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It has previously been shown that bone marrow cells contribute to skeletal muscle regeneration, but the nature of marrow cell(s) involved in this process is unknown. We used an immunocompetent and an immunocompromised model of bone marrow transplantation to characterize the type of marrow cells participating regenerating skeletal muscle fibres. Animals were transplanted with different populations of marrow cells from Green Fluorescent Protein (GFP) transgenic mice and the presence of GFP(+) muscle fibres were evaluated in the cardiotoxin-injured tibialis anterior muscles. GFP(+) muscle fibres were found mostly in animals that received either CD45(−), lineage(−), c-Kit(+), Sca-1(+) or Flk-2(+) populations of marrow cells, suggesting that haematopoietic stem cells (HSC) rather than mesenchymal cells or more differentiated haematopoietic cells are responsible for the formation of GFP(+) muscle fibres. Mac-1 positive population of marrow cells was also associated with the emergence of GFP(+) skeletal muscle fibres. However, most of this activity was limited to either Mac-1(+) Sca(+) or Mac-1(+)c-Kit(+) cells with long-term haematopoietic repopulation capabilities, indicating a stem cell phenotype for these cells. Experiments in the immunocompromised transplant model showed that participation of HSC in the skeletal muscle fibre formation could occur without haematopoietic chimerism.
Numerous studies have reported that bone marrow cells may participate in the formation of skeletal muscle fibres. Although the frequency of these events is reported by some investigators to be low (0·01–0·1%), others have found that up to 5% of the fibres in a muscle stress model are bone marrow-derived (Brazelton et al, 2003; Corbel et al, 2003; Palermo et al, 2005). Our recent work demonstrated that injury was a critical aspect of this phenomenon and by using appropriate transplant variables, including cell dose, radiation dose, and mode of cell delivery, levels of up to 12% were possible (Abedi et al, 2004, 2005). Some investigators have suggested that marrow cells that have homed to muscle follow a biological progression, first forming satellite cells and then fusing to form mature myofibres (LaBarge & Blau, 2002). However, the same group later suggested that marrow cells directly fused to the muscle fibres (Doyonnas et al, 2004). It is not clear whether all bone marrow cells that participate in muscle regeneration follow this progression or if a proportion of cells fuse directly with existing myofibres without an intermediate transformation. A greater understanding of these variables could elucidate the mechanisms by which marrow cells are recruited, incorporated and reprogrammed during this process, and so aid the design of future experiments for the treatment of muscle diseases, such as the muscular dystrophies.
The haematopoietic system is comprised of many different cell types including mesenchymal cells, endothelial precursor cells, haematopoietic stem cells (HSC) and their progenitors, together with the more differentiated (i.e. lineage positive) cells such as monocytes, lymphocytes, granulocytes and erythrocytes. It is not known which subpopulation of marrow cells participate in muscle regeneration and attempts to identify the cell type has resulted in conflicting results suggesting either haematopoietic stem cells, mesenchymal stem cells, myelomonocytic cells, or even macrophages are responsible for this phenomenon (Camargo et al, 2003; Anjos-Afonso et al, 2004; Doyonnas et al, 2004; Dezawa et al, 2005). To address the controversy in the literature regarding the origin of marrow-derived muscle fibres, we conducted a systematic approach by isolating different subpopulations of marrow cells, based on their surface markers, and studied their participation in the formation of muscle fibres. Subpopulations of marrow cells were isolated using fluorescence-activated cell sorting (FACS)-based fractionation of cells from bone marrow, followed by intravenous injection into the radiated immunocompetent mice or by direct intramuscular injection in immunocompromised host. The latter approach makes it possible to examine more mature cells that are not capable of reconstituting the blood.
The present study found that HSC, rather than other marrow cell types (such as mesenchymal or stromal cells), contributed to muscle fibre formation. We further identified that HSC, and not their differentiated derivatives, have the capacity to be incorporated into skeletal muscle fibres.
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This study found that a population of bone marrow stem cells directly participates in the regeneration of muscle fibres. We could not show any such participation from haematopoietic progenitor cells, lineage-differentiated cells or mesenchymal cells. The present study did not address the mechanism of marrow to muscle conversion but focussed on the type of marrow cells responsible for this process, regardless of the mechanism (fusion or direct differentiation of marrow cells to satellite cells), because participation of marrow cells in muscle regeneration would still be scientifically and clinically significant. Furthermore, we have previously shown that, by appropriate conditioning of the host, increasing numbers of marrow cells participated in formation of muscle fibres (Abedi et al, 2005). However, for practical reasons, we used a more simplified protocol (with lower yields for GFP+ muscle fibres) to characterize the type of marrow cells.
