REVIEW Hematopoietic, vascular and cardiac fates of bone marrow-derived stem cells

Transcription

1 (2002) 9, Nature Publishing Group All rights reserved /02 $ REVIEW Hematopoietic, vascular and cardiac fates of bone marrow-derived stem cells KK Hirschi 1 and MA Goodell 2 1 Departments of Pediatrics and Molecular and Cellular Biology, Children s Nutrition Research Center, Baylor College of Medicine, Center for Cell and, Houston, TX, USA; and 2 Department of Pediatrics, Immunology and Human and Molecular Genetics, Baylor College of Medicine, Center for Cell and, Houston, TX, USA Bone marrow contains many cell types, including stroma, vascular cells, adipocytes, osteoblasts and osteoclasts, as well as mesenchymal stem cells and hematopoietic stem cells. It was previously thought that cells within bone marrow solely functioned to regenerate cells within the marrow, as well as all circulating hematopoietic cells in peripheral blood. Recent reports, however, suggest that marrow-derived cells can also regenerate other cell types, including cardiac muscle, liver cell types, neuronal and non-neuronal cell types of the brain, as well as endothelial cells and osteoblasts. These multiple cell types could have originated from either of the stem cell populations within bone marrow or potentially other precursors. Therefore, it is not entirely clear whether each of these distinct cell lineages has a true progenitor within marrow or whether the marrow contains a multipotent population of cells that has been set aside during embryogenesis for postnatal repair and remodeling of a variety of tissues. It is clear, however, that directing the fate of bone marrowderived progenitors (ie toward hematopoietic, vascular or cardiac cell fates) can only be accomplished if the phenotype of the stem cells is defined, and their homing and differentiation programs are elucidated. Much work is focused on these issues, wherein lie the key to harnessing the potential of adult stem cells for autologous cell and gene therapy. (2002) 9, DOI: /sj/gt/ Keywords: bone marrow; hematopoietic stem cells; vascular progenitors; muscle progenitors; stem cell plasticity Introduction Until recently, stem cells from adult tissues have been thought to differentiate exclusively into cell types of their tissue of origin. Evidence is now accumulating that at least some stem cells may differentiate down alternative pathways when introduced into a regenerating environment. One of the first indications that this might occur came from studies in which whole bone marrow was transplanted into lethally irradiated recipients and allowed to engraft and reconstitute the hematopoietic system. After injury introduced into the tissue of interest, donor-derived cells were found to be incorporated into the regenerated tissue. After transplanting bone marrow marked with a LacZ transgene expressed under the control of a muscle-specific promoter, 1 Ferrari et al found galactosidase-positive muscle fibers 2 weeks after a necrotizing injury was induced with cardiotoxin. Although the LacZ-positive muscle fibers were very rare (around %), their data clearly indicated that cells from the bone marrow could migrate to the site of injury and participate in skeletal muscle repair. This work was followed by other reports that cells from the bone marrow could differentiate into cardiac muscle, 2 endothelial cells, 3 osteoblasts, 4 liver cell types, 5 7 and neuronal 8,9 and non-neuronal 10 cell types of the Correspondence: KK Hirschi, Baylor College of Medicine, Center for Cell and, One Baylor Plaza, Houston, TX 77030, USA brain. The bone marrow contains many cell types, and it was not clear which cells could account for the multiple activities. In addition to the differentiated cell types that comprise the bone marrow (eg stroma, vascular cells, adipocytes, osteoblasts and osteoclasts), both mesenchymal stem cells and hematopoietic stem cells (HSC) reside within the marrow and are transferred during bone marrow transplantation. Therefore, these multiple cell types that were found to be donor-derived after bone marrow transplantation could have originated from either of these stem cell types, or potentially other precursors. While these data generated excitement about the possibility that multiple cell types could be generated from one type of stem cell, the HSC, similar data with highly purified stem cells, and ultimately clonal analysis, are necessary to confirm this. Below, we will review the evidence that HSC can generate cardiomyocytes and endothelial cells, in addition to hematopoietic cells. Hematopoietic stem cells HSC have been characterized in mice and humans by cell sorting and transplantation. They reside in the bone marrow in adults and, via a population of committed progenitors, constantly replenish the differentiated cells of the peripheral blood. In the mouse, HSC can be highly enriched or purified using a combination of cell surface markers such as the stem cell antigen, Sca-1, 11 the tyrosine kinase receptor c-kit 12 and low or negative levels of lineage markers (lin /low ). 13 Alternatively, HSC can be

