Will Human Embryonic Stem Cell Therapies Finally Grow Up?

A wealth of evidence attests that the organs of developing embryos, particularly the developing brain, are acutely sensitive to chemical perturbations. However, scientists know very little about how exposures to specific endogenous chemicals actually impact human development or children’s ability to learn. And there are almost no data on how the vast majority of the 84,000 chemicals currently listed in the Toxic Substances Control Act (TSCA) Inventory1—including most of the 201 compounds known to be neurotoxic to adults and the 1,000 chemicals shown to be neurotoxic to animals2—may affect developing infants. It is also unclear whether testing with animals always provides accurate insights into human developmental susceptibility. A new line of research based on human stem cells is providing important insights into how chemicals may affect neonatal development. Stem cells are the master cells capable of producing some or all of the 200-plus different types of cells in the human body. In time, some researchers believe stem cells may enable scientists to amass far more data on how exposure to environmental chemicals affects human development, particularly the development of the brain. Now is a “critical time to be talking about stem cell research in the environmental health context,” says Tracey Woodruff, director of the Program on Reproductive Health and the Environment at the University of California, San Francisco (UCSF) Medical School.

The governance of innovative bioscience could extensively influence the growth of science, economy, society and law of every country. Similar to many countries, Muslim nations are experiencing a variety of socio-cultural and political conflicts relating to emerging bioscience, including human embryonic stem (hES) cell research and therapy. Among Muslims, the discussion of the ethics and regulatory policy related to hES cell research is mostly seen as a restrictive position, however, some Muslim nations take an intermediate stance, e.g. Iran as a leading country among Muslim nations. Islam, similar to other religions, significantly influences the moral, social and legal debates around hES cell science. The main argument is about the level of protection of the human embryo as it is used to obtain stem cells and then destroyed. This essay broadly portrays the fundamental moral constructions of Islamic deliberations on bioscience and technology in general, and particularly hES cell science in Shi’a tradition. The paper helps us to understand how scientific knowledge, bioethical discourses and legal deliberations are produced in the Muslim countries. After introducing some background information, it delves into the core themes framing the discussion, with reference to the moral controversy surrounding hES cell science and Islamic faith. This account is intended to present the main highlighted debates and controversial questions. The essay concludes that given continuing conflicts between science, politics and religion in many countries, in some countries, for example in the Shi’a Iran, hES cell science converges with religious beliefs and government policies.

