100 in 1 PBS for 10 min and blocking with 7.5% bovine serum albumin (BSA) for at least 1 hour. All primary antibodies were diluted in 7.5% BSA, and the primary antibody reactions were incubated at 4C overnight. Then, the cells were washed with 1 PBS for 10 min five times at room temperature. The secondary antibodies with Alexa Fluor 488, Alexa Fluor 555, and Alexa Fluor 647, purchased from Invitrogen, were diluted in 7.5% BSA, and incubation was performed for 1 hour at room temperature, followed by five 10-min washes with 1 PBS. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). The antibodies used in this study are listed in table S2.

Total RNA was extracted using an RNeasy Plus Mini kit (Qiagen). In brief, 1 g of total RNA was used for reverse transcription with an iScript cDNA synthesis kit (Bio-Rad), and the resulting complementary DNA (cDNA) was diluted five times in H2O for PCR. For semiquantitative PCR, 1 l of one-fifth diluted cDNA was used as template for the following PCR program: 95C for 5 min and 35 cycles of 95C for 30 s, 60C for 30 s, and 72C for 30 s, followed by 72C for 10 min. Quantitative PCR was performed following the FAST SYBR Green Master Mix (ABI) protocol. All PCR was performed in triplicate, and the expression of individual genes was normalized to that of Gapdh. The primer sequences are listed in table S3.

Total RNA for each sample was isolated with TRIzol reagent and purified using the RNeasy 23 Mini Kit (Qiagen) according to the manufacturers instructions. RNA quality and quantity were assessed using NanoDrop 2000, Agilent 2100 Bioanalyzer, and Agilent RNA 6000 Nano Kit. RNA library construction and RNA-seq were performed by the Annoroad Gene Technology. Sequencing libraries were generated using the NEB Next Ultra RNA Library Prep Kit for Illumina24 (NEB), and library clustering was performed using HiSeq PE Cluster Kit v4-cBot-HS (Illumina) following the manufacturers recommendations. After cluster generation, the libraries were sequenced on an Illumina platform and 150base pair paired-end reads were generated. The initial data analysis was performed on BMKCloud (www.biocloud.net/).

For MEF preparation, fibroblasts with the desired genotype were cultured in MEF medium until they reached more than 80% confluence. The cells were washed twice with 1 PBS and treated with 0.25% trypsin at 37C for 5 min. After harvesting, the cells were passed through a 70-m filter, washed twice with PBS, and resuspended in precooled buffer (1 PBS, 1.5% FBS, and 0.5% BSA). The cells were incubated with either fluorescein isothiocyanate (FITC)conjugated P75 antibody (Abcam) or isotype control (BD) at the suggested concentrations on ice for 30 min or room temperature for 45 min, followed by six washes with FACS buffer. Cells were then resuspended in FACS buffer and sorted with BD FACSAria II.

Approximately 5 103 ciNCCs were seeded on laminin-coated 24-well tissue culture plates and cultured in N2B27 medium, which contained 1 N2, 1 B27, EGF (10 ng/ml), and bFGF (10 ng/ml) in Neurobasal medium. After 24 hours, cells were subjected to differentiation conditions.

For peripheral neuron differentiation, the medium was switched to neuron differentiation medium [NCC medium without bFGF and EGF, with the addition of 200 M ascorbic acid, 2 M dibutyryl cAMP (db-cAMP), brain-derived neurotrophic factor (BDNF) (25 ng/ml), NT3 (25 ng/ml), and glial cell linederived neurotrophic factor (GDNF) (50 ng/ml)]. Half of the medium was changed every 2 to 3 days. Specific neuron markers were analyzed by day 10 to day 20 after differentiation.

For Schwann cell differentiation, ciNCCs were cultured in N2B27 medium for at least 2 weeks. Differentiation was then induced by culturing in NCC medium without FGF2 and EGF and supplemented with ciliary neurotrophic factor (10 ng/ml), neuregulin (20 ng/ml), and 0.5 mM db-cAMP for 3 to 4 weeks. Media were changed every 2 to 3 days. Cells were then examined for the expression of Schwann cell protein markers by immunostaining.

