Abstract
BackgroundCanine MuStem cells have demonstrated regenerative efficacy in a dog model of muscular dystrophy, and the recent characterization of human counterparts (hMuStem) has highlighted the therapeutic potential of this muscle-derived stem cell population. To date, these cells have only been generated in research-grade conditions. However, evaluation of the clinical efficacy of any such therapy will require the production of hMuStem cells in compliance with good manufacturing practices (GMPs). Because the current use of fetal bovine serum (FBS) to isolate and expand hMuStem cells raises several ethical, safety, and supply concerns, we assessed the use of two alternative xeno-free blood derivatives: human serum (HS) and a human platelet lysate (hPL).MethodshMuStem cells were isolated and expanded in vitro in either HS-supplemented or hPL-supplemented media and the proliferation rate, clonogenicity, myogenic commitment potential, and oligopotency compared with that observed in FBS-supplemented medium. Flow cytometry and high-throughput 3′-digital gene expression RNA sequencing were used to characterize the phenotype and global gene expression pattern of hMuStem cells cultured with HS or hPL.ResultsHS-supplemented and hPL-supplemented media both supported the isolation and long-term proliferation of hMuStem cells. Compared with FBS-based medium, both supplements enhanced clonogenicity and allowed for a reduction in growth factor supplementation. Neither supplement altered the cell lineage pattern of hMuStem cells. In vitro differentiation assays revealed a decrease in myogenic commitment and in the fusion ability of hMuStem cells when cultured with hPL. In return, this reduction of myogenic potential in hPL-supplemented cultures was rapidly reversed by substitution of hPL with HS or fibrinogen-depleted hPL. Moreover, culture of hMuStem cells in hPL hydrogel and fibrinogen-depleted hPL demonstrated that myogenic differentiation potential is maintained in heparin-free hPL derivatives.ConclusionsOur findings indicate that HS and hPL are efficient and viable alternatives to FBS for the preparation of hMuStem cell batches in compliance with GMPs.
Highlights
Canine MuStem cells have demonstrated regenerative efficacy in a dog model of muscular dystrophy, and the recent characterization of human counterparts has highlighted the therapeutic potential of this muscle-derived stem cell population
The in vitro growth ability of hMuStem cells is unchanged by substitution of fetal bovine serum (FBS) with either human serum (HS) or human platelet lysate (hPL) To assess the validity of HS and hPL as alternatives to FBS in hMuStem cell culture, we first quantified proliferation rates of three hMuStem cellsFBS batches cultured in Growth medium (GM) supplemented with either 10% HS or 10% hPL, and compared these results with those obtained in GM supplemented with 10% FBS
HS human serum, hPL human platelet lysate, RFU relative fluorescence units, GM growth medium, Peroxisome proliferator activated receptor gamma (PPARγ) peroxisome proliferator activated receptor gamma, IBSP integrin binding sialoprotein and/or differentiation [70], we investigated the effects of heparin on myogenic commitment in MuStem cells. hMuStem cellsHS were cultured in the presence of increasing doses of heparin (0.5–5 IU/ml), with the highest dose corresponding to that found in hPL-GM
Summary
Canine MuStem cells have demonstrated regenerative efficacy in a dog model of muscular dystrophy, and the recent characterization of human counterparts (hMuStem) has highlighted the therapeutic potential of this muscle-derived stem cell population. To date, these cells have only been generated in research-grade conditions. The use of FBS raises several ethical concerns pertaining to serum harvesting practices and animal suffering [16, 22] Taken together, these issues underscore the need for alternative clinically transferable invitro cell expansion protocols. The combination of PRP and decorin, an inhibitor of transforming growth factor beta-1 (TGF-β1), promotes proliferation and stimulates myogenic commitment in human myoblasts in vitro [52]
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