Human Embryonic Stem Cells . Edited by J. Odorico, S. Zhang and R. Pedersen . (Pp. xxii + 391 , illustrated , ISBN 18599 62785 , £80 hardback.) Abingdon, UK : BIOS Scientific/Garland Science . It is now nearly seven years since the first description of human embryonic stem (ES) cell lines was published by Jamie Thomson. That event, closely apposed to the reports of the cloning of sheep (‘Dolly’), and then mice, by somatic nuclear transfer, catalysed substantial interest in the idea of regenerative medicine and the possibility that stem cells of all sorts might be harnessed for the replacement of tissues lost to accident or disease. At about the same time, a number of papers appeared, reporting that various somatic stem cells – which were previously thought to be committed to lineages from within their tissue of origin – might indeed exhibit substantial plasticity and be able to generate terminally differentiated cells corresponding to a much wider range of cell types. Thus, the current era of stem cell biology was born, though the concept of stem cells and their application in medicine is much older. One consequence of this resurgent interest in stem cell biology has been the publishing of several multi-author volumes devoted to stem cells and their potential. The volume Human Embryonic Stem Cells, edited by J. Odorico, S. Zhang and R. Pedersen, is the latest in a series produced by different publishing houses over the past few years. Paradoxically, however, as Jamie Thomson points out in his foreword to the present volume, progress in the field has been slow over this time. Prospects for adult stem cells have been dimmed by controversy over the phenomenon of ‘plasticity’, with the suggestion that at least some of the reported cases might be artefactual, depending upon cell fusion or rare transdifferentiation events. At the same time, progress with human ES cells was initially hindered by the difficulty of access to established lines, and the complexity of maintaining and expanding the cultures when they could be obtained. Although mouse ES cells have been available for over 20 years, they have mostly been used as tools to produce transgenic mice. With notable exceptions, few have investigated their cell biology for its own sake, and so there has been relatively little experience from studies of mouse ES cells to guide the study of their human counterparts. The development of ES cell lines was a systematic progression from the study of teratocarcinomas in the 1970s, by those who thought that these tumours might provide key insights into the mechanisms of embryonic development. It is then perhaps ironic that clues about how to control the behaviour of human ES cells in culture are now being provided by our detailed knowledge of developmental genetics, especially of the mouse. However, the human ES cell field is changing. Many laboratories in many countries have now derived human ES cell lines. In the current International Stem Cell Initiative, a collaborative venture to compare the properties of human ES cell lines derived worldwide, 75 independent lines derived in 17 laboratories and ten countries have been enrolled in the study. Meanwhile, reports are now beginning to appear in prominent journals, addressing the molecular mechanisms of human ES cell proliferation and differentiation – not merely describing their characteristics and potential for diverse differentiation. The present volume, unlike many of its competitors, focuses explicitly on human ES cells, and may be thought to mark the end of the first phase of human ES cell research. It brings together a series of authors who have contributed significantly to the field and it balances chapters discussing the basic biology of ES cells with others discussing their differentiation along specific lineages and eventual application. Despite the focus on ES cells, two early chapters provide useful comparative reviews of recent studies of adult stem cell plasticity and of mesenchymal stem cells. Further chapters address potential issues that will need to be addressed as derivatives of human ES cells are developed for eventual clinical application. Included here are two interesting and useful reviews of the ethical, legal and intellectual property aspects of human ES cell research and application. Undoubtedly, research in the new field of human ES cell biology is beginning to advance rapidly, so that it might be considered that the present volume will become rapidly obsolete. However, its well-organized and well-written chapters provide a valuable reference to the current state of the field for newcomers and established human ES cell researchers alike. Although our understanding of molecular mechanisms and the means of manipulating human ES cells is likely to change significantly in the future, much of what is contained in this volume will remain fundamental, and so of value to those working in this exciting new area of research for quite a few years to come.

Controversy regarding Human embryonic Stem Cell (hESC) research is evident in the medical and scientific community, legislative, judicial, and executive branches of the government, private companies, religious affiliations, special interest groups, and among the general American public. Stem cells are derived from adult stem cell resources, which are termed non-embryonic or from the cells of immature blastocysts termed embryonic stem cells. Non-embryonic cells do not have the ability to differentiate into other specialized tissues, whereas embryonic cells are capable of differentiating into almost any type of tissue. hESC research, which is the focus of this paper, includes the direct creation and therapeutic cloning of human embryos, known as somatic cell nuclear transfer (SCNT) during the blastocyst-stage of development, the use of surplus embryos from in vitro fertilization, and from tissue derived from extracted embryos through abortion. The purpose of hESC research is threefold: (1) to advance science and medicine, (2) prevent disease, and (3) cure certain diseases. hESC research is an ethical dilemma facing the United States of America. Opponents of hESC often cite the rights of the embryo and define the embryo at conception. Supporters of hESC research include the scientific and medical community, and private hESC research companies. The unique political system of the United States has allowed for a dichotomous policy to arise, where federal funding of hESC is prohibited, but private companies and individual states are able to legally forge a path in hESC research. The prohibition of federal funds has created an abyss between states who choose to fund hESC research and those who prohibit it. It has also set the stage for yet another private corporation to advance in hESC and place profit above the American public. The embryo is the only silent member of this debate.

