Stem cell research: immortality or a healthy old age?

European Journal of Endocrinology (2004) 151 U7 U12 ISSN 0804-4643 Stem cell research: immortality or a healthy old age? Christine Mummery Hubrecht Laboratory, Netherlands Institute for Developmental Biology and the Interuniversity Cardiology Institute of the Netherlands, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands (Correspondence should be addressed to C Mummery; Email: christin@niob.knaw.nl) Abstract Stem cell research holds the promise of treatments for many disorders resulting from disease or trauma where one or at most a few cell types have been lost or do not function. In combination with tissue engineering, stem cells may represent the greatest contribution to contemporary medicine of the present century. Progress is however being hampered by the debate on the origin of stem cells, which can be derived from human embryos and some adult tissues. Politics, religious beliefs and the media have determined society s current perception of their relative value while the ethical antipathy towards embryonic stem cells, which require destruction of a human embryo for their derivation, has in many countries biased research towards adult stem cells. Many scientists believe this bias may be premature and basic research on both cell types is still required. The media has created confusion about the purpose of stem cell research: treating chronic ailments or striving for immortality. Here, the scientific state of the art on adult and embryonic stem cells is reviewed as a basis for a debate on whether research on embryonic stem cells is ethically acceptable. European Journal of Endocrinology 151 U7 U12 Introduction Stem cells are primitive cells with the capacity to selfrenew (divide and produce more of themselves) or to differentiate to specialized cells such as bone, brain and heart cells. Stem cells are often placed in one of two categories: adult stem cells derived from adult or fetal tissues and embryonic stem (ES) cells derived from very early (mouse or human) embryos at the blastocyst stage of development (Fig. 1) prior to implantation in the uterine wall. Stem cells of bone marrow have been used for decades in the successful treatment of blood disorders such as leukemia. Bone marrow transplantation to a patient whose own bone marrow has been destroyed as part of treatment will allow the patient to start regenerating blood cell lineages and forming blood with a normal cellular composition. Likewise, a skin wound will result in stem cells in the basal layers resuming proliferation and differentiating to all cell types present in normal skin in a manner that reconstructs the dermal and epidermal layers. Conventionally, adult or tissue-specific stem cells in adults were regarded as having the ability to proliferate at times of tissue damage and to differentiate to the cell types in the tissue in which they were found. Exceptionally, in sex non-matched organ and bone marrow transplants, where, for example, tissue from a male donor was given to a female recipient, there were occasional reports of some cells being found in multiple organs. They had apparently adopted the phenotype of the local tissue. These observations provided the first evidence of what was referred to as transdifferentiation or adult stem cell plasticity. In 1998, the first stem cell lines were derived from human blastocyst stage embryos (Fig. 1) by Thomson et al. (1). This milestone in cell biological research had been preceded by more than 30 years of research on another very similar cell type, an embryonal carcinoma or teratocarcinoma stem cell, derived from a spontaneous testis tumour found in mice and men (Fig. 1). In mice, teratocarcinomas can be formed experimentally by transferring young embryos to extrauterine sites, for example, under the skin or kidney capsule. Stem cells were derived directly from early mouse embryos as mouse ES cells in 1981, circumventing the necessity of generating a tumour as an intermediate (2, 3). The way was then already open to deriving human ES cell lines as assisted reproduction by in vitro fertilization was becoming clinically routine. Embryos were being discarded even after three to four had been simultaneously transferred to a patient because no methods had been developed for successful freezing. These first attempts were not successful and took place under circumstances where there was no legislation governing human embryo research. In the mid 1980s it became possible to freeze embryos and therefore sources dried up; legislation was developed in several countries that would eventually govern human embryo research. Thomson s article clearly demonstrated that the human ES cell lines that he had derived were immortal and pluripotent, i.e. they could form derivatives of the three primary germ layers and in principle all tissues of the q 2004 Society of the European Journal of Endocrinology Online version via http://

