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Till now I thought that embryonic stem cells have no epigenome as they are pluripotent. (I thought that since epigenome is what gives a cell its identity, no cellular identity means no epigenome) I saw something similar to this on this Wikipedia page. After fertilization, the paternal and maternal genomes are demethylated in order to erase their epigenetic signatures and acquire totipotency.. Other sources mention 'reset' in place of 'erase'. This paper rather suggests that stem cells do have an epigenome. Specifically, genes associated with self-renewal are silenced, while cell-type-specific genes undergo transcriptional activation during differentiation.. I am not very literate in biology, please excuse me if I made a mistake.
So there are a couple of things to bear in mind.
pluripotent does not mean that all genes are active. It means that the stem cells have the ability to form different cell types. However, it still needs to keep the cellular programme of a neuron for example silent. So the epigenome is still present to keep other cell type programmes silent until there is a transition.
DNA methylation is not the only source of epigenetics. Active and inactive genes also correspond to particular post translational modifications on tails of histone proteins. In the cell, DNA is wrapped around histones to form what is known as chromatin.
Hope that is a starting point to answer your question
Single-cell sequencing in stem cell biology
Cell-to-cell variation and heterogeneity are fundamental and intrinsic characteristics of stem cell populations, but these differences are masked when bulk cells are used for omic analysis. Single-cell sequencing technologies serve as powerful tools to dissect cellular heterogeneity comprehensively and to identify distinct phenotypic cell types, even within a ‘homogeneous’ stem cell population. These technologies, including single-cell genome, epigenome, and transcriptome sequencing technologies, have been developing rapidly in recent years. The application of these methods to different types of stem cells, including pluripotent stem cells and tissue-specific stem cells, has led to exciting new findings in the stem cell field. In this review, we discuss the recent progress as well as future perspectives in the methodologies and applications of single-cell omic sequencing technologies.
60-year scientific mystery solved
Over the last 60 years, scientists have been able to observe how and when genetic information was replicated, determining the existence a "replication timing program", a process that controls when and in what order segments of DNA replicate. However, scientists still cannot explain why such a specific timing sequence exists. In a study published today in Science, Dr. David Gilbert and his team have answered this 60-year-old question.
"Why would cells care about the order in which they replicate DNA?" asked lead scientist Dr. Gilbert. "After all - all cells need to replicate all their DNA. Our hypothesis has been that it's not just DNA that replicates, but all of the regulatory molecules that read the DNA replicate as well." Dr. Gilbert further hypothesized that there might be a purpose behind the replication timing program and process because "mother nature would not squander this opportunity to control how the DNA is read."
"The time at which you replicate provides an ideal time at which to choose whether to maintain all the regulatory factors and continue with the same functional interpretation of the information in DNA or change it to elicit new functions," explains Dr. Gilbert.
Over the last 13 years, Dr. Gilbert and his team showed that each type of cell had a unique replication timing program and that diseased cells had distinct alterations in the program. In this study, Dr. Gilbert and his team looked at how changes in the replication timing program impact the packing of DNA with its regulatory factors, collectively known as the epigenome. The epigenome are regulatory factors that are believed to control the "identity" of the cell, and the functions that the cell will perform.
By eliminating a protein called RIF1, that helps to regulate DNA replication, they found that the replication program was severely and sometimes, almost completely gone so that all segments of chromosomes were replicating at different times in different cells. Without RIF1, if cells were prevented from replicating DNA, their epigenomes were fine. However, as soon as the DNA started to replicate, the regulatory molecules that associate with the DNA became incorporated incorrectly and worsened with each round of DNA replication. Eventually, the 3-dimensional folding of the chromosomes was also altered.
Dr. Gilbert suggests that when the epigenome is disrupted by altering the replication timing program, the cells might no longer perform their normal functions, or they may perform inappropriate functions. These inappropriate functions may have a large and negative impact on a person's health.
"We and others have shown previously that the program is altered in many diseases," says Dr. Gilbert. "Our lab recently showed specific patterns of altered timing that were linked statistically to poor outcomes in pediatric leukemia, and in another study to diseases of premature aging."
Thus, the replication timing program provides a whole new genre of molecular pathways and biomarkers that lead to and identify disease states. This could lead to earlier diagnoses and more accurate prognoses for patients.
While Dr. Gilbert's work has answered one important question, he does not plan to stop here. "We think that the epigenome. is not [only] essential for a cell to just maintain its identity, but we hypothesize that it is critical for cells to turn into other cell types."
Testing this hypothesis is crucial for the fields of stem cell research and the therapeutic application of stem cells. Dr. Gilbert is currently using human stem cells to test how a disrupted replication timing affects development of these cells into liver cells, heart cells, and neurons. The results from this study will provide valuable information for human health and disease studies in the future.
This research will appear in the 23rd April 2021 issue of the journal Science, published AAAS, the science society, the world's largest scientific organization.
Disclaimer: AAAS and EurekAlert! are not responsible for the accuracy of news releases posted to EurekAlert! by contributing institutions or for the use of any information through the EurekAlert system.
Stem Cells and Aging – What Happens When Our Stem Cells Get Old and Tired?