S oon the Bush administration will decide the fate of human embryonic stem cell (ESC) research at U.S. government-funded institutions, and the outcome of that decision will greatly influence the role of ESC science in human developmental biology around the world. But although the forces that science brings to this field are powerful, the future of ESC research will largely be determined by other interests: politics, organized religion, commerce, the legal community, and patient advocacy groups. The decision-making process needs to develop a policy that is fact-based and serves the best interests of society as well as science. Broadly, stem cells are rare cells that renew themselves and, in addition, give rise to differentiated cells. Some, from adult tissues, have multiple but usually restricted developmental potency: Hematopoietic stem cells (HSCs), for example, normally make only blood cells; multipotent brain stem cells make only brain cells. ESCs, in contrast, are derived from cells isolated from the inner cavity of the blastocyst: an early embryo that cannot develop further unless it successfully implants in the uterus. These cells are pluripotent: Given an encouraging environment, they can develop into any cell type; although so far in culture they have been unable to form whole organs, much less bodies. It would be convenient if pluripotent cells persisted into adulthood. Unfortunately, most (but not all) published accounts suggesting adult stem cell pluripotency have not successfully established that one type can produce a cell of another tissue type. Indeed, although HSCs capable of regenerating the blood can be isolated from adults or fetuses, so far brain stem cells capable of robust growth and transplantability have come only from fetal or ESC sources. This is likely to be true for a number of tissues; fetal stem cells are much more active than postneonatal cells. A moratorium on research and/or transplantation of fetal stem cells could thus be devastating. As for the search for pluripotent adult stem cells, it is always possible, perhaps even likely, that further research might reveal a source. But that is simply a hope, and it would be foolish to abandon the surer path for the unproven one. The greatest mysteries of human development lie in the time interval between the early blastocyst embryo and the much later point at which organs are formed. What is needed is a thorough understanding of the cellular and genetic events that make the organs and tissues from these pluripotent cells. That can come only from a broad-scale attack on the problem from all kinds of researchers, whether in academic, nonprofit, or commercial organizations. The outcome will surely have profound effects on medicine, science, and translational research. Yet these advances that could be gained from human ESC research are currently restricted in the United States to the single commercial entity that holds the patents for the ESCs developed using its own funding. That is a disturbing anomaly, but it can be remedied by a decision to approve ESC research by government-funded entities. Decisions to approve ESC research have been recently undertaken in several other countries, notably Germany and the United Kingdom, where government sought advice from a broad range of concerned interests. Not only does that provide a constructive example to our administration, it underscores another consequence of the wrong decisions here. Failure to make this research opportunity available in the United States will make those countries centers of the scientific, medical, and commercial advances in which we ought to be a contributing partner. Plainly, scientists alone should not make the decisions about the ethical conduct of their work or about its social applications. It is appropriate that governments, with appropriate public input, define the societal interest in particular lines of research. But in making those policies, the state should minimize purely political considerations and be mindful of the separation of church and state. The wrong action here could close the door to an important avenue of scientific and clinical discovery. The state should not be the barrier to the translation of these potentially revolutionary therapeutic opportunities into real medical advances.

Pluripotent cells have the potential to differentiate into all of the cell types of an animal. This unique cell state is governed by an interconnected network of transcription factors. Among these, Oct4 plays an essential role both in the development of pluripotent cells in the embryo and in the self-renewal of its in vitro counterpart, embryonic stem (ES) cells. Furthermore, Oct4 is one of the four Yamanaka factors and its overexpression alone can generate induced pluripotent stem (iPS) cells. Recent reports underscore Oct4 as an essential regulator of opposing cell state transitions, such as pluripotency establishment and differentiation into embryonic germ lineages. Here we discuss these recent studies and the potential mechanisms underlying these contrasting functions of Oct4.

