Human Cloning and Stem Cells

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.

Stabilization of hESCs in two distinct substates along the continuum of pluripotency.

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.

Distinctive properties of stem cells are not autonomously achieved, and recent evidence points to a level of external control from the microenvironment. Here, we demonstrate that self-renewal and pluripotent properties of human embryonic stem (ES) cells depend on a dynamic interplay between human ES cells and autologously derived human ES cell fibroblast-like cells (hdFs). Human ES cells and hdFs are uniquely defined by insulin-like growth factor (IGF)- and fibroblast growth factor (FGF)-dependence. IGF 1 receptor (IGF1R) expression was exclusive to the human ES cells, whereas FGF receptor 1 (FGFR1) expression was restricted to surrounding hdFs. Blocking the IGF-II/IGF1R pathway reduced survival and clonogenicity of human ES cells, whereas inhibition of the FGF pathway indirectly caused differentiation. IGF-II is expressed by hdFs in response to FGF, and alone was sufficient in maintaining human ES cell cultures. Our study demonstrates a direct role of the IGF-II/IGF1R axis on human ES cell physiology and establishes that hdFs produced by human ES cells themselves define the stem cell niche of pluripotent human stem cells.

We report here a lentiviral vector system for regulated transgene expression. We used the tetracycline repressor fused with a transcriptional suppression domain (tTS) to specifically suppress transgene expression. Human cells were first transduced with a tTS-expressing vector and subsequently transduced with a second lentiviral vector-containing transgene controlled by a regular promoter adjacent to a high-affinity tTS-binding site (tetO). After optimizing the location of the tetO site in the latter vector, we achieved a better inducible transgene expression than the previous lentiviral vectors using the tetracycline repressor systems. In this new system, the transgene transcription from a cellular promoter such as EF1alpha or ubiquitin-C promoter is suppressed by the tTS bound to the nearby tetO site. In the presence of the tetracycline analog doxycycline (Dox), however, the tTS binding is released from the transgene vector and transcription from the promoter is restored. Thus, this system simply adds an extra level of regulation, suitable for any types of promoters (ubiquitous or cell-specific). We tested this tTS-suppressive, Dox-inducible system in 293T cells, human multipotent hematopoietic progenitor cells, and three human embryonic stem cell lines, using a dual-gene vector containing the green fluorescent protein reporter or a cellular gene. We observed a tight suppression in the uninduced state. However, the suppression is reversible, and transgene expression was restored at 5 ng/ml Dox. The lentiviral vectors containing the tTS-suppressive, Dox-inducible system offer a universal, inducible, and reversible transgene expression system in essentially any mammalian cell types, including human embryonic stem cells.

Researchers have cultured cells that look and act like human primordial germ cells (PGCs)—and they have done it reproducibly, in abundance. Analysis of these cells, derived from both human embryonic stem cells and induced pluripotent cells, reveals some of the factors that drive PGC specification and development [1]. Researchers have previously produced mouse PGCs in a dish, but this transformation has proven more recalcitrant from human stem cell lines, which give rise to PGCs only at low frequency. Jacob Hanna and M. Azim Surani overcame this barrier, starting with a protocol they had previously developed to generate embryonic stem cells or induced pluripotent stem cells. Their protocol yields ‘‘naive’’ pluripotent cells that resemble the blastocyst inner cell mass, in contrast with previous methods, which yielded cells with a slightly more differentiated state, akin to cells of the epithelial postimplantation epiblast. The researchers could handily generate PGC-like cells by applying a molecular cocktail similar to that used to derive PGC-like cells from mouse stem cells. The resulting cells had similar RNA profiles as human germline tumor cells and primordial germ cells derived from aborted human fetuses. The cultured cells also showed signs of epigenetic changes—DNA demethylation—known to occur in PGCs. Further analysis, including gene knockout studies, identified some differences and similarities with mouse PGCs. For instance, the researchers found that the transcriptional regulator SOX17 is a key regulator of PGC-like fate in human cells—a role that it does not seem to serve in mice, where it is better known for specifying endoderm. The researchers identified some of the factors downstream of SOX17, including BLIMP1, which seems to repress somatic genes during specification of PGCs. BLIMP1 is also involved in mouse PGC fate. The findings open the door to the possibility of generating sperm and egg cells in cell culture. But for the more immediate future, this experimental system should be invaluable for answering basic questions about human development at its earliest stages.

The term "stem cell exceptionalism" has been used to characterize the policy response to controversies surrounding human embryonic stem cell research. For example, governments and funding agencies have adopted policies governing the derivation and use of human embryonic stem cell lines. These policies have effectively served to fill gaps in existing guidelines and regulations and signal that scientists are committed to a responsible framework for the conduct of research involving human embryos. Recent publications discuss whether ethical and policy issues associated with induced pluripotent cells (iPSCs) from non-embryonic sources create a need for further policy intervention. We suggest many of the issues identified by commentators may be addressed through the application of established policy frameworks governing the use of tissue, human stem cells, and research participation by human research subjects. To the extent, iPSC research intersects with hESC research (e.g. the creation of human gametes and/or embryos), the policy framework governing hESC appears sufficiently robust at this time.

