Nuclear reprogramming: A key to stem cell function in regenerative medicine

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1 PERSPECTIVE Nuclear reprogramming: A key to stem cell function in regenerative medicine Jason Pomerantz and Helen M. Blau The goal of regenerative medicine is to restore form and function to damaged tissues. One potential therapeutic approach involves the use of autologous cells derived from the bone marrow (bone marrow-derived cells, BMDCs). Advances in nuclear transplantation, experimental heterokaryon formation and the observed plasticity of gene expression and phenotype reported in multiple phyla provide evidence for nuclear plasticity. Recent observations have extended these findings to show that endogenous cells within the bone marrow have the capacity to incorporate into defective tissues and be reprogrammed. Irrespective of the mechanism, the potential for new gene expression patterns by BMDCs in recipient tissues holds promise for developing cellular therapies for both proliferative and post-mitotic tissues. Stem cells offer the potential to provide cellular therapies for diseases that are refractory to other treatments. For each stem cell type (for reviews, see refs 1, 2), the ultimate goal is the same: to induce the nucleus to express a specific repertoire of genes, thereby modifying cell identity to maintain, replace, or rescue a particular tissue. Accordingly, stem-cell-mediated therapy ultimately entails nuclear reprogramming the alteration of gene expression patterns unique to cell types in diverse tissues and organs. This review focuses on the recently discovered capacity of BMDCs to reprogram their nuclei for functions other than those of blood cells. For comparison, we draw on historical evidence for reprogramming, nuclear transplantation (cloning) and cell fusion, as a starting point for understanding this nascent field and its potential applications to regenerative medicine. The discovery that BMDCs can contribute to non-haematopoietic tissues may have substantial therapeutic applications for tissue Jason Pomerantz is in the Baxter Laboratory in Genetic Pharmacology and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA. Helen Blau is in the Baxter Laboratory in Genetic Pharmacology and the Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA. hblau@stanford.edu repair. However, major challenges exist in the use of BMDCs in a cell-based therapy for nonhematopoietic tissues, including increasing their efficiency of incorporation into target tissues and demonstrating efficacy in treating tissue malfunction. In addition, the cellular events or mechanisms leading to the contribution of bone marrow to specific tissues in vivo remain a subject of debate and intense research. One mechanism requires that BMDCs first fuse with pre-existing differentiated cells within the target tissue, and nuclei are subsequently reprogrammed in response to intracellular cytoplasmic factors. An alternate mechanism entails nuclear reprogramming in response to extracellular signals in the microenvironment. A combination of the two mechanisms may be used in some tissues. Regardless of mechanism, reprogramming of the nucleus is a key event in all cases of tissue replenishment by BMDCs (Fig. 1). We speculate that the mechanism used to repair diverse tissues by BMDCs would depend on the innate ability of the tissue to self-renew, which is a function of its mitotic activity (Fig. 1). For example, a tissue (such as epidermis) that has a relatively simple structure, is dynamic and undergoes constant renewal 3,4, might readily be regenerated by the provision of cells with the capacity to proliferate and augment cell number. In this case, BMDCs may be expected to transit via an epidermal progenitor in response to extracellular environmental signals on route to becoming mature cells of the skin. In contrast, a highly elaborate cell, such as a Purkinje neuron, which has more than one million synaptic connections to other cells and that is not known to divide or be made anew after birth, might be difficult, if not impossible, to replace in adulthood by a proliferative mechanism. In this case, rescue by cell fusion and subsequent reprogramming of an incorporated nucleus would constitute a more plausible scenario than de novo cell production. The growing excitement regarding the potential of BMDCs including haematopoietic stem cells (HSCs) to contribute to adult tissues, has stimulated growing controversy and attention, especially regarding the underlying mechanisms or impact on normal life. In this review, examples drawn from numerous publications show that the potential exists at the level of the gene, the cell and the organism for cells to assume new functions: gene expression patterns can change, differentiation can be manipulated and cells can incorporate into tissues from which they did not originate. Consideration of the evidence in support of this conclusion will provide a clear reference point for the current state of the field and a determination of whether there are insurmountable blocks to using endogenous genetic or cellular complements (directly, or after ex vivo manipulation) to repair the body. The goal of this review is to stimulate further investigation of an issue central to the use of stem cells, nuclear reprogramming, and the microenvironmental and other variables inherent in its control. 810 NATURE CELL BIOLOGY VOLUME 6 NUMBER 9 SEPTEMBER 2004

