- Email: [email protected]
Harnessing the potential of myogenic satellite cells
Update
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References 1 Beutler, B. (2004) Inferences, questions and possibilities in Toll-like receptor signalling. Nature 430, 257–263 2 O’Neill, L.A. et al. (2003) The Toll–IL-1 receptor adaptor family grows to five members. Trends Immunol. 24, 286–290 3 Ha¨cker, H. et al. (2006) Specificity in Toll-like receptor signalling through distinct effector functions of TRAF3 and TRAF6. Nature 439, 204–207 4 Oganesyan, G. et al. (2006) Critical role of TRAF3 in the Toll-like receptor-dependent and -independent antiviral response. Nature 439, 208–211 5 Chung, J.Y. et al. (2002) All TRAFs are not created equal: common and distinct molecular mechanisms of TRAF-mediated signal transduction. J. Cell Sci. 115, 679–688 6 Takeuchi, M. et al. (1996) Anatomy of TRAF2. Distinct domains for nuclear factor-kB activation and association with tumor necrosis factor signaling proteins. J. Biol. Chem. 271, 19935–19942 7 Kato, H. et al. (2005) Cell type-specific involvement of RIG-I in antiviral response. Immunity 23, 19–28
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8 Hoebe, K. et al. (2003) Upregulation of costimulatory molecules induced by lipopolysaccharide and double-stranded RNA occurs by Trif-dependent and Trif-independent pathways. Nat. Immunol. 4, 1223–1229 9 Le Bon, A. and Tough, D.F. (2002) Links between innate and adaptive immunity via type I interferon. Curr. Opin. Immunol. 14, 432–436 10 Theofilopoulos, A.N. et al. (2005) Type I interferons (a/b) in immunity and autoimmunity. Annu. Rev. Immunol. 23, 307–335 11 Asadullah, K. et al. (2003) Interleukin-10 therapy – review of a new approach. Pharmacol. Rev. 55, 241–269 12 Lomaga, M.A. et al. (1999) TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling. Genes Dev. 13, 1015–1024 13 Naito, A. et al. (1999) Severe osteopetrosis, defective interleukin-1 signalling and lymph node organogenesis in TRAF6-deficient mice. Genes Cells 4, 353–362 1471-4914/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2006.03.008
Harnessing the potential of myogenic satellite cells Richard I. Sherwood1 and Amy J. Wagers2 1
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA Section on Developmental and Stem Cell Biology, Joslin Diabetes Center, Department of Pathology, Harvard Medical School and Harvard Stem Cell Institute, Boston, MA 02215, USA 2
Adult skeletal muscle has remarkable regenerative potential, which is mainly attributable to a small population of undifferentiated skeletal muscle precursors called satellite cells. These cells reside underneath the basal lamina of skeletal myofibers and can be activated to proliferate, differentiate and fuse to form new muscle tissue. Satellite cells have long been considered promising mediators of therapeutic muscle regeneration. However, in practice, the regenerative function of such cells, which in many cases have been derived or expanded by ex vivo cultures, can be surprisingly low. A recent study from Montarras and colleagues has provided new insights into the requirements for efficient muscle engraftment from purified muscle satellite cells, suggesting possible strategies to enhance their therapeutic potential.
Satellite cells and muscle regenerative medicine As a potentially rich and renewable source of muscle regenerative cells, satellite cells hold tremendous promise for the treatment of muscle disease. Progress towards defining the regulators of satellite cell function has been recently made by the demonstration that myogenic and non-myogenic muscle cells can be discriminated and independently isolated by unique cell-surface markers and fluorescence-activated cell sorting (FACS) [1,2]. Using a novel paired-box-gene-3 (Pax3)–green-fluorescentprotein (GFP) reporter mouse, in which expression of Corresponding author: Wagers, A.J. ([email protected]). Available online 3 April 2006 www.sciencedirect.com
GFP is controlled by the Pax3 gene locus [3], a recent study by Montarras et al. [2] has yielded significant insights into the developmental potential and regenerative function of sorted myogenic satellite cells. These findings have significant implications for harnessing the potential of these cells for clinical use in the treatment of acute and degenerative muscle diseases, either by rejuvenation or activation of endogenous cells, or by transplantation to repopulate a depleted or dysfunctional precursor cell pool. Isolating satellite cells from muscle Pax3 and Pax7 are members of the paired-box transcription factor family and are initially expressed early in development by a small population of muscle precursors that delaminate from the dorsal somite and migrate peripherally to establish embryonic myogenesis [3,4]. Most somitederived progenitors that express both Pax3 and Pax7 differentiate to form mature, multinucleated muscle fibers, but some are retained in the muscle as myogenic satellite cells [3–7]. These contribute to natural muscle growth and homeostatic muscle-cell turnover, and to work-induced muscle hypertrophy and muscle repair. Unlike Pax7 [8,9], Pax3 expression is not uniformly maintained in adult satellite cells [2,10]; however, Pax3 is expressed by satellite cells in a subset of adult muscles, which enabled Montarras et al. [2] to isolate by FACS Pax3-expressing GFP-positive cells from Pax3–GFP reporter mice. Analysis of these cells indicated that they act as myogenic precursors, as Pax3-expressing GFP-positive cells generated exclusively
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Quiescent satellite cell M-cadherin(+) CD34(+) β1-integrin(+) CXCR4(+) Syndecan-3 and syndecan-4(+) C-met(+) Pax7(+) Pax3(+/−) CD45(–) Sca1(–) CD31(–) Flk–1(–)
Fusion
Differentiation Wnt?