To differentiate into melanocytes, ciNCCs were cultured in the presence of 5 M RA and Shh (Sonic hedgehog) (200 ng/ml) for 1 day and platelet-derived growth factorAA (PDGF-AA) (20 ng/ml), bFGF (20 ng/ml), and Shh (200 ng/ml) for 3 to 5 days; then, they were cultured in differentiation medium containing T3 (40 ng/ml), Shh (200 ng/ml), 1 nM LDN193189, 5 mM db-cAMP, and NT3 (10 ng/ml) for 8 to 12 days. The medium was refreshed every other day.

For mesenchymal differentiation, ciNCCs were cultured for 3 weeks in -MEM containing 10% FBS, as previously described (57). The differentiation potential of ciNCC-derived mesenchymal stem cells was achieved by incubation with adipogenesis medium, osteogenesis medium, and chondrogenesis medium, respectively (Cyagen Biosciences). The differentiation medium was refreshed every 3 days. The induced cells were stained with Oil Red O stain kit (Solarbio), alizarin red S (Sigma-Aldrich), and Alcian blue (Sigma-Aldrich) after 3 weeks of induction.

Cells were carefully dissociated into single-cell suspensions by trypsin (Gibco), washed twice with PBS, and then fixed overnight with cold 70% ethanol. Fixed cells were washed twice with PBS, followed by ribonuclease (100 g/ml; Sigma-Aldrich) treatment and PI (50 g/ml; Sigma-Aldrich) staining for 30 min at 37C. Approximately 1 106 cells were analyzed using FACSCanto II (Becton Dickinson) to determine the cell cycle distribution pattern. The percentages of cells in the G1, S, and G2-M phases of the cell cycle were analyzed using ModFit 4.1 (Verity Software House).

For generation of teratoma in vivo, 5 106 ciCECs were subcutaneously injected into each recipient NOD/SCID mouse (n = 5). Control NOD/SCID mice (n = 3) were injected with 2 106 mESCs, and teratoma formed from 4 to 8 weeks. Then, images of mice were captured with the cell phone imaging system.

All rabbits weighing 2.0 to 2.5 kg were anesthetized intramuscularly with ketamine hydrochloride (60 mg/kg) and xylazine (10 mg/kg; Bayer). The rabbits were divided into two groups (n = 10 each group), and the right eye was used for this experiment. After disinfection and sterile draping of the operation site, a 6-mm corneal incision centered at 12 oclock was made with a slit knife, and a viscoelastic agent (Healon; Amersham Pharmacia Biotech AB) was infused into the anterior chamber. After the corneal surface had been ruled with a marking pen (Devon Industries Inc.), a 6.0-mm-diameter circular aperture for descemetorhexis was created in the center of the cornea with a 30-gauge needle (Terumo), and Descemets membrane was removed from the anterior chamber of the eye. The corneal endothelium was mechanically scraped from Descemets membrane with a lacrimal passage irrigator (Shandong Weigao) as previously described. Fsp-ciCECs were dissociated using 0.25% trypsin-EDTA, resuspended in basic medium at a density of 1 107 cells/ml, and kept on ice. The anterior chamber was washed with PBS three times. After this procedure, a 26-gauge needle was used to inject 1 106 cultured ciCECs suspended in 100 l of basic DMEM containing 10 M ROCK inhibitor Y-27632 (Selleck) into the anterior chamber of the right eye. Thereafter, rabbits in the ciCEC transplantation groups were kept in the eye-down position for 24 hours under deep anesthesia so the cells could become attached by gravitation. Each surgical eye was checked two or three times a week by external examination, and photographs were taken on days 3, 7, 14, and 28 after injection. Central corneal thickness was measured using the Spectralis BluePeak OCT unit (Heidelberg Engineering, Heidelberg, Germany), and CECs were imaged with confocal scanning laser ophthalmoscopy (HRT3, Heidelberg Engineering, Heidelberg, Germany) on days 0.5, 1, 3, 7, 14, 21, 28, 35, and 42 after surgery. An average of three readings was taken. Corneal transparency was scored using a scale of 0 to 4 as previously described (58), where 0 = completely clear; 1 = slightly hazy, iris and pupils easily visible; 2 = slightly opaque, iris and pupils still detectable; 3 = opaque, pupils hardly detectable; and 4 = completely opaque with no view of the pupils. Photographs of ocular surface were taken with slit-lamp microscopy (SLM-8E, KANGHUA, China) at each time point.