In recent years, multiple reports have shown that marrow cells are involved in the regeneration of skeletal muscle fibres. In these experiments, the investigators used one of several cell tracking methods (i.e. transgenic mice expressing fluorescent proteins, male donors to female recipients, or beta galactosidase transgenic mice) to follow the fate of the donor marrow cells in the recipient mice. The type of marrow cells responsible for this phenomenon, remains unknown. While some researchers showed a mesenchymal origin for marrow-derived muscle fibres (Anjos-Afonso et al, 2004; Dezawa et al, 2005), others have suggested that haematopoietic cells are responsible for regeneration of muscle fibres (Camargo et al, 2003; Doyonnas et al, 2004). Characterization of mesenchymal cells has proved difficult and, at least in the mouse, there are no specific mesenchymal markers, although a combination of both positive and negative markers have been used for their characterization (Krebsbach et al, 1999; Alhadlaq & Mao, 2004). Still, most investigators agree that mesenchymal cells are CD45 (−), c-Kit (−) and Flk-2 (−). Our data showed that CD45(+), c-Kit(+) and Flk-2(+) cells were responsible for the appearance of GFP(+) muscle fibres in recipient mice. Furthermore, transplantation of cells from mesenchymal culture versus cells from the long-term haematopoietic Dexter's culture, shows that the majority of GFP(+) muscle fibres came from the latter. Although it is always difficult to exclude contamination of the transplanted cells with a rare mesenchymal stem cell, our data suggests that, at least in the context of our experimental setting, haematopoietic cells have more potential than mesenchymal cells to be involved in the regeneration of muscle fibres.
It has recently been shown that a single haematopoietic stem cell transplanted to a sublethally-irradiated recipient can give raise to a progeny that reconstitutes the blood and integrates into regenerating myofibres (Camargo et al, 2003; Corbel et al, 2003). However, their data do not show that HSC directly participate in muscle regeneration. In fact, Doyonnas et al (2004) proposed that it is not the marrow stem cells, but a subpopulation of progenitor cells (myelomonocytic cells) that are responsible for this phenomenon. Our results showed that the majority of GFP(+) muscle fibres came from undifferentiated HSC. Progenitor cells, as well as terminally differentiated cells, did not result in GFP(+) muscle fibres. Our experiments in the immunocompromised mouse model (i.e. Beige SCID) also clearly showed that the same populations of marrow stem cells, when directly injected into injured muscle, can result in GFP(+) muscle fibres. This occurred in the absence of any further haematopoietic differentiation of stem cells and haematopoietic chimerism.
Other investigators have suggested that macrophages fusing to other cells resulted in the formation of marrow-derived tissues. For example, fusion of macrophages to the recipient hepatocytes brought about the regeneration of liver from marrow cells (Rizvi et al, 2006 and Willenbring et al, 2004). This argument was based on the use of Mac-1 for the definition of macrophages. Our results showed that surface markers, like Mac-1, could be misleading since a subpopulation of the Mac-1(+) cells, harbouring stem cells markers such as Sca-1 and c-Kit, were able to engraft and repopulate the haematopoietic system in long term experiments and produce high proliferative colonies in HPP assays. In our experiments, only Mac-1 (+) cells with stem cell potential, and not the true macrophages, were responsible for the appearance of GFP (+) muscle fibres.
The discrepancies between our data and those of other investigators results primarily from differences in experimental design. For example, the experiments by Doyonnas et al (2004) were performed by direct injection of different populations of marrow cells into muscle of immunocompetent mice without any systemic irradiation or immunosuppression, while our experiments were performed either after systemic irradiation or in immunosuppressed hosts. Immunoreactivity of GFP (Rosenzweig et al, 2001; Steinbauer et al, 2003; Inoue et al, 2004) may result in different outcomes between the two sets of experiments. In our experience, the infusion of up to 100 × 106 bone marrow cells from GFP transgenic mice (C57BL/6 background) into non-irradiated C57BL/6 mice produced minimal or no chimerism, suggesting the strong immunoreactivity of GFP (data is not shown). Other investigators have shown that mesenchymal cells could integrate into the muscle fibres (Anjos-Afonso et al, 2004; Dezawa et al, 2005) while our experiments showed very little evidence for that. Our previous data showed that marrow stem cells had a tendency to avidly bind to marrow stromal cells (where multiple washes failed to separate them from the stromal cells; Frimberger et al, 2001) and, at least in short term cultures, switching the mesenchymal culture medium (RPMI with 20% fetal calf serum) to a Dexter's culture system will frequently result in the recovery of haematopoiesis in the cultures, suggesting the presence of dormant HSC. One can speculate that the presence of these rare HSCs in the mesenchymal cell preparations might account for some of the reports that showed mesenchymal cells as the origin of marrow-derived muscle fibres. Alternatively, our experiments do not rule out the possibility that, with different experimental designs, mesenchymal cells may also show differentiation to muscle fibres as they do in in-vitro models.
The process that leads to the participation of marrow cells in regeneration of muscle fibres remains unclear. Whether these cells become satellite cells, assume a different stem cell phenotype such as pericytes or mesangioblasts, directly fuse with regenerating myofibres, or employ a combination of some or all of these pathways remains to be determined. Further lineage studies using tools such as genetic markers will be required to define the intermediate cell types that may be involved in appearance of marrow-derived muscle fibres.
We would like to acknowledge Dr Nicola Koutab and his staff for the help with FACS analysis. We also thank Dr Elaine Bearer for help with imaging and microscopy. This publication was made possible by Grant Number 1P20 RR018757-01 from the National Center for Research Resources (NCRR), and by Grant Number 1KO8 HL072332-01 from NHLBI, both are components of the National Institutes of Health (NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NCRR or NIH.