2 Tissue stem cell Proliferation renewel Reprogramming by extracellular signalling Plasticity, transdifferentiation and metaplasia Terminology can hinder progress by providing constraints to the acceptance of novel findings that do not fit a long-held definition. Such semantic problems have contributed to confusion in the stem-cell field, in general, and especially with respect to experiments using BMDCs or the subpopulation of BMDC that are HSCs. Terms such as plasticity, and transdifferentiation have a range of different connotations, and thus clarification based on historical definitions may be useful. The phenotypic conversion of one cell or tissue type into another has been termed metaplasia, and the literature is replete with examples in humans (for a review, see ref. 5). Transdifferentiation, however, is a specific type of metaplasia involving a change in phenotype of a fully differentiated, mature cell to another fully differentiated and mature cell type. As classically defined, the criteria for transdifferentiation of a cell are very specific: initially the cell must exhibit a mature, differentiated, and stable phenotype, followed by a change in phenotype, not necessarily involving an intervening cell division 6,7.As used here, a mature cell refers to a cell type that performs a specialized function, has stable BMDC Reprogramming by fusion and cytoplasmic mixing Epithelium Skeletal muscle Purkinje neuron Figure 1 Schematic representation of the possible mechanisms of BMDC incorporation into nonhaematopoietic tissues. As discussed in the text, not all mechanisms are well established in vivo, and each may occur to a different extent or not at all in different tissues. Thick arrows indicate mechanisms that are better supported in the current literature. First, nuclear reprogramming can occur in cells in response to extracellular signals to yield a mature differentiated cell 69 ; second, nuclear reprogramming may first result in differentiation into a tissue-specific stem cell that has the properties of self-renewal as well as the ability to generate mature, fully differentiated cells 58 ; third, nuclear reprogramming is achieved when the nucleus of the donor cell is exposed to intracellular signals within a mixed cytoplasm following cell fusion. Fusion of BMDCs directly with recipient cells is most clearly shown in Purkinje neuron BMDC heterokaryons, but most probably also occurs in skeletal muscle that exists naturally as a syncytium. and identifiable characteristics, is frequently integrated into a specific organ system to which it contributes, and is often post-mitotic for example, neurons, keratinocytes and cardiomyocytes. Plasticity is exhibited in all examples of nuclear reprogramming, including cloning, heterokaryons, or metaplasia. Simply put, plasticity means the ability to change or adapt. It is a descriptive term that relies on context. To minimize confusion, we propose that the term plasticity always be qualified and used in a specified context; for example, plasticity of nuclear gene expression, plasticity of cell morphology, plasticity of the differentiated state. Nuclear reprogramming by the cytoplasm: cloning of amphibians Nuclear cloning the introduction of a donor cell nucleus into an enucleated oocyte to produce an embryo was first studied in amphibians (Fig. 2a). The results showed that differentiation and development do not require loss or irreversible inactivation of genes 8,9.The disparate interpretations of the early results are instructive vis-a-vis the current controversy surrounding the behaviour of BMDCs, HSCs and their derivatives in different settings. Initial reports by Briggs and King showed that transplantation of nuclei from frog blastulae into enucleated oocytes yields swimming tadpoles. They observed that, irrespective of developmental stage, donor nuclei from a given lineage typically give rise to normal tissues of the same lineage, but to abnormal tissues from other lineages. These experiments led them to conclude that differentiated donor nuclei are restricted in their developmental potential 10. Diberardino demonstrated that differences in cloning efficiency were, in part, related to the cell cycle of the donor nucleus, which had to switch to the rapid cycle typical of the egg 11.Nuclei derived from cells that were further along in development