Aging is an inevitable, unnerving process that confronts us all. Eventually our muscles and immune systems will weaken, our hair will thin, and our minds won&rsquot be as sharp as they once were. But what is the biology that underlies this process? And what if advances in the field of regenerative medicine could counteract this decline and alleviate the symptoms of old age?
Do Our Stem Cells Age as We Do?
Consider the body not as a single entity but as a dynamic multitude of cells growing, changing, dying, and being born. These cells make up and replenish the bodies&rsquo tissues and organs, acting in concert and communicating in fascinating ways to keep the body in good working order. In many tissues, adult stem cells are at the root of this process, tasked with supplying cells to maintain normal tissue function and facilitating regeneration in response to injury. It is logical then to assume that, as our bodies grow older and our organs and faculties begin to degenerate, our stem cells must be failing us.
In fact, much research has gone into uncovering what happens to our stem cells as they age. For example, hematopoietic stem cells, which produce all the cells of the blood and immune system, actually increase in number in aging adults. Unfortunately, the expansion in cell numbers is to compensate for their overall loss in functionality. Ultimately, fewer white blood cells are produced, which contributes to a deficient immune system and diminished resistance to disease and infections in the elderly.
What About Stem Cells in Our Brains?
One fascinating avenue of research focuses on what happens to stem cells in the brain as we age. Until the 1960s it was believed that we are born with our lifetime&rsquos supply of brain cells. This dogma was broken by the discovery of neural stem cells (NSCs), which reside in certain regions of the brain. Now we know that NSCs do have the ability to produce glia and some types of neurons in certain conditions. As NSCs age, however, their ability to regenerate lost or damaged brain cells decreases and they have a significant reduction in the number of neurons they can generate.
Fortunately, the advent of technology to identify and isolate these NSCs means we can study how they change as they age and, armed with this knowledge, begin to innovate ways to halt or reverse the aging process. Recently published research from a group at Stanford University provides fascinating new insights. Using a mouse model, the team investigated the differences in NSCs between young and old mice and found that as NSCs age they do a poor job of clearing away broken proteins that can interfere with the normal functions of the cells. Aged NSCs have an increased accumulation of protein aggregates, or clumps of broken proteins. This is striking as a number of age-related neurodegenerative diseases, such as Alzheimer&rsquos and Parkinson&rsquos disease, are linked to a build-up of proteins that can clog up brain cells and cause them to malfunction or die.
The researchers discovered that the inability of aged NSCs to clear broken proteins impairs their activation and production of new neurons. In the study they found that artificially stimulating the protein clearing system in aged NSCs gave them a new lease on life, restoring their ability to generate neurons, and increasing the number of active NSCs in elderly mouse brains. This type of fundamental research enhances our understanding of the biology of aging and provides the scientific underpinning for potential new treatments that could improve people&rsquos health into old age.
Can We Treat Aging?
Although most research is far from the clinic, new drugs are being developed with the potential to treat degenerative age-related diseases, some by potentially promoting stem cell regeneration. It will take time and controlled clinical trials to determine the safety and efficacy of these treatments. In the meantime, some companies are harvesting and freezing young stem cells, with the hope that the cells will be useful in the future and will be able to delay or reverse aging. While this may sound appealing, at the moment, &ldquo[t]here is no way to extend anybody&rsquos life with stem cells,&rdquo as former ISSCR President Sean Morrison said in a recent interview, and consumers should be wary until further studies have been done.
The study of stem cell aging is a field of research at the cutting edge of biomedical innovation. With incremental progress, research groups around the world are uncovering the biology of why and how stem cell function declines with age. The hope is that one day this fundamental research will be translated into treatments that enhance the health and quality of life for future generations.
Special thanks to Edie Crosse, PhD student at the MRC Centre for Regenerative Medicine, University of Edinburgh, for this guest blog post.
[short-box image="/wp-content/uploads/2018/10/promo-webcasts.jpg" title="Featured On Demand Webcasts" link = "mailto:[email protected]" ] These webcasts are currently unavailable while we transition to another platform. We apologize for the inconvenience. Please contact Jack Mosher with any questions.
Understanding the clinical use of "MSCs"
Dr. Megan Munsie, University of Melbourne Dr. Mark Young, University of Queensland and Queensland Institute of Technology
Stemming Vision Loss: Seeing is Believing
Dr. Peter Coffey, University College London's Institute of Ophthalmology and University of California, Santa Barbara’s Center for Stem Cell Biology and Engineering.
Regenerative Medicine and the Eye
Dr. Brian Ballios, University of Toronto
Regenerative Medicine Efforts for Wounded Warriors
Dr. Anthony Atala, Wake Forest Institute of Regenerative Medicine
III. Embryonic Stem Cell Research
Pluripotent stem cell lines can be derived from the inner cell mass of the 5- to 7-d-old blastocyst. However, human embryonic stem cell (hESC) research is ethically and politically controversial because it involves the destruction of human embryos. In the United States, the question of when human life begins has been highly controversial and closely linked to debates over abortion. It is not disputed that embryos have the potential to become human beings if implanted into a woman’s uterus at the appropriate hormonal phase, an embryo could implant, develop into a fetus, and become a live-born child.
Some people, however, believe that an embryo is a person with the same moral status as an adult or a live-born child. As a matter of religious faith and moral conviction, they believe that “human life begins at conception” and that an embryo is therefore a person. According to this view, an embryo has interests and rights that must be respected. From this perspective, taking a blastocyst and removing the inner cell mass to derive an embryonic stem cell line is tantamount to murder (4).
