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SARS-CoV-2 infection demonstrated in a human lung bronchioalveolar tissue model — ScienceDaily

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Development of an in vitro human-derived tissue model for studying virus infection and disease progression in the alveolar cells of the lungs responsible for oxygen and carbon dioxide exchange with the blood might enable the study of possible therapies for acute respiratory distress syndrome (ARDS) triggered by SARS-CoV-2. Researchers in the Netherlands have demonstrated that the SARS-CoV-2 replicates efficiently in their model resembling the human bronchioalveolar system that is thought to play a critical role in progression of infection towards pneumonia and ARDS.

It is already established that in people infected with COVID-19 or some other respiratory viruses, alveolar injury can trigger a cascade of events that leads to ARDS, restricting transport of oxygen into the blood to dangerously low levels. There is also mounting evidence that the epithelium lining the alveoli plays a major role in progression of COVID-19. However, in vitro models for replicating disease progression in the alveoli of human lungs have proven difficult to establish, especially models that are also permissive to SARS-CoV-2 infection. This has greatly limited our understanding of COVID-19.

The Dutch team has now remedied this deficiency through application of self-renewing organoid models containing stem cells capable of differentiating into relevant cell types for study of disease processes. Organoids are tiny 3D tissues typically around 2 mm in diameter across derived from stem cells to mirror the complex structures of an organ, or at least to express selected aspects of it to meet a given biomedical research objective. Such organoids can then provide continuous sources of 2D tissues that mimic more accurately the geometry or cellular alignment of the structures under study.

A self-renewing organoid model for the epithelium of the airways conducting the gases, has already been developed by the same team, but the alveolar epithelium has proven a greater challenge to generate so far. The Dutch team has overcome this challenge and developed a 2D “air interface” system comprising a basal layer of stem cells in contact with the culture media and a top layer exposed to the air just as it would be in the lungs.

Multiple cultures were generated and infected successfully by SARS-CoV-2 targeting primarily alveolar type-II-like cells, known as ATII-L, confirmed by Transmission Electron Microscopy (TEM), surface marker stainings and single-cell sequencing. The study then shed light on the sequence of events following infection.

The study also identified through messenger RNA expression analysis a cellular immune response to the virus by infected cells. When the cultures were treated with the antiviral signaling molecule interferon lambda early in infection, SARS-CoV-2 replication was almost completely blocked, indicating that — when timed right — interferon lambda could be an effective treatment. These results also indicate that these cultures could be helpful for the development of a therapeutic intervention against acute respiratory distress syndrome (ARDS) from COVID-19.

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Limited rejuvenation of aged hematopoietic stem cells in young bone marrow niche — ScienceDaily

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By transferring mouse aged hematopoietic stem cells (aged HSCs, *1) to the environment of young mice (bone marrow niche, *2), it was demonstrated that the pattern of stem cell gene expression was rejuvenated to that of young hematopoietic stem cells. On the other hand, the function of aged HSCs did not recover in the young bone marrow niche. The epigenome (DNA methylation, *3) of aged HSCs did not change significantly even in the young bone marrow niche, and DNA methylation profiles were found to be a better index than the gene expression pattern of aged HSCs.

A research group led by Professor Atsushi Iwama at the Division of Stem Cell and Molecular Medicine, The Institute of Medical Science, The University of Tokyo (IMSUT) announced these world-first results and was published in the Journal of Experimental Medicine (online) on November 24th.

“The results will contribute to the development of treatments for age-related blood diseases,” states lead scientist, Professor Iwama at IMSUT.

Focus on changes in aged HSCs in the bone marrow niche

The research group investigated whether rejuvenating aged HSCs in a young bone marrow niche environment would rejuvenate.

Tens of thousands of aged hematopoietic stem/progenitor cells collected from 20-month-old mice were transplanted into 8-week-old young mice without pretreatment such as irradiation. After two months of follow-up, they collected bone marrow cells and performed flow cytometric analysis.

The research team also transplanted 10-week-old young mouse HSCs for comparison. In addition, engrafted aged HSCs were fractionated and RNA sequence analysis and DNA methylation analysis were performed.

They found that engrafted aged HSCs were less capable of producing hematopoietic cells than younger HSCs. They also showed that differentiation of aged HSCs into multipotent progenitor cells was persistently impaired even in the young bone marrow niche, and that the direction of differentiation was biased. It was found that the transfer of aged HSCs to the young bone marrow niche does not improve their stem cell function.

