Stem Cell Biology, clinical application, ethical issues and more

Human Molecular Genetics, 2008, Vol. 17, Review Issue 1 doi:10.1093/hmg/ddn074

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The road to pluripotence: the research response to the embryonic stem cell debate Christopher Thomas Scott1, ,{ and Renee A. Reijo Pera2{ 1

Center for Biomedical Ethics, Program on Stem Cells in Society and 2Institute for Stem Cell Biology and Regenerative Medicine, Department of Obstetrics and Gynecology, Stanford University School of Medicine, CA, USA Received January 23, 2008; Revised February 14, 2008; Accepted March 4, 2008

INTRODUCTION Perhaps no biological discovery has prompted more attention and controversy than the isolation of human embryonic stem cells (hESCs). This year marks the 10th anniversary of the Science report by Thomson et al. (1) announcing the first derivation of hESCs; the arc of this discovery has revolutionized a formerly quiet corner of biology and stirred debates in law, ethics and public policy. Meanwhile arguments, about whether cells can be found that can effectively substitute for hESCs derived from 5-day-old embryos, have been strengthened by recent reports of powerful lines made by directly reprogramming somatic cells (2 –4).

The debate in the United States pivots on federal funding restrictions on hESC research. Indeed, on 9 August 2001, President George W. Bush announced that no new hESC lines would be made using government dollars; however the National Institutes of Health (NIH) could support research on lines derived prior to 9 August 2001 (5). In a foreshadowing of President Bush’s mandate, which permits private sector funding of hESC research, lines derived by Thomson et al. (1)

were made possible through use of corporate funds. Following the announcement of August 2001, data quickly accumulated, showing the 20 or so NIH-approved lines derived with older methods were of spotty quality and limited genetic diversity (6 – 16). The stage was set for researchers to clamor for more lines, while endeavoring in parallel to discover substitutes that might circumvent the ethical and political barriers.

THE POLICY WARS: EMBRYOS AND EMBRYONIC STEM CELLS Placing new hESC lines into the hands of researchers has been difficult. National Medal of Science winner Robert Weinberg, writing in the Atlantic in 2002, argued that in the end, politics will settle the question about whether to fund this promising area of science: ‘If the decision is yes, then we will continue to lead the world in a crucial, cutting edge area of biomedical research. If it is no, US biologists will undertake hegiras to laboratories in Australia, Japan, Israel and certain countries

To whom correspondence should be addressed at: Program on Stem Cells in Society, 701 Welch Road, Suite A1105, Palo Alto, CA 94304, USA. Tel: þ1 6507256103; Fax: þ1 6507256111; Email: cscott@stanford.edu Both authors contributed equally to this work.

†

# The Author 2008. Published by Oxford University Press. All rights reserved.

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The controversies surrounding embryonic stem cell research have prompted scientists to invent beyond restrictive national policy and moral concerns. The impetus behind these reports comes from different sources, including individually held moral beliefs, societal pressures and resource constraints, both biological and financial. Along with other contributions to public policy such as advocacy or public testimony, experimentation and scientific curiosity are perhaps more natural responses scientists use to surmount impediments to research. In a research context, we review the history of the first stem cell discoveries, and describe scientific efforts leading up to recent reports of pluripotent lines made without the use of human embryos and eggs. We argue that despite the promise of these new lines, we must not lose sight of fundamental questions remaining at the frontiers of embryology and early human development. The answers to these questions will impact studies of genetics, cell biology and diseases such as cancer, autoimmunity and disorders of development. Human embryonic stem cell research is barely a decade old. The recent pace of discovery—in spite of federal restrictions—is testament to the potential of these cells to uncover some of biology’s most intractable mysteries.

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funding totals are fluid. Adding the approved and pending state initiatives for stem cell research puts future totals in excess of $500 million yearly.) Considering this fact, one could legitimately argue that restrictive federal policy has, paradoxically, been good for stem cell research. Such a view, however, overlooks the fact that the vacuum in Washington has left a prestigious funding agency, the NIH, bereft of hESC expertise, fractured collaborative opportunities and created a muddy regulatory environment. And, emerging evidence suggests the delay in putting money into labs may have hampered American competitiveness (20).

