Stem cells: A dramatic new therapeutic tool*

Stem cells represent a new field of biomedical science which is hardly two decades old but which in the last few years has captured the imagination of the world's scientists. Its emergence as a new biomedical field was rather unanticipated, but it is now very rapidly developing and moving ahead. Stem cells are scientifically exciting and challenging because they appear to offer many new opportunities for novel therapeutic approaches. Embryonic stem cells have the capacity of indefinite self replication and self renewal and, at the same time, they are pluri-or totipotent (i.e. under appropriate conditions, they can differentiate into each and any cell lineage that makes up the mature organism) (Fig. 1). In mammals, such stem cells constitute the inner cell mass of the blastocyst, which is an early developmental stage of the embryo. Pioneering research in stem cell biology was initiated in the early 1980s by Evans and Kaufman1 and Martin2 who independently succeeded in isolating embryonic stem cells from mouse embryos and to propagate them in culture. Crucial for this achievement was the development of a feeder cell layer, consisting of irradiated mouse embryonic fibroblasts. Availability of these totipotent mouse stem cells made possible creative studies of their differentiation and proliferation3 but, obviously, they could not be used for implantation into human patients. Graphic illustration of the development of specialized tissues from an embryonic stem cell. (Courtesy of Prof. Michael A. Rudnicki, Canada Chair in Molecular Genetics, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.) A crucial breakthrough occurred in late 1998 when Thomson et al.4 announced the successful isolation of embryonic stem cells from human embryonic blastocysts. These cells exhibited surface markers characteristic of embryonic stem cells of non-human primates, which they had studied previously.5 They also contained high levels of telomerase activity which suggests immortality. Indeed, these human stem cells retained normal karyotypes for 8 months of continuous culture. When the stem cells were cultured without a mouse embryo fibroblast feeder layer, they promptly differentiated stochastically into cell lineages representing any or all three embryonic germ layers. And when self-replicating cultured human stem cells were injected into immunodeficient (SCID) mice, the animals developed malignant tumors (teratocarcinomas) which included cells representing also all three germ layers. These findings leave little doubt that the cells isolated by Thomson et al.4 were genuine human embryonic stem cells. Since their first isolation in 1998, such cells have already been used in several clinical pilot projects, most of which yielded promising results.6-8 The potential and promise of human embryonic stem cells have evoked profound scientific and clinical enthusiasm but, at the same time, they have raised ethical and legal questions.9-11 The human embryos used by Thomson et al.4 had been produced in vitro for artificial fertilization; leftover embryos were donated by informed individuals and the research had been approved by an institutional review board. In other instances, human embryos became available from early legal abortions. It is not surprising that the US pro-life movement is strongly opposed to this type of scientific research. In response to their political pressure, the USA has imposed major restrictions on research of human embryos and fetuses, at least to the extent that it is supported by government funds, and these restrictions currently are still in force. It is noteworthy that in this regard, other countries, including the UK, Australia and Israel have chosen a remarkably more tolerant policy.12 Fortunately, it is now apparent that this policy dilemma has lost some of its urgency because it can largely be circumvented as the result of two dramatic and entirely unexpected recent discoveries, which are profoundly reorienting stem cell research. The first of these recent discoveries was that stem cells are not only an essential part of the embryo, but that pluripotent stem cells are also present in most organs of the mature adult body. These organ-based stem cells are generating the progenitor cells for the periodic cell turnover of the organs’ tissues. In addition, they are being used for repair or replacement of cells that have been injured, destroyed or undergone apoptosis. Thus, whereas in the embryo, totipotent stem cells provide for the embryo's development, pluripotent stem cells based in mature organs are concerned with tissue maintenance and repair.13 The derivative transition from totipotent embryonic stem cells to pluripotent organ-based stem cells is not yet understood and it is unknown whether in the transition process, the embryonic stem cells are losing some of their differentiation potential. But the organ-based adult stem cells certainly retain their full ability of self-replication which is an essential component of ‘stemness’. In most organs, adult stem cells form small cell aggregates generally located in one of the organ's relatively quiescent parts where the sluggish stem cells slowly and rhythmically self-replicate. Asymmetric division, which yields one differentiating daughter cell and one self-replicated stem cell occurs only to the extent that cell replacement is needed for periodic tissue turnover. In the case of tissue injury, however, this balanced system may be interrupted in order to rapidly provide stem cells and their progenitors needed for repair or replacement of the damaged tissue's cells.14 Among the various organ-based stem cells, the hematopoietic stem cells of the bone-marrow have been studied most