MSCs may be a useful cell type for a variety of applications in regenerative medicine, although there is currently no clear consensus on the best method of sourcing and isolating MSCs for clinical use. In this study, we have evaluated a recently identified population of traumatized muscle-derived MPCs by quantitatively comparing them to a typical population of bone-marrow-derived MSCs. The traumatized muscle tissue is an attractive source of autolgous cells with MSC-like properties, as it is readily obtained in a clinical setting following orthopaedic injury without requiring additional tissue harvesting procedures [31
]. We have demonstrated that MPCs can be harvested at high concentrations from traumatized muscle, and the proliferation rate, cell surface epitope profile and gene expression profile of the MPCs is substantially similar to that of bone marrow-derived MSCs. The MPCs also appear to maintain a higher level of metabolic activity relative to the MSCs. While they differentiate easily into adipocytes, the MPCs exhibit more limited differentiation into osteoblasts and chondrocytes. Finally, the MPCs express factors that modulate inflammatory responses and promote angiogenesis at functional levels that are comparable to the MSCs. Taken together, these results suggest that while traumatized muscle-derived MPCs may not be a direct substitute for bone marrow-derived MSCs, they perform specific trophic functions that will make them useful in regenerative medicine applications.
Several aspects of this study support our conclusions. First, both histological and quantitative analyses of the differentiation assays were included in our investigation. Compared to our previous qRT-PCR study showing the up-regulation of lineage specific genes by MPCs under the appropriate conditions [17
], the protein-level assays reported in this studyprovide a more precise measure of lineage adoption. Using these higher sensitivity assays, we were able to observe the limitations of the MPCs to differentiate into osteoblasts and chondrocytes relative to the MSCs. Second, in addition to performing the in vitro differentiation assays on this novel population of MSC-like cells, we also investigated the trophic function of these cells, which are assumed to constitute at least part of the therapeutic benefit provided by MSCs. In addition to looking at the expression of genes associated with these functions, we performed functional assays to verify the activity of these trophic mechanisms by the traumatized muscle-derived MPCs. Finally, all of the experiments in this study were performed using human cells from a clinically relevant source. As a result, our findings are directly applicable to the development of therapies that can take advantage of this cell type.
Despite these strengths, a few caveats should be noted. First, this study was designed to investigate the spectrum of regenerative functions that MPCs are capable of performing, but it did not generate any insight into the mechanisms by which the MPCs were able perform these functions. Many of these mechanisms have been identified in MSCs, and it is tempting to assume that they are conserved between the two cell types. We are currently performing more comprehensive studies that are will investigate the mechanism of these functions in greater detail using additional experimental outputs. However, the present study was useful in that it provides a broad and quantitative comparison between the MPCs and bone marrow-derived MSCs. Second, all of the experiments in this study were performed in vitro, and further investigation will be necessary to verify that the traumatized muscle-derived MPCs will exhibit the same regenerative functions in vivo. Finally, the MPCs and MSCs were not age and sex matched due to limitations imposed by the demographics of the patients providing our tissue samples, in view of previous reports on the effects of age and sex on stem and progenitor cell function, the differences between the MPCs and MSCs reported here should be evaluated with caution.
Variations in differentiation potential [32
] and trophic function [33
] of MSCs have previously been correlated with the age and sex of the donors from which the cells were harvested. The precise mechanism leading to these changes in MSC function are not completely understood, although age related changes have been attributed to telomere shortening [35
], and the sex dimorphisms appear to be generated by preconditioning of the MSCs by sex hormones prior to harvest [36
]. In the context of these donor effects, the differences in traumatized muscle-derived MPCs and bone-marrow-derived MSCs can be examined in greater detail. The substantially higher yield of harvested MPCs relative to MSCs is not consistent with the changes in cellularity that might be attributable to donor age or sex [37
], suggesting that the difference in cellularity is dominated by the tissue of origin and the cellular responses to injury. However, despite the donor mismatch, no significant differences in the proliferation rate or expression of lineage specific genes were observed between the two cell types. These results suggest that the difference in mean age between the two donor groups had a small effect on these outputs relative to the overall biological variability. We did find evidence of a potential sexual dimorphism in that the MSCs, which contained cells from female donors, appeared to more effectively suppress T-cell proliferation. However, the difference in immunosuppression between the cell types was slight, and only significantly different at two cell concentrations. The angiogenic function of the MPCs and MSCs also appeared to be equivalent. Taken together, the effects of donor sex also appear to be small relative to the biological variability. Finally, the impaired osteogenic and chondrogenic differentiation of the MPCs relative to MSCs might be expected due to the presence of females in the MSC group, but this result is inconsistent with the expected age-related effects [39
]. As a result, these differences in the differentiation potential might be dominated by the difference in cell type.
