Murine and Human Myogenic Cells Identified by Elevated Aldehyde Dehydrogenase Activity: Implications for Muscle Regeneration and Repair

PLOS ONE, Dec 2019

Background 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. 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 Findings Skeletal 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. Conclusions The 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.

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Murine and Human Myogenic Cells Identified by Elevated Aldehyde Dehydrogenase Activity: Implications for Muscle Regeneration and Repair

Huard J (2011) Murine and Human Myogenic Cells Identified by Elevated Aldehyde Dehydrogenase Activity: Implications for Muscle Regeneration and Repair. PLoS ONE 6(12): e29226. doi:10.1371/journal.pone.0029226 Murine and Human Myogenic Cells Identified by Elevated Aldehyde Dehydrogenase Activity: Implications for Muscle Regeneration and Repair Joseph B. Vella 0 Seth D. Thompson 0 Mark J. Bucsek 0 Minjung Song 0 Johnny Huard 0 Pranela Rameshwar, University of Medicine and Dentistry of New Jersey, United States of America 0 1 Department of Orthopedic Surgery, Stem Cell Research Center, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 2 Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 3 McGowen Institute of Regenerative Medicine, University of Pittsburgh , Pittsburgh, Pennsylvania , United States of America Background: 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. 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 Findings: Skeletal 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. Conclusions: The 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. - Funding: This work was supported in part by the Nation Institutes of Health (NIH T32 EB001026 and NIH RO1 DE013420-10), the Department of Defense (Contract #: W81XWH-09-1-0658) the Henry J. Mankin Endowed Chair at the University of Pittsburgh and the William F. and Jean W. Donaldson Endowed Chair at the Childrens Hospital of Pittsburgh. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study. Competing Interests: The authors have read the journals policy and have the following conflicts: The authors wish to disclose that Dr. Johnny Huard received remuneration as a consultant with Cook MyoSite, Inc. during the performance period of this project. None of the other authors have any potential conflicts of interest to disclose. This does not alter the authors adherence to all the PLoS ONE policies on sharing data and materials. 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]. Despite extensive investigation of various approaches to deliver dystrophin to dystrophic muscle, few treatment options for patients with this devastating disease exist [3,4]. 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]. However outcomes of this approach are limited by immune rejection, limited cell migration with (...truncated)


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Joseph B. Vella, Seth D. Thompson, Mark J. Bucsek, Minjung Song, Johnny Huard. Murine and Human Myogenic Cells Identified by Elevated Aldehyde Dehydrogenase Activity: Implications for Muscle Regeneration and Repair, PLOS ONE, 2011, 12, DOI: 10.1371/journal.pone.0029226