Many other people have a different view of the moral status of the embryo, for example that the embryo becomes a person in a moral sense at a later stage of development than fertilization. Few people, however, believe that the embryo or blastocyst is just a clump of cells that can be used for research without restriction. Many hold a middle ground that the early embryo deserves special respect as a potential human being but that it is acceptable to use it for certain types of research provided there is good scientific justification, careful oversight, and informed consent from the woman or couple for donating the embryo for research (5).
Opposition to hESC research is often associated with opposition to abortion and with the “pro-life” movement. However, such opposition to stem cell research is not monolithic. A number of pro-life leaders support stem cell research using frozen embryos that remain after a woman or couple has completed infertility treatment and that they have decided not to give to another couple. This view is held, for example, by former First Lady Nancy Reagan and by U.S. Senator Orrin Hatch.
On his Senate website, Sen. Hatch states: “The support of embryonic stem cell research is consistent with pro-life, pro-family values.
“I believe that human life begins in the womb, not a Petri dish or refrigerator … . To me, the morality of the situation dictates that these embryos, which are routinely discarded, be used to improve and save lives. The tragedy would be in not using these embryos to save lives when the alternative is that they would be discarded” (6).
A. Existing embryonic stem cell lines
In 2001, President Bush, who holds strong pro-life views, allowed federal National Institutes of Health (NIH) funding for stem cell research using embryonic stem cell lines already in existence at the time, while prohibiting NIH funding for the derivation or use of additional embryonic stem cell lines. This policy was a response to a growing sense that hESC research held great promise for understanding and treating degenerative diseases, while still opposing further destruction of human embryos. NIH funding was viewed by many researchers as essential for attracting scientists to make a long-term commitment to study the basic biology of stem cells without a strong basic science platform, therapeutic breakthroughs would be less likely.
President Bush’s rationale for this policy was that the embryos from which these lines were produced had already been destroyed. Allowing research to be carried out on the stem cell lines might allow some good to come out of their destruction. However, using only existing embryonic stem cell lines is scientifically problematic. Originally, the NIH announced that over 60 hESC lines would be acceptable for NIH funding. However, the majority of these lines were not suitable for research for example, they were not truly pluripotent, had become contaminated, or were not available for shipping. As of January 2009, 22 hESC lines are eligible for NIH funding. However, these lines may not be safe for transplantation into humans, and long-standing lines have been shown to accumulate mutations, including several known to predispose to cancer. In addition, concerns have been raised about the consent process for the derivation of some of these NIH-approved lines (7). The vast majority of scientific experts, including the Director of the NIH under President Bush, believe that a lack of access to new embryonic stem cell lines hinders progress toward stem cell-based transplantation (8). For example, lines from a wider range of donors would allow more patients to receive human leukocyte agent matched stem cell transplants (9).
Currently, federal funds may not be used to derive new embryonic stem cell lines or to work with hESC lines not on the approved NIH list. NIH-funded equipment and laboratory space may not be used for research on nonapproved hESC lines. Both the derivation of new hESC lines and research with hESC lines not approved by NIH may be carried out under nonfederal funding. Because of these restrictions on NIH funding, a number of states have established programs to fund stem cell research, including the derivation of new embryonic stem cell lines. California, for example, has allocated $3 billion over 10 yr to stem cell research.
Under President Obama, it is expected that federal funding will be made available to carry out research with hESC lines not on the NIH list and to derive new hESC lines from frozen embryos donated for research after a woman or couple using in vitro fertilization (IVF) has determined they are no longer needed for reproductive purposes. However, federal funding may not be permitted for creation of embryos expressly for research or for derivation of stem cell lines using somatic cell nuclear transfer (SCNT) (10,11).
B. New embryonic stem cell lines from frozen embryos
Women and couples who undergo infertility treatment often have frozen embryos remaining after they complete their infertility treatment. The disposition of these frozen embryos is often a difficult decision for them to make (12). Some choose to donate these remaining embryos to research rather than giving them to another couple for reproductive purposes or destroying them. Several ethical concerns come into play when a frozen embryo is donated, including informed consent from the woman or couple donating the embryo, consent from gamete donors involved in the creation of the embryo, and the confidentiality of donor information.
1. Informed consent for donation of materials for stem cell research.
Since the Nuremburg Code, informed consent has been regarded as a basic requirement for research with human subjects. Consent is particularly important in research with human embryos (13). Members of the public and potential donors of embryos for research hold strong and diverse opinions on the matter. Some consider all embryo research to be unacceptable others only support some forms of research. For instance, a person might consider infertility research acceptable but object to research to derive stem cell lines or research that might lead to patents or commercial products (14). Obtaining informed consent for potential future uses of the donated embryo respects this diversity of views. Additionally, people commonly place special emotional and moral significance on their reproductive materials, compared with other tissues (15).
