How Many Human Embryonic Stem Cell Lines ... - Stem Cells Journals

EDITORIAL How Many Human Embryonic Stem Cell Lines Are Sufficient? A U.S. Perspective INTRODUCTION Pluripotent human embryonic stem cells (hESCs), isolated from the inner cell mass of preimplantation embryonic blastocysts, are able to self-renew and generate every human cell type [1]. Because of the remarkable ability of hESCs to proliferate and differentiate, along with the tremendous impact of research on ESCs from the mouse and other species, hESCs have a tremendously high potential utility for developmental studies in the laboratory and for cell replacement therapies in patients. Current reports comparing the characteristics of the hESC lines approved for use in U.S. federally funded research (the 22 currently available so-called “pre–August 9” 2001 hESC lines) suggest that hESC lines are highly similar to each other in their expression of cell surface antigens and markers characteristic of the ESC state (http://stemcells.nih.gov; [2– 4]). Since the derivation of the first hESC lines by Thomson and colleagues [1], pluripotent ESC lines have been derived by different methods and from embryos at different stages of development [5– 8]. In addition, some success in deriving hESC-like cell lines without embryo destruction has been reported [9 –12]. Recent reports of dramatic success in deriving hESC lines by somatic cell nuclear transfer (SCNT) have been withdrawn, and the actual successes appear to have been restricted to production of SCNT blastocysts but not hESC lines [11]. Thus, the prospects of using SCNT to develop syngeneic (autologous) hESC lines for any given individual remain speculative, although “clinically beneficial” replacement therapy for a genetic defect using SCNT ESCs has been accomplished in mouse models [13]. Success in the hESC field has been paralleled by success using adult stem cell populations. Two advances bear special mention. Multiple groups have reported success in transdifferentiation, in which a stem cell with potential (thought to be) limited to a particular tissue or organ can be “re-programmed” to express properties and markers typical of cells of a different lineage [14]. These results have raised the possibility that less controversial “adult stem cells” might provide an alternative to hESCs for cell replacement therapies. Other investigators have reported success in harvesting ESC-like cells from a variety of tissues [15]. These cells appear to share many of the properties of ESCs, to the extent that they appear to be capable of unlimited self-renewal without senescence (reviewed [16]). In some cases, these cells could be harvested from adults feasibly, and individualized autologous human stem cell products could potentially be made available without either the use of SCNT or the destruction of embryos. The issues of studying existing hESC lines made after August 9, 2001, deriving additional hESC lines, SCNT, and the possibility of generating clones or chimeras of human individuals have raised additional “hot” moral and ethical issues. There is no dominant consensus on these issues internationally or

within the U.S., and groups on both sides of these issues have been quite polarized in their views. Some individuals have suggested that destroying even one human embryo to derive an hESC line is wrong. Others have argued that generation of hESC lines is unnecessary, given the demonstrated potential utility of adult stem cells and the possibility of generating hESC-like cells by novel means without use of human embryos ([17–19] and see above). These individuals clearly advocate that no new hESC lines should be created. At the other extreme, proponents of hESC research and regenerative medicine have proposed that egalitarian personalized medicine will require creation of a personalized line for every individual, or at least a huge bank of histocompatibility antigen-typed cells. In this editorial, we try to summarize the major arguments made for the range of estimated numbers of new hESC lines needed. We are hardly unbiased, as we both strongly favor hESC research, but we welcome comments and corrections from readers if they feel we have not fairly discussed the arguments.

IS ZERO THE OPTIMUM NUMBER LINES NEEDED?