A more detailed analysis may reveal mechanisms that irreversibly affect aged HSC function

Aging studies focusing on HSCs have been actively pursued in mice using a bone marrow transfer model. However, the effect of aging on HSCs remains to be clarified.

Professor Iwama states as follows.”This study has a significant impact because it clarified the effect of aging on HSCs. Our results are expected to contribute to further elucidation of the mechanism of aging in HSCs and understanding of the pathogenic mechanism of age-related blood diseases.”

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New insights into the mechanisms of neuroplasticity — ScienceDaily

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Reactive oxygen molecules, also known as “free radicals,” are generally considered harmful. However as it now turns out, they control cellular processes, which are important for the brain’s ability to adapt — at least in mice. Researchers from the German Center for Neurodegenerative Diseases (DZNE) and the Center for Regenerative Therapies Dresden (CRTD) at TU Dresden published the findings in the journal Cell Stem Cell.

The researchers focused on the “hippocampus,” a brain area that is regarded as the control center for learning and memory. New nerve cells are created lifelong, even in adulthood. “This so-called adult neurogenesis helps the brain to adapt and change throughout life. It happens not only in mice, but also in humans,” explains Prof. Gerd Kempermann, speaker of the DZNE’s Dresden site and research group leader at the CRTD.

A trigger for neurogenesis

New nerve cells emerge from stem cells. “These precursor cells are an important basis for neuroplasticity, which is how we call the brain’s ability to adapt,” says the Dresden scientist. Together with colleagues he has now gained new insights into the processes underlying the formation of new nerve cells. The team was able to show in mice that neural stem cells, in comparison to adult nerve cells, contain a high degree of free radicals. “This is especially true when the stem cells are in a dormant state, which means that they do not divide and do not develop into nerve cells,” says Prof. Kempermann. Current study shows that an increase in the concentration of the radicals makes the stem cells ready to divide. “The oxygen molecules act like a switch that sets neurogenesis in motion.”

Free radicals are waste products of normal metabolism. Cellular mechanisms are usually in place to make sure they do not pile up. This is because the reactive oxygen molecules cause oxidative stress. “Too much of oxidative stress is known to be unfavorable. It can cause nerve damage and trigger aging processes,” explains Prof. Kempermann. “But obviously this is only one aspect and there is also a good side to free radicals. There are indications of this in other contexts. However, what is new and surprising is the fact that the stem cells in our brains not only tolerate such extremely high levels of radicals, but also use them for their function.”

Healthy aging

Radical scavengers, also known as “antioxidants,” counteract oxidative stress. Such substances are therefore considered important components of a healthy diet. They can be found in fruits and vegetables. “The positive effect of antioxidants has been proven and is not questioned by our study. We should also be careful with drawing conclusions for humans based on purely laboratory studies,” emphasizes Kempermann. “And yet our results at least suggest that free radicals are not fundamentally bad for the brain. In fact, they are most likely important for the brain to remain adaptable throughout life and to age in a healthy way.”

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New insights into Fragile X syndrome and the fetal brain — ScienceDaily

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Researchers at Tohoku University have revealed further insight into the fetal development of our brain and the potential causes of Fragile X syndrome (FSX).

During brain development, the fetal period is vital in creating neurons from neural stem cells to build up a functional adult brain. Any impairment in the developmental program could result in severe defects in the brain.

FSX is a genetic disorder characterized by intellectual disability and autistic symptoms. Children with FSX will generally suffer from developmental delays as well as social and behavioral problems.

FSX patients have a defect in the fragile X mental retardation 1 (FMR1) gene, a gene that codes for the fragile X mental retardation protein (FNRP) — the critical factor in normal brain development.

“Our study illustrated the possible molecular mechanism that causes FXS in the fetal brain and furthers our understanding of hereditary developmental disorders in the brain’s developmental stage,” said Noriko Osumi, professor at the Department of Developmental Neuroscience, Tohoku University Graduate School of Medicine.

Using next-generation sequencing, Osumi and her team identified hundreds of FMRP regulated molecules in mice fetal brains. These molecules were associated not only with neurogenesis but also autism and intellectual disability.

Their findings showed that specific groups of molecules were involved in the intracellular signaling pathways such as Ras/MAPK, Wnt/?-catenin, and mTOR.