2. Waiver of consent.
In the United States, federal regulations on research permit a waiver of informed consent for the research use of deidentified biological materials that cannot be linked to donors (16). Thus, logistically it would be possible to carry out embryo and stem cell research on deidentified materials without consent. For example, during IVF procedures, oocytes that fail to fertilize or embryos that fail to develop sufficiently to be implanted are ordinarily discarded. These materials could be deidentified and then used by researchers. Furthermore, if infertility patients have frozen embryos remaining after they complete treatment, they are routinely contacted by the IVF program to decide whether they want to continue to store the embryos (and to pay freezer storage fees), to donate them to another infertile woman or couple, or to discard them. If a patient chooses to discard the embryos, it would be possible to instead remove identifiers and use them for research. Still another possibility involves frozen embryos from patients who do not respond to requests to make a decision regarding the disposition of frozen embryos. Some IVF practices have a policy to discard such embryos and inform patients of this policy when they give consent for the IVF procedures. Again, rather than discard such frozen embryos, it is logistically feasible to deidentify them and give them to researchers.
However, the ethical justifications for allowing deidentified biological materials to be used for research without consent do not always hold for embryo research (13). For example, one rationale for allowing the use of deidentified materials is that the ethical risks are very low there can be no breach of confidentiality, which is the main concern in this type of research. A second rationale is that people would not object to having their materials used in such a manner if they were asked. However, this assumption does not necessarily hold in the context of embryo research. A 2007 study found that 49% of women with frozen embryos would be willing to donate them for research (12). Such donors might be offended or feel wronged if their frozen embryos were used for research that they did not consent to. Deidentifying the materials would not address their concerns.
3. Consent from gamete donors.
Frozen embryos may be created with sperm or oocytes from donors who do not participate any further in assisted reproduction or childrearing. Some people argue that consent from gamete donors is not required for embryo research because they have ceded their right to direct further usage of their gametes to the artificial reproductive technology (ART) patients. However, gamete donors who are willing to help women and couples bear children may object to the use of their genetic materials for research. In one study, 25% of women who donated oocytes for infertility treatment did not want the embryos created to be used for research (17). This percentage is not unexpected because reproductive materials have special significance, and many people in the United States oppose embryo research. Little is known about the wishes of sperm donors concerning research.
There are substantial practical differences between obtaining consent for embryo research from oocyte donors and from sperm donors. ART clinics can readily discuss donation for research with oocyte donors during visits for oocyte stimulation and retrieval. However, most ART clinics obtain donor sperm from sperm banks and generally have no direct contact with the donors. Furthermore, sperm is often donated anonymously to sperm banks, with strict confidentiality provisions.
As a matter of respect for gamete donors, their wishes regarding stem cell derivation should be determined and respected (13). Gamete donors who are willing to help women and couples bear children may object to the use of their genetic materials for research. Specific consent for stem cell research from both embryo and gamete donors was recommended by the National Academy of Sciences 2005 Guidelines for Human Embryonic Stem Cell Research and has been adopted by the California Institute for Regenerative Medicine (CIRM), the state agency funding stem cell research (18,19). This consent requirement need not imply that embryos are people or that gametes or embryos are research subjects.
4. Confidentiality of donor information.
Confidentiality must be carefully protected in embryo and hESC research because breaches of confidentiality might subject donors to unwanted publicity or even harassment by opponents of hESC research (20). Although identifying information about donors must be retained in case of audits by the Food and Drug Administration as part of the approval process for new therapies, concerns about confidentiality may deter some donors from agreeing to be recontacted.
Recently, confidentiality of personal health care information has been violated through deliberate breaches by staff, through break-ins by computer hackers, and through loss or theft of laptop computers. Files containing the identities of persons whose gametes or embryos were used to derive hESC lines should be protected through heightened security measures (20). Any computer storing such files should be locked in a secure room and password-protected, with access limited to a minimum number of individuals on a strict “need-to-know” basis. Entry to the computer storage room should also be restricted by means of a card-key, or equivalent system, that records each entry. Audit trails of access to the information should be routinely monitored for inappropriate access. The files with identifiers should be copy-protected and double encrypted, with one of the keys held by a high-ranking institutional official who is not involved in stem cell research. The computer storing these data should not be connected to the Internet. To protect information from subpoena, investigators should obtain a federal Certificate of Confidentiality. Human factors in breaches of confidentiality should also be considered. Personnel who have access to these identifiers might receive additional background checks, interviews, and training. The personnel responsible for maintaining this confidential database and contacting any donor should not be part of any research team.
hESC research using fresh oocytes donated for research raises several additional ethical concerns as well, as we next discuss (21).
C. Ethical concerns about oocyte donation for research
Concerns about oocyte donation specifically for research are particularly serious in the wake of the Hwang scandal in South Korea, in which widely hailed claims of deriving human SCNT lines were fabricated. In addition to scientific fraud, the scandal involved inappropriate payments to oocyte donors, serious deficiencies in the informed consent process, undue influence on staff and junior scientists to serve as donors, and an unacceptably high incidence of medical complications from oocyte donation (22,23,24). In California, some legislators and members of the public have charged that infertility clinics downplay the risks of oocyte donation (19). CIRM has put in place several protections for women donating oocytes in state-funded stem cell research.
1. Medical risks of oocyte retrieval.
The medical risks of oocyte retrieval include ovarian hyperstimulation syndrome, bleeding, infection, and complications of anesthesia (25). These risks may be minimized by the exclusion of donors at high-risk for these complications, careful monitoring of the number of developing follicles, and adjusting the dose of human chorionic gonadotropin administered to induce ovulation or canceling the cycle (25).
