Expansion Of Muscle-Derived Stem Cells:Implications of Cell Therapy For Muscle Regeneration

Key to advancing stem cell utilization in regenerative medicine and cell-based therapies is the development of systems to expand cells to clinically relevant numbers while maintaining the desired stem cell phenotype. Mathematical growth models play an important role in developing standardized systems, as they are both predictive tools for expansion potential and tools to describe current kinetic parameters of a stem cell population. One disease that may benefit from cell therapy is Duchenne Muscular Dystrophy (DMD), a muscle disease characterized by the lack of dystrophin expression at the sarcolemma of muscle fibers resulting in muscle fiber necrosis and muscle weakness. While transplantation of normal myoblasts into dystrophin-deficient muscle can restore dystrophin, the use of muscle-derived stem cells (MDSC) has enhanced the success of cell transplantation. For these reasons, muscle stem cell isolation and the development of transplantation techniques have garnered increased attention recently. One limitation of MDSC use is the few numbers of cells available from a muscle biopsy, thus presenting the requirement for in vitro expansion. The overall goal of this study was to provide a thorough quantitative examination of the expansion of MDSC populations. In this project, an imaging system was established to analyze stem cell expansion. The applicability of this system was demonstrated in MDSC expansion with cytokine stimulation. It was found that accounting for the proliferative heterogeneity that exists in stem cell populations would allow for more accurate estimations of kinetic parameters. Next, a more sophisticated imaging system was used to further develop an automated system for analysis of MDSC proliferation and behavioral characterization. Finally, an understanding of the limits of expansion was explored. The role of long-term expansion on stem cell phenotype and regeneration capacity was examined to consider the issue of quantity vs. quality of muscle-derived stem cells. This study provided a systematic method for assessing expansion and an in-depth investigation into the natural progression of stem cell expansion. It is expected that these findings will provide a biological understanding of the limits of expansion and a foundation for more standardized methods of expansion of MDSC as MDSC are advanced to a clinical setting

Human muscle-derived cell populations isolated by differential adhesion rates: Phenotype and contribution to skeletal muscle regeneration in Mdx/SCID mice

Muscle-derived stem cells (MDSCs) isolated from murine skeletal tissue by the preplate method have displayed the capability to commit to the myogenic lineage and regenerate more efficiently than myoblasts in skeletal and cardiac muscle in murine Duchenne Muscular Dystrophy mice (mdx). However, until now, these studies have not been translated to human muscle cells. Here, we describe the isolation, by a preplate technique, of candidate human MDSCs, which exhibit myogenic and regenerative characteristics similar to their murine counterparts. Using the preplate isolation method, we compared cells that adhere faster to the flasks, preplate 2 (PP2), and cells that adhere slower, preplate 6 (PP6). The human PP6 cells express several markers of mesenchymal stem cells and are distinct from human PP2 (a myoblast-like population) based on their expression of CD146 and myogenic markers desmin and CD56. After transplantation to the gastrocnemius muscle of mdx/SCID mice, we observe significantly higher levels of PP6 cells participating in muscle regeneration as compared with the transplantation of PP2 cells. This study supports some previous findings related to mouse preplate cells, and also identifies some differences between mouse and human muscle preplate cells

High Harvest Yield, High Expansion, and Phenotype Stability of CD146 Mesenchymal Stromal Cells from Whole Primitive Human Umbilical Cord Tissue

Human umbilical cord blood is an excellent primitive source of noncontroversial stem cells for treatment of hematologic disorders; meanwhile, new stem cell candidates in the umbilical cord (UC) tissue could provide therapeutic cells for nonhematologic disorders. We show novel in situ characterization to identify and localize a panel of some markers expressed by mesenchymal stromal cells (MSCs; CD44, CD105, CD73, CD90) and CD146 in the UC. We describe enzymatic isolation and purification methods of different UC cell populations that do not require manual separation of the vessels and stroma of the coiled, helical-like UC tissue. Unique quantitation of in situ cell frequency and stromal cell counts upon harvest illustrate the potential to obtain high numerical yields with these methods. UC stromal cells can differentiate to the osteogenic and chondrogenic lineages and, under specific culturing conditions, they exhibit high expandability with unique long-term stability of their phenotype. The remarkable stability of the phenotype represents a novel finding for human MSCs, from any source, and supports the use of these cells as highly accessible stromal cells for both basic studies and potentially therapeutic applications such as allogeneic clinical use for musculoskeletal disorders

Neuromuscular Electrical Stimulation as a Method to Maximize the Beneficial Effects of Muscle Stem Cells Transplanted into Dystrophic Skeletal Muscle

Cellular therapy is a potential approach to improve the regenerative capacity of damaged or diseased skeletal muscle. However, its clinical use has often been limited by impaired donor cell survival, proliferation and differentiation following transplantation. Additionally, functional improvements after transplantation are all-too-often negligible. Because the host microenvironment plays an important role in the fate of transplanted cells, methods to modulate the microenvironment and guide donor cell behavior are warranted. The purpose of this study was to investigate whether the use of neuromuscular electrical stimulation (NMES) for 1 or 4 weeks following muscle-derived stem cell (MDSC) transplantation into dystrophic skeletal muscle can modulate the fate of donor cells and enhance their contribution to muscle regeneration and functional improvements. Animals submitted to 4 weeks of NMES after transplantation demonstrated a 2-fold increase in the number of dystrophin+ myofibers as compared to control transplanted muscles. These findings were concomitant with an increased vascularity in the MDSC+NMES group when compared to non-stimulated counterparts. Additionally, animals subjected to NMES (with or without MDSC transplantation) presented an increased maximal specific tetanic force when compared to controls. Although cell transplantation and/or the use of NMES resulted in no changes in fatigue resistance, the combination of both MDSC transplantation and NMES resulted in a faster recovery from fatigue, when compared to non-injected and non-stimulated counterparts. We conclude that NMES is a viable method to improve MDSC engraftment, enhance dystrophic muscle strength, and, in combination with MDSC transplantation, improve recovery from fatigue. These findings suggest that NMES may be a clinically-relevant adjunct approach for cell transplantation into skeletal muscle. © 2013 Distefano et al