The mTOR activity was significant in the fetal brain of FMR1 deficient mice, suggesting that increased mTOR activity may lead to abnormal proliferation and differentiation of neural stem cells in the fetal brain. Ultimately, these molecular mechanisms could be responsible for developing the brain during the fetal period and contribute to the causes of FXS.

The research team hopes this new information will serve as an essential resource for future studies of neurodevelopmental disorders.

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Growing human organs for transplantation with new proof-of-concept — ScienceDaily

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In a new paper published in Stem Cell Reports, Bhanu Telugu and co-inventor Chi-Hun Park of the University of Maryland (UMD) Department of Animal and Avian Sciences show for the first time that newly established stem cells from pigs, when injected into embryos, contributed to the development of only the organ of interest (the embryonic gut and liver), laying the groundwork for stem cell therapeutics and organ transplantation. Telugu’s start-up company, Renovate Biosciences Inc. (RBI), was founded with the goal of leveraging the potential of stem cells to treat terminal diseases that would otherwise require organ transplants, either by avoiding the need for transplants altogether or creating a new pipeline for growing transplantable human organs. With the number of people who suffer from organ failures and the 20 deaths per day in the U.S. alone purely from a lack of available organs for transplant, finding a new way to provide organs and therapeutic options to transplant patients is a critical need. In this paper, Telugu and his team are sharing their first steps towards growing fully transplantable human organs in a pig host.

“This paper is really about using the stem cells from pigs for the first time and showing that they actually can be injected into embryos and only go to the endodermal target organs like the liver, which is very important for delivering safe therapeutic solutions going forward,” says Telugu. “This is an important milestone. It’s a pipe dream in a way because a lot of things need to work out between here and full organ transplantation, but this paper sets the stage for all our future research. We can’t really just go and start working with humans in work like this, so we started with pig-to-pig transfer in this paper, working with the stem cells and putting them back into other pigs to track the process to make sure it is safe for liver production as proof-of-concept.”

Telugu and his team pitched this work at UMD Bioscience Day on behalf of his company, RBI, and received the Inventor Pitch Award and the UMD Invention of the Year Award in 2018. In order to protect the intellectual property, Telugu worked with the UMD Office of Technology Commercialization (OTC) to secure patents and open the work up for additional fundraising to carry this technology through the preclinical and clinical stages. The Maryland Stem Cell Foundation provided some funding to advance this work, and Telugu is thankful that Maryland funds technologies in the human stem cell space.

“There are many terminal cases where people need some sort of an organ replacement, like organ failure and degenerative diseases that cannot be cured by drugs,” explains Telugu. “The traditional paradigm is to find a donor organ, but as of today there are still thousands of patients waiting for transplants, and there is no keeping up with the demand. Researchers have thought for a long time that stem cells could help solve this problem, and these stem cells have the ability to go into a specific organ as opposed to those that go into any lineage. In this case, you can differentiate the cells and place them where they are needed to help rescue a diseased organ, eliminating the need for transplant or at least buying the patient some time. Just making the human liver and collecting them early from a neonatal piglet, the hepatocyte [liver] cells alone are a $3 billion opportunity per year. And in the future, we can move into organ transplantation, first with the liver, and then looking at other organs of interest like the pancreas and lungs.”

According to Telugu, this has distinct advantages over other methods that researchers are currently using to create donor organs in pigs, since the organs Telugu and his team are working with are actually of human origin and are therefore more likely to be accepted when transplanted. “Transplant rejections are pretty common even between humans and humans,” says Telugu, “and if it is such a problem normally, you can imagine how an organ from a pig could be difficult to accept and may not essentially perform the same functions. Pig proteins may not function the same, so that remains a huge barrier for other methods that are not actually growing fully human organs like ours.”

This work has the potential to solve a major problem in the treatment of organ failure and other degenerative diseases, which is what Telugu and his work is all about. “Being a veterinarian by training, we always look at the problem and try to find solutions to them,” says Telugu. “Most animal scientists operate by looking for solutions, so integrating research and entrepreneurship to get this to the market where it is needed is essential. We are one of the few groups on the planet that are working in this space, and we have a great team of embryologists here at Maryland to do this work. We are uniquely positioned to accomplish this with both genome editing and stem cell biology expertise, and being able to prove the concept with this paper is a great first step towards our goals.”

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Materials provided by University of Maryland. Original written by Samantha Watters. Note: Content may be edited for style and length.