Because severe hyperovulation syndrome may require hospitalization or surgery, women donating oocytes for research should be protected against the costs of complications of hormonal stimulation and oocyte retrieval (19). The United States does not have universal health insurance. As a matter of fairness, women who undergo an invasive procedure for the benefit of science and who are not receiving payment beyond expenses should not bear any costs for the treatment of complications. Even if a woman has health insurance, copayments and deductibles might be substantial, and if she later applied for individual-rated health insurance, her premiums might be prohibitive. Compensation for research injuries has been recommended by several U.S. panels (26) but has not been adopted because of difficulties calculating long-term actuarial risk and assessing intervening factors that could contribute to or cause adverse events.
Requiring free care for short-term complications of oocyte donation is feasible. In California, research institutions must ensure free treatment to oocyte donors for direct and proximate medical complications of oocyte retrieval in state-funded research. The term 𠇍irect and proximate” is a legal concept to determine how closely an injury needs to be connected to an event or condition to assign responsibility for the injury to the person who carried out the event or created the condition. Commercial insurance policies are available to cover short-term complications of oocyte retrieval. CIRM allows state stem cell grants to cover the cost of such insurance. The rationale for making research institutions responsible for treatment is that they are in a better position than individual researchers to identify insurance policies and would have an incentive to consider extending such coverage to other research injuries.
2. Protecting the reproductive interests of women in infertility treatment.
If women in infertility treatment share oocytes with researchers𠅎ither their own oocytes or those from an oocyte donor—their prospect of reproductive success may be compromised because fewer oocytes are available for reproductive purposes (21). In this situation, the physician carrying out oocyte retrieval and infertility care should give priority to the reproductive needs of the patient in IVF. The highest quality oocytes should be used for reproductive purposes (21).
As discussed in Section B. 2, in IVF programs some oocytes fail to fertilize, and some embryos fail to develop sufficiently to be implanted. Such materials may be donated to researchers. To protect the reproductive interests of donors, several safeguards should be in place (20). For the donation of fresh embryos for research, the determination by the embryologist that an embryo is not suitable for implantation and therefore should be discarded is a matter of judgment. Similarly, the determination that an oocyte has failed to fertilize and thus cannot be used for reproduction is a judgment call. To avoid any conflict of interest, the embryologist should not know whether a woman has agreed to research donation and also should receive no funding from grants associated with the research. Furthermore, the treating infertility physicians should not know whether or not their patients agree to donate materials for research.
3. Payment to oocyte donors.
Many jurisdictions have conflicting policies about payment to oocyte donors. Reimbursement to oocyte donors for out-of-pocket expenses presents no ethical problems because donors gain no financial advantage from participating in research. However, payment to oocyte donors in excess of reasonable out-of-pocket expenses is controversial, and jurisdictions have conflicting policies that may also be internally inconsistent (27,28).
Good arguments can be made both for and against paying donors of research oocytes more than their expenses (29). On the one hand, some object that such payments induce women to undertake excessive risks, particularly poorly educated women who have limited options for employment, as occurred in the Hwang scandal. Such concerns about undue influence, however, may be addressed without banning payment. For example, participants could be asked questions to ensure that they understood key features of the study and that they felt they had a choice regarding participation (19). Also, careful monitoring and adjustment of hormone doses can minimize the risks associated with oocyte donation (25). A further objection is that paying women who provide research oocytes undermines human dignity because human biological materials and intimate relationships are devalued if these materials are bought and sold like commodities (14,30).
On the other hand, some contend that it is unfair to ban payments to donors of research oocytes, while allowing women to receive thousands of U.S. dollars to undergo the same procedures to provide oocytes for infertility treatment (29). Moreover, healthy volunteers, both men and women, are paid to undergo other invasive research procedures, such as liver biopsy, for research purposes. Furthermore, bans on payment for oocyte donation for research have been criticized as paternalistic, denying women the authority to make decisions for themselves (31). On a pragmatic level, without such payment, it is very difficult to recruit oocyte donors for research.
4. Informed consent for oocyte donation.
In California, CIRM has instituted heightened requirements for informed consent for oocyte donation for research (19). The CIRM regulations go beyond requirements for disclosure of information to oocyte donors (19). The major ethical issue is whether donors appreciate key information about oocyte donation, not simply whether the information has been disclosed to them or not. As discussed previously, in other research settings, research participants often fail to understand the information in detailed consent forms (32). CIRM thus reasons that disclosure, while necessary, is not sufficient to guarantee informed consent. In CIRM-funded research, oocyte donors must be asked questions to ensure that they comprehend the key features of the research (19). Evaluating comprehension is feasible because it has been carried out in other research contexts, such as in HIV prevention trials in the developing world (33). According to testimony presented to CIRM, evaluation of comprehension has also been carried out with respect to oocyte donation for clinical infertility services.
5. Extrinsic regulators
There is strong evidence that the behaviour of stem cells is strongly affected by their local environment or niche ( figureਃ ). Some aspects of the stem cell environment that are known to influence self-renewal and stem cell fate are adhesion to extracellular matrix proteins, direct contact with neighbouring cells, exposure to secreted factors and physical factors, such as oxygen tension and sheer stress (Watt & Hogan 2000 Morrison & Spradling 2008). It is important to identify the environmental signals that control stem cell expansion and differentiation in order to harness those signals to optimize delivery of stem cell therapies.