extensively.15-17 However, as these pluripotent stem cells are few and far between, ranging from one per 103 to 105 marrow cells, special techniques are required to ferret out these precious cells from the bulk of the remaining marrow cells. Among these are electronic cell sorting machines and highly selective binding methods, using specially prepared adhesive plasters. The hematopoietic stem cells of the marrow are the source of at least eight different cell lineages, among them, of course, erythroid, granulocytic and megakaryocytic cells, but also macrophages, monocytes and Langerhans cells.17 In the liver, pluripotent hepatic stem cells appear to be part of, or at least are harbored by, the Canal of Hering.18, 19 This ductular structure is the link between the hepatocyte's canalicular system and the biliary tree. Proliferating liver stem cells give rise to oval cells which are progenitors of mature hepatocytes and biliary epithelial cells (Fig. 2). But the latter two cell types have a very long natural lifespan and, consequently, their population dynamics differ strikingly from those of mature cells in tissues such as bone marrow or intestinal mucosa, which turn over very rapidly. Moreover, in the liver, replacement of physiologically aging hepatocytes, slow as it may be, or of mature cells damaged by infections or toxins, primarily occurs by proliferation of viable hepatocytes in situ. Hepatic stem cells are activated only if liver damage is severe enough that most resident hepatocytes are being destroyed or are unable to enter the growth cycle because of its blockage by hepatotoxic chemicals such as retrorsine.20 It is under such severe circumstances that oval cells become detectable in the liver.21 Schematic diagram of the Canal of Hering, its pluripotent liver stem cells, and their derivative oval cells, hepatocytes and epithelial cells of biliary ducts, pancreas and intestine. Modified after Grisham JW, and Thorgeirsson SS, in Stem Cells, CS Potten, ed. Academic Press, London, 1997, page 247. In contrast to hepatocytes, the mucosal cells, which line the small crypts of the intestine and villi exhibit a very rapid physiological turnover. They migrate in 3–4 days from the base of the crypt where they originate, to the upper part of the villus where they eventually are shed into the intestinal lumen. One or only a very few intestinal stem cells are located in each crypt's base where they predominantly divide asymmetrically (i.e. into one self-replicated stem cell and one progenitor daughter cell). The latter, while moving up along the inside wall of the crypt, undergoes a series of divisions and acquires differential maturity when reaching the base of the villus. Intestinal stem cells yield at least four major cell lineages, including columnar cells, mucin-producing goblet cells, Paneth cells and, probably, selected endocrine cells; the latter arguably could be derived from neural stem cells.22 Thus, in contrast to hepatic stem cells, the primary function of intestinal stem cells appears to be provision of progenitors for replacement of the rapidly turning-over mucosal cells. Neural stem cells seem to have a somewhat similar albeit more complex function. Because mitotic figures are virtually absent from the adult primate neocortex, it traditionally had been assumed that the latter is a structurally stable organ, as neurogenesis and synapsis formation occur only during fetal development. However, this was difficult to reconcile with the demonstrated plasticity of the adult mammalian brain as well as with the neurogenesis commonly observed in the brains of adult birds. This inherent contradiction has now been resolved by the surprising demonstration that in adult subhuman primates, new neurons are continually being added to the prefrontal, inferior temporal and posterior parietal regions of the neocortex.23 These new neurons in all likelihood are derived from neural stem cells, which originate from the neural crest and appear to conglomerate in the subventricular zone where they line the wall of the lateral ventricles. From there, some of the neural stem cells and their progenitors migrate through the white matter to the neocortex where they differentiate into mature neurons.24 It has been suggested that the new neurons continuously added to the neocortex of adult primates may function as a substrate for learning or memory.23 The neural stem cells identified in the subventricular zone appear to represent a mixture of genuine stem cells and of progenitor cells at various stages of differentiation and proliferation, some perhaps already committed to specific cell lineages. This may not be surprising, as cell lineages derived from neural stem cells are engaged all over the body, including, of course, the peripheral nervous system. Accordingly, neural stem cells display an unusually broad capacity for differentiation ranging from neurons and glial cells to melanocytes and a few selected endocrine cells.24 Of particular medical significance may be the Schwann cell lineage because this glial cell's membrane consists of myelin; the cells therefore may be useful for the treatment of demyelinating diseases, such as multiple sclerosis, leukodystrophies or injuries to the brain or spinal cord.25, 26 In addition to bone marrow, liver, intestine and nervous system, adult pluripotent stem cells that initiate progenitor proliferation, have been detected for cell lineages leading to cells of the cornea, skin and hair follicles, skeletal and cardiac muscle, and cartilage, bone and endothelium. (Some of these findings reported up to 1997, are summarized in refs3, 17, 41) The second of the recent crucial discoveries causing reorientation of stem cell research concerns the relative scope of the