U8 C Mummery EUROPEAN JOURNAL OF ENDOCRINOLOGY (2004) 151 Figure 1 Pluripotent human stem cells are found in teratocarcinomas and in the inner cell mass (ICM) of blastocyst stage embryos. human body. The implications for cell-based therapies were immediately obvious and a list of diseases or ailments for which human ES cells might be useful because only one or, at most, a few cell types were affected was easy to draw up (Table 1). The price of this research, however, is destruction of human embryos that have the potential to become new human beings. The fact that these embryos were destined to be discarded and, by implication, destroyed because the gamete donors who had lead to their formation no longer wished to continue with their parent programme, was for many not an argument to redirect their fate towards stem cell derivation however noble the cause of curing chronic disability. The simultaneous revisiting by a number of scientists of the ability of adult stem cells to transdifferentiate may have, in part, derived from the aversion to embryo destruction. It is of note, however, that not all societies or religions adopt the same point of view: while Christian-based ethics believe that life begins at fertilization, other beliefs consider 40 days after conception as the crucial time point in determining the moral status of the embryo. In the following sections, the current status of research on adult and ES cells (Table 2) is reviewed (4). The conclusion is that research on neither adult nor ES cells is sufficiently advanced for a definitive and exclusive choice on whether one is better or worse than the other as a basis for developing a broad range of stem cell therapies. Each is most likely to have its own niche in therapy. In some cases the best option may be combined adult and ES cell therapy. If it were already proven that human ES cells are useful in curing patients it would be much easier to justify sacrificing embryos for those cures. However, it is not proven and it is under those circumstances that we have to decide whether derivation of new human ES cell lines is justified for the purposes of research in the light of Table 1 Examples of ailments for which solutions in stem cell therapy are being sought. Stroke Parkinson s disease Diabetes Heart failure Vascular disease Arthritis Multiple sclerosis Spinal cord lesions In general diseases associated with increasing age (in italics) where one or at most a few cells types have been destroyed or malfunction. Table 2 Stem cell origins. Stem cells are found in: 1. Adult tissues: all tissues and organs that can repair themselves after damage or regenerate have adult stem cells. Examples are: skin, bone marrow/cord blood, intestine, brain, liver, fat. Not ethically sensitive but there are very few cells present in normal tissue and only a few different cell types can be formed. 2. Embryos: embryonic stem cells. Ethically very sensitive as destruction of the embryo is necessary but many cells available in the laboratory and all cells of the human body can be formed.

EUROPEAN JOURNAL OF ENDOCRINOLOGY (2004) 151 Stem cell research U9 the current availability of a number of human ES cell lines already established. Adult stem cells in clinical practice or clinical trials There are already a number of clinical applications for several types of adult stem cells derived from different tissue sources (see Table 3). Some of these are autologous, a patient receiving some of his own stem cells to repair a particular organ, others are heterologous and the stem cells are provided by a donor. Pancreas duct stem cells These stem cells are currently being tested in clinical trials for the treatment of brittle diabetes. This form of diabetes is very severe, difficult to control with insulin and patients are often not aware of an imminent hyperglycemic incident. The pancreatic duct contains a population of stem cells which can be expanded to a limited extent in culture using a protocol developed in Edmonton, Canada which has as a result become known as the Edmonton protocol. This is an example of heterologous transplantation. When first developed, the protocol required the ducts from five deceased donors for isolation, growth and differentiation of cells to b-islet-like colonies for the treatment of just one brittle diabetes patient. This has now improved to two donors for one patient and the current goal is to improve this to one donor for five patients. It will be clear, however, in the light of the high incidence of diabetes worldwide and the scarcity of donors, that this is by definition at present a limited therapy for only the very worst cases. Mesenchymal stem cells ( from bone marrow or fat) for bone and cartilage repair This is in principle an