The proliferation rate of ciCECs cultured in differentiation medium was determined by the Click-iT EdU Alexa Fluor 488 Imaging Kit (Invitrogen) according to the manufacturers instructions. Briefly, passaged CECs were seeded onto a slide at a lower density of 5 103 cells per cm2 and cultured for 24 hours.

For TEM analysis, cells were fixed in 2.5% EM-grade glutaraldehyde (Servicebio) for 2 to 4 hours at 4C, washed with 0.1 M phosphate buffer (pH 7.4), postfixed in 1% osmium tetroxide for 2 to 4 hours at 4C, and washed and then dehydrated in an ethanol series (50 to 100%) to a final rinse in 100% acetone, followed by 2-hour incubations in 1:1 acetone/Pon 812 (SPI) and overnight incubation in 1:2 acetone/Pon 812. The samples were embedded in Pon 812, polymerized for 48 hours at 60C, and then sectioned (60 to 80 nm) with a diamond knife (Daitome). Sections were stained with 2% uranyl acetate, followed by lead citrate, and visualized using an HT7700 transmission electron microscope (HITACHI).

ciCECs and pCECs were cultured for 24 hours and then incubated with Dil-Ac-LDL (10 g/ml) (Invitrogen) in culture medium at 37C for 6 hours. Cells were washed three times with PBS and stained with FITC-lectin (10 mg/ml) (Sigma-Aldrich) at 37C in the dark for 2 hours. Thereafter, cells were fixed with 4% paraformaldehyde for 15 min. The cells were imaged using an inverted fluorescence microscope.

Cells were treated with colcemid (0.1 g/ml) (Gibco) at 37C for 2 hours, trypsinized, resuspended, and incubated in 0.075 M potassium chloride for 15 min at 37C, fixed with 3:1 methanol:acetic acid, and then dropped onto slides to spread the chromosomes. The chromosomes were visualized by Giemsa (Solarbio) staining.

All experiments were independently performed at least three times. The results are expressed as means SD. The data were analyzed by unpaired two-tailed Students t tests for comparisons of two groups and by one-way analysis of variance (ANOVA) with Tukeys test or Dunnetts multiple comparisons test for comparisons of multiple groups. All analyses were performed using SPSS Statistics 19.0 software. P < 0.05 was considered significant. The accession number for the RNA-seq data reported in this paper is National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO): GSE162889.

Acknowledgments: We thank D. Jiang and H. Liu for the computational analysis and H. Li for the support of transplantation in rabbit model. Funding: This study was supported by the Beijing Natural Science Foundation (Z200014), National Key R&D Program of China (2017YFA0105300), National Natural Science Foundation of China (81600749, 81790644, and 81970838), and Zhejiang Provincial Natural Science Foundation of China (LD18H120001LD). Author contributions: Z.-B.J. designed and supervised the study and provided financial supports. S.-H.P. and N.Z. performed transdifferentiation experiments and data analysis. N.Z. and X.F. conducted qRT-PCR experiments, N.Z. performed the reprogramming experiments and compound removal experiments, and X.F. performed the immunostaining. N.Z. worked on the in vivo experiments. Y.J. carried out corneal transparency scaling analysis. S.-H.P. wrote the manuscript. Z.-B.J. and Y.J. revised the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. The Supplemental Information for this article includes seven figures, Supplemental Experimental Procedures, and a Small-Molecule Screening Table and can be found with this article online.