differentiation potential of organ-based pluripotent stem cells. It had been reasonable, if not logical, to assume that organ-based pluripotent stem cells are committed to expression of cell lineages leading to mature cells that are becoming part of their host organ. It had been shown that the number of these cell lineages expressed by stem cells in different organs is variable, ranging from two in the liver to at least eight for hematopoietic stem cells. But it came as a total surprise when in mice, intravenously injected with tagged hematopoietic stem cells, many of these markers appeared in brain cells that exhibited all the attributes of neurons.27, 28 Similarly, injected bone marrow stem cells were found to add new skeletal muscle cells along the anterior tibia, which previously had been chemically injured.29 Several publications reported that marrow stem cells gave rise to mature hepatocytes30 or to their hepatic precursor, oval cells.31 What's more, this generative cross-over activity from the marrow's hematopoietic stem cells to other cell lineages works also in the reverse. For example, in lethally irradiated mice, injection of tagged skeletal muscle stem cells (known also as satellite cells) resulted in high-level engraftment of all major blood cell lineages.32 Similar observations were made when labeled neural stem cells were administered to irradiated mice.33 In fact, neural stem cells of adult mice appear to have a developmental potential that extends to all three germ layers34 (Fig. 3). Most recently, it has been reported that in a complex sequential series of cultures in vitro, mouse embryonic stem cells were coaxed to assemble in cell clusters resembling pancreatic island cells, which in vitro produced detectable amounts of insulin.35 Schematic diagram of the apparent plasticity of adult stem cells. Pluripotent adult stem cells from all mammalian sources studied to date are able to form cell lineages leading to any or all three embryonic cell layers. (Courtesy of Prof. Michael A. Rudnicki.) These striking new findings appear to imply that mature organ-based stem cells functionally are related more closely to embryonic stem cells than had been anticipated (Fig. 4). It is not yet known, however, whether this means that the expression potential of adult organ-based pluripotent stem cells differs little, if at all, from that of embryonal stem cells. Or, as an alternative explanation, whether adult stem cells may retain some kind of memory, which perhaps in response to microenvironmental signals, may re-expand their range of lineage expression. What does seem likely, however, is that signals emanating from the tissue environment are playing a critical role in determining whether mature stem cells are undergoing slow self-replication or are proliferating along directed cell lineages.36 The nature of these signals—whether in vivo derived from the microenvironment or in vitro from additives to cultures—remains to be determined. It is noteworthy, that adult tissues or organs that have been injured or are diseased appear to exert a strong attraction for stem cells, regardless whether they are native or have been injected.29, 32, 36-38 Graphic illustration of lineage differentiation of totipotent embryonic stem cells and pluripotent adult, organ-based stem cells. Dark horizontal arrows indicate the interconversion of adult stem cells. Reflected arrows indicate self-replication. Modified from Ashahara et al., Millennium Review. Gene Therapy 2000; 7: 451–457. For example, in experimental myocardial infarction in mice or rats, injected hematopoietic stem cells selectively migrated to the injured heart where they substantially reduced myocardial damage by preventing apoptosis of ischemic myocytes, lowered collagen deposition, prevented scar formation and improved ventricular function.37, 38 And in several rodents bearing a malignant brain tumor, intravenously administered neural stem cells were found to target and surround the tumor as well as distant infiltrating tumor cells.39, 40 In an expansion of these experiments, the neural stem cells to be injected first were stably transduced with a transgene encoding cytosine deaminase, which converts the non-toxic 5-fluorocytosine to the oncolytic toxin 5-fluorouracil. When the rodents bearing the transfected neural stem cells were treated systemically with 5-fluorocytosine, the brain tumors dramatically shrank by 80% in the course of 2 weeks.39 These two examples strikingly illustrate not only the strong tropic interaction between tissue pathology and stem cells but also the usefulness of stem cells as targeting carriers of therapeutic genes not only for cancer therapy or tissue repair but also for treatment of genetic disorders.41 The term and the concept of pluripotent stem cells residing in the bone marrow originated in the 1960s, largely as the result of the observation that allogeneic bone marrow infusions are beneficial and often curative for patients exposed to excessive radiation (for summaries of earlier publications, see refs.6, 42) Although important progress in stem cell research had been achieved in the subsequent decades,1-3 truly dramatic discoveries were made only in the last 3–4 years when it was found that omnipotent stem cells are not only a crucial part of the embryo, but also occur and function in most of the organs of the adult mammalian organisms. These new horizons beget a fundamental reappraisal of traditional concepts of cell turnover and tissue renewal, tissue repair and organ regeneration. In due time, a plethora of new ideas and novel therapeutic opportunities will emerge, which are likely to profoundly change medical care as it is practised today.

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