The political response from the research community and patient advocates was ferociously intense; not only due to the restrictions themselves but also due to proposed legislation that would criminalize hESC research. The fight, which spanned three legislative sessions, ended in a stalemate last year. The criminal penalties never materialized, and though the United States Congress twice passed measures overturning the policy of President Bush, the measures fell short of enough votes to override his vetoes.

The pace of discovery was also ferocious, even with limited funding (10,12,21). Moreover, recent reports of reprogrammed lines underscore how some researchers have changed strategies in order to surmount ethical concerns. How will these new cell lines compare to other stem cell lines in the laboratory and on the road to the clinic? A review of the research leading to these discoveries—along with their ethical and political implications—will help put this question into perspective.

STEM CELLS All stem cells have two unique qualities, which are dependent on mechanisms of signal transduction. First, they are capable of self-renewal, ensuring a lifetime supply of ancestors for replenishment or repair. Second, they may differentiate into intermediate and downstream types of cells specific to the organ or tissue, including progenitor cells (which can multiply but not renew) and terminally differentiated cells. For example, the blood system has self-renewing long- and shortterm hematopoietic stem cells, which give rise to a number of multipotent progenitors and nine fully differentiated cells of the myeloid and lymphoid lineages (22,23).

Stem cells are comprised of two general types, referred to as tissue- or organ-specific (also called adult or fetal stem cells) and embryonic stem cells (23). Embryonic stem cells are generally derived by removing and then culturing a few dozen cells from the interior of a blastocyst. In mammals, this collection of cells, called the inner cell mass, eventually develops into the epiblast and hypoblast, and subsequent gastrulation forms the germ layers of the animal proper (24). The derivation process destroys the embryo. Approximately 10% of the time, a self-renewing, pluripotent cell line will result, with potential to differentiate into all of the cell and tissue types of the adult animal, including downstream stem and progenitor cells (11,14,25 – 31). Pluripotency of hESCs is confirmed by a combination of criteria, including expression of RNA and protein markers, and differentiation in vitro and in vivo in teratoma assays. Other trademarks include a distinct

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in Europe—an outcome that will leave American science greatly diminished’ (17).

Weinberg’s prediction came on the heels of a tangled and long-simmering controversy about embryo research, human cloning and abortion. It began the year after the 1973 Supreme Court ruling in Roe v. Wade legalized abortion in the first two trimesters of pregnancy. Religious leaders and anti-abortion activists claimed the decision would result in the black-market sale or barter of fertilized eggs and indiscriminate use of embryos for laboratory experiments. Congress halted embryo research until guidelines could be established, and since 1997 has attached a rider on NIH funding—called the Dickey Amendment—expressly prohibiting funding of embryo research. (In 1975, DHEW regulations superseded the moratoria. Incorporated into the Code of Federal Regulations, they prohibited federal funding of IVF experimentation unless approved by an Ethics Advisory Board. Such a board did find some experiments in this arena ethically acceptable in a 1979 report, but the secretary of DHEW did not approve funding, the Board expired, and no human embryo experiments were performed.)

The 1974 action and its sequelae had surprising staying power. With one short-lived exception, the policy has passed its 33rd anniversary. Now, no government funds are allowed for embryo research, an action that swept essential questions about infertility, reproductive medicine and prenatal diagnosis beyond the reach of many American clinicians and scientists.

President Bush’s policy went one step further, mandating that no federal money could be used for hESC research.

The study of human development, the progression of disease and the molecular and cellular events of the first few days of life have a special significance in the context of funding restrictions. While the debate about the medical promise of hESCs takes center stage, it is basic research that stands to benefit now from a more permissive funding environment. The drive to understand the mechanisms of normal development and disease is behind two major efforts. First, supporters sought to fund research in spite of federal restrictions. The second is a consequence of scientists responding to the moral controversies and scientific need. Each effort met with surprising results.

