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Stem cells in the treatment of muscle and connective tissue diseases
329
Stem cells in the treatment of muscle and connective tissue diseases Hairong Peng and Johnny Huardy Current data indicate the existence of two types of postnatal stem cells. Tissue non-specific stem cells are haematopoietic in origin and can differentiate into different blood lineages. In contrast, tissue-specific stem cells preferentially differentiate into cells of the residing tissue, although they also possess a limited ability to turn into other lineages. In terms of therapeutic potential, unmodified muscle-derived stem cells have been proven capable of regenerating dystrophic muscle. Furthermore, when genetically modified to express growth factors, these cells are versatile in promoting bone healing. This also occurs with mesenchymal stem cells, which have been used in an attempt to repair defects of cartilage and ligaments. Thus, stem-cell-based therapy — particularly genetically engineered therapy — holds great potential for the treatment of a variety of disorders and conditions affecting the muscle and connective tissue. Addresses Growth and Development Laboratory, Department of Orthopaedic Surgery, Children’s Hospital of Pittsburgh, University of Pittsburgh, Pittsburgh, PA, USA y Director of the Growth and Developmental Laboratory, Departments of Orthopaedic Surgery, Molecular Genetics and Biochemistry, and Bio-engineering, University of Pittsburgh, 4151 Rangos Research Center, Children’s Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213, USA Correspondence: Johnny Huard; e-mail: jhuardþ@pitt.edu
Current Opinion in Pharmacology 2003, 3:329–333 This review comes from a themed issue on Musculoskeletal pharmacology Edited by Kay E Davies and Kevin Talbot 1471-4892/03/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved.
embryonic stem cells are the most versatile when used for the regeneration or repair of different tissues because of their multipotentiality. However, these cells must be used in the form of allografts unless therapeutic cloning techniques are utilized to generate autologous embryonic stem cells. Postnatal stem cells can be used for autologous transplantation, and thus are the focus of this paper. In the past year, scientists have made remarkable progress in assessing the characteristics of postnatal stem cells and the potential use of these cells for treatment of muscle and connective tissue diseases. This review summarises studies that generated valuable findings concerning the nature of postnatal stem cells and their uses in this exciting field of research.
Plasticity of postnatal stem cells Despite early reports indicating the plasticity of postnatal stem cells, new studies suggest that the interpretation of these dramatic findings warrants caution. Several researchers have suggested that these earlier evaluations of postnatal stem cell plasticity might have mistaken cellfusion for true multipotent differentiation [1,2]. Furthermore, a recent stringent study using single-cell transplantation generated little evidence of differentiation of haematopoietic stem cells into other lineages. The implantation of a single haematopoietic stem cell into lethally irradiated mice led to reconstitution of the haematopoietic system of each recipient; however, researchers observed no appreciable contribution of the donorderived cells to non-haematopoietic tissues, including brain, kidney, gut, liver and muscle [3]. Taken together, these studies indicate that the ability of haematopoietic stem cells to differentiate into cells of non-haematopoietic lineages is very limited.
DOI 10.1016/S1471-4892(03)00051-1
Abbreviations BMP bone morphogenetic protein MDSCs muscle-derived stem cells VEGF vascular endothelial growth factor
Introduction Most of the research on stem cells has focused on either embryonic or postnatal stem cells. Embryonic stem cells are isolated from early embryos and generally possess the potential to differentiate into various lineages. In contrast, postnatal stem cells can be isolated from the tissues of individuals of any age; however, their capacity for multilineage differentiation might be limited, as discussed in this review (Figure 1). From a therapeutic point of view, www.current-opinion.com
It remains unclear whether stem cells isolated from other tissues are tissue-specific or multipotent (i.e. able to differentiate into different lineages). Muscle-derived stem cells (MDSCs) isolated by researchers in our laboratory have been shown to express cellular markers of endothelial or neuronal cells upon stimulation with vascular endothelial growth factor (VEGF) or nerve growth factor, respectively [4]. Additionally, the localisation of b-galactosidase-labelled cells in vascular structures and peripheral nerves following intramuscular implantation of b-galactosidase-labelled MDSCs suggests in vivo differentiation of the implanted MDSCs toward endothelial and neuronal lineages [4]. The ability of these MDSCs to differentiate into other lineages, however, is quite limited compared with their capacity for myogenic differentiation. Current Opinion in Pharmacology 2003, 3:329–333
330 Musculoskeletal
Figure 1
All lineages of the body
Embryonic stem cells
Tissue non-specific stem cells
Post natal stem cells
Stem cells of haematopoietic origin
Haematopoietic lineages Other lineages
Stem cells of mesenchymal origin
Osteogenic, adipogenic, myogenic and chondrogenic lineages
Other lineages Tissue-specific stem cells
Tissue-specific lineages
Main differentiation pathway upon proper induction Minor differentiation pathway upon proper induction Current Opinion in Pharmacology
Stem cells and their possible pathways of differentiation.