Activation FGF and/or HGF?
Activated satellite cell MyoD(+) Myf5(+)
Myoblast Myogenin(+) MRF4(+)
Myofiber Dystrophin(+)
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Figure 1. Adult regenerative myogenesis. In response to extrinsic signals including exercise, muscle damage or degenerative disease, normally quiescent skeletal muscle satellite cells (green) become activated and differentiate to produce myoblasts, which subsequently fuse to generate multinucleated myofibers. Some previously activated satellite cells might return to quiescence to re-establish the satellite cell pool. At each stage of muscle regeneration, differentiating myogenic cells express specific phenotypic markers, some of which are indicated here. Most markers of quiescent satellite cells are not unique to the quiescent state and are also expressed by activated satellite cells [17]. Activated satellite cells can be distinguished from quiescent cells by their induction of MyoD and Myf5. Differentiation into myoblasts is accompanied by both induced expression of differentiation markers (Myogenin and myogenic regulatory factor MRF4) and loss or alteration of expression of at least some satellite cell markers (e.g. CD34, Pax3 and Pax7). Fusion of mononucleated myoblasts generates multinucleated myotubes and myofibers, which induce transcripts that are important for skeletal muscle function such as the structural protein dystrophin. C, expressed by most or all cells; C/K, expressed by a subset of cells. K, not expressed.
myogenic colonies in vitro and O90% of them co-expressed the satellite cell marker Pax7. Notably, !10% of Pax3expressing GFP-positive cells expressed MyoD, a marker of satellite cell activation, indicating that isolation itself does not activate these cells. Consistent with previous reports [1], myogenic satellite cells that were identified by Pax3–GFP expression lacked hematopoietic and endothelial cell surface markers [CD45, CD31, foetal-liver kinase 1 (Flk1) and stem cell antigen-1 (Sca-1)], but expressed the satellite-cell marker CD34 (Figure 1). To evaluate the regenerative capacity of freshly isolated satellite cells in vivo, Montarras et al. [2] isolated Pax3expressing GFP-positive cells from the diaphragm (where the majority of satellite cells expresses Pax3) and transplanted them directly into the tibialis anterior (TA) muscles of heavily irradiated, immunodeficient, dystrophic recipient mice (mdx nu/nu). These mice harbor a point mutation in the muscle structural protein dystrophin, which results in loss of dystrophin expression in myofibers [11], and enables contributions of donor cells to myofiber regeneration in engrafted mdx nu/nu recipients to be measured by restoration of dystrophin expression. In addition, in this mouse model, host immunodeficiency and local destruction of resident satellite cells by muscle irradiation is likely to facilitate engraftment of transplanted cells. Remarkably, intramuscular delivery of only 20 000 freshly isolated Pax3-expressing GFP-positive cells yielded O500 dystrophin-expressing myofibers within 3 weeks, and injection of as few as 1000 Pax3-expressing GFP-positive cells produced 160 dystrophin-expressing myofibers [2]. This high efficiency of myofiber formation www.sciencedirect.com
from freshly sorted Pax3-expressing GFP-positive cells stands in marked contrast to previous studies that employed heterogeneous populations of unsorted cells that were harvested by enzymatic digestion of whole muscle [12] or of cultured muscle-derived cell populations [13], where up to 25 times as many cells were required to achieve similar engraftment rates. Transplantation of Pax3-expressing GFP-positive cells that were isolated from the diaphragm also contributed to regeneration of the satellite cell pool in radiation-damaged TA muscles of mdx nu/nu mice. Three weeks after grafting, Pax3-expressing GFP-positive cells were recovered from TA muscles and shown to maintain characteristic properties of activated satellite cells including association with muscle fibers, expression of Pax7 and MyoD and differentiation in culture to form myotubes [2]. Thus, consistent with recent studies that tracked the fate of satellite cells that are associated with grafted single myofibers [14], these data [2] clearly indicate the ability of satellite cells to contribute both to the formation of new myofibers and to the renewal of the satellite cell pool. However, direct clonal analysis is still required to demonstrate the ability of individual satellite cells to fulfil both self-renewal and differentiation functions (the hallmark of tissue stem cells) and to assay the frequency at which these stem cell activities are present in the satellite cell pool. Interestingly, at least a subset of transplanted Pax3-expressing GFP-positive cells that re-entered the satellite cell compartment in TA muscles retained Pax3 expression, despite the fact that Pax3 is not normally expressed in these muscles [2,10]. These data suggest that Pax3 expression might be cell autonomous. However, it is