On 2 November 2004, the citizens of California passed Proposition 71, a law that effectively established the California Stem Cell Research and Cures Initiative to create the California Institute for Regenerative Medicine, a granting agency charged with distributing $3 billion for stem cell research over the next decade to California laboratories (18). The sheer size of the funding initiative not only sent a message about the promise of stem cells but also underscored how expensive biomedical research can be and how distant therapies may be from human use. Now, California stands to compete alongside nations who support embryo research and the full spectrum of stem cell biology. The measure is designed to outlast several presidential administrations and insulate the state from the vagaries of Washington politics.

Since the passage of the California bond measure, several states have put forward funding initiatives, with different degrees of success (19). The end result is that by the end of the decade, state funding for hESC research could outpace NIH allocations by a margin of tenfold or more. (State

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in vitro morphology, cell surface markers and distinctive gene signatures (32).

In contrast, tissue-specific stem cells appear sequentially during fetal development, and are more limited in their developmental potential (33). Pluripotent stem cells develop into more differentiated types through gene signaling and epigenetic changes that progressively act to restrict cell fate (22,34). As a result, stem cells isolated from a fully developed animal are already programmed to become brain, blood, bone or other cells of the resident tissue or organ. Generally, adult stem cells are few in number, hard to isolate, and with the exception of some neural types, difficult to culture and expand (23,35– 37).

NEW DISCOVERIES; NEW CONTROVERSIES

embryos (44). Surprisingly, in 2007 others confirmed that the remaining fraudulent SCNT line was in fact parthenogenic; and that year another group reported successful derivation of lines from parthenotes (44,45).

The South Korean fiasco was a watershed moment for stem cell research. It laid bare the difficulties of moving from an animal to a human system and catalyzed fears that scientists were running unchecked into a dystopian future. More than anything, these events fueled efforts to find non-controversial substitutes for embryos and eggs: a case of how ethics and politics can drive a search for solutions at the bench, rather than in the halls of Congress.

One early concept was quickly reduced to practice. A nonscientist member of the President’s Commission on Bioethics suggested disabling a gene necessary for the formation of the placenta and amniotic tissues, Cdx2. Such an embryo would not survive implantation, and could not be used for reproductive purposes. As proof of the concept, MIT researchers disrupted mouse Cdx2, used nuclear transfer to make a cell line and then restored the gene’s function with a Cre-Lox recombination system once the cells were in culture (46). The results were distinguished not so much by their scientific import as by the heated policy discussion that followed. Some hESC researchers criticized the technique (called Altered Nuclear Transfer or ANT), as a ‘flawed proposal’ or ‘playing politics for the sake of science’ (47,48). In contrast, others defended the work as an inventive way around moral objections (49).

Philosophers argued the results did not solve the moral problem for ‘do-no-harm’ hard liners, who objected to engineering a ‘disabled’ human being, only to kill it to obtain the cells inside.

ANT was not the only study designed with ethics and social issues in mind. Scientists at Advanced Cell Technology published two reports demonstrating that a blastomere removed from a preimplantation human embryo—the morula—could be used to derive lines with embryonic cell-like qualities (29,30). Press reports from the company hailed the discovery as a moral breakthrough but critics differed; they reiterated that even if the study used a cell selection technique that was considered safe in the context of IVF (pre-implantation genetic diagnosis or PGD), objections from those who would protect the embryo at any cost would remain (50,51).

Even questions about the fate of cells at the earliest stages of embryonic life have ethical resonance. Recent research in mice and Drosophila suggested that cells of the first few divisions are not created equal: cell polarity and epigenetic effects may be responsible for differing cell fates (52). In contrast, others maintain that any cell in an eight-cell embryo can develop fully, based on mammalian experiments of the 1980s (53). If a blastomere has the potential to develop into a newborn, the reasoning goes, then destroying it is as ethically troublesome as destroying an embryo. This point was made not by a philosopher or theologian, but by an embryologist (54).