Tissue-specific and non-specific stem cells in postnatal tissues At least two populations of stem cells are thought to exist in most types of postnatal tissue. The first stem cell population appears to be present in all tissues, and is most likely haematopoietic in origin. The second population comprises tissue-specific stem cells, which preferentially differentiate into cells of the same type as those constituting the tissue in which they reside, although these stem cells also possess a limited ability to transdifferentiate into cells of other lineages (Figure 1). In muscle, for example, stem cells of haematopoietic origin are CD45þ and have the capacity to differentiate into blood cells; these cells undergo very limited myogenic differentiation in vivo and no myogenic differentiation in vitro [5,6]. These so-called side population cells can be isolated using FACS by virtue of dye exclusion [5,6]. Side population cells have been identified in a variety of postnatal tissues, including tissues of the brain, heart, lung, spleen, kidney, small intestine and sketetal muscle [7]. In contrast to these non-tissue-specific MDSCs, tissue-specific cells are CD45– and have the ability to differentiate into myogenic lineage both in vitro and in vivo [5]. The CD45 MDSCs differ from satellite cells (a group of quiescent myogenic progenitors that normally reside underneath the basement membrane of myofibers) [4,6], although the latter also display the capacity to differentiate Current Opinion in Pharmacology 2003, 3:329–333
into cells other than myoblasts, such as osteoblasts and adipocytes [8]. Interestingly, undifferentiated progenitor cells derived from single satellite cells co-express the transcription factors MyoD and Runx2, which are thought to determine myogenic and osteogenic differentiation, respectively. Whether this finding indicates the intrinsic potential of satellite cells to undergo either osteogenic or myogenic differentiation, or a shared regulatory mechanism between these two phenotypically different pathways, remains to be determined.
Stem cell therapy to improve bone healing Significant progress has been made in the application of stem cells — most notably MDSCs — to promote or improve bone healing. MDSCs transduced with a retroviral vector expressing bone morphogenetic protein (BMP)4 undergo osteogenic differentiation both in vitro and in vivo, and can heal bone defects in immunocompetent animals [9]. Bone formation and healing elicited by stem cells expressing BMP4 can be enhanced if the bone defect is also treated using stem cells expressing VEGF (Figure 2) [10]. Intriguingly, the synergistic effect of these two growth factors depends critically on their ratio, with an improper ratio leading to detrimental effects on the bone healing process [10]. The feasibility of using transduced marrow stromal cells expressing BMP4 to enhance bone healing has been confirmed recently [11]. In addition, new scaffold materials, such as atelopeptide type I collagen, www.current-opinion.com
Stem cells in the treatment of muscle and connective tissue diseases Peng and Huard 331
Figure 2
(b)
Relative bone area
(a)
B4
B4+Ve
∗
30 20 10 0
B4 BMP4
B4+Ve
BMP4+VEGF
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
7d
10d
10d
14d
VEGF enhances endochondral bone formation elicited by MDSCs expressing BMP4. (a) Radiograph shows augmented bone formation in the BMP4þVEGF site (B4þVe) compared with the BMP4 site (B4) four weeks after implantation. (b) Quantitative analysis shows that the BMP4þVEGF group produced significantly more bone than the BMP4 group (*P < 0.01, n¼4). (c–f) Alcian Blue staining shows enhanced cartilage formation (purple) at 7 days, and accelerated cartilage resorption at 10 days in the BMP4þVEGF group compared with the BMP4 group. (g–j) von Kossa staining shows more extensive cartilage mineralization (black) at 10 days, and increased mineralized bone formation (black) at 14 days in the BMP4þVEGF group compared with the BMP4 group. Magnification: (c,d) and (g–j), 100x; (e,f), 200x. Republished with permission from The Journal of Clinical Investigation.
have been utilised in conjunction with marrow stromal cells to evaluate whether this combination bolsters the osteogenic effects of BMP2 [12]. However, it is worth noting that stem cells alone, either MDSCs or marrow stromal cells, cannot improve the healing of critical-sized bone defects [10,11]. This limitation can apparently be overcome through genetic engineering of the stem cells to express specific growth factors. www.current-opinion.com
Stem-cell-based gene therapy has proven to be a powerful method to investigate the mechanisms of bone formation and healing using both gain-of-function and loss-of-function approaches. A loss-of-function design was used to demonstrate that VEGF activity is important for endochondral bone formation elicited by BMP4. Stem cells expressing a VEGF specific antagonist, soluble Flt1, inhibited ectopic bone formation induced by BMP4 by Current Opinion in Pharmacology 2003, 3:329–333
332 Musculoskeletal
both reducing cartilage formation and delaying cartilage resorption [10]. Utilisation of a gain-of-function approach revealed a synergistic relationship between BMP4 and VEGF in stimulating bone formation, a process found to involve multiple mechanisms in endochondral pathways (Figure 2). These include an increase in stem cell recruitment to enhance cartilage formation, enhanced angiogenesis to accelerate cartilage resorption, and improved cell survival at the bone formation site [10]. An increase in the proliferation potential of stromal stem cells can also lead to improved bone formation. Like other types of stem cells (e.g. haematopoietic stem cells), marrow stromal stem cells have a relatively short life-span. This limitation has recently been overcome through forced expression of telomerase transcriptase in stromal cells [13]. Following implantation into immunodeficient animals, these transfected cells produce more bone than do non-transfected cells. Interestingly, such enhanced bone formation correlated with an increase in the expression of STRO-1, an early pre-osteogenic stem cell marker, in the transfected cells. These results indicate that optimal bone formation depends on a high capacity for cell proliferation and preservation of progenitor cell characteristics.