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Conversion of mouse embryonic fibroblasts into neural crest cells and functional corneal endothelia by defined small molecules - Science Advances

Global Stem Cells Market Regulations and Competitive Landscape Outlook, 2020 to 2025 The Courier – The Courier

Global Stem Cells Market 2020 by Company, Regions, Type and Application, Forecast to 2025 recently published by MarketQuest.biz, contains important market data that is collected from two or three sources, and the models. A loyal team of experienced forecasters, well-versed analysts, and knowledgeable researchers have worked painstakingly. The report involves six major parameters namely market analysis, market definition, market segmentation, key developments in the market, competitive analysis, and research methodology. Different markets, marketing strategies, future products, and emerging opportunities are taken into account while studying the global Stem Cells market and preparing this report.

The report presents a great understanding of the current market situation with the historic and upcoming market size based on technological growth, value and volume, projecting cost-effective and leading fundamentals in the market. The research report gives essential statistics on the market status of producers as well as offers beneficial advice and direction for businesses and individuals interested in the global Stem Cells industry.

NOTE: Our report highlights the major issues and hazards that companies might come across due to the unprecedented outbreak of COVID-19.

DOWNLOAD FREE SAMPLE REPORT: https://www.marketquest.biz/sample-request/31756

Market Scope And Segments:

It provides market size (value and volume), market share, growth rate by types, applications, and combines both qualitative and quantitative methods to make micro and macro forecasts in different regions or countries. The global Stem Cells market is segmented on the basis of product, application, and leading regions. The report brings together granular experiences with enormous demand drivers, headway opportunities, pay prospects, and massive challenges and dangers that have a significant effect on the expansion of the company space.

The top players listed in the market report are:

CCBC, Beikebiotech, Vcanbio, Boyalife

Based on type, the report split into:

Umbilical Cord Blood Stem Cell, Embryonic Stem Cell, Adult Stem Cell, Other

Based on application market is segmented into:

Diseases Therapy, Healthcare

According to the regional segmentation, the market provides the information covers the following regions:

North America (United States, Canada and Mexico), Europe (Germany, France, UK, Russia and Italy), Asia-Pacific (China, Japan, Korea, India and Southeast Asia), South America (Brazil, Argentina, etc.), Middle East & Africa (Saudi Arabia, Egypt, Nigeria and South Africa)

This report aims to give emerging as well as established industry players a strategic edge by allowing them to better grasp industry events and gather insights on past and current industry happenings that are expected to affect the global Stem Cells markets growth in the coming years. The study provides an up-to-date overview of the emerging global business situation, as well as the most recent developments and factors, as well as the overall market climate. This report makes it easy to know about the market strategies that are being adopted by the competitors and leading organizations.

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The market study report can help to understand the market and strategize for business expansion. In the strategy analysis, the report throws light on insights from marketing channel and market positioning to potential growth strategies, providing in-depth analysis for new entrants or exists competitors in the global Stem Cells industry.

The Market Research/analysis Report Contains Answers To Your Following Questions:

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Global Stem Cells Market Regulations and Competitive Landscape Outlook, 2020 to 2025 The Courier - The Courier

Global cell isolation market was valued at USD7013.71 million in 2020 and is anticipated to reach USD15529.45 million by 2026 – Yahoo Finance

by registering a CAGR of 15. 25% until 2026. Cell isolation is a technique of isolating cells for diagnosis and analysis of a particular type of cell. The market growth can be attributed to the rising demand for drugs, vaccines and other related products, as they are manufactured with the assistance of cell isolation technique.

New York, June 03, 2021 (GLOBE NEWSWIRE) -- Reportlinker.com announces the release of the report "Global Cell Isolation Market - Competition Forecast & Opportunities, 2026" - https://www.reportlinker.com/p06089447/?utm_source=GNW Increasing popularity of precision medicines is also working in the favor of the market growth.