2 highly enriched using fluorescent vital dyes such as Hoechst which are actively effluxed from primitive cells, 14 thus defining the so-called side population, or SP. Using these purification strategies alone or in combination, populations of highly purified and homogeneous murine HSC can be obtained, and the hematopoietic potential of these populations has been extensively characterized. Human HSC have been isolated primarily through their expression of the marker CD34, 15 lack of lineage markers and low expression levels of Thy1, 16 although there is also evidence for a CD34-negative HSC precursor. 17,18 Although over the years, the ability of HSC to give rise to stromal cell types resident in the marrow has been debated, the dogma that HSC can only give rise to hematopoietic cells has stood until recently. Vascular fates of circulating and marrowderived precursors Unlike the continuous process of blood regeneration in adults, the generation of new blood vessels is thought to occur only in response to tissue injury or growth, such as during wound healing, and in pregnancy and lactation. Pathological processes including tumorigenesis and atherosclerosis, on the other hand, are associated with the synthesis, mobilization and/or activation of growth factors, cytokines and proteases that stimulate abnormal vascular cell replication and migration. The ensuing pathological vascularization plays a central role in the progression of such prevalent diseases. 19 It was previously thought that new blood vessels formed during postnatal neovascularization, in normal and pathological conditions, were solely derived from the pre-existing vasculature. The process of sprouting or remodeling of established vessel structures, to form new ones, is referred to as angiogenesis. 20 Recent studies, however, indicate that vascular cell progenitors are present in blood circulation 21,22 and resident in bone marrow, 23,24 and contribute to blood vessel formation during tissue repair and in pathological conditions. Furthermore, the neovascularization process that involves the coalescence of circulating and marrow-derived precursors, is thought to more closely resemble embryonic vasculogenesis rather than angiogenesis. 25 Endothelial cells Precursors to vascular endothelial cells in adults were first identified in blood circulation 26 and have been shown to engraft into vascular endothelium to a level of ~10%. 27 Progenitors isolated from bone marrow have reported incorporation rates ranging from ~3 23 to 25%. 24 Such contributions of adult vascular progenitors to injury-induced neovascularization have been associated with enhanced blood flow, 28 and improved function 24 of ischemia-damaged tissues. The apparent variation of engraftment efficiency of isolated progenitors can be due to the fact that the majority of the precursor populations in blood and bone marrow are heterogeneous, and some likely contain a higher proportion of cells committed to a vascular lineage versus hematopoietic or other cell lineage. Interestingly, more heterogeneous populations tend to engraft to a higher degree into vessel structures. Therefore, it may also be that highly purified populations do not contain other cell types that provide instructive signals to the multipotent progenitors. Thus, the molecular phenotype of true vascular progenitor cells, and the mechanisms by which they are induced to become vascular cells in vivo, remain to be determined. Such insights are necessary to effectively direct the fate of adult stem cells for vascular cell and gene therapy. Toward this goal, we examined the vascular potential of SP cells, highly enriched for HSC, 14 from adult mouse bone marrow. After bone marrow transplantation and cardiac injury, we found LacZ-marked endothelial progeny of SP cells in vessels in the region of injury repair. 23 This is intriguing given that developmental studies suggest hematopoietic and endothelial cells share a common precursor, 29 and may suggest such relationships can be retained in the adult. To prove definitively that a single SP cell can generate both hematopoietic and endothelial cells, clonal analysis of transplanted cells, 30 or retroviral lineage marking 31,32 must be performed. In support of the potential relationship between marrow SP cells and endothelial progenitors, we have found some similarities in the phenotype of adult bone marrow SP cells 23 and embryonic hemangioblasts. 33 Importantly, bone marrow-derived SP cells do not express genes reflective of mature endothelial cells (Flt-1, VE-cadherin, von Willebrand factor, factor VIII), smooth muscle cells (SM- -actin, calponin, SM22 ) or cardiac muscle cells ( actinin, desmin). 23 Bone marrow-derived SP cells do, however, express some genes shared by embryonic hematopoietic and vascular cell progenitors, including PE- CAM-1 34 and Tal Surprisingly, other genes thought to be specifically reflective of an embryonic blood and endothelial cell progenitor, such as CD34 36 and tyrosine kinase receptor Flk-1 33 are not expressed in SP cells. However, expression of the Flk-1 ligand, VEGF-A, is detected. 