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also possible that with longer exposures (O3 weeks) to the TA muscle environment, the donor cells might lose Pax3 expression and adopt a myogenic expression profile that is more typical of their new environment. Fresh is best for satellite cell transplant Many previous studies and several clinical trials that have tested the potential of cell therapy in muscle regeneration have employed ex vivo expanded myoblasts for transplantation. To evaluate the effects of cell culture on the in vivo regenerative function of isolated satellite cells, Montarras et al. [2] directly compared myofiber engraftment from freshly isolated Pax3-expressing GFP-positive cells with that from Pax3-expressing GFP-positive cells that were cultured for three days before grafting. Remarkably, ten times as many previously cultured cells as freshly isolated cells were required to achieve equivalent numbers of myofibers in vivo, indicating a dramatic loss of regenerative activity associated with satellite cell culture. This key finding indicates that ex vivo expansion of myogenic cells does not necessarily translate into expansion of regenerative function, and highlights the crucial importance of direct purification of myogenic satellite cells in studies of their regenerative activity. Notably, a similar loss of regenerative function has been observed in vitro in cultures of other adult tissue precursor cells, including bone marrow-derived hematopoietic stem cells [15]. However, whether this detrimental effect of cell culture is a general property of all adult progenitor cells or depends on the particular cell population under investigation remains to be determined. Although the reason for culture-induced loss of satellite cell function remains unclear, it might be caused, at least in part, by in vitro conditions that promote myogenic differentiation rather than self-renewal of immature precursor cells. Clonal assays comparing freshly isolated Pax3-expressing GFP-positive cells with the progeny of these cells generated by short-term culture showed decreased proliferative capacity and an increased fraction of differentiated cells [2], which possibly limited their in vivo expansion and long-term regenerative function upon transplant. Interestingly, most cells that initially express Pax3–GFP seem to retain Pax3–GFP expression after culture [16]. Although the factors that are responsible for mediating satellite cell self-renewal and maintaining satellite cell function are still incompletely defined, several candidate pathways, including Notch, hepatocyte growth factor (HGF) and fibroblast growth factor (FGF) signaling [17], have been implicated in these processes and might be altered by the culture environment. In vitro conditions also might decrease the ability of cultured myogenic cells to survive or to re-enter their physiological niche following intramuscular transfer, as suggested by Beauchamp et al. [18], who reported that 99% of labeled, cultured myogenic cells are lost from the muscle within 24 hours after intramuscular injection. Although the cause of this loss remains unclear, it might relate to immune rejection [19], culture-induced differentiation, cellular stress, or downregulation of survival factors [e.g. B-cell leukemia/lymphoma 2 (Bcl-2)] or growth factor and adhesion receptors {e.g. M-cadherin, c-met or chemokine (CXC motif) receptor 4 (CXCR4) [17]}. www.sciencedirect.com
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Box 1. Outstanding questions † What are the factors that are involved in satellite cell selfrenewal? † What is responsible for the reduced myogenicity of cultured satellite cells? † What is the developmental relationship between satellite cells and other myogenic cells that are isolated from skeletal muscles? † Do myogenic populations that are distinct from satellite cells also lose muscle regenerative potential during cell culture? † Do sorted human satellite cells behave similarly to mouse cells in culture and following transplantation?
This downregulation might predispose cells to apoptosis or inhibit myogenic cell proliferation, differentiation or re-entry into the satellite cell niche upon in vivo injection. Concluding remarks In summary, the work of Montarras et al. [2] emphasizes two considerations that are essential for the success of myogenic cell transplantation: the choice and purity of the cell population that is transplanted and the influence of pre-transplant manipulations. Long-lasting cell therapy for muscle disease will require cells that are capable of long-term muscle formation. Although there are reports of distinct populations with some degree of myogenic potential either in vitro or in vivo [20–22], sublaminar satellite cells seem to be the most abundant cells in muscle that are capable of such regeneration. The precise relationships among muscle satellite cells and these distinct cell populations, as well as the molecular signals that regulate satellite cell activation and differentiation remain important areas for investigation (Box 1). Moreover, the findings of Montarras et al. [2] strongly encourage the use of freshly purified satellite cells in new transplantation strategies, and indicate the urgent need for a more complete understanding of satellite cell self-renewal and the development of culture conditions that maintain mouse, and eventually human, satellite cell regenerative function. In addition, it will be essential to define more clearly the physiological regulators of satellite cell maintenance and function in vivo, as these might represent targets for manipulation to induce or enhance endogenousmuscle regenerative function. Such strategies might provide a necessary alternative to cell therapy via intramuscular delivery of precursor cells, which in many cases might be too cumbersome for clinical practice. The ability to isolate satellite cells with high purity [1,2] will greatly accelerate the understanding of how these cells are regulated and how they can be used for regenerative medicine.
Acknowledgements The authors acknowledge support from a National Science Foundation Graduate Research Fellowship (R.I.S.), the Burroughs Wellcome Fund (A.J.W.) and Harvard Stem Cell Institute (A.J.W.).