lost totipotency, presumably because they were more differentiated, had slower cell cycles and were therefore more difficult to activate. In other nuclear transplant experiments, Gurdon showed that when tadpoles were used as the source, some normal adult frogs could be produced using nuclei derived from differentiated intestinal endoderm 12. In contrast with Briggs and King, Gurdon heralded these findings as strong evidence for the plasticity of gene expression, and concluded that genes are not permanently inactivated, but instead can be re-awakened, even in differentiated cells. However, some caveats remained: attempts to generate adult frogs from nuclei derived from adult animals were unsuccessful. King favoured the interpretation that reprogramming of differentiated nuclei was limited, and that Gurdon s results most probably represented contaminated transplants that included less differentiated cells 11. Nonetheless, Gurdon s bold interpretation stimulated thinking in the field of differentiation. Remarkably, confirmation of these results in mammals was not forthcoming for decades. Nuclear reprogramming by the cytoplasm: mammalian cells fused in heterokaryons in vitro Despite the experiments in amphibians, 20 years later their relevance to mechanisms of differentiation in mammalian cells had still not gained acceptance. Studies using synkaryons (cell hybrids that proliferate and exhibit nuclear fusion) first showed the existence of trans-acting repressors and activators, but gene activation was only transient because of cell division and the consequent loss of chromosomes These studies were extended by fusion of cultured cells to form multinucleate stable heterokaryons in which each nucleus remained distinct and intact. Such non-dividing cell fusion products allowed changes in gene expression to be monitored over time. Gene expression patterns depended on the nuclear ratio or relative NATURE CELL BIOLOGY VOLUME 6 NUMBER 9 SEPTEMBER

3 a b c Blastula Intestine Oocyte Figure 2 Reprogramming gene expression by nuclear transplantation (cloning) and by experimentally induced heterokaryon formation. (a) Schematic representation of amphibian cloning. Transplantation of nuclei from frog blastulae (Rana pipiens) or from intestinal epithelium (Xenopus laevis) into enucleated oocytes yields tadpoles and, eventually, adult frogs, providing evidence that genetic material is not lost during development. (b) A single heterokaryon, a stable non-dividing mammalian cell hybrid in which the nuclei remain distinct and intact following fusion in culture (top), demonstrates plasticity of gene expression in differentiated mammalian cells in culture. A heterokaryon is shown with human fibroblasts (five diffusely stained nuclei) and mouse muscle (one punctate nucleus). The heterokaryon is stained with an antibody that recognizes human, but not mouse, NCAM, a muscle gene product expressed by the reprogrammed fibroblast nucleus in response to factors in mixed cytoplasm 92. (c) A mouse cloned using the nucleus of an olfactory neuron, a definitive demonstration that nuclei from terminally differentiated cells retain the capacity to be reprogrammed to totipotency 37. gene dosage, and the consequent balance of factors present in the cytoplasm at any given time, showing that differentiation is dynamic and continuously regulated 16,17. In early work with heterokaryons, Ringertz showed that fusion of rat myoblasts and chick erythrocytes caused the nuclei of the erythrocytes to swell and chromatin to become diffuse, presaging reprogramming 18. Primary mouse muscle cells were later fused in tissue culture with human primary diploid cells derived from all three embryonic lineages: endoderm (hepatocytes), ectoderm (keratinocytes) and mesoderm (fibroblasts) (Fig. 2b). Notably, nuclei from each of these cell types were capable of activating a number of previously silent muscle genes 19 22, indicating that the differentiated state could be altered, even in specialized cells derived from adult humans. These experiments provided definitive findings because they used nontransformed cells and incorporated new techniques to inhibit cell division. Similar results were also obtained using other differentiated cell types In vitro studies with heterokaryons established that the expression of genes that had been previously silenced during development and differentiation could be reactivated by cytoplasmic factors present in somatic cells, without the passage of nuclei through the oocyte or embryogenesis that is typical of cloning. That similar fusion events occur naturally in mammals (see below), was not predicted two decades ago. Nuclear reprogramming by the cytoplasm: mammalian cloning Micromanipulation of the much smaller, more delicate mammalian oocyte was pivotal to the success of nuclear transplantation in mammals. Techniques for injecting oocytes, rather than zygotes, and