23 Bone marrow SP cells, as HSC, are predisposed to differentiate into hematopoietic cells, but with some lower efficiency can generate endothelial cells after an injury. If cells with similar potential can be reproducibly isolated from adult human bone marrow and their vascular potential enhanced by ex vivo manipulation, the development of autologous cell and gene therapy strategies to modulate postnatal neovascularization and hematopoiesis will be accelerated. Smooth muscle cells Evidence that endothelial cell progenitors reside in blood circulation and bone marrow is increasing. Such evidence of the existence of similar mural cell (smooth muscle cell and pericyte) progenitors has been slower to accumulate, perhaps reflecting a lower frequency. Vascular smooth muscle cells and pericytes were previously thought to be recruited via endothelial-derived signals 37,38 from local mesenchyme in the region of neovascularization, or recruited along with endothelial cells from pre-existing vessels 39 to form the surrounding vessel wall as new vessels sprout. However, recent findings suggests that blood circulation also contains a smooth muscle cell progenitor population. 40 The molecular identity of the circulating mural cell precursor remains to be determined. It must also be determined whether endothelial and mural cells arise from a common progenitor in blood and bone marrow. A recent study of embryonic stem cells provides evidence that this 649

3 650 may be the case. Yamashita and coworkers 34 found that embryonic stem cells expressing the VEGF receptor Flk- 1, which is expressed early in vascular development by hemangioblasts, as well as mature endothelial cells, 33 give rise to vascular smooth muscle cells in vivo and in vitro. These studies suggest that stem cells identified in adult tissues as endothelial cell precursors may serve as smooth muscle cell precursors, as well. Isolation and characterization of distinct progenitor cell types, along with investigation of their vascular potential in vivo, are needed to address this issue. Cardiac fates of circulating and marrowderived precursors Relative to the regeneration of blood and blood vessels, regeneration of cardiac tissue is far less frequent. The heart is one of the few organs of the body that does not appear to contain a resident stem cell population on call to repair damage, although there is some evidence to the contrary. 41 Perhaps this is why cardiovascular disease, resulting in chronic ischemia and myocyte degeneration, is among the leading causes of death. Since adult cardiac tissue appears unable to adequately repair itself in response to injury, several attempts have been made to use other cell types to generate adult cardiac myocytes. The cell populations examined include fetal and neonatal cardiac myocytes, skeletal myoblasts, 45 and cardiomyocytes differentiated from embryonic stem cells. 46 These pioneering studies using skeletal myoblasts or neonatal myocytes directly injected into or grafted on to injured hearts improved contractile strength, but failed to produce cardiac myocytes that are electrically coupled to pre-existing muscle fibers. 45 While grafted mouse fetal cardiomyocytes appeared to form intercalated disks with adjacent cells and were electrically coupled, they are not a practical source of cardiac tissue for clinical use. 42 More recent studies have focused on the use of bone marrow-derived cells as a renewable and readily accessible source for generating cardimyocytes. In Orlic and coworkers studies, 47 GFP-marked bone marrow cells enriched for HSC were injected directly into heart tissue that had immediately before undergone coronary artery ligation, producing an ischemic region. Nine days after transplantation, the injected regions displayed large numbers of GFP-marked cells which expressed cardiac myosin and some of which had markers of immature cardiomyocytes. In another study involving the direct injection of marrow-derived progenitors into injured hearts, 24 decreased apoptosis of cardiomyocytes and improved cardiac function was reported. In this case, the beneficial effects of the transplanted marrow-derived cells were attributed to enhanced neovascularization, and not direct regeneration of cardiac myocytes. In our studies, LacZ-marked bone marrow SP cells were first stably engrafted into the hematopoietic system via bone marrow transplantation. Two to 4 weeks after cardiac injury consisting of coronary artery occlusion followed by reperfusion, sections of the heart revealed LacZpositive cells that lacked hematopoietic markers and instead expressed -actinin, indicative of a cardiac muscle fate. 23 As discussed above, marked cells were also found in vascular endothelium. Engraftment was primarily found in the repairing region adjacent to the induced fibrotic scar. While these studies suggest that SP cells or their progeny can migrate to the site of injury and participate in cardiac repair, further studies are needed to assess both the functional integration of the participating cells and the exact cell types involved, whether migrating HSC or other progenitors. Engraftment levels observed in our model were low: around 0.02% of cardiomyocytes were derived from the SP cells, which is too low to significantly contribute to functional restoration of the injured tissue. However, methods that increase the presence of the progenitors at the site of injury, or increase the differentiation to cardiomyocytes may prove useful as adjunct therapy to existing strategies. Along these lines, Orlic and co-workers reported that G-CSF administered several days before and following myocardial infarction in mice reduced both infarct size and mortality. 