Cartilage repair using stem cells Research into stem-cell-based cartilage repair has focused primarily on two areas: firstly, the construction of tissueblocks that simulate cartilage; and secondly, the identification of growth factors with the potential to induce chondrogenic differentiation, and the characterisation of gene expression profiles during this process in vitro. In the first of these approaches, different polymers were used to fabricate three-dimensional constructs, which were subsequently seeded with mesenchymal stem cells and cultured in chondrogenic medium to induce chondrogenic differentiation. Human mesenchymal stem cells seeded in constructs created by press-coating D,D-L,Lpolylactic acid polymer blocks were found to undergo chondrogenic differentiation when cultured in chondrogenic medium for three weeks [14]. More importantly, the cell layers present in the construct were well organised, with the superficial layer resembling compact hyaline cartilage [14]. However, it remains to be determined whether such a construct formed in vitro could integrate with surrounding tissues and maintain the structure to a degree comparable to that of normal articular cartilage following implantation into a cartilage defect. The other primary aim of stem-cell-related cartilage research is to identify growth factors with the potential to promote chondrogenic differentiation. BMP2 and BMP9 have been shown to induce chondrogenic differentiation of human mesenchymal stem cells, as indicated by the expression of type II mRNA and the increased expression of aggrecan and cartilage oligomeric matrix proteins [15]. Current Opinion in Pharmacology 2003, 3:329–333
Such an increase in the expression of chondrogenic proteins is probably attributable to the activation of Sox 9, a transcription factor involved in the regulation of chondrogenesis. These growth factors are also able to partially block the inhibitory effects on chondrogenic differentiation mediated by interleukin-1, a major cytokine implicated in the pathogenesis of osteoarthritis [15]. Gene expression profiles related to the in vitro chondrogenic differentiation of human adult bone marrow stromal stem cells have been investigated recently [16]. However, it is unknown whether these in vitro results accurately portray in vivo occurrences.
Muscle regeneration using stem cells Finding a way to treat severe muscle diseases, most notably muscular dystrophy, continues to pose a formidable challenge. Direct gene-transfer mediated by viral vectors (particularly adeno-associated viral vectors) offers the greatest potential as treatment for Duchenne Muscular Dystrophy, the most common muscle disease. However, stem-cell-mediated therapy could also play an important role in the treatment of patients during later stages of the disease, which are characterised by depletion of endogenous MDSCs, secondary to repeated injury and regeneration. Researchers in our laboratory have recently isolated a novel population of MDSCs that proliferate in vitro for a prolonged period of time and can differentiate into muscle, neural and endothelial lineages both in vitro and in vivo [4]. More importantly, these cells are able to restore dystrophin expression upon implantation into MDX mice (an animal model of Duchenne Muscular Dystrophy) with a degree of efficiency unrivaled by myoblast or satellite cell transplantation [4]. This superior transplantation capability of MDSCs was shown to be a result of their immune privilege (failure to trigger a strong immune response compared to myoblasts [4]), self-renewal ability and multipotential differentiation [4]. In addition to the isolation of long-term proliferating cells in our laboratory, efforts have been made to identify cellular properties that can predict heightened transplantation efficiency, which is indicated by the ability of cells to reconstitute dystrophin expression in muscle of MDX mice. We have determined that CD34þ MDSCs displaying delayed adhesion to collagen type I have a better regeneration capacity than do either CD34þ MDSCs characterised by early adhesion to collage type 1, or CD34 MDSCs displaying delayed adhesion [17]. This superior restoration capacity correlates positively with the proliferation potential of MDSCs [17].
