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Fusion of bone marrow–derived stem cells with striated muscle may not be sufficient to activate muscle genes
Giulio Cossu
Stem Cell Research Institute, DIBIT, Milan, Italy.
Address correspondence to: Giulio Cossu, Stem Cell Research Institute, DIBIT, H. San Raffaele, 58 Via Olgettina, 20132 Milan, Italy. Phone: 3902-2156-0250; Fax: 3902-2156-0220; E-mail: cossu.giulio@hsr.it.
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Published December 1, 2004 - More info
J Clin Invest. 2004;114(11):1540–1543. https://doi.org/10.1172/JCI23733.
© 2004 The American Society for Clinical Investigation
Bone marrow–derived side population (BM-SP) stem cells have the ability to repopulate the hematopoietic system (1) and to colonize, at low frequencies, many different tissues, including skeletal (2) and cardiac muscle (3). The δ-sarcoglycan–null mouse is a model of cardiomyopathy and muscular dystrophy (corresponding to a human limb-girdle myopathy) and develops microinfarcts in heart and skeletal muscle (4). Muscle cell death in this model is related to the lack of δ-sarcoglycan, which causes loss of the other sarcoglycans (α, β, and γ) and in turn disassembly of the dystroglycan complex (Figure 1), which is composed of several proteins that link the cytoskeleton to the basal lamina and reduce membrane stress during contraction. Absence or reduction of the dystroglycan complex results in increased fragility of the membrane and increased chance of damage to the muscle cell. Resident cells capable of repairing injured skeletal muscle (satellite cells) are well characterized (5), and despite the long-held belief that heart cells cannot regenerate, evidence for the existence of cells with a similar reparative function in the heart has only begun to accumulate during the last 2 years (6, 7). This observation suggests that injured δ-sarcoglycan–deficient tissues should recruit both local and blood-born stem cells that may contribute to regeneration.
Figure 1Schematic representation of the dystroglycan complex at the membrane of the muscle cell. A number of transmembrane proteins, including dystroglycan (DG), sarcospan (SP), and sarcoglycans (α, β, γ, and δ), connect the actin cytoskeleton to the basal lamina by binding dystrophin (in turn linked to actin) on the cytoplasmic side and laminin on the extracellular side of the muscle plasma membrane. Other functionally important proteins such as nitric oxide synthetase (NOS) are maintained at the cytoplasmic side of the membrane by a different protein complex also bound to dystrophin.
In this issue of the JCI, Lapidos et al. (8) transplanted BM-SP stem cells from wild-type male mice into female δ-sarcoglycan–null mice, and their results, consistent with those from previous studies of stem cell–mediated skeletal and cardiac muscle regeneration, demonstrated the presence of these donor cells, determined by the presence of a Y chromosome inside cardiac and skeletal muscle at the expected frequency. A fraction of the nuclei of these Y chromosome–positive donor cells was unequivocally shown to be present inside the cytoplasm of several differentiated cardiomyocytes and myofibers. Surprisingly, exhaustive examination of the quadriceps muscles and of the hearts of the 14 recipient mice (representing many thousands of muscle cells) detected only 2 sarcoglycan-positive fibers; all cardiomyocytes containing donor-derived nuclei were sarcoglycan negative. The presence of only 2 sarcoglycan-positive fibers is important, as it confirms that BM-SP stem cells can produce δ−sarcoglycan but do so at a negligible frequency. The absence of sarcoglycan expression from wild-type donor cells indicates impaired muscle differentiation of donor-derived nuclei, thereby limiting the regenerative potential of these cells. Appropriate controls demonstrate that transplantation of primary myoblasts isolated and cultured from wild-type male mice into δ-sarcoglycan–deficient female mice restore expression of all sarcoglycans in regenerated myofibers, which confirms that the sarcoglycan complex could be restored through transplantation in these mice. These data raise the question: Why do BM-SP stem cells fail to express δ-sarcoglycan even after exposure to myogenic transcription factors?
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Muscle differentiation is a multistep process
We are inclined to think that once differentiation is triggered either by the binding of soluble molecules to the cell surface or by transcription factors from neighboring nuclei within the same cytoplasm (in the case of cell fusion), it will automatically lead to the full expression of the complete genetic repertoire of a differentiated tissue. Yet we know from decades of tissue culture studies that this is not true. In vitro, skeletal myoblasts differentiate into multinucleated myotubes upon depletion of growth factors in the medium and express a large number of muscle-specific genes (12). However the genes encoding adult isoforms of sarcomeric proteins as well as other “late” genes characteristic of mature muscle fibers in vivo are not expressed in vitro in the absence of nerve and hormones (13). δ-Sarcoglycan is only expressed during the formation of the dystroglycan complex (Figure 1) and thus may represent a “late” muscle gene (14) that could not be expressed by BM-SP stem cell nuclei (Figure 2), possibly due to a failure to reprogram gene expression. From the experimental design of the Lapidos study (8), it was not possible to know to what degree the donor-derived nuclei have entered into the myogenic pathway (all muscle genes other than δ-sarcoglycan may in fact derive from host nuclei) and whether δ-sarcoglycan may be detectable at a later developmental stage, even though a longer time interval after transplantation was not sufficient to detect its expression. In this context it would be interesting to repeat the experiments conducted by Lapidos et al., obtaining BM-SP stem cells from mice that express a LacZ reporter gene under the control of a myogenic determination gene (i.e., Myf5 or MyoD) or of a muscle gene expressed early after differentiation, such as MLC1/3F (11). I would predict that LacZ would be expressed at variance with δ-sarcoglycan. If this proves to be the case, then previous conflicting reports could be reconciled.
Figure 2Different degrees of reprogramming of the nucleus of a wild-type BM-SP stem cell from a male mouse once inside a cardiomyocyte or a myofiber of a female δ-sarcoglycan_null mouse. The model proposes that the Y chromosome_positive (green) donor nucleus (gray) will be exposed to transcription factors from recipient nuclei (cream) as a consequence of either fusion or transdifferentiation. The donor nucleus may then be subject to different fates: no reprogramming at all (I); transcription of myogenic determination genes but not of muscle structural genes (II); transcription of myogenic determination genes and of muscle structural genes expressed during early differentiation only (III); or transcription of all muscle genes (IV). The last event is ruled out in the study of Lapidos et al. (8) by the lack of δ-sarcoglycan expression observed after transplantation of wild-type BM-SP stem cells into δ-sarcoglycan_null mice.
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