autologous treatment, for which there are several spectacular anecdotal clinical examples, several of which have been used in combination with tissue engineering to create three-dimensional structures. One example is of a boy born lacking the left side of his ribcage so that there was little structure to protect his heart from injury. Tissue engineers constructed a ribcage using an artificial bone matrix composed of hydroxy-appetite. This was seeded with stem cells Table 3 Clinical applications or ongoing clinical trials with adult stem cells. Pancreas duct stem cells for brittle diabetes. Mesenchymal stem cells (from bone marrow or fat) for bone and cartilage repair. Skeletal muscle stem cells for heart attack (but drugs are necessary to control heart rate). Fresh bone marrow injected directly into heart or intravenously for treating heart attack (mechanism unknown and unproven in humans but no evidence of transdifferentiation to heart cells in animals). Skin stem cells for skin transplantation. derived from his own bone marrow and implanted; bone fide bone then developed and functioned as a normal ribcage. A second example concerned a young woman who underwent surgery for removal of a large tumour in her jaw. This left her with a serious facial disfigurement. Again, tissue engineers reconstructed a bone matrix but now in the form of a jaw. Her stem cells were seeded to the matrix and bone developed in situ of such quality that artificial teeth could be implanted and the jaw functioned essentially as normal. This is an area being very actively pursued. The main drawback is an age limitation of the suitability and numbers of stem cells. Above the age of 50 years, the method generally does not work. Another drawback is the size of the engineered implant; the seeded cells require adequate nutrients and blood supply, and therefore only tend to survive well in the outermost regions of the bone matrix. Strategies are being developed to encourage the matrices to vascularize by also seeding with vascular endothelial cells. Skeletal muscle stem cells for treatment following heart attack Skeletal muscle has an undifferentiated population of stem cells known as satellite cells that retain the capacity to differentiate to skeletal muscle. In the case of cardiac patients, satellite cells are isolated from their muscle, expanded in culture and transplanted into the ischemic regions of the heart. While the transplanted cells do beat in situ, they fail to couple to the host myocardium and have been known to induce fatal arrythmias. The arrythmias could sometimes be treated by drugs but the effects were severe enough for one clinical trial to be terminated prematurely. Retrospectively, experiments in rats showed that there was no functional coupling between transplanted and host muscle (5; references in review 4). Fresh bone marrow injected directly into heart or intravenously for treating heart attack The mechanism of how this might work is unknown and the effects are essentially unproven in humans because no large-scale randomized trial has yet been carried out. Most results are anecdotal. The original experiment upon which these trials were based was carried out in mice several years ago (6) but in more than 80 independent attempts, others have not been able to reproduce the finding. Two very recent publications clearly demonstrated that the original results are likely to have been an artifact of the experimental method (7, 8). There is no evidence of transdifferentiation of bone marrow stem cells to heart cells in animals but it has clearly been shown that bone marrow stem cells can fuse with host cells in heart, liver and brain and adopt those phenotypes with low frequency. This is probably the explanation of the data described above concerning sex non-matched donors; transplanted stem cells fuse locally at low incidence giving the impression that transdifferentiation has taken

U10 C Mummery EUROPEAN JOURNAL OF ENDOCRINOLOGY (2004) 151 place. Such a mechanism is unlikely to be of benefit since there will be no net increase in cardiac cell numbers to replace those cells lost as a result of ischemic damage. More importantly if nuclei also fuse during cell fusion, tetraploid nuclei may arise with an increased tumorigenic potential. It is of note, however, that in experiments in pigs with a myocardial infarction into which genetically marked bone marrow was injected, the marked cells contributed to neovascularization in the infarct region; no marked cardiomyocytes were found but there were no significant numbers of vascular endothelial cells. Such a mechanism could be beneficial post-infarction but more experiments in animals are necessary to investigate and optimize this possibility. The bone marrow contains several different subpopulations of cells defined on the basis of how they are selected. It is not