Other connective tissues Recently, a study was conducted to investigate the possible development of a synthetic structure that could be used to repair injured ligament. The creation of such an implant poses an enormous challenge, as the material would need to sustain the tremendous mechanical stress www.current-opinion.com
Stem cells in the treatment of muscle and connective tissue diseases Peng and Huard 333
Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration. J Cell Biol 2002, 157:851-864.
typically absorbed by normal ligament and, at the same time, be able to integrate with the existing ligament or bone. In an attempt to develop materials that could be used for reconstruction of the anterior cruciate ligament, researchers seeded a silk-fibre matrix with human bone marrow stromal cells [18]. In vitro culture indicated that this new matrix would provide sufficient support during the attachment, proliferation and differentiation of the cells; however, the in vivo performance of ligaments engineered by this method has not been evaluated.
McKinney-Freeman SL, Jackson KA, Camargo FD, Ferrari G, Mavilio F, Goodell MA: Muscle-derived hematopoietic stem cells are hematopoietic in origin. Proc Natl Acad Sci USA 2002, 99:1341-1346. By separating MDSCs into CD45þ and CD45 populations, the authors demonstrate that CD45þ stem cells can differentiate into haematopoietic lineages but with limited myogenic potential. In contrast, CD45 MDSCs preferentially differentiate into myogenic lineage. 6.
Asakura A, Seale P, Girgis-Gabardo A, Rudnicki MA: Myogenic specification of side population cells in skeletal muscle. J Cell Biol 2002, 159:123-134.
Conclusions
7.
Asakura A, Rudnicki MA: Side population cells from diverse adult tissues are capable of in vitro hematopoietic differentiation. Exp Hematol 2002, 30:1339-1345.
8.
Wada MR, Inagawa-Ogashiwa M, Shimizu S, Yasumoto S, Hashimoto N: Generation of different fates from multipotent muscle stem cells. Development 2002, 129:2987-2995.
9.
Wright V, Peng H, Usas A, Young B, Gearhart B, Cummins J, Huard J: BMP4-expressing muscle-derived stem cells differentiate into osteogenic lineage and improve bone healing in immunocompetent mice. Mol Ther 2002, 6:169-178.
Significant progress has been made in researching the potential application of postnatal stem cells for treatment of muscle and connective tissue diseases. Current data support the hypothesis that at least two types of stem cells are present in postnatal tissues. The tissue non-specific stem cells exist in most tissues, can differentiate into different blood lineages, and are most likely haematopoietic in origin. In contrast, the tissue-specific stem cells differentiate preferentially into the main lineages of the residing tissue, although they also possess limited ability to differentiate into other lineages. In line with this new hypothesis, it is not difficult to understand the therapeutic limitation of postnatal stem cells. Fortunately, the potential of these stem cells can be greatly enhanced following genetic engineering. This has been elegantly demonstrated using genetically engineered MDSCs. Future research should illustrate the relationship between different postnatal stem cell populations, their therapeutic potential and limitations, and how genetic engineering can expand their application in the treatment of muscle and connective tissue diseases.
Update Recently, implantation of muscle-derived cells has been shown to improve the healing of articular cartilage defects, with a capacity similar to chondrocyte implantation [19].
Acknowledgements We wish to thank Ryan Sauder for his excellent editorial assistance with the manuscript.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Ying QL, Nichols J, Evans EP, Smith AG: Changing potency by spontaneous fusion. Nature 2002, 416:545-548.
2.
Terada N, Hamazaki T, Oka M, Hoki M, Mastalerz DM, Nakano Y, Meyer EM, Morel L, Petersen BE, Scott EW: Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 2002, 416:542-545.
3.
4.
Wagers AJ, Sherwood RI, Christensen JL, Weissman IL: Little evidence for developmental plasticity of adult hematopoietic stem cells. Science 2002, 297:2256-2259. Qu-Petersen Z, Deasy B, Jankowski R, Ikezawa M, Cummins J, Pruchnic R, Mytinger J, Cao B, Gates C, Wernig A, Huard J:
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5.