other conditions for cloning mammals were progressively improved in the 1980s and 1990s 27,28, culminating in 1997 with the successful cloning of Dolly, a sheep derived from the transplantation of an adult mammary epithelial cell nucleus into a mammalian oocyte 29.Mammalian cloning has now been replicated in a number of species, including humans, owing to a progression of technological advances The low efficiency of mammalian cloning (less than 1% of all nuclear transfers produce live offspring) left open the question of the cell of origin of successful clones, with the possibility that tissue-specific stem cells were the source of the effective nuclei. That concern was addressed first in cloning experiments using mature B and T lymphocytes, and has recently been laid to rest with the generation of live mice from unambiguous, fully differentiated, post-mitotic olfactory neurons 36,37.These experiments leave no doubt that even terminally differentiated cells in mammals have nuclei that retain the remarkable capacity to be reprogrammed to totipotency and generate all the cell types necessary to form an intact animal (Fig. 2c). From a practical standpoint, the efficiency of mammalian cloning has not improved since the first cloned mammals were reported. In addition, surviving cloned animals often continue to have a high incidence of defects; for example, large offspring, placental problems and arthritis 28.These abnormal phenotypes are most probably the result of epigenetic, rather than genetic, abnormalities, because they are not passed on to the offspring of affected cloned animals. Accordingly, a major effort is being directed at identifying the factors that are responsible for promoting the orderly silencing and reactivation of genes. In summary, we now know that the cytoplasm has the capacity to direct reprogramming and that, at least in oocytes, reprogramming to a totipotent state can occur. Therapeutic cloning through the derivation of embryonic stem cells derived from somatic nuclear transfer into oocytes has already proven to be a viable approach to cell therapy with the cure of Rag2-deficient mice 38. Oocytes have also been produced from existing embryonic stem cell lines, potentially bypassing the need to repeatedly obtain oocytes from live humans for the purpose of cloning, and also avoiding the associated ethical concerns 39. Roughly half a century has passed since the pioneering work of Briggs and King, and that of Gurdon, and although the practical limitations of low efficiency, high cost 40 and an incomplete understanding of reprogramming mechanisms must be overcome, it seems probable that therapeutic cloning will have a major role in regenerative medicine. 812 NATURE CELL BIOLOGY VOLUME 6 NUMBER 9 SEPTEMBER 2004

4 Metaplasia in vivo: evidence from diverse phyla Evidence that nuclear programming occurs naturally in vivo is important, because both cloning and PEG-induced heterokaryon formation are examples of extreme, experimental manipulations. Examples of tissue metaplasia in humans include bone formation in scars and muscle, as well as conversion of one type of epithelium to another in the respiratory tract, the urinary bladder, and in the oesophagus (Barrett s metaplasia) 41.Metaplasia can also be induced experimentally. For example, pancreas tissue can be converted into liver in vitro and in vivo in response to diverse factors In Drosophila melanogaster, conversion of one imaginal disc to another in response to changes in homeotic gene expression is another example of metaplasia 46,47.In urodeles, a differentiated limb muscle cell can de-differentiate and yield muscle, cartilage and connective tissue progeny 48,49, and seminal studies of newt lens regeneration (for a review, see ref. 50) provide the best known examples of transdifferentiation. Like urodeles, zebrafish also have a marked capacity for regeneration, but, as in humans, rigorous documentation of cellular metaplasia in zebrafish by tracking labelled cells, has yet to be reported 51. Finally, in axolotls, individually labelled spinal cord glial cells can de-differentiate after tail amputation, and give rise to mature mesodermal (and neural) progeny, a definitive germ layer switch 52.Thus, in response to injury, there are cells in differentiated tissues that can change and exhibit different phenotypes in lower animals as well as mammals, and the mechanisms may be evolutionarily conserved. Notably, in humans, metaplasia usually occurs in response to pathological conditions (for example, gastroesophageal reflux in Barrett s oesophagus). Indeed, it is unclear whether there are examples in humans of metaplasia as a component of normal development, and the cells responsible for tissue metaplasia in mammals have not been defined. It is possible that reserve cells for each different cell type are present as distinct entities in metaplastic tissues. In contrast, in