Global cell isolation market has been segmented into product, cell type, source, technique, application, end-user, company and region.Based on technique, the market is further fragmented into centrifugation-based cell isolation, surface-marker based cell isolation and filtration-based cell isolation, amongst which, centrifugation-based cell isolation segment occupied the largest market share in 2020 as it finds extensive applications in various end user sectors such as academic institutes, research laboratories, etc.

Based on application, the market is further divided into biomolecule isolation, cancer research, stem cell research, in vitro diagnostics and others.Among these, cancer research and stem cell research are projected to be the lucrative segments of the market in the forecast period.

Increase in the research activities by biopharma companies and laboratory is the key factor for the growth of the segments.

Based on regional analysis, Asia-Pacific is expected to grow at the highest CAGR during the forecast period.The high CAGR of the region can be attributed to the relaxation in the stringent rules and regulations laid down by the government for drug development.

Another factor that can be held responsible for the fastest growth of the region is the availability of competent researchers and personnel who can carry out cell isolation techniques along with a wide genome pool.

Major players operating in the global cell isolation market include GE Healthcare Inc., Stemcell Technologies Inc., Danaher Corporation (Beckman Coulter Inc.), Becton, Dickinson and Company, Merck KGaA, Thermo Fisher Scientific Inc., Bio-Rad Laboratories Inc., Terumo Corporation, Sartorius AG, Cell Biolabs Inc., Miltenyi Biotec GmbH, F. Hoffmann-La Roche AG, Corning Inc, Akadeum Life Sciences, Inc., Invent Biotechnologies, Inc. and others. The market players are focusing on research and development activities in order to enhance their product portfolios and strengthen their position across the global market. For instance, the major pharmaceutical companies worldwide are making substantial investments in R&D to introduce new drugs in the market. Such investments are expected to increase the demand for cell isolation products over the coming years. In addition to this, new product developments help vendors to expand their product portfolio and gain maximum share in the sector. For example, Thermo Scientifics Medifuge is a benchtop centrifuge which is having a unique hybrid rotor as well as an interchangeable swing-out buckets and fixed-angle rotors to facilitate rapid & convenient applications on a single platform. Moreover, collaborations, mergers & acquisitions and regional expansions are some of the other strategic initiatives taken by major companies for serving the unmet needs of their customers.

Years considered for this report:

Historical Years: 2016-2019 Base Year: 2020 Estimated Year: 2021 Forecast Period: 2022-2026

Objective of the Study:

To analyze the historical growth in the market size of global cell isolation market from 2016 to 2020. To estimate and forecast the market size of global cell isolation market from 2021 to 2026 and growth rate until 2026. To classify and forecast global cell isolation market based on product, cell type, source, technique, application, end-user, company and region. To identify dominant region or segment in the global cell isolation market. To identify drivers and challenges for global cell isolation market. To examine competitive developments such as expansions, new product launches, mergers & acquisitions, etc., in global cell isolation market. To conduct pricing analysis for global cell isolation market. To identify and analyze the profile of leading players operating in global cell isolation market. To identify key sustainable strategies adopted by market players in global cell isolation market. The analyst performed both primary as well as exhaustive secondary research for this study.Initially, the analyst sourced a list of companies and laboratories using cell isolation techniques across the globe.

Subsequently, the analyst conducted primary research surveys with the identified companies.While interviewing, the respondents were also enquired about their competitors.

Through this technique, the analyst could include the companies and laboratories using cell isolation techniques which could not be identified due to the limitations of secondary research. The analyst examined the companies and laboratories using cell isolation techniques and presence of all major players across the globe. The analyst calculated the market size of global cell isolation market using a bottom-up approach, wherein data for various end-user segments was recorded and forecast for the future years. The analyst sourced these values from the industry experts and company representatives and externally validated through analyzing historical data of these product types and applications for getting an appropriate, overall market size.

Various secondary sources such as company websites, news articles, press releases, company annual reports, investor presentations and financial reports were also studied by the analyst.