clear which population might be most useful if neovascularization were the essential mechanism behind any effect. The endothelial precursor cell population may well be the one of choice. In this area there is particularly sensitive discord between scientists and physicians. While scientists prefer to optimize and understand the mechanism before considering application, physicians faced sometimes with terminal patients do not necessarily regard understanding how something works as a high priority. That it might work and is not dangerous is enough. For review see Hassink et al. (9). Skin stem cells for skin transplantation This technology is making a significant contribution, particularly to the treatment of burns patients and patients with varicose ulcers. Stem cells are taken from skin biopsies, expanded in culture and grown on rafts of extracellular matrix proteins at an air water interface. In the presence of a growth factor cocktail and ascorbic acid, they will differentiate into several layers of skin types, which can be transferred as sheets to large areas devoid of skin. Together, these form the major areas in which adult stem cells have proven or are proving their worth. Research on neural stem cells derived from adult or fetal brain is also actively being carried out. However, the results of clinical trials on the usefulness, for example, of human fetal brain stem cells for the treatment of Parkinson s disease, have been somewhat contradictory with positive and no effect outcomes being reported. There is little evidence as yet that cord blood will be useful for more than the treatment of blood disorders, including leukemia, as it is now. Human ES cells Human ES cells are derived from a small group of cells present in the early pre-implantation embryo at the blastocyst stage (Fig. 1). After isolation from the embryo, ES cells can grow indefinitely in culture in a primitive or undifferentiated state provided they are supplied with a so-called feeder cells. These cells provide them with as yet unknown factors that encourage selfrenewal without differentiation. Most of the human ES cells to date and all of those on a list approved by President Bush for government (NIH) funding, have been derived and cultured using feeder cells from mouse embryos. They are therefore perceived as presenting a xenorisk (such as cross-species recombination of viruses) to patients and are unlikely to be approved for clinical use. This is currently one of the driving forces behind the wish to derive new cell lines from human embryos surplus to in vitro fertilization requirements. There are estimated to be hundreds of thousands of these worldwide; 400 000 in the US alone. ES cells show a strong resemblance to teratocarcinoma (EC) stem cells. Many markers have been developed by pathologists to determine whether there was a residual stem cell population in teratocarcinomas (which in turn determines whether it is malignant or not, and thus the patient s prognosis and treatment). The cells are immortal and, just as cancer cells, express telomerase activity that controls the telomere length protecting the ends of chromosomes at every cell division. Human ES cells form derivatives of all three embryonic germ layers in vitro and in teratocarcinomas in mice in vivo. This means that it is most likely that they can form all, 200 cell types of the human body. It also means, however, that they may form teratomas posttransplantation in patients. Rigorous animal experimentation will be required to establish their safety; i.e. to determine whether they are likely to form tumors or not in the event that undifferentiated cells contaminate the differentiated cell populations to be transplanted. A variety of protocols have been useful in driving the differentiation of mouse ES cells to particular lineages, the most useful of which has been the growth of the cells as aggregates in suspension as so-called embryoid bodies. Addition of various trophic or growth factors has resulted in cells becoming biased towards particular lineages. It has thus been possible to derive cell populations enriched with neural cell precursors or cardiac cell precursors. Pure populations of differentiated cells are, however, never obtained without some methods for selection of the required phenotype. These may include using the promoter of a lineage or cell type specific gene coupled to an antibiotic resistance gene for example. Addition of an antibiotic then results in the death of all cells not expressing the gene since they are not protected. The required cell population becomes selected in culture. Copying the signals used by an embryo to create different cell types from common precursors could lead to new methods for deriving cells suitable for transplantation. We have used this approach to derive cardiomyocytes (heart cells) from human ES cells.