OF

NEW HESC

Some opponents of hESC research believe, generally as a matter of religious faith, that no hESC line derivation can be morally acceptable if it involves the destruction of a human embryo. Such beliefs are based on the sanctity of human life and the time during embryogenesis when individuality or the soul is felt to originate. As a prominent example, current Catholic dogma holds that life begins at conception and that destruction of a human blastocyst is as objectionable as abortion and similar to murder. Others have raised analogies to current ethical bans on conducting experimental clinical trials in or harvesting organs from criminals condemned to death, without their informed consent. Proponents of hESC research have countered that the definition of the start of human life, individuation, and the soul are somewhat arbitrary and certainly not constant across religions (or even among Catholics) and that this plurality of belief and opinion must be respected by society. Furthermore, proponents have argued that preservation of human life is not always society’s dominant goal [20, 21]. They note that societies have sentenced traitors and murderers to death, sent soldiers to battle to defend property or ideologies, allowed euthanasia and “do not resuscitate” orders, permitted abortions, and tolerated embryo disposal as a byproduct of in vitro fertilization (IVF). Some hESC research proponents posit that, even if one accorded a blastocyst the full rights of an individual, societies might still apply cost-benefit arguments to the issue of embryo destruction in the derivation of hESC lines.

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Stem cell research proponents have also pointed out that, as a compromise, hESC derivation might be restricted to use only embryos already targeted for disposal. Opponents of hESC research, in turn, respond that we do not permit harvesting of organs from condemned criminals (without their consent). Stem cell research proponents have rebutted that the parent-owners of the embryos, not the society, should have the authority to make this decision for their own embryos. It appears from at least some survey results that the majority of the U.S. population does not support an absolute ban on creation of additional hESC lines. However, in certain countries (e.g., Ireland, France, Norway, and Germany), laws have been enacted forbidding generation of new hESC lines. In contrast, there is governmental approval of generation of new hESC lines in some other countries (e.g., United Kindgom, Israel, China, Singapore, and Australia).

IS ONE

OR

TWO HESC LINES ENOUGH?

Some have argued that, whereas zero is not a reasonable number, perhaps one or two hESC lines may be enough. Their rationale is that hESC lines are quite similar and, specifically, that they all appear able to self-renew indefinitely. Therefore, adequate numbers of cells could theoretically be obtained from a single, well-characterized hESC line for all possible experimental and therapeutic purposes. hESC subclones might be developed that had characteristics that were important for development of a specific lineage or use in a particular disease. Homologous recombination technologies could generate knockout or knockin lines involving all the major histocompatibility genes, to address clinical donor tissue requirements. Furthermore, given that germ cells can be generated from ESCs, perhaps in the future, new hESC lines could be generated by “IVF.” Finally, given that it has been shown that hESC lines contain a re-programming capacity similar to that of oocytes, it may become possible to produce transdifferentiated or re-programmed adult cell lines using just one or two hESC lines. Notably, the entire scientific community currently works with only approximately one or two well characterized mouse ESC lines, and new lines are derived as they become necessary. Although all of these arguments are reasonable, they involve several issues that must be clarified or validated. Perhaps the most important issue is allelic variability among humans. Humans show tremendous individual variability (e.g., in susceptibility to disease, resistance to infection, and response to drugs). Using inbred mouse strains, it is clear that phenotypes are often strain-specific. Thus, no single hESC line, or even a small number of hESC lines, can be truly representative of this genetic diversity among humans. The second important issue is that many of the futuristic techniques mentioned above have not been shown to work routinely and reliably. For example, although homologous recombination in human hESCs has been successfully accomplished, it is currently a tour de force that is far less efficient to accomplish than in mouse ESCs; and targeting of multiple alleles has not been accomplished. Likewise, IVF from gametes derived from hESC differentiation has not yet been performed successfully [22]. A third issue is that hESCs lines are not absolutely stable and hence cannot be truly propagated indefinitely without risk of changes in properties. All cell lines in culture undergo muwww.StemCells.com

tational changes at some stochastic rate. Genetic drift has been shown to occur in hESC lines by meiotic recombination, accumulation of DNA damage, oxidative damage, erosion of telomeres, acquisition of mitochondrial mutations, and loss of imprinting [23–26]. Thus, although one or two hESC lines might be a theoretically reasonable number, we feel this is currently too low because of feasibility issues. Instead, we believe that (at the least) laboratory research will require scientists to periodically replace hESC lines with new stocks, as has been done in mouse ESC research.