Considerable progress has been made in directing ES cells to differentiate along specific lineages in vitro (Conti et al. 2005 Lowell et al. 2006 Izumi et al. 2007) and there are many in vitro and murine models of lineage selection by adult tissue stem cells (e.g. Watt & Collins 2008). It is clear that in many contexts the Erk and Akt pathways are key regulators of cell proliferation and survival, while pathways that were originally defined through their effects in embryonic development, such as Wnt, Notch and Shh, are reused in adult tissues to influence stem cell renewal and lineage selection. Furthermore, these core pathways are frequently deregulated in cancer (Reya et al. 2001 Watt & Collins 2008). In investigating how differentiation is controlled, it is not only the signalling pathways themselves that need to be considered, but also the timing, level and duration of a particular signal, as these variables profoundly influence cellular responses (Silva-Vargas et al. 2005). A further issue is the extent to which directed ES cell differentiation in vitro recapitulates the events that occur during normal embryogenesis and whether this affects the functionality of the differentiated cells (Izumi et al. 2007).
For a more complete definition of the stem cell niche, researchers are taking two opposite and complementary approaches: recreating the niche in vitro at the single cell level and observing stem cells in vivo. In vivo tracking of cells is possible because of advances in high-resolution confocal microscopy and two-photon imaging, which have greatly increased the sensitivity of detecting cells and the depth of the tissue at which they can be observed. Studies of green fluorescent protein-labelled haemopoietic stem cells have shown that their relationship with the bone marrow niche, comprising blood vessels, osteoblasts and the inner bone surface, differs in normal, irradiated and c-Kit-receptor-deficient mice (Lo Celso et al. 2009 Xie et al. 2009). In a different approach, in vivo bioluminescence imaging of luciferase-tagged muscle stem cells has been used to reveal their role in muscle repair in a way that is impossible when relying on retrospective analysis of fixed tissue (Sacco et al. 2008).
The advantage of recreating the stem cell niche in vitro is that it is possible to precisely control individual aspects of the niche and measure responses at the single cell level. Artificial niches are constructed by plating cells on micropatterned surfaces or capturing them in three-dimensional hydrogel matrices. In this way, parameters such as cell spreading and substrate mechanics can be precisely controlled (Watt et al. 1988 Théry et al. 2005 Chen 2008). Cells can be exposed to specific combinations of soluble factors or to tethered recombinant adhesive proteins. Cell behaviour can be monitored in real time by time-lapse microscopy, and activation of specific signalling pathways can be viewed using fluorescence resonance energy transfer probes and fluorescent reporters of transcriptional activity. It is also possible to recover cells from the in vitro environment, transplant them in vivo and monitor their subsequent behaviour. One of the exciting aspects of the reductionist approach to studying the niche is that it is highly interdisciplinary, bringing together stem cell researchers and bioengineers, and also offering opportunities for interactions with chemists, physicists and materials scientists.
Applying gene therapy to optic nerve regeneration
The researchers tested their approach on cells in the central nervous system because it is the first part of the body affected by aging. After birth, the ability of the central nervous system to regenerate declines rapidly.
To test whether the regenerative capacity of young animals could be imparted to adult mice, the researchers delivered the modified three-gene combination into retinal ganglion cells of adult mice with optic nerve injury.
For the work, Lu and Sinclair collaborated with Zhigang He, HMS professor of neurology and of ophthalmology at Boston Children’s Hospital, who studies optic nerve and spinal cord development and regeneration.
The treatment resulted in a two-fold increase in the number of surviving retinal ganglion cells after the injury and a five-fold increase in nerve regrowth.
“At the beginning of this project, many of our colleagues said our approach would fail or would be too dangerous to ever be used,” said Lu. “Our results suggest this method is safe and could potentially revolutionize the treatment of the eye and many other organs affected by aging.”
Mesenchymal stem cells (MSCs) reside in many tissues during development, and their differentiation produces specific phenotypes such as osteoblasts, chondrocytes, adipocytes, and myoblasts. In recent years, MSCs have been considered an important source for cell therapy and tissue regeneration in many clinical applications. Tissue damage is followed by an inflammatory response, and pro-inflammatory factors can rescue MSCs and start the repair process. The process of regeneration is quite complex MSCs interact with stromal and inflammatory cells, and derived factors play an important role in this process. MSCs are also involved in immunosuppression via inhibition of T cells, B cells, dendritic cells and natural killer cells[2-5], and they may exert immunomodulatory activity during the co-transplantation process.
In addition to cell therapy, MSCs are also a promising choice for other clinical applications because they can be used as diagnostic tools. The involvement of MSCs in many physiological or physiopathological aspects offers the possibility of targeting these cells, and their related molecular products, circulating in peripheral blood to obtain an early diagnosis by a non-invasive approach.