Embryonic stem (ES) cells are derived from the epiblast of the inner cell mass (ICM) of a blastocyst. These are pluripotent cells and can give rise to all derivatives of the three primary germ layers ectoderm, endoderm, and mesoderm. ES cells require specific signals for specific lineage of differentiation if injected directly into another body, ES cells will differentiate into many different types of cells, causing a teratoma (Wu et al., 2007). The techniques for culturing mouse embryonic stem (ES) cells from the inner cell mass of the preimplantation blastocyst were first done in 1981 (Martin, 1981), and versions of these standard procedures are used today in laboratories throughout the world. The first successful derivation of human ES (hES) cells was reported by Thomson et al. (Thomson et al., 1998). They isolated and plated the cells onto mitotically inactivated MEF (mouse embryonic fibroblast) cells. In 2000, Reubinoff et al. confirmed that hES cells could be efficiently derived from surplus embryos and possess the differentiation potential under in vitro conditions. Since then, there has been rapid progress made and numerous studies have described the derivation of new hES cell lines including methods of growing both undifferentiated hES cells and their differentiated progeny. In last 6 years, there has been exponential increase in methods to improve culture conditions, differentiation patterns to produce human cells for transplantation and drug testing (Trounson, 2006, Gepstein, 2002) and genetic manipulation (Draper et al., 2004, Zwaka and Thomson, 2003). Generating cultures of mouse or human ES cells that remain in a proliferating and undifferentiated state is multistep process. Typically, the inner cell mass of a preimplantation blastocyst is removed from the trophectoderm that surrounds it and cultured in the small plastic culture dishes containing growth medium supplemented with fetal calf serum. The culture dishes are sometimes coated with a layer of nondividing cells, which are often MEF cells that have been chemically inactivated so they will not divide. Mouse ES cells can be grown in vitro without feeder layers if the cytokine leukemia inhibitory factor (LIF) is added to the culture medium, but human ES cells do not respond to LIF. The process of generating an embryonic stem cell line is somewhat difficult, so lines are not produced every time from the preimplantation-stage embryo maintained in a culture dish. However, if the plated cells survive, divide and multiply enough to crowd the dish, they are removed, and plated into several fresh culture dishes. The process of re-plating or

This paper examines the influences that congressional staff people viewed as important in shaping legislators' voting decisions on the human embryonic stem (ES) cell research bill in the 109th Congress, the first legislation vetoed by President George W. Bush. The analysis illuminates factors that impact congressional decision making on a salient issue with a strong moral component. Constituent concerns, ideology, and a desire to make good public policy all centrally affected members' choices; however, moral overtones permeated considerations relevant to the human ES cell research question. In addition, at least three influences that directly reflect or relate to members' moral claims - religious convictions, personal connections to potential beneficiaries of human ES cell research, and moral pressure from outside interests - were important also. The analysis draws on data gathered from interviews with congressional aides.

Human and mouse embryonic stem (ES) cells have the capacity to differentiate into derivatives of all three germ layers, suggesting novel therapies for degenerative, metabolic, and traumatic disorders. ES-based regenerative medicine will be further advanced by the development of reliable methods for transgene introduction and expression. Here, we show infection of human and mouse embryonic stem (ES) cells with two of the most popular vectors in gene transfer, adenovirus type 5 (Ad5) and adeno-associated virus (AAV; serotypes 2, 4, and 5). All vectors express the nuclear-localized marker gene beta-galactosidase expressed from the Rous Sarcoma Virus long terminal repeat (RSV-LTR). Both Ad5 and AAV2 infected human and mouse ES cells and gave rise to beta-galactosidase expression. AAV4 and 5 did not yield detectable levels of beta-galactosidase expression. Quantitative PCR analysis of virally infected human and mouse ES cells revealed that only Ad5 and AAV2 are capable of transducing both cell-types. No viral DNA was detected in cells infected with either AAV4 or AAV5. Infection and subsequent differentiation of mouse and human ES cells with Ad5 showed that beta-galactosidase-expressing cells were restricted to cells in the interior of the embryoid body mass. No beta-galactosidase expression was observed in AAV-infected cells following differentiation. There was no difference in morphology or differentiation patterns between infected and noninfected differentiating mouse and human ES cells. Differentiation of hES cells prior to infection led to transduction of neuronally differentiated cells with good efficiency using all vectors. These data show that Ad5- and AAV2-based vectors are capable of infecting both human and mouse ES cells, in both their undifferentiated and differentiated states, whereas AAV4 and AAV5 can infect human and mouse ES cells only following differentiation.