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Surprisingly simple method could provide a new tool for producing specialized cytoplasm for reproductive medicine — ScienceDaily

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In a new study published in the journal Nature, researchers in Japan report that activating just eight genes for producing gene-controlling proteins is enough to convert mouse stem cells directly into oocyte-like cells that mature and can even be fertilized like egg cells.

On top of providing new insights into the mechanisms of egg cell development, the research may lead to a simple route for generating highly specialized substances unique to oocytes for use in reproductive biology and medicine.

Stored in the body until they mature into egg cells ready for fertilization, oocytes represent the very first step in the creation a new human life.

Oocytes are extremely unique because of their ability to bring forth the over two hundred kinds of highly differentiated cells needed to create an individual person, and one key to this ability is the complex mixture of substances within the fluid-like cytoplasm filling the cells.

So extraordinary are oocytes and their cytoplasm that replacing an oocyte’s DNA-containing nucleus with that of a body cell — a process called somatic cell nuclear transfer — can produce a new life, as famously demonstrated with Dolly the sheep.

Thus, a fundamental understanding of oocytes and their development is important for both advancing reproductive medicine and better grasping how life propagates, but knowledge of the many genes that orchestrate oocyte development is still far from complete.

Analyzing the development of oocytes from mice, researchers led by Katsuhiko Hayashi, professor at Kyushu University’s Faculty of Medical Sciences, have now identified eight genes for gene-triggering proteins known as transcription factors that not only are necessary for oocyte growth but also can directly convert mouse stem cells into oocyte-like cells.

“I was initially in complete disbelief to see mouse stem cells so quickly and easily take the form of oocytes based on introducing just a handful of factors, but repeated experiments proved it was true,” says Nobuhiko Hamazaki, first author on the study reporting the results and assistant professor at Kyushu University at the time of the research. “To find that eight transcription factors could lead to such big changes was quite astonishing.”

Working in collaboration with researchers at RIKEN, Hayashi’s group found that both mouse embryonic stem cells and induced pluripotent stem (iPS) cells — which can be created from adult body cells — consistently converted into oocyte-like cells when forced to produce the set of eight transcription factors, with only four factors being sufficient in some cases though with worse reproducibility.

“That stem cells can be directly converted into oocyte-like cells without following the same sequence of steps that happen naturally is remarkable,” says Hayashi.

When grown in the presence of other cells usually found around oocytes, the oocyte-like cells developed structures similar to mature egg cells but with an abnormal chromosome structure. Despite this, the mature oocyte-like cells could be fertilized in vitro and exhibited early development, with some even progressing to an eight-cell stage.

Though the modified nuclei of the oocyte-like cells may not be useable in the long run, this is no problem for applications needing mainly the oocyte cytoplasm, such as for studies of reproductive biology and for treatments like mitochondrial replacement therapy, in which parts of oocytes are replaced to prevent mothers from passing to their children diseases related to the mitochondria.

“Cytoplasm from oocytes is an invaluable resource in reproductive biology and medicine, and this method could provide a novel tool for producing large amounts of it without any invasive procedures,” comments Hayashi. “While the processes could still be much more complex for humans, these initial results in mice are very promising.”

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Scientists modified ‘mesenchymal stem cells’ to carry anti-cancer drugs and deliver them to target cancers — ScienceDaily

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As humans evolve, cancer also evolves in parallel, making the race for finding efficient treatment methods for cancer patients challenging and constant. In addition to designing drugs for treatment, the delivery of these drugs to targeted organs is also a major challenge faced by the cancer research community.

Many research groups have tried to develop techniques to efficiently deliver anti-cancer drugs to tumors. An interesting way utilizes a distinct group of cells in our body, the mesenchymal stem cells (MSCs), which have a special ability to find and move towards tumors. This means that theoretically, we can load these “tumor-homing” MSCs with anti-cancer drugs and use them to hinder cancer progression. However, pilot studies show that the anti-cancer drug loading capacity of MSCs is limited, and they tend to lose their ability to target and reach tumor cells upon drug loading.

In a recent study published in Journal of Controlled Release, researchers from Japan, led by Dr. Kosuke Kusamori and Professor Makiya Nishikawa from Tokyo University of Science, sought to find out how to modify MSCs to bypass these problems. Dr. Kusamori, Assistant Professor in the University’s Department of Pharmacy says, “We wondered if the answer to our dilemma of modifying mesenchymal stem cells with an anticancer drug was to exploit the property of mesenchymal stem cells to accumulate in tumor tissues.”