the mammalian lung, electron microscopy has shown that morphological characteristics and organelles of two different cell types can appear in one cell during metaplastic conversion 53,suggesting that in some cases multiple gene expression patterns are available to a particular cell in response to environmental changes. Taken together, the many examples of metaplasia are a testament to the intrinsic ability of cells to alter significant patterns of the genes they express in response to their in vivo environment. Metaplasia in vivo: BMDC contribution to non-muscle tissues The mammalian examples of metaplasia described above do not involve transcendence of embryonic lineages. Intense investigation in mammals has recently been directed at seeking evidence of metaplasia across embryonic lineages both for fundamental interest and potential therapeutic benefit, as immunological and ethical problems associated with using tissue from another individual could be avoided. This research has primarily focused on BMDCs in adults, which have efficient access to all tissues of the body through the circulation. When the first report appeared demonstrating that bone marrow cells could contribute to muscle in mice, it was unclear whether this was a sporadic event or one of fundamental physiologic significance. Using bone marrow from a transgenic animal expressing the myosin light chain enhancer driving β-galactosidase (β-gal), muscle fibres expressing the reporter gene were found in transplant recipients (Fig. 3a) 54.A number of subsequent reports showed similar findings, using marrow cells marked genetically with β- gal, Y-chromosome and green fluorescent protein. Such cells were reported to be present in the brain, liver, heart, skeletal muscle and epithelia of the kidney, lung and skin The incidence was generally low ( % of total cells) and many of the initial observations were not reproduced 71.Although much of the debate seems to be steeped in semantics, there is justifiable concern about the validity of reported findings, and each should be met with appropriate scepticism, as well as rigorous examination of the stringency of the methodologies used and accuracy of the conclusions drawn. To be conclusive, experimental findings must be replicated by others. The emerging picture is that the contribution of BMDCs to non-haematopoietic tissues is a rare but real event under normal physiological conditions. This contribution can be significantly increased in response to tissue stress or damage; for example, in the liver 59.In another case, damage to muscle either by local toxin injection 61,62 or exercise 58 results in a markedly higher BMDC contribution, and it may be possible to increase this frequency further. Although recognizing that other excellent examples exist, we focus below on two that are illustrative, skeletal muscle and Purkinje neurons, which have been extensively studied by our group. The identification of one cell type within bone marrow with the capacity to contribute to muscle was a critical finding. Because the bone marrow contains stromal cells with the ability to differentiate into various tissues 72,73, it was not clear whether stromal cells were responsible for the BMDC contributions to liver and skeletal muscle after bone marrow transplant (BMT). Single-cell transplants have now shown definitively that the progeny of ckit + Sca1 + Lin HSCs not only reconstitute all lineages of the blood, but also incorporate into muscle in lethally irradiated mice 61,62.Whether additional cells in bone marrow share this capacity and can transit naturally to other tissues through the circulation remains to be determined. BMDCs also contribute to a population of cells that are unique to the cerebellum 65,66, In both mice and humans, BMT-derived nuclei can be found in Purkinje neurons (Fig. 3b). In mice, this is a low frequency event that increases over time after BMT, and most such Purkinje neurons exist as binucleate heterokaryons 79,80. The use of transgenic mice that harbour the Purkinjespecific promoter, L7, showed that BMDC nuclei that are present in heterokaryons exhibit dispersed chromatin and express the L7 promoter 79.This example constitutes the first evidence of in vivo nuclear reprogramming in heterokaryons of the mammalian brain. Heterokaryon formation also occurs in damaged multinucleated muscle fibres. These findings suggest an intriguing mechanism by which complex, post-mitotic structures may incorporate genomic material from endogenous cells in adulthood. The contribution of bone marrow to other tissues seems to represent an additional example of metaplasia in mammals; however, the available data do not address the question of whether BMDC contribution to other tissues is functionally significant in normal life. From a therapeutic standpoint, proof of principle has already been achieved with the survival of tyrosinaemic FAH mice