Embryonic stem cells are pluripotent and capable of unlimited self-renewal. Elucidation of the underlying molecular mechanism may contribute to the advancement of cell-based regenerative medicine. In the present work, we performed a large scale analysis of the phosphoproteome in mouse embryonic stem (mES) cells. Using multiplex strategies, we detected 4581 proteins and 3970 high confidence distinct phosphosites in 1642 phosphoproteins. Notably, 22 prominent phosphorylated stem cell marker proteins with 39 novel phosphosites were identified for the first time by mass spectrometry, including phosphorylation sites in NANOG (Ser-65) and RE1 silencing transcription factor (Ser-950 and Thr-953). Quantitative profiles of NANOG peptides obtained during the differentiation of mES cells revealed that the abundance of phosphopeptides and non-phosphopeptides decreased with different trends. To our knowledge, this study presents the largest global characterization of phosphorylation in mES cells. Compared with a study of ultimately differentiated tissue cells, a bioinformatics analysis of the phosphorylation data set revealed a consistent phosphorylation motif in human and mouse ES cells. Moreover, investigations into phosphorylation conservation suggested that phosphoproteins were more conserved in the undifferentiated ES cell state than in the ultimately differentiated tissue cell state. However, the opposite conclusion was drawn from this conservation comparison with phosphosites. Overall, this work provides an overview of phosphorylation in mES cells and is a valuable resource for the future understanding of basic biology in mES cells.

Embryonic stem (ES) cells require a network of transcription factors and signaling molecules to maintain their pluripotency. The transcription factor Oct4, one of pluripotency genes, is required for self-renewal and maintains the undifferentiated state of ES cells. Loss of expression of Oct4 initiates ES cell differentiation. Germ cell nuclear factor (GCNF), an orphan nuclear receptor, binds to a DR0 site within Oct4 gene promoter in human ES cells. Retinoic acid induces expression of GCNF, which binds the promoter of Oct4 gene, and accelerates the decrease of Oct4 gene expression and human ES cells differentiation. Expression of other pluripotency genes, such as Sox2, also decrease during human ES cell differentiation induced with retinoic acid. These findings increase our understanding of the mechanism of regulation of gene expression and in maintaining human ES cell pluripotence and during the differentiation process. Results 1: Tera-2 cells and human ES (H9) cells were induced to differentiate by Retinoic Acid (1μM). Expression of GCNF, Oct4, Sox2, Dax1, LRH1, SF1, Actin was determined by RT-PCR (A), and by western blot (B) in Tera-2 cells and human ES cells. GCNF expression increased by day 2 of differentiation in Tera-2 cells and human ES cells when treated with Retinoic Acid (1μM). GCNF expression is transient and decreased earlier in Tera-2 cells than in human ES cells. Expression of Oct4, Sox2 and SF-1 was gradually repressed during differentiation. Expression of the orphan receptor LRH1 increased when Tera-2 and human ES cells differentiated, and subsequently decreased. DAX1 expression was up-regulated when Tera-2 cells and human ES cells differentiate, and maintained the same level until day 6 in Tera-2 cells but gradually increased in human ES cells. Result 2: GCNF binding the DR0 site within the Oct4 promoter in Tera-2 cells and human ES cells is induced upon differentiation and is transient in nature. Detection of GCNF binding to the Oct4 promoter in vivo using Chromatin immunoprecipitation (ChIP) assays: Tera-2 cells and human ES cells were treated with Retinoic Acid (1 μM) and induced to differentiate. GCNF binding to the DR0 site of Oct4 promoter can be detected in vivo when Tera-2 cell and human ES cells differentiate, and then decreases at later stages of differentiation. Result 3: Analysis of GCNF binding to the DR0 element in the Oct4 promoter by electrophoretic mobility shift assays (EMSAs). No GCNF binding to the DR0 element was detected at day 0 in the Tera-2 cells and human ES cells. GCNF has been at the highest level of binding to the DR0 site at day 3 of RA-induced differentiation (arrow) and is shifted (arrow head) when GCNF-specific antibody is added to the EMSAs. GCNF DNA binding activity disappeared by day 5 of differentiation in Tera-2 cells. In human ES cells, GCNF displayed a higher level of binding to the DR0 site at day 3 of differentiation and decreased at day 5 of RA treatment. This result shows that GCNF directly binds to the conserved DR0 element in the Oct4 promoter in Tera-2 cells and human ES cell. (platform)