ARE THE CURRENT 22 U.S. FEDERALLY APPROVED “PRE–AUGUST 9” HESC LINES SUFFICIENT? Currently, only 22 lines can be used by U.S. federally funded investigators, as opposed to the more than 70 initially expected (http://stemcells.nih.gov). Still, although it is clear that neither 22 nor 70 hESC lines would suffice for all proposed uses of hESCs, 22 good hESC lines might more than suffice for most current laboratory research. However, recent data have suggested that some hESC lines have acquired genetic changes and that not all hESC lines differentiate into all lineages equally well. In addition, current U.S. federal restrictions do not permit replacement of “approved” hESC lines as they are lost to genetic drift or contamination or to fulfill the tissue culture precept of avoiding high-passage cell lines. Thus, the existing 22 hESC lines are, at best, adequate only for the short run. Nevertheless, the 22 pre–August 9 hESC lines currently provide scientists a reasonable base for examining allelic variability and provide sufficient diversity for comparative gene expression, drug discovery assays, proteomic and genomic analyses, and many developmental biology investigations. Indeed, there has been considerable progress using the pre–August 9 hESC lines, as evidenced by the number of papers published, patents filed, and novel results reported. However, to sustain this progress, it will soon be necessary to replenish these 22 hESC lines with new low-passage hESC lines; this could be done from hESC lines available from non–U.S. federally funded sources. (It is estimated that there are approximately 200 such post– August 9 hESC lines available currently.)

WILL 100 –1,000 HESC LINES BE NECESSARY? There are also multiple experiments and potential clinical uses that will require additional hESC lines [27], even if the existing 22 have the right characteristics (e.g., lineage differentiation capacity) and are not lost or are permitted to be replaced. A potential use of hESCs that has widespread support is cell replacement therapy (“regenerative medicine”). Optimal transplantation of human embryonic stem– derived cells will, for the foreseeable future, require availability of histocompatible hESC lines for every patient. In a recent report [28], the number of hESC lines needed to achieve varying degrees of HLA match was estimated using a series of 10,000 consecutive cadaveric organ donors in the U.K. The authors determined that 150 consecutive donors provided a full match at HLA-A, HLA-B, and HLA-DR for organ transplant of a minority of recipients (and a single HLA-DR match or better for 85%). If one were to prospectively identify hESC lines that could serve a larger fraction of patients, such as lines that were homozygous for common HLA types, then as few as 10 such lines might be

Embryonic Stem Cell Number

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sufficient to provide a organ donations for 68% of the U.K. population. However, an analysis of the HLA phenotype of the existing hESC lines has so far revealed no line homozygous for HLA phenotype (Richardson et al., American Type Culture Collection, Manassas, VA, http://www.atcc.org, personal communication). Furthermore, providing compatible cell lines for the remaining potential recipients would require many more hESC lines. Even this number is a low estimate that matches only the major HLA types; if a more complete match were needed (as is preferred in blood and marrow transplantation), many thousands of hESC lines would need to be available. It is also important to note that the U.S. population is ethnically much more diverse than the U.K. population, and therefore the estimates used may be low. We also note that the current hESC lines were obtained from only a few localities and thus are unlikely to reflect the ethnic diversity of the U.S. population pool. It is unclear whether any hESC lines were made from Hispanic, African-American, or Asian donors, for example. A separate estimate of numbers of hESC lines required is based on the recognition that lines derived from patients/donors who carry a disease-associated mutation may have tremendous utility in discovering the pathophysiology of that disease. Researchers have shown that prenatal genetic diagnosis (PGD) allows identification of IVF-generated embryos that carry disease-associated mutations [8]. These embryos, which have always been discarded, could potentially be used to generate hESC lines carrying a specific genetic defect (however, note that this activity is restricted by current U.S. federal policy). Indeed, hundreds of such lines have been made already [8]. One can estimate that many more unique “disease” lines will be generated. In our view, the derivation of these “disease” hESC lines is even less morally debatable than derivations from frozen embryos already targeted for destruction, because these “disease” embryos could never produce a healthy human. Scientists could use many such “disease” hESC lines, perhaps several for each human disease-associated gene (including potential disease-modifying genes), adding up to a large total (though limited to cases that would be typed by PGD for clinical reasons).