following HSC transplantation (Fig. 3c) 59,63,82. Thus, the degree to which this phenomenon can be manipulated for therapeutic goals is an important line of investigation. Reprogramming in vivo through extracellular signalling or by fusion and cytoplasmic mixing Two different mechanisms have been proposed whereby BMDCs contribute to nonhaematopoietic tissues. Either mechanism ultimately involves nuclear reprogramming (Fig. 1). Initial reports of plasticity in adult stem cells suggested that a developmentally immature BMDC could alter its typical course of differentiation to that of non-haematopoietic tissues 1,73,83. Therefore BMDCs could function as traditional stem cells for other tissues. This first mechanism implies a response NATURE CELL BIOLOGY VOLUME 6 NUMBER 9 SEPTEMBER

5 a b c Figure 3 Reprogramming of bone marrow in non-haematopoietic tissues in vivo. (a) β-gal-positive nuclei in skeletal muscle fibres of a mouse transplanted with marrow from a mouse transgenic for Lac-Z under the control of a muscle-specific promoter, from the first report demonstrating that BMDC can incorporate into skeletal muscle and express a muscle gene in vivo 54. (b) A Purkinje neuron BMDC heterokaryon formed in vivo. The Purkinje neuron in the cerebellum is green owing to green fluorescent protein (GFP) expressed in a mouse transplanted with marrow from a mouse transgenic for GFP (courtesy of C. Johansson). In such binucleate heterokaryons a previously silent Purkinje-promoter reporter gene is activated 79. The inset highlights the presence of two distinct nuclei, as occurs in each Purkinje fusion product. (c) Y-chromosomes detected in the liver of a female Fah / female mouse after HSC transplantation, providing the first evidence that nuclear reprogramming of bone marrow-derived cells can rescue a lethal defect. Top left panel, a section of a liver nodule stained for X-gal; top right panel, fumarylacetoacetate hydrolase (FAH) staining; bottom panel, Y-chromosome fluorescent in situ hybridization 59. to extracellular signals that are detected by the BMDCs and result in an alteration of gene expression and differentiation along an alternative developmental path. Such a process could, in theory, result in the production of mononuclear cells, either directly or through a tissue stem cell intermediate. Examples that have been reported include: BMDC-derived kidney epithelium 56, pulmonary epithelium 56,84 and pancreatic islets 55.Three groups have also reported the discoverey of BMDCs in the satellite cell position (underneath the basal lamina) expressing muscle stem cell markers 58,85,86.Although skeletal muscle may be the most amenable system for discerning stem cell intermediates, it also has inherent complexities, as the end state via stem cell or direct fusion is the same: a multinucleated fibre. The strongest evidence so far that BMDCs can be reprogrammed as mononuclear cells has recently been shown in pulmonary epithelium using the Cre/lox system to exclude fusion 84. As evidence for the second mechanism, fusion, the heterokaryons formed between BMDCs and muscle or BMDCs and Purkinje neurons are perhaps the clearest examples. In the case of fusion, reprogramming is achieved by cytoplasmic factors that may be similar to those associated with reprogramming during cloning, or in the nuclei of heterokaryons formed in vitro.initial reports of contribution by BMDCs to non-haematopoietic tissues by fusion described the phenomenon as mere 87, or random 61.However, time has granted a new respect for fusion. It is becoming clear that the fusion of BMDCs with other tissues is a specific process limited to particular donor and recipient cells. Moreover, it occurs naturally in vivo.a number of molecules have been implicated in cell-cell fusion in eukaryotic species, some of which may function in BMDC fusion. It will be interesting to determine whether fusion mechanisms are evolutionarily conserved, and if cells that are involved in BMDC contribution to other tissues fuse by similar mechanisms. In mammals, why fusion has remained a property of some cells except in the case of muscle, sperm or egg cells, is unclear. We propose that the observed fusion of BMDCs with cells of other tissues may represent a way for cells from different parts of the body with diverse histories to contribute to one another. For example, one could envision a situation where a post-mitotic cell suffers a deleterious loss-of-function mutation and is rescued by expression of a reprogrammed, wild-type copy present in a donated nucleus. If such scenarios occur, a provocative and as yet unanswered question arises: if a BMDC exists, such as myeloid cells 88 90, that can self-renew and yield progeny that are amenable to fusion, reprogramming and rescue of a defunct Purkinje nucleus is it functioning as a Purkinje stem cell? Conclusions Beginning with amphibian research, the past five decades