References 1 Sherwood, R.I. et al. (2004) Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle. Cell 119, 543–554
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2 Montarras, D. et al. (2005) Direct isolation of satellite cells for skeletal muscle regeneration. Science 309, 2064–2067 3 Relaix, F. et al. (2005) A Pax3–Pax7-dependent population of skeletal muscle progenitor cells. Nature 435, 948–953 4 Gros, J. et al. (2005) A common somatic origin for embryonic muscle progenitors and satellite cells. Nature 435, 954–958 5 Mauro, A. (1961) Satellite cell of muscle skeletal fibers. J. Biophys. Biochem. Cytol. 9, 493–495 6 Armand, O. et al. (1983) Origin of satellite cells in avian skeletal muscles. Arch. Anat. Microsc. Morphol. Exp. 72, 163–181 7 Yablonka-Reuveni, Z. et al. (1987) Isolation and clonal analysis of satellite cells from chicken pectoralis muscle. Dev. Biol. 119, 252–259 8 Seale, P. et al. (2000) Pax7 is required for the specification of myogenic satellite cells. Cell 102, 777–786 9 Zammit, P.S. et al. (2004) Muscle satellite cells adopt divergent fates: a mechanism for self-renewal? J. Cell Biol. 166, 347–357 10 Buckingham, M. et al. (2003) The formation of skeletal muscle: from somite to limb. J. Anat. 202, 59–68 11 Sicinski, P. et al. (1989) The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science 244, 1578–1580 12 Morgan, J.E. et al. (1993) Long-term persistence and migration of myogenic cells injected into pre-irradiated muscles of mdx mice. J. Neurol. Sci. 115, 191–200 13 Qu-Petersen, Z. et al. (2002) Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration. J. Cell Biol. 157, 851–864
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14 Collins, C.A. et al. (2005) Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell 122, 289–301 15 Kondo, M. et al. (2003) Biology of hematopoietic stem cells and progenitors: implications for clinical application. Annu. Rev. Immunol. 21, 759–806 16 Relaix, F. et al. (2006) Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells. J. Cell Biol. 172, 91–102 17 Dhawan, J. and Rando, T.A. (2005) Stem cells in postnatal myogenesis: molecular mechanisms of satellite cell quiescence, activation and replenishment. Trends Cell Biol. 18 Beauchamp, J.R. et al. (1999) Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source. J. Cell Biol. 144, 1113–1122 19 Huard, J. et al. (1994) Gene transfer into skeletal muscles by isogenic myoblasts. Hum. Gene Ther. 5, 949–958 20 Lee, J.Y. et al. (2000) Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing. J. Cell Biol. 150, 1085–1100 21 De Angelis, L. et al. (1999) Skeletal myogenic progenitors originating from embryonic dorsal aorta coexpress endothelial and myogenic markers and contribute to postnatal muscle growth and regeneration. J. Cell Biol. 147, 869–878 22 Asakura, A. et al. (2002) Myogenic specification of side population cells in skeletal muscle. J. Cell Biol. 159, 123–134 1471-4914/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2006.03.002
Ions, energy and axonal injury: towards a molecular neurology of multiple sclerosis Stephen G. Waxman Department of Neurology, LCI 707, Yale Medical School, P.O. Box 208018, New Haven, CT 06520, USA
It is now clear that effective treatments for nervous system disorders such as multiple sclerosis (MS) represent achievable objectives. Molecular mechanisms of axonal degeneration – a major pathological substrate for disability in MS – have been identified, pointing to the possibility of neuroprotection. Although previous studies were mainly carried out in laboratory models, recent analyses of human MS tissue have identified molecular targets that are related to mitochondrial function and specific isoforms of ion channels as contributors to axonal degeneration in MS. Taken together, the observations in model systems and in human tissue converge on the identification of a group of molecules that are related to ion fluxes and energetics as significant actors in the axonal-injury cascade, and suggest a set of molecular targets that might be useful in the development of new therapies.