The year 2007 may be recalled as one of the most productive periods for stem cell research. Researchers in Oregon showed that nuclear transfer could yield cloned rhesus ESC lines, taking one step closer to the cloned human lines falsely claimed by the South Koreans (55). Nonetheless, national ethics guidelines assert that women should

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The report of derivation of hESCs came on the heels of another announcement, a cloned sheep named Dolly (38).

Dolly’s appearance heralded the intersection of two technologies with profound biomedical consequences. The same method used to produce an animal clone—somatic cell nuclear transfer (SCNT)—could theoretically be used to make a cloned line of human cells with a near genetic match to any person who needed them. The nucleus from a somatic donor cell would be inserted into an egg stripped of its nucleus. Then, just as in animal cloning, chemical activation of the egg would prompt it to divide, and a human embryo might be cultured to the blastocyst stage for subsequent derivation of an hESC line (39,40). To date, blastocysts have been obtained from cloned human embryos; however, no hESC lines have been derived (41).

Reprogrammed lines derived by SCNT were hypothesized to be powerful tools to study the genetic basis of human development and disease and to serve as platforms for drug discovery. In the most optimistic scenario, a lifetime supply of genetically matched cells for personally tailored cell replacement therapy could be used with minimal worry of immune rejection. As in other dimensions of stem cell research, the promise has proved difficult to realize due to both moral and technical obstacles. Conservative opponents of hESC research described the method—referred to as therapeutic cloning—as equivalent to cloning and then killing a human being, though the embryo to be used was no more than 5 days old. Though all sides agreed that human reproductive cloning was morally repugnant, nuclear transfer became fully embroiled in the embryo debate.

The technical barriers became clear during a widely publicized South Korean stem cell scandal. A research team announced in 2004 and 2005 in two separate reports in Science that they had succeeded with somatic cell nuclear transfer and established the first patient-specific hESC lines; moreover, they claimed to have accomplished the cloning with astounding efficiencies, dampening worries that hundreds or thousands of human eggs would be needed (42,43). Subsequently, it was revealed that thousands of eggs were indeed used, and some were unethically obtained from women working in the laboratories. Moreover, the lines were not made by nuclear transfer; they were later shown to be derived from parthenotes or possibly directly from IVF

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IS HESC RESEARCH STILL IMPORTANT? While we may be years away from widespread stem cell therapies, these discoveries and others have opened up new horizons for basic and translational research. Over the last century, studies of the biological basis of human development and disease have been limited to primary or immortalized differentiated cell types in culture and/or to correlative studies of human epidemiology and genetics that pursue evidence through statistical and/or linkage analysis. With the advent of advances in assisted reproduction, the culture of human embryos and the derivation of hESC lines, we have entered an era of direct experimentation on differentiating hESC to extra-embryonic, germ line and somatic lineages. Yet we still know remarkably little regarding the specifics of human development and gene action during the earliest days and stages in vivo.

Our lack of knowledge might be overlooked in the face of moral and ethical concerns. Clearly, however, there are severe consequences to human health and development, most specifically to human reproductive and fetal health, as well as to the future use of pluripotent stem cells from embryos or alternative sources for novel cell-based therapeutics. Below, we discuss several areas where key knowledge

gleaned from human embryo and hESC research would be most beneficial in the short term. These areas are in addition to the pure scientific knowledge that we could obtain regarding the development of the first human cells and cell lineages.