Diagnostic application of MSCs
In recent years, MSCs have been considered important 𠇋iomarkers” for a non-invasive prenatal diagnosis. Accurate prenatal diagnoses without fetal damage are needed to prevent genetic diseases, and MSCs have been identified in fetal blood during the first trimester, albeit at low concentrations. Despite 20 years of research in this area, technical challenges have produced many obstacles to reproducible fetal MSC isolation, and culture strategies have been developed. However, fetal MSCs have been observed in maternal peripheral blood, suggesting that fetal surface antigens could be considered promising biomarkers for a noninvasive prenatal diagnosis, and the analysis of neural and MSCs in the amniotic fluid represents a useful tool for the identification of neural tube defects.
Recruitment of progenitor cells in the blood stream in response to skeletal damage has been reported, and we have shown that circulating MSCs are increased in patients affected by osteoporosis as a consequence of the impairment of osteoblast differentiation. Furthermore, the molecular analyses of genes involved in the differentiation process have revealed an abnormal level of expression of the transcription factor runx2, the master gene of osteogenic differentiation. These data suggest the possibility of using MSC analysis for the evaluation of bone diseases, and the identification of circulating MSCs could be proposed as a noninvasive diagnostic tool.
Circulating stem cells have been observed after ischemic stroke in patients with myocardial infarction[11,12], and this finding suggests that stem cells in the peripheral blood may provide a potential cell marker for prognosis and risk evaluation in patients with cardiac diseases.
Kim et al showed that CD105 MSCs were mobilized in patients with cerebrovascular stroke, and, in particular, the authors demonstrated that the percentage of apoptotic CD105 cells, based on annexin V expression, was higher in patients with cerebral infarcts than that in normal control subjects.
The epithelial mesenchymal transition that occurs during cellular neoplastic transformation makes MSCs an important target for the diagnosis and prognosis of malignant tumors. Because cancer may originate from the neoplastic transformation of progenitors or related early differentiated cells, stem cell-like markers expressed in tumor cells could be of interest for biomarker identification.
New technologies for stem cells
Recent technologies have made possible the study of the molecular biology of cells at different levels (i.e., the genome, epigenome, transcriptome, small RNAome and metabolome). However, the study of stem cells is still a major challenge due to their low numbers and their potency and plasticity. Currently, most stem cell studies reported in the literature apply microarray technology and focus on the gene expression profile analysis of MSCs[7,15-19], cancer stem cells[20-24], tumor-initiating cells and embryonic stem cells. The study of gene expression at the global level is generally a pitfall and an ordeal due to the presence of low expression levels of many genes, which occurs because stem cells need to be ready to undertake a variety of differentiation programs. Accordingly, the study of different individual gene expression profiles is very difficult. Such issues cause the true gene expression signal to be confounded by background noise. At present, more recent Next Generation Sequencing (NGS) technologies are entering the stem cell arena. NGS is based on massive sequencing, and it can be applied for several purposes, such as the detection of transcripts and estimation of their levels (transcriptome), the identification of genome sequence variants (exome, genome) or the examination of modified methylated nucleotides (methylome). It is noteworthy that epigenomic NGS applications can be used to study stem cells because they can be used to investigate gene regulation and genome function during the differentiation and reprogramming processes. Therefore, the Epigenomics Roadmap consortium has been formed by the NIH to create extensive maps of genomic and epigenomic elements in stem cells and ex vivo tissue. NGS methods can be used to sequence single cells (DNA and/or RNA) opening avenues to thoroughly investigate how differential gene expression in individual cells defines cellular differentiation, function and physiology, making it possible to study rare stem cell populations and to investigate the prevalence and differences of potential stem cell subpopulations in cancers or other types of tissue.
GENOME STABILITY OF STEM CELLS
Plant lifespan is characterized by a rudimentary body plan, modular growth, and disparity between cell death and death of the organism (Watson and Riha, 2011 ). Plants exhibit a wide range of lifespans from a few weeks in monocarpic annuals to as long as millennia in long-lived perennials, which harbor meristematic cells that undergo thousands of divisions. In addition, plants being sessile organisms, environmental stresses increase DNA damage in stem cells therefore, how efficient the DNA repair mechanisms are in long-lived plant species and what the difference is between repair mechanisms in plants and animals are interesting questions to be answered.
Previous work focusing on animal aging highlighted the positive correlation between increased copy number of DNA repair genes and longevity in mammals (Tian et al., 2017 ). The naked mole-rat, the longest-lived rodent with a maximum lifespan of 32 years, has a higher copy number of genes for CCAAT/enhancer binding protein-γ (CEBPG), a regulator of DNA repair, and TERF1-interacting nuclear factor 2 (TINF2), a protector of telomere integrity, than short-lived rodent species (MacRae et al., 2015 ). Another long-lived mammal, the African elephant, encodes 20 copies of the tumor suppressor gene TP53, which induces apoptosis or senescence programs in response to DNA damage (Sulak et al., 2016 ). Analyses of genomes of two other long-lived species, the bowhead whale and bat, showed the signature of positive selection of multiple DNA repair and DNA damage-signaling genes (Keane et al., 2015 Zhang et al., 2013 ). These reports suggest the importance of genome maintenance mechanisms for longevity. However, in plants, no studies have yet employed comparative genome analyses to identify DNA repair genes associated with the evolution of longevity. Thanks to substantial progress in the elucidation of DNA damage signaling and repair mechanisms in Arabidopsis (Manova and Gruszka, 2015 ), it has become evident that most of the major DNA repair pathways are conserved in plants. Our plant stem cell project aims to systematically compare the DNA repair systems of diverse plant species and uncover their effects on organismal phenotypes such as mutation rates, lifespan, and adaptation to extreme environments, thereby identifying the role of DNA repair mechanisms in stem cell maintenance.
