Abstract
BackgroundDespite the initial promise of myoblast transfer therapy to restore dystrophin in Duchenne muscular dystrophy patients, clinical efficacy has been limited, primarily by poor cell survival post-transplantation. Murine muscle derived stem cells (MDSCs) isolated from slowly adhering cells (SACs) via the preplate technique, induce greater muscle regeneration than murine myoblasts, primarily due to improved post-transplantation survival, which is conferred by their increased stress resistance capacity. Aldehyde dehydrogenase (ALDH) represents a family of enzymes with important morphogenic as well as oxidative damage mitigating roles and has been found to be a marker of stem cells in both normal and malignant tissue. In this study, we hypothesized that elevated ALDH levels could identify murine and human muscle derived cell (hMDC) progenitors, endowed with enhanced stress resistance and muscle regeneration capacity.Methodology/Principal FindingsSkeletal muscle progenitors were isolated from murine and human skeletal muscle by a modified preplate technique and unfractionated enzymatic digestion, respectively. ALDHhi subpopulations isolated by fluorescence activate cell sorting demonstrated increased proliferation and myogenic differentiation capacities compared to their ALDHlo counterparts when cultivated in oxidative and inflammatory stress media conditions. This behavior correlated with increased intracellular levels of reduced glutathione and superoxide dismutase. ALDHhi murine myoblasts were observed to exhibit an increased muscle regenerative potential compared to ALDHlo myoblasts, undergo multipotent differentiation (osteogenic and chondrogenic), and were found predominately in the SAC fraction, characteristics that are also observed in murine MDSCs. Likewise, human ALDHhi hMDCs demonstrated superior muscle regenerative capacity compared to ALDHlo hMDCs.ConclusionsThe methodology of isolating myogenic cells on the basis of elevated ALDH activity yielded cells with increased stress resistance, a behavior that conferred increased regenerative capacity of dystrophic murine skeletal muscle. This result demonstrates the critical role of stress resistance in myogenic cell therapy as well as confirms the role of ALDH as a marker for rapid isolation of murine and human myogenic progenitors for cell therapy.
Highlights
Duchenne muscular dystrophy is a degenerative muscle disease caused by a mutation of the gene encoding dystrophin, a protein that anchors the myofiber cytoskeleton to the basal lamina, resulting in muscle fiber necrosis and progressive weakness [1,2]
The methodology of isolating myogenic cells on the basis of elevated Aldehyde dehydrogenase (ALDH) activity yielded cells with increased stress resistance, a behavior that conferred increased regenerative capacity of dystrophic murine skeletal muscle. This result demonstrates the critical role of stress resistance in myogenic cell therapy as well as confirms the role of ALDH as a marker for rapid isolation of murine and human myogenic progenitors for cell therapy
Isolation of ALDHlo and ALDHhi populations of skeletal muscle derived cells Cells with elevated ALDH levels become fluorescent when exposed to boron-dipyrromethene (BODIPY) labeled amino acetaldehyde (Aldefluor, StemCell Technologies) and can be isolated using fluorescence activated cell sorting (FACS)
Summary
Duchenne muscular dystrophy is a degenerative muscle disease caused by a mutation of the gene encoding dystrophin, a protein that anchors the myofiber cytoskeleton to the basal lamina, resulting in muscle fiber necrosis and progressive weakness [1,2]. Myoblast transfer therapy, defined as the transplantation of normal myoblasts into dystrophin-deficient muscle, has been shown to transiently deliver dystrophin to dystrophic myofibers as well as improve muscle contraction force [5]. Outcomes of this approach are limited by immune rejection, limited cell migration with the formation of cell pockets, and poor cell survival rates, which is perhaps the most important barrier to efficacious myogenic cell therapy [6,7,8]. Despite the initial promise of myoblast transfer therapy to restore dystrophin in Duchenne muscular dystrophy patients, clinical efficacy has been limited, primarily by poor cell survival post-transplantation. We hypothesized that elevated ALDH levels could identify murine and human muscle derived cell (hMDC) progenitors, endowed with enhanced stress resistance and muscle regeneration capacity
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