have provided us with a wealth of information, and have demonstrated extraordinary technical achievements. We have learned that: first, genetic material is generally not lost during development and differentiation; second, the cytoplasm of the oocyte as well as somatic cells has the ability to reprogram gene expression; third, most genes, even in terminally differentiated cells, can be reactivated; fourth, a single somatic nucleus has the replicative capacity to yield enough tissue for a whole new organism; and finally, during the lifetime of an organism, cells change their phenotypes, and in some cases nuclear gene expression is reprogrammed across lineages. These findings indicate that the theoretical roadblocks formed on the basis of long-held dogma and old paradigms that might have precluded the use of cells from one source to contribute to another, have been overcome. Nevertheless, the efficiency with which these events occur remains low, and will not be of clinical interest until the incidence of incorporation as well as reprogramming can be increased. Thus, the practical utility of BMDCs and their advantages and disadvantages will become clear, relative to embryonic stem cells and other stem cells, with further experimentation. Another unresolved question regarding the current understanding of the in vivo contribution of bone marrow to other tissues involves the potential for proliferation of reprogrammed cells. The different mechanisms by which BMDCs may contribute to non-haematopoietic tissues are each potentially useful, but have different implications. Differentiation through a stem cell intermediate implies the ability to expand reprogrammed cells. In contrast, direct fusion with post-mitotic cells that cannot proliferate, but form stable heterokaryons, could function as an alternative means of tissue repair. Fusion to form heterokaryons presents the intriguing possibility that a structurally 814 NATURE CELL BIOLOGY VOLUME 6 NUMBER 9 SEPTEMBER 2004

6 complex, non-dividing cell could be rescued by a cell from another tissue. Nuclear donation and subsequent reprogramming may already constitute an elegant solution used by nature in humans and mice, and one that could be capitalized on for therapeutic purposes. In one potential application relevant to either mechanism, BMDCs could be engineered to function as cellular delivery vehicles for therapeutic genes to non-haematopoietic tissues 91. A distinction between the potential mechanisms required for nuclear reprogramming is important to consider when drawing parallels between cloning and heterokaryons formed in vivo.non-dividing heterokaryons observed in vivo are not subject to many of the demands that cloned nuclei are. In addition, the more limited gene expression patterns required by in vivo reprogramming (BMDCs only need to express genes of one different cell type) may have different mechanistic requirements. Whereas the gold standard test for reprogramming in nuclear transplantation is the generation of an animal, the requirements for reprogramming BMDC nuclei are less extensive if not less stringent. If the existence of technological hurdles and questions of degree or low frequency had daunted researchers of the last half century, then cloning would not have persisted beyond a dream or the subject of science fiction. Perhaps the progress of cloning research, resulting in therapeutic (not reproductive) cloning may predict the future with respect to the field of nuclear reprogramming and its applications to regenerative medicine. Similar to cloning, we should approach in vivo reprogramming of BMDCs with vigour, rigor and persistence. ACKNOWLEDGEMENTS We thank M. LaBarge, A. Palermo, R. Doyonnas, T. Brazelton, D. Spiegel and other members of the Blau laboratory for helpful discussions and critical reading of the manuscript. We especially thank C. Johansson for contributing the Purkinje cell image. We apologize to those whose important work we were not able to cover owing to space and reference limitations. J.P. is supported by an NIH training grant (HD 07249) as a postdoctoral fellow at Stanford University, and is a resident in the Division of Plastic and Reconstructive Surgery, Department of Surgery at the University of California, San Francisco (U.C.S.F.). H.M.B. is supported by: NIH grants AG , AG , HD , Ellison AG-SS-0817, the McKnight Endowment Fund for Neuroscience and the Baxter Foundation. 1. Blau, H. M., Brazelton, T. R. & Weimann, J. M. The evolving concept of a stem cell: entity or function? Cell 105, (2001). 2. Weissman, I. L. Stem cells: units of development, units of regeneration, and units in evolution. Cell 100, (2000). 3. Alonso, L. & Fuchs, E. Stem cells of the skin epithelium. Proc. Natl Acad. Sci. USA 100, Watt, F. M. & Hogan, B. L. Out of Eden: stem cells and their niches. Science 287, (2000). 5. 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