Key Target Audience:

Companies and laboratories using cell isolation techniques, research labs, end users and other stakeholders Government bodies such as regulating authorities and policy makers Organizations, forums and alliances related to cell isolation Market research and consulting firms The study is useful in providing answers to several critical questions that are important for the industry stakeholders such as research labs, end users, etc., besides allowing them in strategizing investments and capitalizing on market opportunities.

Report Scope:

In this report, global cell isolation market has been segmented into the following categories, in addition to the industry trends which have also been detailed below: Global Cell Isolation Market, By Product: o Consumables o Instruments Global Cell Isolation Market, By Cell Type: o Human Cells o Animal Cells Global Cell Isolation Market, By Source: o Bone Marrow o Cord Blood/Embryonic Stem Cells o Adipose Tissue Global Cell Isolation Market, By Technique: o Centrifugation-Based Cell Isolation o Surface Marker-Based Cell Isolation o Filtration-Based Cell Isolation Global Cell Isolation Market, By Application: o Biomolecule Isolation o Cancer Research o Stem Cell Research o In Vitro Diagnostics o Others Global Cell Isolation Market, By End-User: o Biotechnology and Biopharmaceutical Companies o Research Laboratories and Institutes o Hospitals and Diagnostic Laboratories o Cell Banks Global Cell Isolation Market, By Region: o North America United States Mexico Canada o Europe Germany United Kingdom France Italy Spain o Asia-Pacific China Japan India South Korea Australia o South America Brazil Argentina Colombia o Middle East and Africa South Africa Saudi Arabia UAE

Competitive Landscape

Company Profiles: Detailed analysis of the major companies present in global cell isolation market.

Available Customizations:

With the given market data, we offers customizations according to a companys specific needs. The following customization options are available for the report:

Company Information

Detailed analysis and profiling of additional market players (up to five). Read the full report: https://www.reportlinker.com/p06089447/?utm_source=GNW

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Global cell isolation market was valued at USD7013.71 million in 2020 and is anticipated to reach USD15529.45 million by 2026 - Yahoo Finance

Human blastocyst-like structures generated entirely from pluripotent stem cells. Shifting the paradigm of developmental biology? – ESHRE

Human blastocyst-like structures generated entirely from pluripotent stem cells. Shifting the paradigm of developmental biology?

Four research groups have in the past few weeks independently reported that they have grown clusters of cells which mimic the function of human blastocysts. Susana Chuva de Sousa Lopes, co-ordinator of ESHREs SIG Stem Cells, and deputy Mina Popovic describe how these breakthroughs might extend the present limits on human embryo research.

Until now knowledge of early mammalian development has relied heavily on observing and manipulating human and animal embryos directly. Nevertheless, the relatively short timeframe available for analysis, coupled with the inaccessibility of research material, has inherently limited our understanding. Fired by their ambitious quest to elucidate the cellular and molecular complexities of human embryos, researchers are currently rethinking the way in which we study early human development in vitro.

Embryo-like structures are taking precedence. Unlike embryos resulting from the process of fertilisation, these structures are formed by stem cell coaxing, providing a novel, scalable platform for interrogating developmental pathways.

Human pluripotent stem cells (hPSCs) have been extremely valuable for understanding aspects of early human development. However, hPSCs have thus far only been successfully applied for capturing early human post-implantation development, recapitulating aspects of epiblast, trophoblast and amniotic cavity formation, and some features of axis development and gastrulation.(1) Traditional culture systems have lacked the complexity to model the spatio-temporal dynamics of a blastocyst.

Now, in a recent breakthrough, two research groups have harnessed the synergy between stem cell and developmental biology to generate the first human blastocyst-like structures, termed blastoids.(2.3) These papers were accompanied by two additional preliminary reports describing similar results.(4,5). Yu et al and Liu et al applied a 3D-microwell system and specific culture media to support the differentiation of hPSC towards structures that resembled human blastocysts in terms of their morphology, size and cell number.