10. Peng H, Wright V, Usas A, Gearhart B, Shen HC, Cummins J, Huard J: Synergistic enhancement of bone formation and healing by stem cell-expressed VEGF and bone morphogenetic protein-4. J Clin Invest 2002, 110:751-759. Using genetically engineered MDSCs, the authors demonstrate how the therapeutic potential of stem cells can be further enhanced through genetic engineering to express specific growth factors. This study reveals that VEGF enhances bone formation and healing elicited by BMP4 through multiple mechanisms involving enhancement of cartilage formation and accelerated cartilage resorption. Intriguingly, such a synergistic effect depends critically on the correct ratio between VEGF and BMP4. 11. Gysin R, Wergedal JE, Sheng MH, Kasukawa Y, Miyakoshi N, Chen ST, Peng H, Lau KH, Mohan S, Baylink DJ: Ex vivo gene therapy with stromal cells transduced with a retroviral vector containing the BMP4 gene completely heals critical size calvarial defect in rats. Gene Ther 2002, 9:991-999. 12. Ikeuchi M, Dohi Y, Horiuchi K, Ohgushi H, Noshi T, Yoshikawa T, Yamamoto K, Sugimura M: Recombinant human bone morphogenetic protein-2 promotes osteogenesis within atelopeptide type I collagen solution by combination with rat cultured marrow cells. J Biomed Mater Res 2002, 60:61-69. 13. Shi S, Gronthos S, Chen S, Reddi A, Counter CM, Robey PG, Wang CY: Bone formation by human postnatal bone marrow stromal stem cells is enhanced by telomerase expression. Nat Biotechnol 2002, 20:587-591. 14. Noth U, Tuli R, Osyczka AM, Danielson KG, Tuan RS: In vitro engineered cartilage constructs produced by press-coating biodegradable polymer with human mesenchymal stem cells. Tissue Eng 2002, 8:131-144. 15. Majumdar MK, Wang E, Morris EA: BMP-2 and BMP-9 promotes chondrogenic differentiation of human multipotential mesenchymal cells and overcomes the inhibitory effect of IL-1. J Cell Physiol 2001, 189:275-284. 16. Sekiya I, Vuoristo JT, Larson BL, Prockop DJ: In vitro cartilage formation by human adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chondrogenesis. Proc Natl Acad Sci USA 2002, 99:4397-4402. 17. Jankowski RJ, Deasy BM, Cao B, Gates C, Huard J: The role of CD34 expression and cellular fusion in the regeneration capacity of myogenic progenitor cells. J Cell Sci 2002, 115:4361-4374. 18. Altman GH, Horan RL, Lu HH, Moreau J, Martin I, Richmond JC, Kaplan DL: Silk matrix for tissue engineered anterior cruciate ligaments. Biomaterials 2002, 23:4131-4141. 19. Adachi N, Sato K, Usas A, Fu FH, Ochi M, Han CW, Niyibizi C, Huard J: Muscle derived cell based ex vivo gene therapy for treatment of full thickness articular cartilage defects. J Rheumatol 2002, 29:1920-1930. Current Opinion in Pharmacology 2003, 3:329–333
Stem cells in the treatment of muscle and connective tissue diseases Hairong Peng and Johnny Huardy Current data indicate the existence of two types of postnatal stem cells. Tissue non-specific stem cells are haematopoietic in origin and can differentiate into different blood lineages. In contrast, tissue-specific stem cells preferentially differentiate into cells of the residing tissue, although they also possess a limited ability to turn into other lineages. In terms of therapeutic potential, unmodified muscle-derived stem cells have been proven capable of regenerating dystrophic muscle. Furthermore, when genetically modified to express growth factors, these cells are versatile in promoting bone healing. This also occurs with mesenchymal stem cells, which have been used in an attempt to repair defects of cartilage and ligaments. Thus, stem-cell-based therapy — particularly genetically engineered therapy — holds great potential for the treatment of a variety of disorders and conditions affecting the muscle and connective tissue. Addresses Growth and Development Laboratory, Department of Orthopaedic Surgery, Children’s Hospital of Pittsburgh, University of Pittsburgh, Pittsburgh, PA, USA y Director of the Growth and Developmental Laboratory, Departments of Orthopaedic Surgery, Molecular Genetics and Biochemistry, and Bio-engineering, University of Pittsburgh, 4151 Rangos Research Center, Children’s Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213, USA Correspondence: Johnny Huard; e-mail: jhuardþ@pitt.edu
Current Opinion in Pharmacology 2003, 3:329–333 This review comes from a themed issue on Musculoskeletal pharmacology Edited by Kay E Davies and Kevin Talbot 1471-4892/03/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved.
embryonic stem cells are the most versatile when used for the regeneration or repair of different tissues because of their multipotentiality. However, these cells must be used in the form of allografts unless therapeutic cloning techniques are utilized to generate autologous embryonic stem cells. Postnatal stem cells can be used for autologous transplantation, and thus are the focus of this paper. In the past year, scientists have made remarkable progress in assessing the characteristics of postnatal stem cells and the potential use of these cells for treatment of muscle and connective tissue diseases. This review summarises studies that generated valuable findings concerning the nature of postnatal stem cells and their uses in this exciting field of research.
Plasticity of postnatal stem cells Despite early reports indicating the plasticity of postnatal stem cells, new studies suggest that the interpretation of these dramatic findings warrants caution. Several researchers have suggested that these earlier evaluations of postnatal stem cell plasticity might have mistaken cellfusion for true multipotent differentiation [1,2]. Furthermore, a recent stringent study using single-cell transplantation generated little evidence of differentiation of haematopoietic stem cells into other lineages. The implantation of a single haematopoietic stem cell into lethally irradiated mice led to reconstitution of the haematopoietic system of each recipient; however, researchers observed no appreciable contribution of the donorderived cells to non-haematopoietic tissues, including brain, kidney, gut, liver and muscle [3]. Taken together, these studies indicate that the ability of haematopoietic stem cells to differentiate into cells of non-haematopoietic lineages is very limited.
DOI 10.1016/S1471-4892(03)00051-1
Abbreviations BMP bone morphogenetic protein MDSCs muscle-derived stem cells VEGF vascular endothelial growth factor
Introduction Most of the research on stem cells has focused on either embryonic or postnatal stem cells. Embryonic stem cells are isolated from early embryos and generally possess the potential to differentiate into various lineages. In contrast, postnatal stem cells can be isolated from the tissues of individuals of any age; however, their capacity for multilineage differentiation might be limited, as discussed in this review (Figure 1). From a therapeutic point of view, www.current-opinion.com
It remains unclear whether stem cells isolated from other tissues are tissue-specific or multipotent (i.e. able to differentiate into different lineages). Muscle-derived stem cells (MDSCs) isolated by researchers in our laboratory have been shown to express cellular markers of endothelial or neuronal cells upon stimulation with vascular endothelial growth factor (VEGF) or nerve growth factor, respectively [4]. Additionally, the localisation of b-galactosidase-labelled cells in vascular structures and peripheral nerves following intramuscular implantation of b-galactosidase-labelled MDSCs suggests in vivo differentiation of the implanted MDSCs toward endothelial and neuronal lineages [4]. The ability of these MDSCs to differentiate into other lineages, however, is quite limited compared with their capacity for myogenic differentiation. Current Opinion in Pharmacology 2003, 3:329–333
330 Musculoskeletal
Figure 1
All lineages of the body
Embryonic stem cells
Tissue non-specific stem cells
Post natal stem cells
Stem cells of haematopoietic origin
Haematopoietic lineages Other lineages
Stem cells of mesenchymal origin
Osteogenic, adipogenic, myogenic and chondrogenic lineages
Other lineages Tissue-specific stem cells
Tissue-specific lineages
Main differentiation pathway upon proper induction Minor differentiation pathway upon proper induction Current Opinion in Pharmacology
Stem cells and their possible pathways of differentiation.
Tissue-specific and non-specific stem cells in postnatal tissues At least two populations of stem cells are thought to exist in most types of postnatal tissue. The first stem cell population appears to be present in all tissues, and is most likely haematopoietic in origin. The second population comprises tissue-specific stem cells, which preferentially differentiate into cells of the same type as those constituting the tissue in which they reside, although these stem cells also possess a limited ability to transdifferentiate into cells of other lineages (Figure 1). In muscle, for example, stem cells of haematopoietic origin are CD45þ and have the capacity to differentiate into blood cells; these cells undergo very limited myogenic differentiation in vivo and no myogenic differentiation in vitro [5,6]. These so-called side population cells can be isolated using FACS by virtue of dye exclusion [5,6]. Side population cells have been identified in a variety of postnatal tissues, including tissues of the brain, heart, lung, spleen, kidney, small intestine and sketetal muscle [7]. In contrast to these non-tissue-specific MDSCs, tissue-specific cells are CD45– and have the ability to differentiate into myogenic lineage both in vitro and in vivo [5]. The CD45 MDSCs differ from satellite cells (a group of quiescent myogenic progenitors that normally reside underneath the basement membrane of myofibers) [4,6], although the latter also display the capacity to differentiate Current Opinion in Pharmacology 2003, 3:329–333
into cells other than myoblasts, such as osteoblasts and adipocytes [8]. Interestingly, undifferentiated progenitor cells derived from single satellite cells co-express the transcription factors MyoD and Runx2, which are thought to determine myogenic and osteogenic differentiation, respectively. Whether this finding indicates the intrinsic potential of satellite cells to undergo either osteogenic or myogenic differentiation, or a shared regulatory mechanism between these two phenotypically different pathways, remains to be determined.
Stem cell therapy to improve bone healing Significant progress has been made in the application of stem cells — most notably MDSCs — to promote or improve bone healing. MDSCs transduced with a retroviral vector expressing bone morphogenetic protein (BMP)4 undergo osteogenic differentiation both in vitro and in vivo, and can heal bone defects in immunocompetent animals [9]. Bone formation and healing elicited by stem cells expressing BMP4 can be enhanced if the bone defect is also treated using stem cells expressing VEGF (Figure 2) [10]. Intriguingly, the synergistic effect of these two growth factors depends critically on their ratio, with an improper ratio leading to detrimental effects on the bone healing process [10]. The feasibility of using transduced marrow stromal cells expressing BMP4 to enhance bone healing has been confirmed recently [11]. In addition, new scaffold materials, such as atelopeptide type I collagen, www.current-opinion.com
Stem cells in the treatment of muscle and connective tissue diseases Peng and Huard 331
Figure 2
(b)
Relative bone area
(a)
B4
B4+Ve
∗
30 20 10 0
B4 BMP4
B4+Ve
BMP4+VEGF
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
7d
10d
10d
14d
VEGF enhances endochondral bone formation elicited by MDSCs expressing BMP4. (a) Radiograph shows augmented bone formation in the BMP4þVEGF site (B4þVe) compared with the BMP4 site (B4) four weeks after implantation. (b) Quantitative analysis shows that the BMP4þVEGF group produced significantly more bone than the BMP4 group (*P < 0.01, n¼4). (c–f) Alcian Blue staining shows enhanced cartilage formation (purple) at 7 days, and accelerated cartilage resorption at 10 days in the BMP4þVEGF group compared with the BMP4 group. (g–j) von Kossa staining shows more extensive cartilage mineralization (black) at 10 days, and increased mineralized bone formation (black) at 14 days in the BMP4þVEGF group compared with the BMP4 group. Magnification: (c,d) and (g–j), 100x; (e,f), 200x. Republished with permission from The Journal of Clinical Investigation.
have been utilised in conjunction with marrow stromal cells to evaluate whether this combination bolsters the osteogenic effects of BMP2 [12]. However, it is worth noting that stem cells alone, either MDSCs or marrow stromal cells, cannot improve the healing of critical-sized bone defects [10,11]. This limitation can apparently be overcome through genetic engineering of the stem cells to express specific growth factors. www.current-opinion.com
Stem-cell-based gene therapy has proven to be a powerful method to investigate the mechanisms of bone formation and healing using both gain-of-function and loss-of-function approaches. A loss-of-function design was used to demonstrate that VEGF activity is important for endochondral bone formation elicited by BMP4. Stem cells expressing a VEGF specific antagonist, soluble Flt1, inhibited ectopic bone formation induced by BMP4 by Current Opinion in Pharmacology 2003, 3:329–333
332 Musculoskeletal
both reducing cartilage formation and delaying cartilage resorption [10]. Utilisation of a gain-of-function approach revealed a synergistic relationship between BMP4 and VEGF in stimulating bone formation, a process found to involve multiple mechanisms in endochondral pathways (Figure 2). These include an increase in stem cell recruitment to enhance cartilage formation, enhanced angiogenesis to accelerate cartilage resorption, and improved cell survival at the bone formation site [10]. An increase in the proliferation potential of stromal stem cells can also lead to improved bone formation. Like other types of stem cells (e.g. haematopoietic stem cells), marrow stromal stem cells have a relatively short life-span. This limitation has recently been overcome through forced expression of telomerase transcriptase in stromal cells [13]. Following implantation into immunodeficient animals, these transfected cells produce more bone than do non-transfected cells. Interestingly, such enhanced bone formation correlated with an increase in the expression of STRO-1, an early pre-osteogenic stem cell marker, in the transfected cells. These results indicate that optimal bone formation depends on a high capacity for cell proliferation and preservation of progenitor cell characteristics.
Cartilage repair using stem cells Research into stem-cell-based cartilage repair has focused primarily on two areas: firstly, the construction of tissueblocks that simulate cartilage; and secondly, the identification of growth factors with the potential to induce chondrogenic differentiation, and the characterisation of gene expression profiles during this process in vitro. In the first of these approaches, different polymers were used to fabricate three-dimensional constructs, which were subsequently seeded with mesenchymal stem cells and cultured in chondrogenic medium to induce chondrogenic differentiation. Human mesenchymal stem cells seeded in constructs created by press-coating D,D-L,Lpolylactic acid polymer blocks were found to undergo chondrogenic differentiation when cultured in chondrogenic medium for three weeks [14]. More importantly, the cell layers present in the construct were well organised, with the superficial layer resembling compact hyaline cartilage [14]. However, it remains to be determined whether such a construct formed in vitro could integrate with surrounding tissues and maintain the structure to a degree comparable to that of normal articular cartilage following implantation into a cartilage defect. The other primary aim of stem-cell-related cartilage research is to identify growth factors with the potential to promote chondrogenic differentiation. BMP2 and BMP9 have been shown to induce chondrogenic differentiation of human mesenchymal stem cells, as indicated by the expression of type II mRNA and the increased expression of aggrecan and cartilage oligomeric matrix proteins [15]. Current Opinion in Pharmacology 2003, 3:329–333
Such an increase in the expression of chondrogenic proteins is probably attributable to the activation of Sox 9, a transcription factor involved in the regulation of chondrogenesis. These growth factors are also able to partially block the inhibitory effects on chondrogenic differentiation mediated by interleukin-1, a major cytokine implicated in the pathogenesis of osteoarthritis [15]. Gene expression profiles related to the in vitro chondrogenic differentiation of human adult bone marrow stromal stem cells have been investigated recently [16]. However, it is unknown whether these in vitro results accurately portray in vivo occurrences.