Axons: targets for neuroprotection Damage to the brain and spinal cord have been Corresponding author: Waxman, S.G. ([email protected]). Available online 30 March 2006 www.sciencedirect.com
classically regarded as irreparable – a nihilistic doctrine that engendered pessimism over the search for therapeutic interventions. Nevertheless, neuroprotective therapies that prevent loss of neurons and thus preserve function after injury to the CNS have been sought for many years. Over the past decade, many studies have focused on dissecting the molecular cascade that produces axonal injury – a major substrate for clinical disability – in multiple sclerosis (MS), which is the most common neurological cause of disability in young adults. These investigations, which were largely carried out in laboratory models, have set the stage for a molecularly-targeted search of neuroprotective approaches that might preserve function in MS. Recent studies have extended the search for tractable molecular targets in human MS tissue. These studies have added a new level of specificity to the identification of ion channels that contribute to axonal injury in MS, and have identified another group of molecular targets that are related to neuronal energetics as potentially significant contributors to axonal injury in MS. Here, we briefly survey these recent findings and
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References 1 Beutler, B. (2004) Inferences, questions and possibilities in Toll-like receptor signalling. Nature 430, 257–263 2 O’Neill, L.A. et al. (2003) The Toll–IL-1 receptor adaptor family grows to five members. Trends Immunol. 24, 286–290 3 Ha¨cker, H. et al. (2006) Specificity in Toll-like receptor signalling through distinct effector functions of TRAF3 and TRAF6. Nature 439, 204–207 4 Oganesyan, G. et al. (2006) Critical role of TRAF3 in the Toll-like receptor-dependent and -independent antiviral response. Nature 439, 208–211 5 Chung, J.Y. et al. (2002) All TRAFs are not created equal: common and distinct molecular mechanisms of TRAF-mediated signal transduction. J. Cell Sci. 115, 679–688 6 Takeuchi, M. et al. (1996) Anatomy of TRAF2. Distinct domains for nuclear factor-kB activation and association with tumor necrosis factor signaling proteins. J. Biol. Chem. 271, 19935–19942 7 Kato, H. et al. (2005) Cell type-specific involvement of RIG-I in antiviral response. Immunity 23, 19–28
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8 Hoebe, K. et al. (2003) Upregulation of costimulatory molecules induced by lipopolysaccharide and double-stranded RNA occurs by Trif-dependent and Trif-independent pathways. Nat. Immunol. 4, 1223–1229 9 Le Bon, A. and Tough, D.F. (2002) Links between innate and adaptive immunity via type I interferon. Curr. Opin. Immunol. 14, 432–436 10 Theofilopoulos, A.N. et al. (2005) Type I interferons (a/b) in immunity and autoimmunity. Annu. Rev. Immunol. 23, 307–335 11 Asadullah, K. et al. (2003) Interleukin-10 therapy – review of a new approach. Pharmacol. Rev. 55, 241–269 12 Lomaga, M.A. et al. (1999) TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling. Genes Dev. 13, 1015–1024 13 Naito, A. et al. (1999) Severe osteopetrosis, defective interleukin-1 signalling and lymph node organogenesis in TRAF6-deficient mice. Genes Cells 4, 353–362 1471-4914/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2006.03.008
Harnessing the potential of myogenic satellite cells Richard I. Sherwood1 and Amy J. Wagers2 1
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA Section on Developmental and Stem Cell Biology, Joslin Diabetes Center, Department of Pathology, Harvard Medical School and Harvard Stem Cell Institute, Boston, MA 02215, USA 2
Adult skeletal muscle has remarkable regenerative potential, which is mainly attributable to a small population of undifferentiated skeletal muscle precursors called satellite cells. These cells reside underneath the basal lamina of skeletal myofibers and can be activated to proliferate, differentiate and fuse to form new muscle tissue. Satellite cells have long been considered promising mediators of therapeutic muscle regeneration. However, in practice, the regenerative function of such cells, which in many cases have been derived or expanded by ex vivo cultures, can be surprisingly low. A recent study from Montarras and colleagues has provided new insights into the requirements for efficient muscle engraftment from purified muscle satellite cells, suggesting possible strategies to enhance their therapeutic potential.
Satellite cells and muscle regenerative medicine As a potentially rich and renewable source of muscle regenerative cells, satellite cells hold tremendous promise for the treatment of muscle disease. Progress towards defining the regulators of satellite cell function has been recently made by the demonstration that myogenic and non-myogenic muscle cells can be discriminated and independently isolated by unique cell-surface markers and fluorescence-activated cell sorting (FACS) [1,2]. Using a novel paired-box-gene-3 (Pax3)–green-fluorescentprotein (GFP) reporter mouse, in which expression of Corresponding author: Wagers, A.J. ([email protected]). Available online 3 April 2006 www.sciencedirect.com
GFP is controlled by the Pax3 gene locus [3], a recent study by Montarras et al. [2] has yielded significant insights into the developmental potential and regenerative function of sorted myogenic satellite cells. These findings have significant implications for harnessing the potential of these cells for clinical use in the treatment of acute and degenerative muscle diseases, either by rejuvenation or activation of endogenous cells, or by transplantation to repopulate a depleted or dysfunctional precursor cell pool. Isolating satellite cells from muscle Pax3 and Pax7 are members of the paired-box transcription factor family and are initially expressed early in development by a small population of muscle precursors that delaminate from the dorsal somite and migrate peripherally to establish embryonic myogenesis [3,4]. Most somitederived progenitors that express both Pax3 and Pax7 differentiate to form mature, multinucleated muscle fibers, but some are retained in the muscle as myogenic satellite cells [3–7]. These contribute to natural muscle growth and homeostatic muscle-cell turnover, and to work-induced muscle hypertrophy and muscle repair. Unlike Pax7 [8,9], Pax3 expression is not uniformly maintained in adult satellite cells [2,10]; however, Pax3 is expressed by satellite cells in a subset of adult muscles, which enabled Montarras et al. [2] to isolate by FACS Pax3-expressing GFP-positive cells from Pax3–GFP reporter mice. Analysis of these cells indicated that they act as myogenic precursors, as Pax3-expressing GFP-positive cells generated exclusively
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Quiescent satellite cell M-cadherin(+) CD34(+) β1-integrin(+) CXCR4(+) Syndecan-3 and syndecan-4(+) C-met(+) Pax7(+) Pax3(+/−) CD45(–) Sca1(–) CD31(–) Flk–1(–)
Fusion
Differentiation Wnt?