EUROPEAN JOURNAL OF ENDOCRINOLOGY (2004) 151 Stem cell research U11 Reasoning that the heart develops adjacent to the invaginating gut and using evidence from species such as chick and frog, where defects in endoderm development result in cardiac defects, we co-cultured human ES cells with visceral endoderm-like cells and obtained beating muscle cells with a phenotype resembling human fetal ventricle (10, 11). We have recently transplanted these to the hearts of immuno-compromised but otherwise healthy mice and shown long-term survival and functional integration of the human ES derivatives. We do not yet know whether these cells would cure a mouse with a myocardial infarction or ischemic damage. These experiments thus join several others that have shown successful transplantation to mice or rats. Human ES cell derived neurons survive transplantation to the brains of young mice; endothelial cells from human ES cells have now been transplanted into three-dimensional matrices then into immunodeficient mice where they form capillaries through which mouse blood circulates (reviewed in reference 1). Otherwise, transplantation studies with human ES cells to date are still fairly limited. Exceptionally, in the US, neural cells derived from teratocarcinoma stem cells were transplanted to the brains of 11 stroke patients in a phase I trial (safety). In 1999, the transplanted cells were still detectable by positron emission tomography (PET) scan in all patients and in 2000, tumour development was not detected in any of the 11 patients. In 6 patients, symptoms improved ; in 2, this improvement was reported as significant (12). Nevertheless, such experiments on humans are regrettable. If only a few undifferentiated cells had been present the patients may have developed teratomas or, worse, teratocarcinomas. The whole field would have been brought into discredit before it had even begun. Insulin-producing cells, bone cells, blood cells and liver cells have all been reported to develop from human ES cells but have not yet transplanted to animal models. The majority of transplantation studies and the most spectacular evidence of control of selfrenewal and differentiation have been carried out only on mouse ES cells. This is largely where over interpretation by the media originates since the extrapolation from mouse to human is (too) easily made. A simple case in point; the self-renewal factor from feeder cells for mouse ES cells is leukemia inhibitory factor (LIF) but this does not work on human ES and the feeder factor remains unknown to date. The indications are, however, that the same transcription factors in both species are involved in control of self-renewal. While it is unquestionable that human ES cells have a much wider differentiation potential than adult stem cells, it is still fairly difficult to grow them and to control their differentiation. Questions that need to be addressed before human ES cells can be used clinically include the following. How can we derive pure populations of cells in sufficient numbers for transplantation? For example, for myocardial infarction 10 8-10 9 cells would probably require replacement. Here, bioreactors in combination with (genetic) selection are likely to be useful. How can requirements of good medical practice (GMP) be achieved (i.e. no mouse feeder cells, no fetal calf serum, etc.)? Some human ES cell lines have already been derived on human feeder cells but it is hoped that, in the near future, the relevant factors for self-renewal will have been identified. Most of the necessary ingredients are available now at GMP grade. How can potential problems of tissue rejection be resolved? This should be no problem for transplantation to the brain/central nervous system, but what about other organs? Immunosuppressive drugs and tissue matching as carried out for whole organ transplants are likely to be the most useful option. With this in mind a stem cell bank has already been established by the Medical Research Council in the UK and Sweden. Immunotolerance is another option. It has been known for many years that if a patient receives bone marrow from a kidney donor prior to transplantation, chimeric bone marrow can develop in vivo and the patient is later immunotolerant for the transplanted kidney. Finally therapeutic cloning has been discussed as an option. Proof of principle was recently reported by a group in Korea (13). They showed that they could remove a nucleus from a human oocyte (egg cell), replace it by a somatic cell and allow the reconstructed embryo to grow to the blastocyst stage. From this blastocyst they could isolate human ES cells containing the derivatives of the somatic nucleus. If the somatic nucleus were from a patient, for example from the skin, then the human ES cell derivatives would be recognized as self and would not be rejected at transplantation. In summary, human ES cells are at present an exciting research challenge but we have now entered a phase that should include quiet, in-depth research to solve the many scientific problems that still bar the way to clinical application. This is the light in which their current value should be debated. Conclusions There are at present three main conclusions: (i) Rapid advances are being made in both adult and ES cell research. However, a great deal more research is necessary before either of these becomes part of therapy in mainstream medicine, apart for the treatment of blood disorders. (ii) It is a pity that interest in stem cell research has become hyped in the media. The practical and ethical problems are far from solved (14). Nevertheless, the potential of stem cells in the development of therapies for chronic disorders remains and they could represent a major contribution