In Arabidopsis, stem cells highly express DNA repair genes, such as RADIATION SENSITIVE 51 (RAD51) and BREAST CANCER SUSCEPTIBILITY 1 (BRCA1), which maintain genome integrity (Yadav et al., 2009 ). However, severe DNA damage induces selective death of stem cells, but not of other somatic cells, in a programmed manner, and stem cells are replenished by activation of cell division in the adjacent organizing center (Fulcher and Sablowski, 2009 Furukawa et al., 2010 ). In mammals, cell death induction is a common strategy to cope with DNA damage, suggesting that plants trigger cell death in a stem cell-specific manner to prioritize the avoidance of unexpected destruction of developing tissues caused by disordered cell death. In spite of such a unique feature, information about stem cell death in plants is fragmentary: DNA damage-induced cell death is suppressed in Arabidopsis mutants of the brassinosteroid receptor BRI1 and the transcription factors ANAC044 and ANAC085, which are involved in cell cycle arrest (Chen et al., 2017 Lozano-Elena et al., 2018 Takahashi et al., 2019 ), although the link between brassinosteroid signaling and the cell cycle remains elusive. By contrast, the mechanism of stem cell replenishment has been uncovered in a recent study of the root stem cell niche the transcription factor ERF115, which is induced by brassinosteroid, promotes quiescent center cell division, thereby providing new stem cells after DNA damage (Heyman et al., 2013 ). Interestingly, ERF115 also triggers cell division adjacent to collapsed differentiated cells in roots (Canher et al., 2020 Heyman et al., 2016 ), suggesting that an ERF115-mediated pathway is a common system promoting cell division next to dead cells and regenerating tissues. Our focus is on how stem cell replenishment is fine-tuned to properly reconstitute the stem cell niche and how genome stability is preserved in stem cells. By answering these questions, we will better understand how plant longevity is guaranteed under fluctuating environmental conditions and what its essential difference is from animals.
Adult stem cells that do not age
Biomedical researchers at the University at Buffalo have engineered adult stem cells that scientists can grow continuously in culture, a discovery that could speed development of cost-effective treatments for diseases including heart disease, diabetes, immune disorders and neurodegenerative diseases.
UB scientists created the new cell lines -- named "MSC Universal" -- by genetically altering mesenchymal stem cells, which are found in bone marrow and can differentiate into cell types including bone, cartilage, muscle, fat, and beta-pancreatic islet cells.
The researchers say the breakthrough overcomes a frustrating barrier to progress in the field of regenerative medicine: The difficulty of growing adult stem cells for clinical applications.
Because mesenchymal stem cells have a limited life span in laboratory cultures, scientists and doctors who use the cells in research and treatments must continuously obtain fresh samples from bone marrow donors, a process both expensive and time-consuming. In addition, mesenchymal stem cells from different donors can vary in performance.
The cells that UB researchers modified show no signs of aging in culture, but otherwise appear to function as regular mesenchymal stem cells do -- including by conferring therapeutic benefits in an animal study of heart disease. Despite their propensity to proliferate in the laboratory, MSC-Universal cells did not form tumors in animal testing.
"Our stem cell research is application-driven," says Techung Lee, PhD, UB associate professor of biochemistry and biomedical engineering in the School of Medicine and Biomedical Sciences and the School of Engineering and Applied Sciences, who led the project. "If you want to make stem cell therapies feasible, affordable and reproducible, we know you have to overcome a few hurdles. Part of the problem in our health care industry is that you have a treatment, but it often costs too much. In the case of stem cell treatments, isolating stem cells is very expensive. The cells we have engineered grow continuously in the laboratory, which brings down the price of treatments."
UB has applied for a patent to protect Lee's discovery, and the university's Office of Science, Technology Transfer and Economic Outreach (UB STOR) is discussing potential license agreements with companies interested in commercializing MSC-Universal.
Stem cells help regenerate or repair damaged tissues, primarily by releasing growth factors that encourage existing cells in the human body to function and grow.
Lee's ongoing work indicates that this feature makes it feasible to repair tissue damage by injecting mesenchymal stem cells into skeletal muscle, a less invasive procedure than injecting the cells directly into an organ requiring repair. In a rodent model of heart failure, Lee and collaborators showed that intramuscular delivery of mesenchymal stem cells improved heart chamber function and reduced scar tissue formation.
UB STOR commercialization manager Michael Fowler believes MSC-Universal could be key to bringing new regenerative therapies to the market. The modified cells could provide health care professionals and pharmaceutical companies with an unlimited supply of stem cells for therapeutic purposes, Fowler says.
Lee says his research team has generated two lines of MSC-Universal cells: a human line and a porcine line. Using the engineering technique he and colleagues developed, scientists can generate an MSC-Universal line from any donor sample of mesenchymal stem cells, he says. "I imagine that if these cells become routinely used in the future, one can generate a line from each ethnic group for each gender for people to choose from," Lee says.
The research was funded by the National Institutes of Health and New York State Stem Cell Science (NYSTEM).
Materials provided by University at Buffalo. Note: Content may be edited for style and length.