Activation FGF and/or HGF?
Activated satellite cell MyoD(+) Myf5(+)
Myoblast Myogenin(+) MRF4(+)
Myofiber Dystrophin(+)
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Figure 1. Adult regenerative myogenesis. In response to extrinsic signals including exercise, muscle damage or degenerative disease, normally quiescent skeletal muscle satellite cells (green) become activated and differentiate to produce myoblasts, which subsequently fuse to generate multinucleated myofibers. Some previously activated satellite cells might return to quiescence to re-establish the satellite cell pool. At each stage of muscle regeneration, differentiating myogenic cells express specific phenotypic markers, some of which are indicated here. Most markers of quiescent satellite cells are not unique to the quiescent state and are also expressed by activated satellite cells [17]. Activated satellite cells can be distinguished from quiescent cells by their induction of MyoD and Myf5. Differentiation into myoblasts is accompanied by both induced expression of differentiation markers (Myogenin and myogenic regulatory factor MRF4) and loss or alteration of expression of at least some satellite cell markers (e.g. CD34, Pax3 and Pax7). Fusion of mononucleated myoblasts generates multinucleated myotubes and myofibers, which induce transcripts that are important for skeletal muscle function such as the structural protein dystrophin. C, expressed by most or all cells; C/K, expressed by a subset of cells. K, not expressed.
myogenic colonies in vitro and O90% of them co-expressed the satellite cell marker Pax7. Notably, !10% of Pax3expressing GFP-positive cells expressed MyoD, a marker of satellite cell activation, indicating that isolation itself does not activate these cells. Consistent with previous reports [1], myogenic satellite cells that were identified by Pax3–GFP expression lacked hematopoietic and endothelial cell surface markers [CD45, CD31, foetal-liver kinase 1 (Flk1) and stem cell antigen-1 (Sca-1)], but expressed the satellite-cell marker CD34 (Figure 1). To evaluate the regenerative capacity of freshly isolated satellite cells in vivo, Montarras et al. [2] isolated Pax3expressing GFP-positive cells from the diaphragm (where the majority of satellite cells expresses Pax3) and transplanted them directly into the tibialis anterior (TA) muscles of heavily irradiated, immunodeficient, dystrophic recipient mice (mdx nu/nu). These mice harbor a point mutation in the muscle structural protein dystrophin, which results in loss of dystrophin expression in myofibers [11], and enables contributions of donor cells to myofiber regeneration in engrafted mdx nu/nu recipients to be measured by restoration of dystrophin expression. In addition, in this mouse model, host immunodeficiency and local destruction of resident satellite cells by muscle irradiation is likely to facilitate engraftment of transplanted cells. Remarkably, intramuscular delivery of only 20 000 freshly isolated Pax3-expressing GFP-positive cells yielded O500 dystrophin-expressing myofibers within 3 weeks, and injection of as few as 1000 Pax3-expressing GFP-positive cells produced 160 dystrophin-expressing myofibers [2]. This high efficiency of myofiber formation www.sciencedirect.com
from freshly sorted Pax3-expressing GFP-positive cells stands in marked contrast to previous studies that employed heterogeneous populations of unsorted cells that were harvested by enzymatic digestion of whole muscle [12] or of cultured muscle-derived cell populations [13], where up to 25 times as many cells were required to achieve similar engraftment rates. Transplantation of Pax3-expressing GFP-positive cells that were isolated from the diaphragm also contributed to regeneration of the satellite cell pool in radiation-damaged TA muscles of mdx nu/nu mice. Three weeks after grafting, Pax3-expressing GFP-positive cells were recovered from TA muscles and shown to maintain characteristic properties of activated satellite cells including association with muscle fibers, expression of Pax7 and MyoD and differentiation in culture to form myotubes [2]. Thus, consistent with recent studies that tracked the fate of satellite cells that are associated with grafted single myofibers [14], these data [2] clearly indicate the ability of satellite cells to contribute both to the formation of new myofibers and to the renewal of the satellite cell pool. However, direct clonal analysis is still required to demonstrate the ability of individual satellite cells to fulfil both self-renewal and differentiation functions (the hallmark of tissue stem cells) and to assay the frequency at which these stem cell activities are present in the satellite cell pool. Interestingly, at least a subset of transplanted Pax3-expressing GFP-positive cells that re-entered the satellite cell compartment in TA muscles retained Pax3 expression, despite the fact that Pax3 is not normally expressed in these muscles [2,10]. These data suggest that Pax3 expression might be cell autonomous. However, it is
