This study exploits the novel application of stem cell therapy specific for COL6 CMD. We tested the hypothesis that collagen VI secreted by ADSC in the ECM can ameliorate the genetic impairment of skeletal muscles in the COL6 CMD mouse model. We developed a novel procedure for isolation of ADSC from neonatal human skin discarded after newborn circumcision and demonstrated that established cultures represent a morphologically homogeneous population of cells with phenotypic and functional features of mesenchymal progenitors. Also, we conducted a comprehensive gene expression profile of ADSC with special emphasis on ECM and adhesion molecules. Furthermore, we showed for the first time that locally transplanted ADSC are capable of donating collagen VI protein into muscle of Col6a1
mice under physiological and CTX-injury/regeneration conditions.
Adult stem cells represent a diverse group of stem cells, which are characterized by their ability to self-renew and their ability to differentiate along multiple lineage pathways. These unique features make these cells an attractive therapeutic option for transplantation and regeneration of damaged tissues. The most common type of adult stem cells is multipotent MSC, which can be isolated from several sources, including bone marrow, adipose tissue, dental pulp, placenta, umbilical cord and fallopian tube . MSC isolated originally from bone marrow are defined as fibroblast-like cells with extensive proliferation capacity in culture and the ability to differentiate in vitro into various connective tissue cell types [28, 29]. Recent advances in stem cell biology have shown that adult stem cells isolated from adipose tissue exhibit a number of properties suggesting the feasibility of their use as therapeutic cells . The most important features of adipose tissue are its abundance in the body, relatively simple isolation procedures, expandability to relatively large quantities under minimal conditions and the ability to engraft after local or systemic reintroduction [22, 23].
Here, we developed a novel procedure for the isolation of ADSC from neonatal human skin discarded after newborn circumcision and demonstrated that established cultures represent population of cells with features of mesenchymal progenitors. The developed isolation protocol proved to be a reliable approach to prepare a homogeneous fraction of human ADSC. Our antigenic cell surface profile is consistent with a previously published immunophenotypic analysis available for adult stem cells . A hallmark characteristic of stem cells is their self-renewal potential and ability to differentiate into several cell lineages in vitro with the appropriate inductive media. The established ADSC cultures were able to differentiate toward adipogenic, osteogenic and chondrogenic lineages in vitro in the presence of lineage specific differentiation factors. Successful differentiation was confirmed by morphological changes demonstrated using lineage specific staining and gene expression analysis of genetic markers for the cell type of interest. Overall, these results are consistent with previously published works.
Multiple gene expression profiling studies have shown that adult stem cells, in particular MSC, can produce a vast array of proteins, including growth factors, cytokines, ECM and adhesion molecules . However, no data are available regarding the expression of ECM and related genes by human neonatal ADSC. Our comprehensive gene expression analysis has shown that neonatal ADSC express a plethora of various ECM molecules, including many collagens and laminins, as well as adhesion molecules and other ECM-related genes. Of special interest to this study is collagen VI, ultimate cause of COL6 CMD. We show that ADSC produce a significant level of all three collagen VI mRNAs, in particular COL6A1. The use of ADSC for the treatment of COL6 CMD thus seems to be a logical choice since the cells are able to supply continuously the missing normal collagen VI protein.
Cell-based therapy has been explored for different types of muscular dystrophies, in particular, Duchenne muscular dystrophy [19, 31]. The majority of these studies evaluated muscle derived progenitor or stem cells, such as myoblasts, side population cells, myogenic endothelial cells and mesoangioblasts. Several studies have tested stem cells derived from non-muscle tissues [32–34]. The transplanted cells were found to have limited ability to regenerate muscle fibers but were capable of reducing inflammation through trophic effects produced by transplanted cells [33, 35]. Thus, even though a range of therapeutic approaches have been investigated for traditional muscular dystrophies, there is a need to develop treatment strategies that specifically target muscle ECM alterations. In the skeletal muscle, collagen VI is synthesized by muscle fibroblasts rather than muscle cells . This is in stark contrast to most other muscular dystrophies in which the gene mutations usually involve intracellular or cell surface proteins produced by muscle cells. Therefore, the ability of stem cells to differentiate into muscle cells is not crucial for the therapeutic intervention of COL6 CMD. Moreover, the multi-lineage differentiation capacity of these cells is ideally suited for therapeutic intervention of COL6 CMD in which different connective tissue cell types need to be replenished. Also, apart from the cell regeneration capacity, an emerging therapeutic application of the stem cell therapy takes advantage of their ability to secrete cytokines and growth factors that are anti-apoptotic, pro-angiogenic, anti-inflammatory and anti-fibrotic . Increased muscle cell apoptosis and interstitial fibrosis are typical pathological features of CMDs; therefore, stem cell transplantation likely will be beneficial even without cell differentiation and regeneration.