U12 C Mummery EUROPEAN JOURNAL OF ENDOCRINOLOGY (2004) 151 to treatment of diseases likely to increase in the coming decennia as the general population ages. (iii) Adult stem cells are preferable for ethical reasons and because they can, in principle, be autologous. However, present scientific evidence is that they are unlikely to be broadly applicable because their developmental potential is limited, their numbers are extremely low and only few have proven amenable to ex vivo expansion. Commercial companies offering services to freeze cord blood or bone marrow for the donors own future use should thus be viewed with caution. Most important is that open dialog, faithfully reflecting the scientific facts and as far as possible including consistent ethical values without political bias, is possible in this highly sensitive field. Elizabeth Blackburn, a scientist highly respected for her work on telomerase, was sacked from President Bush s Council for Bioethics for criticizing a report (Report on Monitoring Stem Cell Research (15)): the scientific results have been manipulated to bias away from ES cells towards adult stem cell research. This is counterproductive for scientists, society and politicians alike. The future? Hopefully healthy old age and not immortality. Acknowledgements I thank Dorien Ward, Robert Passier, Stieneke van den Brink, Leon Tertoolen and Rutger Hassink for their contributions to the work on cardiomyocytes from human ES cells, Alan Trounson and Martin Pera for providing the human ES cell lines used and Embryonic Stem Cell International for financial support. References 1 Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS & Jones JM. Embryonic stem cell lines derived from human blastocysts. Science 1998 282 1145 1147. 2 Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. PNAS 1981 78 7634 7638. 3 Evans MJ & Kaufmann MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981 292 154 156. 4 Passier R & Mummery CL. The origin and use of embryonic and adult stem cells in differentiation and tissue repair. Cardiovascular Research 2003 58 324 335. 5 Menasche P, Hagege AA, Scorsin M, Pouzet B, Desnos M, Duboc D, Schwartz K, Vilguin JT & Mardlean JP. Myoblast transplantation for heart failure. Lancet 2001 357 279 280. 6 Orlic D, Kajstura J, Chimenti S et al. Bone marrow cells repair the infarcted myocardium. Nature 2001 410 701 705. 7 Murry CE, Soonpaa MH, Reinecke H, Nakajima H, Nakajima HO, Rubart M, Pasumarthi KB, Virag JI, Bartelmez SH, Poppa V, Bradford G, Dowell JD, Williams DA & Field LJ. Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature 2004 428 664 668. 8 Balsam LB, Wagers AJ, Christensen JL, Kofidis T, Weissman I & Robbins RC. Haematopoietic stem cells adopt mature haematopoietic fates in ischemic myocardium. Nature 2004 428 668 670. 9 Hassink RJ, Brutel de la Riviere A, Mummery CL & Doevendans PA. Transplantation of cells for cardiac repair. Journal of the American College of Cardiologists 2003 41 711 717. 10 Mummery CL, Ward D, van den Brink CE, Bird SD, Doevendans PA, Opthof T, Brutel de la Riviere A, Tertoolen L, van der Heyden M & Pera M. Cardiomyocyte differentiation of mouse and human embryonic stem cells. Journal of Anatomy 2002 2000 233 242. 11 Mummery CL, Ward-van Oostwaard D, Doevendans PA, Spijker R, van den Brink S, Hassink R, van der Heyden M, Opthof T, Pera M, Brutel de la Riviere A, Passier R & Tertoolen L. Differentiation of human embryonic stem cells to cardiomyocytes: the role of coculture with visceral endoderm-like cells. Circulation 2003 107 2733 2740. 12 Kondziolka D, Weschler L, Goldstein S, Meltzer C, Thulborn KR, Gebel J, Jannetta P, DeCesare S, Elder EM, McGrogan M, Reitman MA & Bynum L. Transplantation of cultured human neuronal cells for patients with stroke. Neurology 2000 55 565 569. 13 Hwang WS, Ryu YJ, Park JH, Park ES, Lee EG, Koo JA, Chun HY, Lee BC, Kang SK, Kim SJ, Ahn C, Hwang JH, Park KY, Cibelli JB & Moon SY. Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst. Science 2004 303 1669 1674. 14 de Wert G & Mummery CL. Human embryonic stem cells: research ethics and policy. Human Reproduction 2003 58 324 335. 15 President s Council on Bioethics (2004): Monitoring stem cell research. http://bioethics.gov/reports/stemcells/index Received 18 May 2004 Accepted 10 August 2004