48 They propose that this is due to G-CSFmediated mobilization of a stem cell population from the bone marrow to the site of injury. However, secondary effects of G-CSF administration, such as perhaps increased numbers of red blood cells and hence oxygen perfusion of the tissue, could have similar effects. The use of mobilized stem cells that are marked with a transgene or retrovirus should reveal the basis of the G-CSF effect. Regardless of its mechanism of action, since G-CSF is already approved for clinical use, its use in patients at high risk for myocardial infarction is already being explored in clinical trials. While several stem cell sources have now been shown to be capable of generating cardiomyocytes, their clinical utility will depend on a variety of factors, including efficiency of generation of tissue and accessibility of the stem cell population. The isolation of human embryonic stem cells 49 and their propensity to differentiate into cardiac muscle 50 make these cells an important potential source. In addition, marrow-derived hematopoietic progenitors, as well as mesenchymal stem cells 51 are potential sources. Further characterization of both the stem cells and the engrafted cells is essential, as well as an evaluation of the contribution of engrafted cells to improved function. Harnessing the potential of adult stem cells for autologous cell and gene therapy Adult stem cells are, by definition, capable of becoming a variety of differentiated cell types in vivo. Although stem cells derived from adult tissues have not been definitively shown to exhibit pluripotent capabilities like their embryonic counterparts, their accessibility for autologous therapies in adult patients offers them a distinct advantage over embryonic stem cells. The keys to harnessing the potential of these progenitors will be reproducible isolation, characterization, and direction toward specific fates in vitro and in vivo. In addition, the engraftment levels currently observed will have to be enhanced for maximum utility. This will likely come from better understanding of both the environmental factors that allow engraftment in an injured site, as well as signals that draw stem cells into the regeneration process, either from the circulation or from local residence. In addition, improvements in efficiency of engraftment can result from modification of the stem cells; understanding the mechanisms involved may allow us to selectively direct the fate to specific lineages of interest.

4 Directing the fate of multipotent bone marrow-derived progenitors specifically toward hematopoietic, vascular or cardiac cell fates can only be accomplished if the phenotype of the stem cells is defined, and their homing and differentiation programs are elucidated. Although much has been learned in recent years about the genes that regulate these cell lineages in the embryo, virtually nothing is known about the molecular profile of progenitors in the adult or whether insights gained from embryonic systems can be directly applied to the manipulation of adult progenitors. Finally, manipulation of stem cells ex vivo offers the opportunity of introducing genes of therapeutic value before transplantation. Some stem cell types may be more readily transducible than others, so study in this area is also warranted. We may ultimately have several powerful options for cadiovascular cell and gene therapy. Acknowledgements MAG is a Scholar of the Leukemia and Lymphoma Society. We thank members of the Hirschi and Goodell laboratories for helpful discussions. References 1 Ferrari G et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 1998; 279: Bittner RE et al. Recruitment of bone-marrow-derived cells by skeletal and cardiac muscle in adult dystrophic mdx mice. Anat Embryol (Berl) 1999; 199: Shi Q et al. Evidence for circulating bone marrow-derived endothelial cells. Blood 1998; 92: Horwitz EM et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 1999; 5: Petersen BE et al. Bone marrow as a potential source of hepatic oval cells. Science 1999; 284: Theise ND et al. Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation. Hepatology 2000; 31: Theise ND et al. Liver from bone marrow in humans. Hepatology 2000; 32: Brazelton TR, Rossi FM, Keshet GI, Blau HM. From marrow to brain: expression of neuronal phenotypes in adult mice. Science 2000; 290: Mezey E et al. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 2000; 290: Eglitis MA, Mezey E. Hematopoietic cells differentiate into both microglia and macroglia in the brains of adult mice. Proc Natl Acad Sci USA 1997; 94: Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells (published erratum appears in Science 1989; 244: 1030). Science 1988; 241: Ogawa M et al. Expression and function of c-kit in hemopoietic progenitor cells. J Exp Med 1991; 174: Uchida N, Weissman IL. Searching for hematopoietic stem cells: evidence that Thy-1.1lo Lin- Sca-1+ cells are the only stem cells in C57BL/Ka-Thy-1.1 bone marrow. J Exp Med 1992; 175: Goodell MA et al. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 1996; 183: Berenson RJ. Transplantation of CD34+ hematopoietic precursors: clinical rationale. Transplant Proc 1992; 24: Baum CM et al. Isolation of a candidate human hematopoietic stem-cell population. Proc Natl Acad Sci USA 1992; 89: Goodell MA et al. Dye efflux studies suggest the existence of CD34-negative/low hemaptopoietic stem cells in multiple species. Nat Med 1997; 3: Bhatia M et al. A newly discovered class of human hematopoietic cells with SCID- repopulating activity. Nat Med 1998; 4: Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000; 407: Risau W. Differention of endothelium. FASEB J 1995; 9: Murohara T et al. Tranplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization. J Clin Invest 2000; 105: Lin Y, Weisdorf DJ, Solovey A, Hebbel RP. Origins of circulating endothelial cells and endothelial outgrowth from blood. J Clin Invest 2000; 105: Jackson KA et al. Regeneration of cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 2001; 107: Kocher AA et al. Neovascularization of ischemic myocardium by human bone marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 2001; 7: Springer ML et al. VEGF gene delivery to muscle: potential role for vasculogenesis in adults. Cell 1998; 2: Asahara T et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997; 275: Crosby JR et al. Endothelial cells of hematopoietic origin make a significant contribution to adult blood vessel formation. Circ Res 2000; 87: Schatteman GC et al. Blood-derived angioblasts accelerate blood flow restoration in diabetic mice. J Clin Invest 2000; 106: Keller G. The hemangioblast. In: Marshak DR, Gardner RL, Gottlieb D (eds). Stem Cell Biology. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 2001, pp Osawa M, Hanada K, Hamada H, Nakauchi H. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 1996; 273: Jordan CT, Lemischka IR. Clonal and systemic analysis of longterm hematopoiesis in the mouse. Genes Dev 1990; 4: Lemischka IR, Raulet DH, Mulligan RC. Developmental potential and dynamic behavior of hematopoietic stem cells. Cell 1986; 45: Drake CJ, Fleming PA. Vasculogenesis in the day 6.5 to 9.5 mouse embryo. Blood 2000; 95: Yamashita J et al. Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature 2000; 408: Kallianpur AR, Jordan JE, Brandt SJ. The SCL/Tal-1 gene is expressed in progenitors of both the hematopoietic and vascular systems during embryogenesis. Blood 1994; 83: McClanahan T, Dalrymple S, Barkett M, Lee F. Hematopoietic growth factor receptor gene as markers of lineage commitment during in vitro development of hematopoietic cells. Blood 1993; 81: Hirschi KK, Rohovsky SA, D Amore PA. PDGF, TGF-ß and heterotypic cell-cell interactions mediate the recruitment and differentiation of 10T1/2 cells to a smooth muscle cell fate. J Cell Biol 1998; 141: Hirschi KK et al. Endothelial cells modulate the proliferation of mural cell precursors via PDGF-BB and heterotypic cell contact. Circ Res 1999; 84: Lindahl P, Hellstrom M, Kalen M, Betsholtz C. Endothelial-pervascular cell signaling in vascular development: lessons from knockout mice. Curr Opin Lipid 1998; 9: Shimizu K et al. Host bone marrow cells are a source of donor intimal smooth muscle-like cells in murine aortic transplant arteriopathy. Nat Med 2001; 7: Beltrami AP et al. Evidence that human cardiac myocytes divide after myocardial infarction. N Engl J Med 2001; 344: Soonpaa MH, Koh GY, Klug MG, Field LJ. Formation of nascent 651

5 652 intercalated disks between grafted fetal cardiomyocytes and host myocardium. Science 1994; 264: Koh GY et al. Stable fetal cardiomyocyte grafts in the hearts of dystropic mice and dogs. J Clin Invest 1995; 96: Zhang M et al. Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies. J Mol Cell Cardiol 2001; 33: Reinecke H, MacDonald GH, Hauschka SD, Murry CE. Electromechanical coupling between skeletal and cardiac muscle: implications for infarct repair. J Cell Biol 2000; 149: Klug MG, Soonpaa MH, Koh GY, Field LJ. Genetically selected cardiomyocytes from differentiation embryonic stem cells form stable intracardiac grafts. J Clin Invest 1996; 98: Orlic D et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001; 410: Orlic D et al. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci USA 2001; 98: Thomson JA et al. Embryonic stem cell lines derived from human blastocysts. Science 1998; 282: Kehat I et al. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest 2001; 108: Pittenger MF et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284: