- Email: [email protected]
SP-litting the Satellite Niche to Repopulate Muscle
Cell Stem Cell
Previews of precipitation and mask or destroy epitopes, both of which could result in a failure to detect modified regions. On the other hand, under native conditions, histone-modifying enzymes may remain active throughout handling, and if unchecked can degrade samples and obscure profiling. Whatever the explanation, confirmation and extension of the LOCK hypothesis is now eagerly awaited as these molecular studies offer to throw new light on enduring ideas proposed by Spemann more than 70 years ago.
REFERENCES Guelen, L., Pagie, L., Brasset, E., Meuleman, W., Faza, M.B., Talhout, W., Eussen, B.H., de Klein, A., Wessels, L., de Laat, W., et al. (2008). Nature 453, 948–951. Liu, Y., Mochizuki, K., and Gorovsky, M.A. (2004). Proc. Natl. Acad. Sci. USA 101, 1679–1684. Meshorer, E., Yellajoshula, D., George, E., Scambler, P.J., Brown, D.T., and Misteli, T. (2006). Dev. Cell 10, 105–116. Mikkelsen, T.S., Ku, M., Jaffe, D.B., Issac, B., Lieberman, E., Giannoukos, G., Alvarez, P., Brockman, W., Kim, T.K., Koche, R.P., et al. (2007). Nature 448, 553–560.
Mohn, F., Weber, M., Rebhan, M., Roloff, T.C., Richter, J., Stadler, M.B., Bibel, M., and Schubeler, D. (2008). Mol. Cell 30, 755–766. O’Neill, L.P., and Turner, B.M. (2003). Methods 31, 76–82. Reik, W. (2007). Nature 447, 425–432. Spemann, H. (1938). Embryonic Development and Induction (New York: Hafner Publishing Co.). Stock, J.K., Giadrossi, S., Casanova, M., Brookes, E., Vidal, M., Koseki, H., Brockdorff, N., Fisher, A.G., and Pombo, A. (2007). Nat. Cell Biol. 9, 1428–1435. Wen, B., Wu, H., Shinkai, Y., Irizarry, R.A., and Feinberg, A.P. (2009). Nat. Genet. 41, 246–250.
SP-litting the Satellite Niche to Repopulate Muscle Stefani Fontana1 and Ronald D. Cohn1,* 1Department of Pediatrics and Neurology, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, 733 North Broadway, BRB 529 Baltimore, MD 21205, USA *Correspondence: [email protected] DOI 10.1016/j.stem.2009.02.002
Multiple myogenic populations have been highlighted in past publications. In this issue of Cell Stem Cell, Tanaka et al. (2009) advance our understanding of the cells that contribute to muscle regeneration by identifying an ABCG2-expressing population that exhibits excellent engraftment potential, particularly within the satellite cell niche. Tissue-specific stem cells are as sought after as they are elusive in the pursuit of effective clinical cell therapy. Empirically, cell therapy would be the most effective in tissues that are continually regenerating, with blood, liver, and muscle cells being a few that are immediately evident. While hematopoietic cells have already been accepted as a therapeutically useful tissue-specific stem cell, its equivalent has yet to be identified for repopulating damaged or diseased muscle. Satellite cells are myogenic precursor cells located beneath the basal lamina in myofibers (Mauro, 1961) and have been shown to exhibit archetypal stem cell properties (Collins et al., 2005). However, the engraftment efficiency and capability of isolated satellite cells has thus far proven insufficient to improve muscle function and regeneration. In this issue of Cell Stem Cell, Tanaka et al. (2009) very elegantly define a novel population of myogenic precursor cells
that not only possess the ability to differentiate and fuse with myotubes, but when transplanted, can also regenerate and maintain a substantial, functional population of muscle progenitor cells. Muscle regeneration has proven to be a complex process involving multiple cell lineages. Satellite cells are most commonly defined by their location within the basal lamina and their expression of various myogenic markers such as paired box gene, Pax7, myoD, and myogenin. Another population of cells shown to contribute to regenerating myofibers are side population (SP) cells, defined by their dye exclusion properties and which are found in the interstitium. The majority of SP cells express the ATP-binding cassette transporter, ABCG2 (Goodell et al., 1996). Acknowledging the relationship between satellite cells and SP cells, given that a coculture of the two will initiate myogenesis, Tanaka et al. (2009) sorted a popula-
194 Cell Stem Cell 4, March 6, 2009 ª2009 Elsevier Inc.
tion of cells that coexpress both Pax7 and ABCG2. The sort criteria also enriched for CD34 expression, which is a marker that identifies cells that are more likely to engraft when transplanted (Montarras et al., 2005; Cerletti et al., 2008; Sacco et al., 2008). Combined, cells expressing these three markers were enriched within the Syndecan-3/4-positive pool, and the population, coined satellite SP cells by the authors, was shown to spontaneously differentiate into myotubes in culture. Additionally, when GFP-marked wild-type cells were transplanted into mdx mice, an animal model for dystrophin-deficient muscular dystrophy, which demonstrates constant cycles of muscle degeneration and regeneration, transplanted cells were able to contribute to sarcolemmal dystrophin expression, fusion of myotubes, and engraftment into the satellite cell niche. The donor cells engrafted with such high efficiency that after intramuscular
Cell Stem Cell
Previews transplantation, 2500 sorted cells gave rise to 75% of the satellite cells present in the tibialis anterior muscle of recipients. By demonstrating the ability to mature into terminally differentiated muscle tissue and repopulation of the stem cell niche, the ABCG2+/Pax7+/CD34+/Syndecan-3/ 4+ cells defined in this paper by Olwin and colleagues embody the ideal characteristics of the archetypal tissue specific satellite cell. Another striking characteristic of tissue stem cell populations is their ability to mediate multiple rounds of regeneration. Myogenic stem cells must have the capacity to undergo asymmetric division, thus producing daughter cells capable of terminal differentiation into myotubes while also maintaining the population of muscle-specific stem cells. When mdx mice engrafted with satellite-SP cells were reinjured, donor-derived myofibers as well as satellite cells were observed at a very high frequency, indicative of the ability of this population to regenerate mature muscle while simultaneously repopulating the progenitor cell pool. The cells described in this paper exhibit more robust potential as functional muscle-specific stem cells relative to the muscle progenitor cells highlighted in previous publications (Kuang et al., 2008), at least in the mdx mouse injury model. The fact that donor cells were capable of repopulating 75% of mature myofibers opens up new avenues of research in the future. It is interesting to note that Tanaka et al. did not observe any signs of rejection despite their transplantation into immunocompetent mice. These observations
require further studies to explore whether the transplanted myogenic cells are sufficiently immature such that they do not express markers that antagonize the immune system. If this speculation proves true, the fact that the immune system does not appear to target their mature progeny raises speculation as to the influence of the environment over the differentiation and maturation of the donor myogenic population. Moreover, it will be important to study the expression pattern and physiological profile of the satellite-SP cells isolated from a variety of inherited and acquired forms of muscle degeneration, regeneration, and atrophy. The findings observed in these future studies may unveil further insights into the functional role of the satellite-SP cell per se, as well as the molecular pathogenesis underlying muscle regeneration in general. Finally, let us consider a more basic vignette. The most rudimentary functional assay for any muscle progenitor cell candidate is to demonstrate the potential for spontaneous differentiation into myofibers in culture. One important question that needs to be addressed in the future is to characterize the molecular pathways underlying the processes of myoprogenitor proliferation and differentiation. While spontaneous differentiation has long been the accepted assay, a deeper understanding of the signals that initiate this process would move the field closer to an understanding of muscle regeneration. The isolation of the robust, well-defined myogenic progenitor population described by Tanaka et al. offers an improved
opportunity to conduct such mechanistic studies. There has been a flurry of recent papers highlighting various precursor populations that contribute to muscle formation, all of which are indeed capable of giving rise to myotubes, though very few of which appear to be the sought after ‘‘satellite cell.’’ As we put together the pieces of the complex puzzle of muscle regeneration, each cell type identified brings us closer to understanding the complex mechanisms underlying muscle regeneration and repair, which will ultimately enable us to develop safe and efficient cell-based therapies for a variety of myopathic conditions. REFERENCES Cerletti, M., Jurga, S., Witczak, C.A., Hirshman, M.F., Shadrach, J.L., Goodyear, L.J., and Wagers, A.J. (2008). Cell 134, 37–47. Collins, C.A., Olsen, I., Zammit, P.S., Heslop, L., Petrie, A., Patridge, T.A., and Morgan, J.E. (2005). Cell 122, 289–301. Goodell, M.A., Brose, K., Paradis, G., Conner, A.S., and Mulligan, R.C. (1996). J. Exp. Med. 183, 1797–1806. Kuang, S., Gillespie, M.A., and Rudnicki, M.A. (2008). Cell Stem Cell 2, 22–31. Mauro, A. (1961). J. Biophys. Biochem. Cytol. 9, 493–496. Montarras, D., Morgan, J., Collins, C., Relaix, F., Zaffran, S., Cumano, A., Partridge, T., and Buckingham, M. (2005). Science 309, 2064–2067. Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S., and Blau, H.M. (2008). Nature 456, 502–506. Tanaka, K.K., Hall, J.K., Troy, A.A., Cornelison, D.D.W., Majka, S.M., and Olwin, B.B. (2009). Cell Stem Cell 4, this issue, 217–225.
Cell Stem Cell 4, March 6, 2009 ª2009 Elsevier Inc. 195
Previews of precipitation and mask or destroy epitopes, both of which could result in a failure to detect modified regions. On the other hand, under native conditions, histone-modifying enzymes may remain active throughout handling, and if unchecked can degrade samples and obscure profiling. Whatever the explanation, confirmation and extension of the LOCK hypothesis is now eagerly awaited as these molecular studies offer to throw new light on enduring ideas proposed by Spemann more than 70 years ago.
REFERENCES Guelen, L., Pagie, L., Brasset, E., Meuleman, W., Faza, M.B., Talhout, W., Eussen, B.H., de Klein, A., Wessels, L., de Laat, W., et al. (2008). Nature 453, 948–951. Liu, Y., Mochizuki, K., and Gorovsky, M.A. (2004). Proc. Natl. Acad. Sci. USA 101, 1679–1684. Meshorer, E., Yellajoshula, D., George, E., Scambler, P.J., Brown, D.T., and Misteli, T. (2006). Dev. Cell 10, 105–116. Mikkelsen, T.S., Ku, M., Jaffe, D.B., Issac, B., Lieberman, E., Giannoukos, G., Alvarez, P., Brockman, W., Kim, T.K., Koche, R.P., et al. (2007). Nature 448, 553–560.
Mohn, F., Weber, M., Rebhan, M., Roloff, T.C., Richter, J., Stadler, M.B., Bibel, M., and Schubeler, D. (2008). Mol. Cell 30, 755–766. O’Neill, L.P., and Turner, B.M. (2003). Methods 31, 76–82. Reik, W. (2007). Nature 447, 425–432. Spemann, H. (1938). Embryonic Development and Induction (New York: Hafner Publishing Co.). Stock, J.K., Giadrossi, S., Casanova, M., Brookes, E., Vidal, M., Koseki, H., Brockdorff, N., Fisher, A.G., and Pombo, A. (2007). Nat. Cell Biol. 9, 1428–1435. Wen, B., Wu, H., Shinkai, Y., Irizarry, R.A., and Feinberg, A.P. (2009). Nat. Genet. 41, 246–250.
SP-litting the Satellite Niche to Repopulate Muscle Stefani Fontana1 and Ronald D. Cohn1,* 1Department of Pediatrics and Neurology, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, 733 North Broadway, BRB 529 Baltimore, MD 21205, USA *Correspondence: [email protected] DOI 10.1016/j.stem.2009.02.002
Multiple myogenic populations have been highlighted in past publications. In this issue of Cell Stem Cell, Tanaka et al. (2009) advance our understanding of the cells that contribute to muscle regeneration by identifying an ABCG2-expressing population that exhibits excellent engraftment potential, particularly within the satellite cell niche. Tissue-specific stem cells are as sought after as they are elusive in the pursuit of effective clinical cell therapy. Empirically, cell therapy would be the most effective in tissues that are continually regenerating, with blood, liver, and muscle cells being a few that are immediately evident. While hematopoietic cells have already been accepted as a therapeutically useful tissue-specific stem cell, its equivalent has yet to be identified for repopulating damaged or diseased muscle. Satellite cells are myogenic precursor cells located beneath the basal lamina in myofibers (Mauro, 1961) and have been shown to exhibit archetypal stem cell properties (Collins et al., 2005). However, the engraftment efficiency and capability of isolated satellite cells has thus far proven insufficient to improve muscle function and regeneration. In this issue of Cell Stem Cell, Tanaka et al. (2009) very elegantly define a novel population of myogenic precursor cells
that not only possess the ability to differentiate and fuse with myotubes, but when transplanted, can also regenerate and maintain a substantial, functional population of muscle progenitor cells. Muscle regeneration has proven to be a complex process involving multiple cell lineages. Satellite cells are most commonly defined by their location within the basal lamina and their expression of various myogenic markers such as paired box gene, Pax7, myoD, and myogenin. Another population of cells shown to contribute to regenerating myofibers are side population (SP) cells, defined by their dye exclusion properties and which are found in the interstitium. The majority of SP cells express the ATP-binding cassette transporter, ABCG2 (Goodell et al., 1996). Acknowledging the relationship between satellite cells and SP cells, given that a coculture of the two will initiate myogenesis, Tanaka et al. (2009) sorted a popula-
194 Cell Stem Cell 4, March 6, 2009 ª2009 Elsevier Inc.
tion of cells that coexpress both Pax7 and ABCG2. The sort criteria also enriched for CD34 expression, which is a marker that identifies cells that are more likely to engraft when transplanted (Montarras et al., 2005; Cerletti et al., 2008; Sacco et al., 2008). Combined, cells expressing these three markers were enriched within the Syndecan-3/4-positive pool, and the population, coined satellite SP cells by the authors, was shown to spontaneously differentiate into myotubes in culture. Additionally, when GFP-marked wild-type cells were transplanted into mdx mice, an animal model for dystrophin-deficient muscular dystrophy, which demonstrates constant cycles of muscle degeneration and regeneration, transplanted cells were able to contribute to sarcolemmal dystrophin expression, fusion of myotubes, and engraftment into the satellite cell niche. The donor cells engrafted with such high efficiency that after intramuscular
Cell Stem Cell
Previews transplantation, 2500 sorted cells gave rise to 75% of the satellite cells present in the tibialis anterior muscle of recipients. By demonstrating the ability to mature into terminally differentiated muscle tissue and repopulation of the stem cell niche, the ABCG2+/Pax7+/CD34+/Syndecan-3/ 4+ cells defined in this paper by Olwin and colleagues embody the ideal characteristics of the archetypal tissue specific satellite cell. Another striking characteristic of tissue stem cell populations is their ability to mediate multiple rounds of regeneration. Myogenic stem cells must have the capacity to undergo asymmetric division, thus producing daughter cells capable of terminal differentiation into myotubes while also maintaining the population of muscle-specific stem cells. When mdx mice engrafted with satellite-SP cells were reinjured, donor-derived myofibers as well as satellite cells were observed at a very high frequency, indicative of the ability of this population to regenerate mature muscle while simultaneously repopulating the progenitor cell pool. The cells described in this paper exhibit more robust potential as functional muscle-specific stem cells relative to the muscle progenitor cells highlighted in previous publications (Kuang et al., 2008), at least in the mdx mouse injury model. The fact that donor cells were capable of repopulating 75% of mature myofibers opens up new avenues of research in the future. It is interesting to note that Tanaka et al. did not observe any signs of rejection despite their transplantation into immunocompetent mice. These observations
require further studies to explore whether the transplanted myogenic cells are sufficiently immature such that they do not express markers that antagonize the immune system. If this speculation proves true, the fact that the immune system does not appear to target their mature progeny raises speculation as to the influence of the environment over the differentiation and maturation of the donor myogenic population. Moreover, it will be important to study the expression pattern and physiological profile of the satellite-SP cells isolated from a variety of inherited and acquired forms of muscle degeneration, regeneration, and atrophy. The findings observed in these future studies may unveil further insights into the functional role of the satellite-SP cell per se, as well as the molecular pathogenesis underlying muscle regeneration in general. Finally, let us consider a more basic vignette. The most rudimentary functional assay for any muscle progenitor cell candidate is to demonstrate the potential for spontaneous differentiation into myofibers in culture. One important question that needs to be addressed in the future is to characterize the molecular pathways underlying the processes of myoprogenitor proliferation and differentiation. While spontaneous differentiation has long been the accepted assay, a deeper understanding of the signals that initiate this process would move the field closer to an understanding of muscle regeneration. The isolation of the robust, well-defined myogenic progenitor population described by Tanaka et al. offers an improved
opportunity to conduct such mechanistic studies. There has been a flurry of recent papers highlighting various precursor populations that contribute to muscle formation, all of which are indeed capable of giving rise to myotubes, though very few of which appear to be the sought after ‘‘satellite cell.’’ As we put together the pieces of the complex puzzle of muscle regeneration, each cell type identified brings us closer to understanding the complex mechanisms underlying muscle regeneration and repair, which will ultimately enable us to develop safe and efficient cell-based therapies for a variety of myopathic conditions. REFERENCES Cerletti, M., Jurga, S., Witczak, C.A., Hirshman, M.F., Shadrach, J.L., Goodyear, L.J., and Wagers, A.J. (2008). Cell 134, 37–47. Collins, C.A., Olsen, I., Zammit, P.S., Heslop, L., Petrie, A., Patridge, T.A., and Morgan, J.E. (2005). Cell 122, 289–301. Goodell, M.A., Brose, K., Paradis, G., Conner, A.S., and Mulligan, R.C. (1996). J. Exp. Med. 183, 1797–1806. Kuang, S., Gillespie, M.A., and Rudnicki, M.A. (2008). Cell Stem Cell 2, 22–31. Mauro, A. (1961). J. Biophys. Biochem. Cytol. 9, 493–496. Montarras, D., Morgan, J., Collins, C., Relaix, F., Zaffran, S., Cumano, A., Partridge, T., and Buckingham, M. (2005). Science 309, 2064–2067. Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S., and Blau, H.M. (2008). Nature 456, 502–506. Tanaka, K.K., Hall, J.K., Troy, A.A., Cornelison, D.D.W., Majka, S.M., and Olwin, B.B. (2009). Cell Stem Cell 4, this issue, 217–225.
Cell Stem Cell 4, March 6, 2009 ª2009 Elsevier Inc. 195