Using the well-known “avidin-biotin complex” (ABC) method, the researchers used liposomes — cellular lipid bags popularly used as drug delivery systems — to carry the anti-cancer drug doxorubicin (DOX) to the surface of specific mouse MSCs. They named these lipid bags carrying DOX “DOX-Lips.”

The researchers found that the MSCs loaded with DOX-Lips could carry and selectively target mouse colon cancer cells growing in artificial cultures in the lab. These modified MSCs could not only carry a significant amount of the drug, but also efficiently deliver it inside the target cancer cells. To test if this feature is replicated in a living system, they used a mouse model with skin and lung cancer. They found that in both cases, DOX-Lips were able to efficiently reach cancer cells and release the drug into the cytoplasm of the cancer cells. The researchers concluded that the modified MSCs could completely suppress tumor growth in mouse models.

There are several advantages to this new method. First, the process is relatively faster than previously known methods. As Yukiya Takayama, a doctoral student in Professor Nishikawa’s lab and a co-author of the study observes, “The relatively short duration of the ABC method made it possible to quickly modify the cell surface with DOX-Lips and avoid cell damage.” Second, this method did not affect the process of cell attachment to cancer cells, thereby ensuring maximum efficiency in drug delivery. Third, contrary to previous observations that lipid bags of only a certain size can be used to deliver drugs, this new study suggests that the size of the lipid bags very likely does not affect drug delivery; this finding can be exploited to deliver many different doses of drugs as well.

The combination of the ABC method and the use of Lips thus seems to be the answer to the researchers’ dilemma. Professor Nishikawa is excited about these results. “We have succeeded in developing a new targeted cancer therapy,” he observes. “Mesenchymal stem cells can migrate to brain tumors and minute cancer lesions that are otherwise inaccessible to conventional drug delivery systems. Our method may thus be effective against intractable cancers,” Nishikawa says.

This study is thus a promising advancement in the field of cancer research; the new method identified by the researchers may prove to be the answer on how to send the treating drug to cancer’s doorstep.

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Research dispels fears human stem cells contain cancer-causing mutations — ScienceDaily

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Pioneering new research has made a pivotal breakthrough that dispel concerns that human stem cells could contain cancer-causing mutations.

A team of scientists from the University of Exeter’s flagship Living Systems Institute has shown that stem cells contain no cancer mutations when they are grown in their most primitive or naïve state.

The ground-breaking advances made by the research team should help allay fears surrounding recent controversy about the genetic stability of human embryonic stem cells.

The study is published in the peer-reviewed journal Cell Stem Cell on Monday, December 14th 2020.

Human embryonic stem cells offer great promise for regenerative medicine because they can be turned into every type of cell in our bodies — such as neurons, heart, pancreatic, and liver cells.

As a result, they represent a significant potential source of cells that could be used to replace those lost through damage or disease.

A major concern, however, had been whether embryonic stem cells acquire cancer-causing mutations.

Recent studies had indicated that human pluripotent stem cells had shown the potential for increased frequency of serious cancer-causing mutations.

However, the new research, led by Senior Research Fellow Dr Ge Guo from the University of Exeter has shown that there is no increased frequency of mutations in cancer-related genes found in these cells.

Analysing RNA-sequencing data from human naïve pluripotent stem calls, the research team found that the actual incidence of cancer-causing mutations were closer to zero.

Dr Guo, who has pioneered research into human naïve embryonic stem cells and is part of the University of Exeter’s College of Medicine and Health said: “Our study corrects misinformation in the field and encourages us to continue exploring the potential of naïve stem cells.”

Professor Austin Smith co-author of the paper and Director of the Living Systems Institute added: “I am delighted to see Dr Guo launch her team in LSI by publishing these significant results.”

Dr Guo’s research is focussed on mammalian pluripotent stem cells and cell fate transition during early embryo development.

Key research areas in the lab include understanding the developmental plasticity of human naïve stem cells; Modelling early human embryo development ex vivo by reconstruction of embryo structures; and establishing pluripotent stem cells from various mammalian species and elucidation of shared and distinct gene regulatory features.

The research was funded by the Medical Research Council.

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Development of new stem cell type may lead to advances in regenerative medicine — ScienceDaily

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A team led by UT Southwestern has derived a new “intermediate” embryonic stem cell type from multiple species that can contribute to chimeras and create precursors to sperm and eggs in a culture dish.