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also possible that with longer exposures (O3 weeks) to the TA muscle environment, the donor cells might lose Pax3 expression and adopt a myogenic expression profile that is more typical of their new environment. Fresh is best for satellite cell transplant Many previous studies and several clinical trials that have tested the potential of cell therapy in muscle regeneration have employed ex vivo expanded myoblasts for transplantation. To evaluate the effects of cell culture on the in vivo regenerative function of isolated satellite cells, Montarras et al. [2] directly compared myofiber engraftment from freshly isolated Pax3-expressing GFP-positive cells with that from Pax3-expressing GFP-positive cells that were cultured for three days before grafting. Remarkably, ten times as many previously cultured cells as freshly isolated cells were required to achieve equivalent numbers of myofibers in vivo, indicating a dramatic loss of regenerative activity associated with satellite cell culture. This key finding indicates that ex vivo expansion of myogenic cells does not necessarily translate into expansion of regenerative function, and highlights the crucial importance of direct purification of myogenic satellite cells in studies of their regenerative activity. Notably, a similar loss of regenerative function has been observed in vitro in cultures of other adult tissue precursor cells, including bone marrow-derived hematopoietic stem cells [15]. However, whether this detrimental effect of cell culture is a general property of all adult progenitor cells or depends on the particular cell population under investigation remains to be determined. Although the reason for culture-induced loss of satellite cell function remains unclear, it might be caused, at least in part, by in vitro conditions that promote myogenic differentiation rather than self-renewal of immature precursor cells. Clonal assays comparing freshly isolated Pax3-expressing GFP-positive cells with the progeny of these cells generated by short-term culture showed decreased proliferative capacity and an increased fraction of differentiated cells [2], which possibly limited their in vivo expansion and long-term regenerative function upon transplant. Interestingly, most cells that initially express Pax3–GFP seem to retain Pax3–GFP expression after culture [16]. Although the factors that are responsible for mediating satellite cell self-renewal and maintaining satellite cell function are still incompletely defined, several candidate pathways, including Notch, hepatocyte growth factor (HGF) and fibroblast growth factor (FGF) signaling [17], have been implicated in these processes and might be altered by the culture environment. In vitro conditions also might decrease the ability of cultured myogenic cells to survive or to re-enter their physiological niche following intramuscular transfer, as suggested by Beauchamp et al. [18], who reported that 99% of labeled, cultured myogenic cells are lost from the muscle within 24 hours after intramuscular injection. Although the cause of this loss remains unclear, it might relate to immune rejection [19], culture-induced differentiation, cellular stress, or downregulation of survival factors [e.g. B-cell leukemia/lymphoma 2 (Bcl-2)] or growth factor and adhesion receptors {e.g. M-cadherin, c-met or chemokine (CXC motif) receptor 4 (CXCR4) [17]}. www.sciencedirect.com
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Box 1. Outstanding questions † What are the factors that are involved in satellite cell selfrenewal? † What is responsible for the reduced myogenicity of cultured satellite cells? † What is the developmental relationship between satellite cells and other myogenic cells that are isolated from skeletal muscles? † Do myogenic populations that are distinct from satellite cells also lose muscle regenerative potential during cell culture? † Do sorted human satellite cells behave similarly to mouse cells in culture and following transplantation?
This downregulation might predispose cells to apoptosis or inhibit myogenic cell proliferation, differentiation or re-entry into the satellite cell niche upon in vivo injection. Concluding remarks In summary, the work of Montarras et al. [2] emphasizes two considerations that are essential for the success of myogenic cell transplantation: the choice and purity of the cell population that is transplanted and the influence of pre-transplant manipulations. Long-lasting cell therapy for muscle disease will require cells that are capable of long-term muscle formation. Although there are reports of distinct populations with some degree of myogenic potential either in vitro or in vivo [20–22], sublaminar satellite cells seem to be the most abundant cells in muscle that are capable of such regeneration. The precise relationships among muscle satellite cells and these distinct cell populations, as well as the molecular signals that regulate satellite cell activation and differentiation remain important areas for investigation (Box 1). Moreover, the findings of Montarras et al. [2] strongly encourage the use of freshly purified satellite cells in new transplantation strategies, and indicate the urgent need for a more complete understanding of satellite cell self-renewal and the development of culture conditions that maintain mouse, and eventually human, satellite cell regenerative function. In addition, it will be essential to define more clearly the physiological regulators of satellite cell maintenance and function in vivo, as these might represent targets for manipulation to induce or enhance endogenousmuscle regenerative function. Such strategies might provide a necessary alternative to cell therapy via intramuscular delivery of precursor cells, which in many cases might be too cumbersome for clinical practice. The ability to isolate satellite cells with high purity [1,2] will greatly accelerate the understanding of how these cells are regulated and how they can be used for regenerative medicine.
Acknowledgements The authors acknowledge support from a National Science Foundation Graduate Research Fellowship (R.I.S.), the Burroughs Wellcome Fund (A.J.W.) and Harvard Stem Cell Institute (A.J.W.).