5 ISSN 1746-0751 10.2217/RME.09.84 © 2010 Future Medicine Ltd Regen. Med. (2010) 5(1), 5–6 “To further the advancement of responsible stem cell research, the Canadian Stem Cell Foundation ... released the Stem Cell Charter in September 2009 ... The promulgation and promotion of this Charter aims to prospectively frame stem cell research and to serve as a benchmark for the guidance of international and national initiatives in both scientific research and policymaking.” Bartha Maria Knoppers Author for correspondence: Director, Centre of Genomics & Policy, Faculty of Medicine, Department of Human Genetics, McGill University, 740 Dr. Penfield Avenue, Suite 5214, Montreal, Quebec, H3A 1A4 Canada Tel.: +1 514 398 8866; Fax: +1 514 398 8954; bartha.knoppers@mcgill.ca

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.

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)

Stem cell gene transfer—efficacy and safety in large animal studies

Human Thrombopoietin Knockin Mice Efficiently Support Human Hematopoiesis In Vivo

Human pluripotential stem cells, including both embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), possess self-renewing potency or the ability to differentiate into virtually any type of somatic cell. These features make them particularly advantageous as sources from which to generate specific types of human tissue cells in vitro for use in drug development and regenerative medicine. In most cases, however, human pluripotential cell lines and especially human ES cells can only be used in cell-based applications because of ethical issues. Animal models are therefore sought as an alternative to using human cells. An increasingly popular non-human primate model is the common marmoset (Callithrix jacchus). The recent successful creation of lentivirus-mediated transgenic marmosets provides a new animal model for human disease offering the powerful advantage of a close genetic relationship with humans (Sasaki et al., 2009), though this technique is not yet sufficiently developed for common use, and the numbers of monkeys available for experiments are limited. Mouse ES cells, meanwhile, still have great value as a research tool, even after the development of human pluripotent cells, as they can be used to create chimeric mice, achieve germline transmission, and generate normal offspring. The available genetic engineering technologies often employ an embryonic manipulation approach in mice, using mouse ES cells to examine the gain-of-function or loss-of-function effects associated with certain chromosomal regions in vivo. Recently, we have developed chromosome elimination cassettes (CEC) using a Cre-inverted loxP system that was first used in mouse ES cells. In this system, transient cre expression can initiate immediate chromosomal loss over the course of a few cell cycles in the recombinant cells. This technology was developed to clarify chromosomal function through observing loss-offunction at the chromosomal level. In mammalian cells, chromosome composition and gene dosage are kept stable because large chromosome-wide deletions are usually fatal. Accordingly, we first applied the Cre-inverted loxP system to tetraploid cells composed of mouse ES cells fused with mouse somatic cells to generate conditions conducive to largescale chromosomal imbalance. The CEC-tagged chromosomes could be targets of Credependent chromosome elimination. In addition, we have demonstrated that the Creinverted loxP system enhances cohesion between a loxP site and an adjacent inverted loxP,

Human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons hold potential for treating Parkinson's disease (PD) through cell replacement therapy. Generation of DA neurons from hESCs has been achieved by coculture with the stromal cell line PA6, a source of stromal cell-derived inducing activity (SDIA). However, the factors produced by stromal cells that result in SDIA are largely undefined. We previously reported that medium conditioned by PA6 cells can generate functional DA neurons from NTera2 human embryonal carcinoma stem cells. Here we show that PA6-conditioned medium can induce DA neuronal differentiation in both NTera2 cells and the hESC I6 cell line. To identify the factor(s) responsible for SDIA, we used large-scale microarray analysis of gene expression combined with mass spectrometric analysis of PA6-conditioned medium (CM). The candidate factors, hepatocyte growth factor (HGF), stromal cell-derived factor-1 α (SDF1α), secreted frizzled-related protein 1 (sFRP1), and vascular endothelial growth factor D (VEGFD) were identified, and their concentrations in PA6 CM were established by immunoaffinity capillary electrophoresis. Upon addition of SDF1α, sFRP1, and VEGFD to the culture medium, we observed an increase in the number of cells expressing tyrosine hydroxylase (a marker for DA neurons) and βIII-tubulin (a marker for immature neurons) in both the NTera2 and I6 cell lines. These results indicate that SDF1α, sFRP1, and VEGFD are major components of SDIA and suggest the potential use of these defined factors to elicit DA differentiation of pluripotent human stem cells for therapeutic intervention in PD.

Hypoxic-ischemic (HI) brain injury in newborn infants represents a major cause of cerebral palsy, development delay, and epilepsy. Stem cell-based therapy has the potential to rescue and replace the ischemic tissue caused by HI and to restore function. However, the mechanisms by which stem cell transplants induce functional recovery are yet to be elucidated. In the present study, we sought to investigate the efficacy of human neural stem cells derived from human embryonic stem cells in a rat model of neonatal HI and the mechanisms enhancing brain repair. The human neural stem cells were genetically engineered for in vivo molecular imaging and for postmortem histological tracking. Twenty-four hours after the induction of HI, animals were grafted with human neural stem cells into the forebrain. Motor behavioral tests were performed the fourth week after transplantation. We used immunocytochemistry and neuroanatomical tracing to analyze neural differentiation, axonal sprouting, and microglia response. Treatment-induced changes in gene expression were investigated by microarray and quantitative polymerase chain reaction. Bioluminescence imaging permitted real time longitudinal tracking of grafted human neural stem cells. HI transplanted animals significantly improved in their use of the contralateral impeded forelimb and in the Rotorod test. The grafts showed good survival, dispersion, and differentiation. We observed an increase of uniformly distributed microglia cells in the grafted side. Anterograde neuroanatomical tracing demonstrated significant contralesional sprouting. Microarray analysis revealed upregulation of genes involved in neurogenesis, gliogenesis, and neurotrophic support. These results suggest that human neural stem cell transplants enhance endogenous brain repair through multiple modalities in response to HI.