The findings, published online this week in Cell Stem Cell, could lead to a host of advances in basic biology, regenerative medicine, and reproductive technology.

Cells in early embryos have a range of distinct pluripotency programs, all of which endow the cells to create various tissue types in the body, explains study leader Jun Wu, Ph.D., assistant professor of molecular biology. A wealth of previous research has focused on developing and characterizing “naïve” embryonic stem cells (those about four days post-fertilization in mice) and “primed” epiblast stem cells (about seven days post-fertilization in mice, shortly after the embryo implants into the uterus).

However, says Wu, there’s been little progress in deriving and characterizing pluripotent stem cells (PSCs) that exist between these two stages — largely because researchers have not been able to develop a paradigm for maintaining cells in this intermediate state. Cells in this state have been thought to possess unique properties: the ability to contribute to intraspecies chimeras (organisms that contain a mix of cells from different individuals of the same species) or interspecies chimeras (organisms that contain a mix of cells from different species) and the ability to differentiate into primordial germ cells in culture, the precursors to sperm and eggs.

For this study, the researchers successfully created intermediate PSCs, which they named “XPSCs” from mice, horses, and humans.

Wu says that these results could eventually lead to an array of advances in both basic and applied research. For example, looking at gene activity in XPSCs from different species and interspecies chimeras could help researchers understand which signatures have been conserved through evolution. Examining the communication between cells in chimeras may help scientists identify strategies that could be used to accelerate the development of tissues and organs from stem cells used for transplantation. And using chimera-derived primordial germ cells to create sperm and eggs could aid in preserving endangered animal species and advancing infertility treatments.

“These XPSCs have enormous potential. Our study helps open the door to each of these possibilities,” says Wu, who is a Virginia Murchison Linthicum Scholar in Medical Research.

Wu notes that developing XPSCs presented a special challenge because the conditions that keep naïve PSCs in a stable state are exactly the opposite from those that stabilize primed PSCs. While culture conditions for naïve PSCs must activate a WNT cell-signaling pathway and suppress the FGF and TGF-ß pathways, the conditions to maintain primed PSCs must suppress WNT and activate FGF and TGF-ß.

Aiming for the preferred environment for XPSC derivation, Wu and his colleagues placed cells from early mouse embryos into cultures containing chemicals and growth factors that activate all three pathways. These lab-grown cells were extremely stable in culture and able to multiply without developing any further for approximately two years.

Additional experiments showed that these cells met the expectations researchers have long strived to meet of contributing to chimeras and directly differentiating into primordial germ cells. Wu and his colleagues made intraspecies chimeras of mice using cells derived from mice with different coat colors by injecting the cells into early mouse embryos. They also tracked the contributions of the XPSCs by tagging the cells with a fluorescent protein and then identifying them throughout the resulting offspring’s body.

Wu’s team made interspecies chimeras by injecting horse XPSCs into early mouse embryos and allowing the embryos to develop in mice for several days. Surprisingly, although horses have a comparatively long gestational period — nearly a year — the researchers found that these foreign cells had contributed to mouse organ development, indicating that signals from the mouse cells determine organ developmental timelines.

Like XPSCs from other species, the human cells showed that they were capable of differentiating into a variety of tissues if culture conditions allowed them to progress in development, as well as directly form primordial germ cells in a dish.

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Tagging produces detailed catalog of transcription factors key to making each cell type — ScienceDaily

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A team of biomedical engineers at Duke University has created a new way to turn stem cells into a desired cell type by mastering the language of gene regulatory networks.

Programming stem cells into other cell types is not a new idea. Several methods already exist, but the results have left something to be desired. Often, programmed stem cells do not mature correctly when cultured in the lab, so researchers seeking adult neuron cells for an experiment might end up with embryonic neurons, which won’t be able to model late-onset psychiatric and neurodegenerative conditions.

“The cells might seem right at first glance,” said Josh Black, the Duke Ph.D. student who led the work in Charles Gersbach’s lab, “but they are often missing some of the key properties you want in those cells.”

Using CRISP gene editing, the lab led by Gersbach, The Rooney Family Associate Professor of Biomedical Engineering and the Director of the Center for Advanced Genomic Technologies, created a method to identify which transcription factors — master controllers of gene activity — were essential to making a good neuron.

Their work, appearing Dec. 1 in Cell Reports, demonstrates the potential of the approach to make mature adult neurons, but it could be applied to program any cell type.