References 1 Sherwood, R.I. et al. (2004) Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle. Cell 119, 543–554
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2 Montarras, D. et al. (2005) Direct isolation of satellite cells for skeletal muscle regeneration. Science 309, 2064–2067 3 Relaix, F. et al. (2005) A Pax3–Pax7-dependent population of skeletal muscle progenitor cells. Nature 435, 948–953 4 Gros, J. et al. (2005) A common somatic origin for embryonic muscle progenitors and satellite cells. Nature 435, 954–958 5 Mauro, A. (1961) Satellite cell of muscle skeletal fibers. J. Biophys. Biochem. Cytol. 9, 493–495 6 Armand, O. et al. (1983) Origin of satellite cells in avian skeletal muscles. Arch. Anat. Microsc. Morphol. Exp. 72, 163–181 7 Yablonka-Reuveni, Z. et al. (1987) Isolation and clonal analysis of satellite cells from chicken pectoralis muscle. Dev. Biol. 119, 252–259 8 Seale, P. et al. (2000) Pax7 is required for the specification of myogenic satellite cells. Cell 102, 777–786 9 Zammit, P.S. et al. (2004) Muscle satellite cells adopt divergent fates: a mechanism for self-renewal? J. Cell Biol. 166, 347–357 10 Buckingham, M. et al. (2003) The formation of skeletal muscle: from somite to limb. J. Anat. 202, 59–68 11 Sicinski, P. et al. (1989) The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science 244, 1578–1580 12 Morgan, J.E. et al. (1993) Long-term persistence and migration of myogenic cells injected into pre-irradiated muscles of mdx mice. J. Neurol. Sci. 115, 191–200 13 Qu-Petersen, Z. et al. (2002) Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration. J. Cell Biol. 157, 851–864
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14 Collins, C.A. et al. (2005) Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell 122, 289–301 15 Kondo, M. et al. (2003) Biology of hematopoietic stem cells and progenitors: implications for clinical application. Annu. Rev. Immunol. 21, 759–806 16 Relaix, F. et al. (2006) Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells. J. Cell Biol. 172, 91–102 17 Dhawan, J. and Rando, T.A. (2005) Stem cells in postnatal myogenesis: molecular mechanisms of satellite cell quiescence, activation and replenishment. Trends Cell Biol. 18 Beauchamp, J.R. et al. (1999) Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source. J. Cell Biol. 144, 1113–1122 19 Huard, J. et al. (1994) Gene transfer into skeletal muscles by isogenic myoblasts. Hum. Gene Ther. 5, 949–958 20 Lee, J.Y. et al. (2000) Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing. J. Cell Biol. 150, 1085–1100 21 De Angelis, L. et al. (1999) Skeletal myogenic progenitors originating from embryonic dorsal aorta coexpress endothelial and myogenic markers and contribute to postnatal muscle growth and regeneration. J. Cell Biol. 147, 869–878 22 Asakura, A. et al. (2002) Myogenic specification of side population cells in skeletal muscle. J. Cell Biol. 159, 123–134 1471-4914/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2006.03.002
Ions, energy and axonal injury: towards a molecular neurology of multiple sclerosis Stephen G. Waxman Department of Neurology, LCI 707, Yale Medical School, P.O. Box 208018, New Haven, CT 06520, USA
It is now clear that effective treatments for nervous system disorders such as multiple sclerosis (MS) represent achievable objectives. Molecular mechanisms of axonal degeneration – a major pathological substrate for disability in MS – have been identified, pointing to the possibility of neuroprotection. Although previous studies were mainly carried out in laboratory models, recent analyses of human MS tissue have identified molecular targets that are related to mitochondrial function and specific isoforms of ion channels as contributors to axonal degeneration in MS. Taken together, the observations in model systems and in human tissue converge on the identification of a group of molecules that are related to ion fluxes and energetics as significant actors in the axonal-injury cascade, and suggest a set of molecular targets that might be useful in the development of new therapies.
Axons: targets for neuroprotection Damage to the brain and spinal cord have been Corresponding author: Waxman, S.G. ([email protected]). Available online 30 March 2006 www.sciencedirect.com
classically regarded as irreparable – a nihilistic doctrine that engendered pessimism over the search for therapeutic interventions. Nevertheless, neuroprotective therapies that prevent loss of neurons and thus preserve function after injury to the CNS have been sought for many years. Over the past decade, many studies have focused on dissecting the molecular cascade that produces axonal injury – a major substrate for clinical disability – in multiple sclerosis (MS), which is the most common neurological cause of disability in young adults. These investigations, which were largely carried out in laboratory models, have set the stage for a molecularly-targeted search of neuroprotective approaches that might preserve function in MS. Recent studies have extended the search for tractable molecular targets in human MS tissue. These studies have added a new level of specificity to the identification of ion channels that contribute to axonal injury in MS, and have identified another group of molecular targets that are related to neuronal energetics as potentially significant contributors to axonal injury in MS. Here, we briefly survey these recent findings and