Muscle regeneration using stem cells Finding a way to treat severe muscle diseases, most notably muscular dystrophy, continues to pose a formidable challenge. Direct gene-transfer mediated by viral vectors (particularly adeno-associated viral vectors) offers the greatest potential as treatment for Duchenne Muscular Dystrophy, the most common muscle disease. However, stem-cell-mediated therapy could also play an important role in the treatment of patients during later stages of the disease, which are characterised by depletion of endogenous MDSCs, secondary to repeated injury and regeneration. Researchers in our laboratory have recently isolated a novel population of MDSCs that proliferate in vitro for a prolonged period of time and can differentiate into muscle, neural and endothelial lineages both in vitro and in vivo [4]. More importantly, these cells are able to restore dystrophin expression upon implantation into MDX mice (an animal model of Duchenne Muscular Dystrophy) with a degree of efficiency unrivaled by myoblast or satellite cell transplantation [4]. This superior transplantation capability of MDSCs was shown to be a result of their immune privilege (failure to trigger a strong immune response compared to myoblasts [4]), self-renewal ability and multipotential differentiation [4]. In addition to the isolation of long-term proliferating cells in our laboratory, efforts have been made to identify cellular properties that can predict heightened transplantation efficiency, which is indicated by the ability of cells to reconstitute dystrophin expression in muscle of MDX mice. We have determined that CD34þ MDSCs displaying delayed adhesion to collagen type I have a better regeneration capacity than do either CD34þ MDSCs characterised by early adhesion to collage type 1, or CD34 MDSCs displaying delayed adhesion [17]. This superior restoration capacity correlates positively with the proliferation potential of MDSCs [17].
Other connective tissues Recently, a study was conducted to investigate the possible development of a synthetic structure that could be used to repair injured ligament. The creation of such an implant poses an enormous challenge, as the material would need to sustain the tremendous mechanical stress www.current-opinion.com
Stem cells in the treatment of muscle and connective tissue diseases Peng and Huard 333
Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration. J Cell Biol 2002, 157:851-864.
typically absorbed by normal ligament and, at the same time, be able to integrate with the existing ligament or bone. In an attempt to develop materials that could be used for reconstruction of the anterior cruciate ligament, researchers seeded a silk-fibre matrix with human bone marrow stromal cells [18]. In vitro culture indicated that this new matrix would provide sufficient support during the attachment, proliferation and differentiation of the cells; however, the in vivo performance of ligaments engineered by this method has not been evaluated.
McKinney-Freeman SL, Jackson KA, Camargo FD, Ferrari G, Mavilio F, Goodell MA: Muscle-derived hematopoietic stem cells are hematopoietic in origin. Proc Natl Acad Sci USA 2002, 99:1341-1346. By separating MDSCs into CD45þ and CD45 populations, the authors demonstrate that CD45þ stem cells can differentiate into haematopoietic lineages but with limited myogenic potential. In contrast, CD45 MDSCs preferentially differentiate into myogenic lineage. 6.
Asakura A, Seale P, Girgis-Gabardo A, Rudnicki MA: Myogenic specification of side population cells in skeletal muscle. J Cell Biol 2002, 159:123-134.
Conclusions
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Asakura A, Rudnicki MA: Side population cells from diverse adult tissues are capable of in vitro hematopoietic differentiation. Exp Hematol 2002, 30:1339-1345.
8.
Wada MR, Inagawa-Ogashiwa M, Shimizu S, Yasumoto S, Hashimoto N: Generation of different fates from multipotent muscle stem cells. Development 2002, 129:2987-2995.
9.
Wright V, Peng H, Usas A, Young B, Gearhart B, Cummins J, Huard J: BMP4-expressing muscle-derived stem cells differentiate into osteogenic lineage and improve bone healing in immunocompetent mice. Mol Ther 2002, 6:169-178.
Significant progress has been made in researching the potential application of postnatal stem cells for treatment of muscle and connective tissue diseases. Current data support the hypothesis that at least two types of stem cells are present in postnatal tissues. The tissue non-specific stem cells exist in most tissues, can differentiate into different blood lineages, and are most likely haematopoietic in origin. In contrast, the tissue-specific stem cells differentiate preferentially into the main lineages of the residing tissue, although they also possess limited ability to differentiate into other lineages. In line with this new hypothesis, it is not difficult to understand the therapeutic limitation of postnatal stem cells. Fortunately, the potential of these stem cells can be greatly enhanced following genetic engineering. This has been elegantly demonstrated using genetically engineered MDSCs. Future research should illustrate the relationship between different postnatal stem cell populations, their therapeutic potential and limitations, and how genetic engineering can expand their application in the treatment of muscle and connective tissue diseases.
Update Recently, implantation of muscle-derived cells has been shown to improve the healing of articular cartilage defects, with a capacity similar to chondrocyte implantation [19].
Acknowledgements We wish to thank Ryan Sauder for his excellent editorial assistance with the manuscript.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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