CRISPR technology is most often used for editing DNA sequences, known as “genome editing,” in which the Cas9 protein is bound to a guide RNA that directs Cas9 to cut the DNA at a specific location, leading to changes in the DNA sequence. “DNA editing has been widely used to alter gene sequences, but that doesn’t help in situations where the gene is turned off,” Gersbach said.

A deactivated Cas9 (dCas9) protein, though, will attach to the DNA without cutting it. In fact, it typically won’t do anything without another molecule attached or recruited to it. Gersbach and his colleagues have previously reported a variety of methods to attach different molecular domains to the dCas9 protein can that will tell a cell to turn on a gene and remodel chromatin structure.

When Black joined Gersbach’s lab, he was interested in using these tools to turn on genes that could convert one cell type into another to create better disease models.

In 2016, Black and Gersbach reported an approach to use the CRISPR-based gene activators to turn on gene networks that would convert fibroblasts, an easily accessible cell type that makes up connective tissue, to neuronal cells. This study targeted gene networks that were known to be associated with neuronal specification, but did not generate cells with all of the properties needed to make effective disease models. However, the right gene networks to generate those desired cells were unknown, and there are were thousands of possibilities encoded in the human genome. So Black and Gersbach devised a strategy to test all of the networks in a single experiment.

They started with pluripotent stem cells, since this cell type should be able to become any other cell in the human body. To make mature neurons from stem cells, the team engineered stem cells that fluoresced red once they became neuronal. The brighter the fluorescence, the stronger the push towards a neuronal fate. Then they made a pooled library of thousands of guide RNAs targeted to all of the genes that encode transcription factors in the human genome. Transcription factors are the master regulators of gene networks, so to make the desired neurons, they have to get all of the right transcription factors turned on.

They introduced the CRISPR gene activator and guide RNA library into the stem cells so that each cell only received a single guide RNA, and therefore turned on its particular corresponding transcription factor gene target. Then they sorted the cells based on how red they became and sequenced the guide RNAs in the most and least red cells, which told them which genes, when turned on, made the cells more or less neuronal.

When they profiled the gene expression from the stem cells engineered with the guide RNAs, the results suggested that the corresponding cells generated more specific and more mature types of neurons. They also found genes that worked together when targeted simultaneously. Moreover, the experiment revealed factors that antagonized the neuronal commitment of the stem cells, and when they used CRISPR-based repressors of those genes, they could also enhance the neuronal specification.

However, these results were all just measuring markers of neurons. To know if these engineered cells truly recapitulated the function of more mature neurons, they needed to test their ability to transmit electrical signals.

For this, they turned to Professor Scott Soderling, the George Barth Geller Distinguished Professor for Research in Molecular Biology and Chair of the Duke Department of Cell Biology. Shataakshi Dube, a grad student in Soderling’s lab, used a technique known as patch clamp electrophysiology to measure the electrical signals inside the newly formed neurons. By poking a tiny hole in the cell with a very small pipette, she could look inside the neuron and see if it was transmitting electrical signals known as action potentials. If so, the team knew the neuronal cell had properly matured. In fact, the neurons engineered to activate a particular pair of transcription factor genes were more functionally mature, emitting more action potentials more frequently.

“I was curious but skeptical on how neuronal these stem cells could become,” Dube said, “but it was remarkable to see how much these programmed cells looked just like normal neurons.”

The process from stem cell to mature neuronal cell took seven days, dramatically shortening the timeframe compared to other methods that take weeks or months. This faster timeline has the potential to significantly accelerate the development and testing of new therapies for neurological disorders.

Creating better cells will help researchers in a number of ways. Diseases like Alzheimer’s disease, Parkinson’s disease, and schizophrenia most often occur in adults and are difficult to study because making the right cells in the lab is challenging. This new method can allow researchers to better model these diseases and others. It can also help with drug screening, as different cells respond to drugs differently.

More broadly, the same method for screening transcription factor genes and gene networks could be used to improve methods to make any cell type, which could be transformative for regenerative medicine and cell therapy.

For example, Gersbach’s group reported a method for using CRISPR-based gene activation to convert human stem cells into muscle progenitor cells that could regenerate damaged skeletal muscle tissue earlier this year.

“The key to this work is developing methods to use the power and scalability of CRISPR-based DNA targeting to program any function into any cell type,” Gersbach said. “By leveraging the gene networks already encoded in our genome, our control over cell biology is dramatically improved.”

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