SHOULD NO UPPER LIMIT ON THE NUMBER OF HESC LINE DERIVATIONS BE CONSIDERED? Proponents of hESC research have suggested that a major strength of hESC research is that one may be able to derive lines, using SCNT, that are clonally identical to the donor of the somatic cell nucleus. These lines could be derived on demand, and theoretically one could develop as many lines as necessary. These personalized hESC lines would be syngeneic to the clinical regenerative medicine recipient and would not require immune suppression for use in clinical transplantation. However, there is considerable concern as to the numbers of hESC lines implicit in this idea. An open-ended number of hESC lines could theoretically reach into the millions if hESC therapies live up to their potential. This is perhaps the most controversial of all issues related to hESC research, with scientists and ethicists on both sides. Adding to the controversy is the recent revelation that the high efficiencies recently reported for SCNT (⬃10%) were based on fraudulent data [11]. hESC research proponents, however, have argued that SCNT technology and generating cell lines for personalized medicine does not automatically imply destruction of millions

of blastocysts. As technology improves, the use of blastocysts might be avoided totally. On the other hand, SCNT research will provide important fundamental information on the processes of transdifferentiation, dedifferentiation, and imprinting, in addition to providing useful cell lines for clinical transplantation therapies. We note that the basic information on the process of re-programming gleaned from research using hESCs can also be applied to adult stem cell re-programming and may enhance the therapeutic utility of adult stem cells. Opponents remain convinced that SCNT and personalized medicine will inevitably lead to destruction of large numbers of embryos, and therefore they favor banning SCNT altogether. Opponents also point to the risk of a “black market” in human eggs. They opine that abuses have occurred already in Korea, where some young female researchers appear to have been paid to donate eggs for research and may have been coerced. Many physicians, scientists, and ethicists agree that paying research egg donors should not be permitted, although some argue that modest payments to women who undergo this uncomfortable procedure is in line with current clinical practice in IVF. The very potential of this technology will inevitably lead to its abuse, argue opponents. Proponents of SCNT counter that good laws can prevent such abuses. Another potential concern that justifies a ban on SCNT, opponents argue, is the idea of cloning humans. Opponents have argued that there is nothing to prevent rogue scientists from transferring such an SCNT-modified blastocyst into a woman’s uterus and thereby generating clonally identical individuals. Because this has been done with mammals, it certainly will someday become technically possible. Proponents of SCNT suggest criminalizing uterine transfer of SCNT hESCs to prevent cloning of human beings.

CONCLUSION Both proponents and opponents of hESC research have made many sound arguments, and there is perhaps more consensus than generally thought. First, there is widespread consensus that cloning human beings (“reproductive cloning”) does not make scientific sense and is medically unsafe and morally unacceptable. Second, it appears that the majority of physicians, scientists, and ethicists agree that “therapeutic cloning” is ethically distinct from “reproductive cloning” and that a broad ban on SCNT would block promising research. Third, the majority of U.S. citizens, including most physicians, scientists, and ethicists, agree that the U.S. federal guidelines restricting derivation of hESC lines should be loosened considerably to allow derivation of new hESC lines from extra embryos generated in the course of IVF in which the parent-owners consent to having their extra embryos used for research. A compromise might limit and monitor the numbers of such hESC lines that are created, perhaps following the U.K. example [29, 30]. It is important to note that all investigations involving humans involve some risk, and a worthy goal is to minimize these risks without blocking scientific progress.

ACKNOWLEDGMENTS This work was supported by Invitrogen Corporation and the CNS and Christopher Reeves Foundations (M.S.R.) and by grants from NIH (CA070970) and a Fellow award from the

Rao, Civin National Foundation for Cancer Research (C.I.C.). We thank all the members of our laboratories, as well as numerous other scientists and ethicists, for many stimulating discussions on these issues. M.S.R. gratefully acknowledges the contributions of Dr. S. Rao that made this project possible.

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Dickinson and Company; and Baxter HealthCare Corporation. The terms of this arrangement are being managed by the Johns Hopkins University in accordance with its conflict-of-interest policies.

DISCLOSURES The Johns Hopkins University holds patents on CD34 monoclonal antibodies and inventions related to stem cells. C.I.C. is entitled to a share of the sales royalty received by the university under licensing agreements between the university; Becton,

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