In this study, the therapeutic potential of neonatal ADSC was examined after local intramuscular injection into the GCM of Col6a1
mice, allowing human cell transplantation without immune rejection. Our study has shown for the first time that ADSC contributed robustly to the collagen VI-deficient GCM by providing efficient engraftment, wide-distribution of therapeutic cells in muscle tissue, continuous production of collagen VI protein by engrafted cells throughout the observation period and proper assembly of collagen VI microfibrils in the interstitial connective tissue of muscle, thus achieving restoration of the affected muscle basement membrane. One of the striking observations is the location of donor cells within the muscle tissue at initial stages of transplantation. During the first two weeks, the vast majority of ADSC was preferentially detected in perimysial space with very few cells in endomysium. Such unique locale could be explained by the fact that the muscle ECM, which is rich in connective tissue and adhesion proteins, may provide a proper environment for firm anchorage/attachment of ADSC. Once cells are acclimated to the perimysial environment, they start to migrate along endomysium, possibly in response to pro-migratory factors secreted by resident interstitial fibroblasts and muscle cells. Importantly, the number of α1(VI) collagen-positive myofibers was markedly increased (two-fold) at week six as compared to the first week of transplantation, suggesting the possibility of long-term commitment of ADSC in the muscle environment.
The capacity of neonatal ADSC to participate in the regeneration of skeletal muscle was further evaluated using the CTX-induced myonecrosis model with actively ongoing regeneration and remodeling of muscle tissue. Several important observations were made during these studies. It is believed that the depletion of the satellite cell pool with consequent irreversible muscle degeneration is responsible for terminal muscle failure in DMD, COL6 CMD and, possibly, other muscular dystrophies [37, 38]. Restoration of the regeneration potential of muscle tissue by transplanted stem cells may provide long-term efficacy for muscle homeostasis. This notion is strongly supported by a recent observation that transplantation of wild-type fibroblasts into Col6a1 null mice was able to ameliorate regeneration and satellite cell homeostasis by restoring muscle mechanical properties and satellite cell activity . However, these effects may be somehow limited by the fact that the grafted cells are differentiated fibroblasts. Our data clearly demonstrate that ADSC are able to colonize in muscle, sustaining their long-term maintenance and the continuous replenishment of collagen VI. However, our data demonstrated that ADSC do not directly participate in the repair of CTX-damaged muscle fibers. Immunofluorescent analysis did not show any ADSC entering the satellite niche, as judged by the position of lamin A/C-positive cells in newly regenerated myofibers. In addition, double-immunostaining of CTX-treated biopsies with lamin A/C (donor human cells) and Pax7 (a marker for both quiescent and activated satellite cells) antibodies did not show any overlapping signals. Taken together, these data suggest that ADSC may not possess cell-autonomous properties supporting myogenic differentiation in vivo. Also, it may suggest that CTX treatment alone does not induce or enhance production of supporting factors to provide a more conducive environment for ADSC to go through myogenic commitment. Nevertheless, CTX-treated muscles receiving the ADSC transplant showed a significant increase (four-fold) in the number of collagen VI-positive myofibers at the sixth week compared to the first week of transplantation and a two-fold increase compared to transplanted muscle without CTX-injury at the sixth week. It is important to note that ADSC engraftment was not dependent on ablative injury, as cells also contributed at high efficiency (approximately 20%) after transplantation into the GCM and sustained at this level throughout the study. However, it is interesting to note that after CTX-injury collagen VI-donating ADSC were able to migrate intramuscularly from their initial perimysial locale shortly after transplantation to the CTX-injected site, and eventually repopulated a significant area of the damaged tissue and adjacent myofibers. This directional migration from perimysium to endomysium could possibly be induced in response to pro-migratory factors released in the course of CTX-injury and regeneration, elicited by various mechanisms. As one explanation, it is likely that pro-inflammatory cytokines, chemokines and growth factors that typically result in homing of immune cells to a damaged site are released from muscle resident cells, stimulating directional migration of ADSC within muscle tissue. In fact, a prominent feature of CTX-injured muscle is a striking inflammatory infiltrate of immune cells, such as macrophages and neutrophils. Also, it is well known that CCR2 and CXCR2 are major regulators of induced macrophage and neutrophils trafficking in vivo[39, 40]. In addition, the CXCR4-CXCL12 chemotactic axis was shown to play a significant role in regulating migration of both proliferating and terminally differentiated muscle cells . Based on our assessment of chemokine receptors on ADSC, it is plausible that ADSC expressing those receptors can be recruited to the CTX-damaged site by the chemotactic mechanisms of inflammatory and satellite cells, respectively. Also, it is possible that abnormal signaling due to the deficient ECM leads to secretion of various chemokines, which may activate corresponding receptors on ADSC and facilitate migration toward the highest concentration of chemokine(s). Together, these observations suggest that mobilization of ADSC in CTX-damaged muscle may highly depend on the local inflammatory state rather than other factors. The ability of ADSC to donate the protein of interest to the injured muscle strongly supports the possibility of stem cell therapy for COL6 CMD, which exhibits substantially more severe myopathology than the mouse model.