Stem Cell Policy Exceptionalism: Proceed with Caution

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.

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.

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

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.

Human embryonic stem (ES) cell lines have opened great potential and expectation for cell therapy and regenerative medicine. Monkey and human ES cell lines, which are very similar to each other, have been established from monkey blastocysts and surplus human blastocysts from fertility clinics.Nonhuman primate ES cell lines provide important research tools for basic and applicative research. Firstly, they provide wider aspects of investigation of the regulative mechanisms of stem cells and cell differentiation among primate species. Secondly, their usage does not need clearance or permission from the regulative rules in many countries that are associated with the ethical aspects of human ES cells, although human and nonhuman embryos and fetuses are very similar to each other. Lastly and most importantly, they are indispensable for animal models of cell therapy to test effectiveness, safety, and immunological reaction of the allogenic transplantation in a setting similar to the treatment of human diseases.So far, ES cell lines have been established from rhesus monkey (Macaca mulatta), common marmoset (Callithrix jacchus), and cynomolgus monkey (Macaca fascicularis), using blastocysts produced naturally or by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). These cell lines seem to have very similar characteristics. They express alkaline phosphatase activity and stage-specific embryonic antigen (SSEA)-4 and, in most cases, SSEA-3. Their pluripotency was confirmed by the formation of embryoid bodies and differentiation into various cell types in culture and also by the formation of teratomas that contained many types of differentiated tissues including derivatives of three germ layers after transplantation into the severe combined immunodeficiency (SCID) mice.The noneffectiveness of the leukemia inhibitory factor (LIF) signal makes culture of primate and human ES cell lines prone to undergo spontaneous differentiation and thus it is difficult to maintain these stem cell colonies. Also, these ES cells are more susceptible to various stresses, causing difficulty with subculturing using enzymatic treatment and cloning from single cells. However, with various improvements in culture methods, it is now possible to maintain stable colonies of monkey ES cells using a serum-free medium and subculturing with trypsin treatment. Under such conditions, cynomolgus monkey ES cell lines can be maintained in an undifferentiated state with a normal karyotype and pluripotency even after prolonged periods of culture over 1 year. Such progress should facilitate many aspects of stem cell research using both nonhuman primate and human ES cell lines.

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.

Scientists at Duke University Medical Center have demonstrated they can grow human stem cells in the laboratory by blocking an enzyme that naturally triggers stem cells to mature and differentiate into specialized cells. The discovery may enable scientists to rapidly grow stem cells and transplant them into patients with blood disorders, immune defects and select genetic diseases, said the Duke researchers.Stem cells are the most flexible cells in the body, continually dividing into new stem cells or into specialized cells that carry out specific roles in the body. But little is known about how stem cells choose their fate. The Duke team focused on "hematopoietic" or blood stem cells.In their study, the investigators discovered that an enzyme, aldehyde dehydrogenase (ALDH), stimulates hematopoietic stem cells to mature and transform into blood or immune cells, a process called differentiation. They inhibited this enzyme in stem cell cultures and successfully increased the number of stem cells by 3.4 fold. Moreover, they demonstrated the new stem cells were capable of fully rebuilding the blood-forming and immune systems of immune-deficient mice.Results of the study are published on line and will be published in the August 1, 2006, issue of the Proceedings of the National Academy of Sciences."Our ability to treat human diseases is limited by our knowledge of how human stem cells determine their fate -- that is, whether they maintain their ability to self-renew or whether they go on to become specialized cells," said John Chute, M.D., associate professor of medicine in the Duke Adult Bone Marrow and Stem Cell Transplant Program. "Unraveling the pathways that regulate self-renewal or differentiation in human stem cells can facilitate our ability to expand the growth of human stem cells for therapeutic uses."Currently, patients who require stem cell transplants are given either bone marrow from adult donors, umbilical cord blood derived from newborn babies, or stem cells from blood. But stem cells are scarce, representing less than 0.01 percent of the bone marrow cell population. Likewise, cord blood units frequently lack sufficient numbers of stem cells to rebuild a patient's decimated immune system.Efforts to grow human hematopoietic stem cells in the laboratory have proven extraordinarily difficult, Chute said, because growth factors in culture make stem cells rapidly differentiate. The scientists searched for ways to block a stem cell's natural propensity to differentiate without promoting uncontrolled growth. The researchers focused on the ALDH enzyme because it is a telltale "marker" that distinguishes stem cells from other blood and immune cells. Moreover, it is known to play an essential role in the body's production of retinoic acids, which regulate cell differentiation in a variety of tissues. Yet how ALDH functions in stem cells remained unknown, Chute said.The scientists began by analyzing how stem cells behave under normal circumstances when grown in culture. They mixed together purified human stem cells with growth factors that induce stem cells to mature and differentiate. As expected, the stem cells showed a marked decline in number as they differentiated into other types of specialized cells. By day seven, all stem cells had disappeared from culture.The scientists then added an inhibitor of ALDH to the stem cell cultures, and they found that half of the stem cells maintained their immature and undifferentiated status. Moreover, adding the inhibitor caused a 3.4-fold increase in stem cell numbers within seven days.Next, the scientists transplanted the cultured stem cells into immune-deficient mice to determine how the stem cells would behave. The new population of stem cells migrated to the bone marrow as expected and successfully "engrafted," or took hold in the bone marrow, where they began to produce new blood and immune cells. "ALDH appears to play a fundamental role in the differentiation program of human hematopoietic stem cells," Chute said. "Inhibition of this enzyme facilitates the expansion of human hematopoietic stem cells in culture." Chute said their results reveal a unique role for both ALDH and the process of retinoic acid signaling in human stem cells. Chute and colleague DonaldMcDonnell, Ph.D., professor of pharmacology and cancer biology, are currently testing whether they can directly block the retinoic acid receptors in stem cells and produce a comparable expansion of human stem cells.The investigators plan to develop a clinical trial to test their approach to expand human stem cells for therapeutic purposes.For more information contact: Becky Levine; Tel.: 919.660.1308 or 919.684.4148; Email: levin005@mc.duke.edu