Federally funded human embryonic stem cell research can move ahead unimpeded because of a Supreme Court decision announced last week. The Court declined to hear a case, which has dragged on for more than three years, challenging the legality of such stem cell research funding. The original lawsuit against the National Institutes of Health was filed in 2009 by two scientists who work with adult stem cells. They claimed that the agency’s policy for funding human embryonic stem cell research violates the Dickey-Wicker Amendment, an obscure law that prohibits federal funding of any research that destroys human embryos. Human embryos are destroyed in order to derive such stem cells. In 2010, the case temporarily halted government-funded research on human embryonic stem cells. A federal appeals court lifted the injunction, however, and subsequently ruled that conducting research on these cells is not the same as deriving them. The plaintiffs appealed to ...

Human embryonic stem (ES) cells originate from the inner cell mass of the blastocyst, and retain in culture the properties of pluripotent cells of the early embryo. The study aim was to determine whether the open pulled straw (OPS) vitrification method, which is highly effective for the cryopreservation of embryos, might be also efficient for human ES cells. All human ES cell clumps that were vitrified by the OPS method could be recovered upon thawing, and gave rise to ES cell colonies after plating. Vitrified colonies were significantly smaller and showed an increased level of background differentiation compared with control colonies. However, these unwanted effects could be overcome by additional cultivation of the colonies for 1 and 2 days respectively. The vitrified human ES cells were cultivated for prolonged periods and retained the properties of pluripotent cells, including a normal karyotype, expression of the transcription factor Oct-4 and surface markers that are characteristic to human ES cells. When grafted into SCID mice, the vitrified cells gave rise to teratomas containing derivatives of all three embryonic germ layers. Vitrification by the OPS method is reliable and effective for the cryopreservation of human pluripotent embryonic stem cells.

The advantages and problems in cell transplantation depend on the type of stem cells used. Blood stem cells have been used in clinical treatment for more than 40 years. Mesenchymal stem cells are already used in a large number of clinical trials. They are used in severe graft versus host reaction, and in forming bone, cartilage and muscle. Pluripotent stem cells, human embryonic stem cells (ESC) and induced pluriotent cells (hiPSC) have not yet been used clinical trials, but the first trials in spinal cord injury has been planned. Safety issues are the concerns in stem cell transplantation. Risks of infection are known, and they can be tackled by careful testing of the donor and careful handling of the cells. A particular quality system, GMP, has been developed for this purpose. Immunogenicity is a problem in all situation in which patient’s own cells cannot be used. For tackling immunogenicity, options are matching the tissue types of the donor and the recipient, immunosuppressive medication, possible modulation of the immune response, and the use of patient-specific stem cells induced from the recipient’s somatic cells. Substances of animal origin in cultures may promote immune reactions. They can be avoided by using only xeno-free cultures. Tumorigenicity of cells differentiated from human embryonic and pluripotent stem cells is a risk if cells differentiated form such pluripotent cells are transplanted. Pluripotent cells need to be eliminated by using effective differentiation protocols and by removing the possible remaining pluripotent cells from the population aimed at transplantation.KeywordsHuman embryonic stem cellsInduced pluripotent cellsCell therapyTumorigenicityGood manufacturing practice

Stem cell research: win, win, win by keeping politics out.