Other progenitor cells with properties similar to MSCs have been harvested from human skeletal muscle tissue. Using immunoselection techniques, a population of cells can be isolated from digested muscle tissue that can differentiate into osteoblasts, adipocytes and chondrocytes, as well as into myoblasts [40
]. Termed myoendothelial cells due to the myogenic (CD56) and endothelial cell (CD34 and CD144) markers that distinguish this cell type, they also promote regeneration of skeletal [16
] and cardiac [41
] muscle tissue, in part by secreting pro-angiogenic and pro-survival factors that promote the endogenous repair mechanisms. These cells demonstrate many characteristics of the muscle-derived stem cells (MDSCs), which can be isolated from murine skeletal muscle [42
], although a direct equivalent of MDCSs has not been identified in human muscle tissue. It is noteworthy that the MDSC cell type is isolated on the basis of its slow adherence to tissue culture plastic (TCP) during the harvesting procedure [43
], in contrast to the traumatized-muscle-derived MPCs, which rapidly adhere to the TCP in less than 2 hours.
In humans, it is assumed that the myoendothelial cells are related to pericytes, as both cell types share the perivascular niche. The pericytes also closely resemble MSCs in vitro
, and there has been recent, compelling evidence that pericytes can be harvested from various tissues throughout the body and induced to exhibit MSC characteristics [18
]. The MPCs may be an activated descendent of the myoendothelial/pericyte cell types that have (1) been activated in response to traumatic injury, (2) down-regulated the expression of surface proteins required for their vascular niche [44
], and (3) began to proliferate in the tissue [22
]. There is also recent evidence indicating that a multipotential progenitor cell population may be derived from the vasculature via epithelial to mesenchymal transition in response to injury [45
]. Taken together, these studies suggest that the MPCs may arise from the vasculature in large numbers following trauma, and they may participate in the wound healing process by secreting trophic factors to promote tissue regeneration by mechanisms similar to those used by the myoendothelial cells and pericytes [46
The MPCs may also be descendants of bone marrow-derived MSCs that entered the traumatized tissue via the bloodstream while homing to the site of injury [47
]. Some differences were detected between the MPCs and bone marrow-derived MSCs, and these differences may reflect the tissues from which they were harvested and the extracellular environment immediately prior to harvest. The MPCs exhibited higher metabolic activity than the MSCs, which may indicate that they undergo mitochondrial biogenesis in response to injury and to prepare them for their contribution to the wound healing response [48
]. The differences in the baseline gene expression profile may also be justified given that MPCs were in the regenerating muscle tissue at the time of harvest, whereas the MSCs were in their bone marrow niche [20
]. For example, the MSCs expressed higher levels of VCAM1
, which is characteristic of genes associated with bone physiology and maintenance by the marrow stroma [49
], whereas the MPCs expressed higher levels of genes that indicate neuromuscular differentiation: THY1
] and NES
]. It was not possible to definitively trace the origin of the MPCs to the muscle tissue or the bone marrow without using an in vivo
injury model. However, it is likely that there are multiple sources of stem and progenitor cells that converge into the MPC phenotype once they are exposed to the biochemical milieu of the traumatized muscle tissue, and this heterogeneity could account for differences between MPCs and the more homogeneous population of bone marrow-derived MSCs.
Regardless of their origin, it appears that the muscle-derived MPCs may be a clinically useful population of autologous cells for regenerative medicine, particularly in orthopaedic applications [52
]. The typical standard of care for musculoskeletal injuries is to debride to the wound margin until definitive closure is possible [31
]. The results of this study indicate that the viable portions of debrided tissue, typically discarded as surgical waste, might instead be harvested to obtain MPCs, which can be used augment the wound healing process. The MPCs exhibit the trophic functions that are characteristic of bone marrow-derived MSCs, and they can be harvested without the need for additional procedure, such as bone marrow aspiration, which may be painful and exposes patient to additional surgical risks. Although the immunoregulatory functions of MPCs may be less effective than MSCs on a per cell basis, the MPCs can be harvested from traumatized muscle tissue at concentrations that are orders of magnitude higher than MSCs from bone marrow (). Therefore, the overall immunoregulatory function of MPCs harvested per gram of muscle tissue should be at least as effective as the MSCs harvested per gram of bone marrow (). Furthermore, the MPCs may be harvested at sufficient numbers for immediate clinical use without the need for ex vivo
expansion. We are currently evaluating the use of autologous MPCs to promote regeneration of muscle, as well as other tissues that are typically damaged as a result of orthopaedic trauma, such as bone, nerve and blood vessels [53
]. MPCs may also be useful to manage GvHD following transplant of orthopaedic tissues [55
In this study, we have evaluated the traumatized muscle-derived MPCs by quantitatively comparing their differentiation potential and trophic properties to bone marrow-derived MSCs. The two cell types share many similarities, although the extent to which the MPC population is able to undergo osteogenic and chondrogenic differentiation appears to be limited. Both cell types appear to exhibit immunoregulatory and pro-angiogenic trophic functions, which are an important component of the regenerative benefit of MSCs. Therefore, the traumatized muscle-derived MPCs appear to be an alternative source of autologous cells that are capable of performing the trophic functions that enhance tissue regeneration. The MPCs may have an advantage over bone marrow-derived cells in cellular therapy applications that follow orthopaedic trauma since they may be harvested without performing an additional surgical procedure, they do not require ex vivo expansion, and they appear activated by the trauma to participate in the wound healing response. We are continuing to investigate the mechanisms that mediate these trophic abilities in the MPCs and their function during wound healing. Simultaneously, we are developing regenerative medicine and tissue engineering strategies that take advantage of these functions to promote tissue regeneration.