Of the countless interviews I have conducted with scientists over the years, only once has a question prompted something of a striptease. In December of 1999, I found myself in the elegant parlor of the Union Club in New York City, chatting with a biologist named Leonard Hayflick. Although hardly a household name to the general public, Hayflick is that rare scientist whose name is permanently attached to a biological phenomenon. It is known as the “Hayflick limit,” and it derives from experiments he did in the late 19505 and early 19605 showing that human cells grown in Petri dishes will predictably replicate for a certain number of cell divisions, but then hit a wall and stop dividing. This has obvious implications for cell biology, aging, and immortality (of the in vitro sort), and indeed the Hayflick limit has been the seed around which a spirited biological debate about the biology of aging has swirled, without definitive resolution, for about four decades now. Because of this history, Hayflick has closely followed the recent work on the biology of aging and regenerative medicine, which in turn has made him a front-row spectator in the more recent controversies involving human embryonic stem cell research and “therapeutic cloning.” At the time of my conversation with Hayflick, his longtime friend Michael West was attempting to obtain human embryonic stem cells through cloning—in a particularly controversial way, by putting human cells into egg cells from ... cows. Almost as an aside, I asked Hayflick what he thought about West's experiments. Hayflick replied by rolling up his pants leg. He bared enough skin to be able to point out a tiny dimple on his right knee. “The human cells he's using for the cow work came from here,” he said. I had to stand up and lean over to see it, but there was undeniably a tiny divot in Hayflick's skin. The implications were stunning: Leonard Hayflick, the father of cellular senescence and one of the elder statesmen of gerontology, was allowing himself, in a manner of speaking, to be cloned. In addition to making the obvious point that even the most innocuous question can elicit a startling answer, Hayflick's reply offered another lesson, too: that colorful characters can provide a narrative thread for bringing a controversy to life.

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.

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

In 2003, Ruth Faden and eighteen other colleagues argued that a "problem of unequal biological access" is likely to arise in access to therapies resulting from human embryonic stem cell research. They showed that unless deliberate steps are taken in the United States to ensure that the human embryonic stem cell lines available to researchers mirrors the genetic diversity of the general population, white Americans will likely receive the benefits of these therapies to the relative exclusion of minority ethnic groups.Over the past five years the problem of unequal biological access has not received much attention from politicians, bioethicists and even many researchers in the United States, in spite of the widely held belief in the country that there is an obligation to prevent and correct ethnic disparities in access to medical care. The purpose of this paper is to increase awareness of the problem of unequal biological access and of the need to do more than is currently being done to ensure that ethnic disparities in access to human embryonic stem cell-based therapies do not arise.Specifically, this paper explains why the problem of unequal biological access will likely arise in the United States in such a way that white Americans will disproportionately receive most of the benefits of the therapies resulting from human embryonic stem cell research. It also argues for why there is an obligation to prevent these ethnic disparities in access from happening and outlines four steps that need to be taken towards meeting this obligation.

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.

Stem cells and cloning: The fuss, revisited.

Remarkably little is known about the transcriptional profiles of human embryonic stem (ES) cells or the molecular mechanisms that underlie their pluripotency. To identify commonalties among the transcriptional profiles of different human pluripotent cells and to search for clues into the genesis of human germ cell tumors, we compared the expression profiles of human ES cell lines, human germ cell tumor cell lines and tumor samples, somatic cell lines, and testicular tissue samples by using cDNA microarray analysis. Hierarchical cluster analysis of gene expression profiles showed that the five independent human ES cell lines clustered tightly together, reflecting highly similar expression profiles. The gene expression patterns of human ES cell lines showed many similarities with the human embryonal carcinoma cell samples and more distantly with the seminoma samples. We identified 895 genes that were expressed at significantly greater levels in human ES and embryonal carcinoma cell lines than in control samples. These genes are candidates for involvement in the maintenance of a pluripotent, undifferentiated phenotype.

The work on human embryonic stem cells is being performed with a variety of cell lines using a variety of culture conditions; a situation that makes standardisation between projects and publications very difficult. Clearly the consequence of using such cells would be wasted time and resources but, more seriously, the generation of erroneous data in the literature which could both confuse and delay scientific progress in this area. This guidance document represents the outcome of the first meeting of the group named The International Stem Cell Banking Initiative held in October 2007. The document has been prepared from the perspective of hESC culture but, in many respects, is broadly applicable to all human stem cell lines including induced pluripotent stem cell lines.