First, it is well known that even though IVF is routinely used to assist infertile men and women in achieving parenthood, many aspects of the treatment have not been optimized (61,62). This is in spite of the fact that infertility is a particularly common health problem affecting 10– 15% of reproductive-age couples. The latest data collected in the United States reveals that in 2004 more than 127 000 IVF cycles were performed and nearly a million embryos were produced (63). Nonetheless, in that year and in all other years recorded, the vast majority of embryos failed to develop properly in vitro and/or in vivo (63,64). This failure must be attributed, at least in part, to our lack of knowledge of requirements for optimal human embryo culture. In the absence of optimized culture conditions, we also lack methods to distinguish embryos most suitable for transfer to the uterus and viable pregnancy (65 – 69). Traditionally, grading of embryos is based on simple morphological observations such as the presence of uniformly sized, mononucleate blastomeres and assessment of cellular fragmentation (67,70). Yet, evidence suggests that these criteria are not enough. For example, assessing morphological and growth on day 3 does not accurately predict blastocyst formation much less development following transfer (67,70,71). In one study, embryologists were asked to choose two embryos with optimal potential using traditional criteria of assessment in vitro. Results showed that in 39% of cycles, neither did an embryo picked on day 3 result in blastocyst growth; in 38% of cycles one pick resulted in growth to blastocyst; and in just 23% of cycles were both picks successful in reaching the blastocyst stage (70). These results mirror other studies where an equally low probability of predicting embryo outcomes on day 3 was observed at day 5 (67).

Thus, in order to circumvent lack of knowledge and simultaneously enhance the chance of pregnancy, the transfer of multiple embryos, especially when deemed to be of suboptimal quality, is routine (68,69). The end result is an increased risk of adverse outcomes that include embryo loss, miscarriage, low and very low birth weight and multiple pregnancies (68,72).

In addition to adverse outcomes of embryo loss, miscarriage, low birth weight and multiple pregnancies, there is a need for knowledge of human imprinting and early development. Recently, several reports demonstrate an association between assisted reproduction and increased risk for epigenetic errors of imprinting leading to Beckwith– Wiedemann syndrome, Angelman syndrome and retinoblastoma (73 – 75).

No conclusions can be made regarding the true incidence of errors in imprinting associated with assisted reproduction, due either to culture conditions or genetically inherited defects leading to poor gamete formation, yet the evidence that such errors are routine is daunting and accumulating.

Limitations on embryo research have undoubtedly hampered efforts to examine fundamental properties of human embryo development including gene activation, imprinting, genomewide demethylation and methylation, histone modification, chromosome segregation and X chromosome inactivation/ reactivation (62,65,76 – 79).

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not be compensated for eggs obtained for hESC research, notwithstanding the practice of paying for eggs offered by fertility clinics. So, obtaining enough eggs for cloning work is a dilemma for nuclear transfer research. This served as the primary impetus for experiments that transferred chromosomes as an alternate method of nuclear transfer or explored the use of oocytes that would otherwise be discarded from the IVF clinic (56,57). Other laboratories have explored the potential of unique fetal stem cell sources as possible therapeutic substitutes, such as amniotic stem cells (58).

Finally in 2007, reports have described the direct reprogramming of human somatic cells and generated tremendous interest and publicity in the scientific community and beyond. Using viral vectors to deliver transcription factors found in mammalian embryos or oocytes (Oct-3/4, Sox2, c-Myc, KLF4, Lin28, Nanog, and telomerase), several research groups have reprogrammed fetal and adult cultured and primary cells (2 – 4). The new lines have normal karyotypes and express intracellular and cell surface markers and genes indicative of ESCs; they also possess the ability to re-differentiate into adult cell types. Thus, if these lines are found to be functionally equivalent to embryonic cells, then they could—in principle—replace nuclear transfer as a means of generating pluripotent lines that genetically match the patient. The ethical and political consequences of these findings are profound, because no eggs or embryos would be required to make stem cell lines. The senior author of one paper, Shinya Yamanaka, has said he attempted the research in part as a response to moral worries, though he concedes more study on embryonic stem cells is still needed (59). Similarly, James Thomson, who conducted another reprogramming study, broke a long silence about his personal feelings and discomfort about embryo research, and hailed his results as the beginning of the end of the controversy (60).

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Other daunting challenges remain. Although direct reprogramming may be the panacea for those opposed to embryo research, time will tell whether we can harness the power of this new technology. Properties of induced pluripotent cells are routinely compared with those of hESCs, and native human embryo development is the gold standard. Clearly, there are intriguing similarities but substantial differences cannot be ignored (2– 4). It would be premature to abandon hESC and embryo research given current uncertainties and technology.

