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Induced differentiation of large yellow croaker muscle satellite cells

ABSTRACT: Traditional differentiation of myoblasts is usually induced by serum starvation method, in which 2%HS is used to induce myoblasts to differentiate into myotubes. But, traditional serum starvation method is difficult to differentiate large yellow croaker muscle satellite cells efficiently. Although myotubes were induced within 2-3 days with medium containing 2% horse serum (HS), they detached shortly and died soon after. To improve differentiation efficiency and survival rate, different combinations of basal medium, serum ratios and myogenic factors were evaluated. Finally, the F12 medium containing 8% HS, 10 ng/ml IGF-1, 50 nM necrosulfonamide and 200 μM ascorbic acid was identified as an effective recipe for myogenic differentiation. On day 3 of culture in this differentiation medium, elongated myotubes began to appear, and on day 6, striations similar to skeletal muscle were observed in some of myotubes. But it is still inefficient, the fusion index (the proportion of nuclei in multinucleated myotubes) was 6.39% on day 3 and 1.30% on day 6 (1.30%) .Therefore, we need to find a more efficient method to induce myogenic differentiation. We then performed gene expression profiling analysis using data obtained from RNA-seq of Large yellow croaker muscle satellite cells during differentiation at three time points(day0,day3,day6).

ORGANISM(S): Larimichthys crocea

PROVIDER: GSE212904 | GEO | 2023/08/08

REPOSITORIES: GEO

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Project description:Purpose: The population of muscle-derived stem cells called MuStem cells is presented as promising candidate for cell-based therapy of muscle diseases. To validate if this agent can be really presented as therapeutic product and so to be eligible to a future clinical use, it is now required to demonstrate beforehand an efficacy with cells prepared in compliance with good manufacturing practices (GMPs). The aim of the current study was to evaluate the use of two xeno-free blood derivatives corresponding to human serum (HS) and human platelet lysate (hPL) as alternatives to controverted but until now used fetal bovine serum (FBS) for isolation and expansion of human MuStem (hMuStem) cells. Methods: A comparative study was performed with hMuStem cells isolated and in vitro expanded by using commercially available HS and hPL to determine its impact on their proliferation rates, clonogenicity, myogenic commitment level and oligopotency with regard to results obtained under FBS-based medium. Also, their respective phenotype and global gene expression patterns were investigated by flow cytometry and high throughput 3’ digital gene expression RNA-sequencing in order to define a possible differential impact of the human nutrients tested. Results: Comparatively to FBS-based medium, use of HS- and hPL-supplemented ones efficiently supported long-term proliferation of hMuStem cells and enhanced clonogenicity, without main modification of their expression profile and allowing besides limiting the supplementation in growth factors. In vitro differentiation assay combined to transforming growth factor β1 (TGF-β1)-depletion experiments showed a lower myogenic commitment level as well as fusion ability of hMuStem cells when cultured with hPL-based medium according to a TGF-β1-independent process. Use of hPL-derived 3D hydrogel or fibrinogen-depleted hPL demonstrated that heparin-free hPL derivatives maintain consequent myogenic differentiation potential. In addition, the reduced myogenicity was shown to be rapidly reversible following replacement of hPL by HS or fibrinogen-depleted hPL. Conclusions: All together, our original findings position HS and hPL as efficient and suitable alternatives to FBS for preparation of hMuStem cell batch in compliance with GMPs.

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Induced differentiation of large yellow croaker muscle satellite cells

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