In both studies, ~20% of the cell aggregates formed blastoids after 6-8 days. Detailed gene expression analysis revealed the presence of distinct embryonic lineages; however, the blastoids also contained many unidentified cell types. The authors thus cultured the blastocyst-like structures beyond the implantation stages in vitro. Interestingly, a small portion of outgrowths revealed phenotypes akin to the epiblast and amniotic cavity. Nevertheless, these findings warrant careful interpretation. Ultimately, thorough characterisation remains a challenge, as there are currently no optimal culture systems to mimic human peri-implantation in vitro.

With advances in high resolution genetic analysis and imaging technologies, research using blastoids certainly holds promise. For instance, blastoids may be generated in large numbers, allowing sufficient material for in-depth assays and high-throughput screens. Furthermore, they are more amenable to rapid genetic modifications than in experiments involving (natural) human embryos. However, as Heuser and Streeter elegantly wrote in 1941: The embryo is a machine that needs to function while it is being built.

Accordingly, it is important to appreciate that these models do not capture the full complexity of human blastocysts. Blastoids do not have a zona pellucida and, while some primitive endoderm (PE)-like cells were present, a defined PE cell layer could not be observed. Furthermore, immunofluorescence staining and transcriptomic analysis show inconsistencies for trophectoderm markers, while many of the blastoid cells cannot be correlated to in vivo counterparts. Several further limitations persist, such as poor efficiency and heterogeneity within and between different blastoids. Most importantly, the developmental potential of human blastoids remains to be determined. At present, blastoids generated from mouse PSCs do not have the capacity to develop beyond the early post-implantation stages.

Alongside scientific innovation, harnessing the full potential of human blastoids will also require urgent ethical reflection. While blastoids may overcome the destruction of human embryos, their genome is not individually unique, but rather represents a genetic clone of the stem cells or donor cells of origin. Hence, the legal and ethical implications associated with informed consent for the application of hPSCs will require revision. For instance, a donor may agree to his/her stem cells being used to generate tissues, but not for the creation of cloned embryos.

In addition, evaluating the extent to which the use of blastoids raises ethical concerns typical of human embryo research, such as the 14-day rule, will be crucial. If these structures were to acquire functionality, the definition of an embryo will require careful rethinking. Perhaps in the future, some of the ethical and legal restraints imposed on human embryo research may be overcome in blastoids by ensuring non-viability. For instance, gene editing may be used to introduce a necessary lethal mutation in the donor hPSCs.

In this context, the multidisciplinary approach offered by stem-cell based embryo models does provide a new edge. Depending on their functionality and moral status, blastoids may prove valuable in complementing human blastocysts for research. This integrated approach will be important not only for addressing fundamental biological questions, but perhaps also for improving ART, for studying implantation, modelling specific diseases related to early pregnancy and improving embryo selection. Armed with this potential, we are undoubtedly facing thrilling times ahead in human embryo research.

1. Rossant J, Tam PP. Opportunities and challenges with stem cell-based embryo models. Stem Cell Reports 2021; doi:10.1016/j.stemcr.2021.02.002. 2. Yu L, Wei Y, Duan J, et al. Blastocyst-like structures generated from human pluripotent stem cells. Nature 2021; 591: 620-626. doi:10.1038/s41586-021-03356-y 3. Liu X, Tan JP, Schrder J, et al. Modelling human blastocysts by reprogramming fibroblasts into iBlastoids. Nature 2021; 591: 627-632. doi:10.1038/s41586-021-03372-y 4. Fan Y, Min ZY, Alsolami S, et al. Generation of human blastocyst-like structures from pluripotent stem cells. bioRxiv 2021; preprint at doi:10.1101/2021.03.09.434313 5. Sozen B, Jorgensen V, Zhu M, et al. Reconstructing human early embryogenesis in vitro with pluripotent stem cells. bioRxiv 2021; preprint at doi.org/10.1101/2021.03.12.435175

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Human blastocyst-like structures generated entirely from pluripotent stem cells. Shifting the paradigm of developmental biology? - ESHRE