SCIENCE POLICY BY PRACTICING SCIENCE

scientists engage in policy-making by explaining clearly and evenhandedly to lawmakers and citizens the reasons stem cell research is important from a scientific and medical standpoint. But moving from making policy to political advocacy carries risks. Scientists who participate in political debates are in conflict between advocating for their research on one hand and professional detachment on the other. Yet, they are also members of a pluralistic society, with a right to weigh in, if they choose, on deeply held beliefs. In sum, the stem cell debate has engaged scientists in the political process in unimagined ways, with a vigor and intensity not seen since the policy debates surrounding recombinant DNA technologies of the early 1970s.

CONCLUSIONS The examples presented above show how the controversies surrounding stem cell research have prompted scientists to ‘invent beyond’ restrictive federal policy and moral concerns.

The impetus behind these discoveries comes from different sources, including individually held moral beliefs, societal pressures and resource constraints, both biological and financial. Along with other contributions to public policy such as advocacy or public testimony, experimentation and scientific curiosity are perhaps more natural responses to surmounting impediments to research.

Despite the promise of derived pluripotent lines without the use of embryos or eggs we must not lose sight of fundamental questions remaining at the frontiers of embryology and early human development. The answers to these questions will impact studies of genetics, cell biology and diseases such as cancer, autoimmunity and disorders of development. hESC research is barely a decade old. The pace of discovery and innovation is testament to the potential of these cells to uncover some of biology’s most intractable mysteries.

Thanks to state funding programs and the tenacious efforts of individual scientists, a widespread Diaspora of the mosttalented scientific experts to other shores has not come to pass. Like anything else in the sciences, the pace of gaining new knowledge is dependent on substantial resources applied across a broad spectrum of research that is agnostic to cell type. Only then can we realize the full potential of this promising and exciting field.

Conflict of Interest statement. None declared.

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Principally, scientists impact public policy through the power of their professional action. The German philosopher Hans Jonas maintained there is a cognitive side to moral action, not just the good will exemplified by charity, honesty, justice and virtue (80). Knowledge in this context becomes a prime duty. This is predicated on the argument there are obligations to conduct research. Freedom of inquiry thus becomes a compelling reason for anyone who believes there are moral duties to help others, and to do one’s part. These beliefs spring from Judeo-Christian ethics and the notion of stewardship— humans are creatures put on earth with special abilities and responsibilities including the duty to heal. If one can save a life, one must save a life. If one can heal, one must heal (81). There are many ways to exercise the ethical principle of doing good—known as beneficence—including the impulse to experiment. While striving for beneficence may be a weaker duty than preventing harm, it has a positive, compelling force. Many biomedical scientists do their work because of the social benefits that may result (82). In turn, society and governments have an obligation to create and maintain an environment that encourages these activities.

In light of funding restrictions for hESC research, scientists are faced with two choices. One is radical: moving to other countries or states with permissive research environments, or, more drastically, abandoning their preferred lines of research. The other comes more naturally, and springs from the impulse to experiment: treat social barriers as experimental ones. Research that attempts to hurdle a social or moral barrier—though it may not be the most direct or efficient route to hoped-for answers—contributes to the overall good by increasing knowledge.

Should scientists ‘vote their conscience’ by proposing experiments that align with their personal values or the values of others? As long as all avenues of inquiry can be freely explored and offered to society, we believe that they can, and should. If the science is important, adds to the field, and passes the rigors of peer-review, there is little reason to think that research done in response to ethical or societal concerns is in some way inferior. Most importantly, it is far too early to call the question about which discovery (or which cell type) will be the most useful for research and medicine. These discoveries cannot stand alone as solutions, and must join other advances in the search for treatments and cures. This is the central reason why we should aggressively pursue all avenues of stem cell research.

Besides the act of researching and publishing, scientists can participate in public policy in other ways. For example,

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