Human serum and heparin-free platelet lysate as appropriate xeno-free alternatives for production of human MuStem cell batches

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. mRNA profile of hMuStem cells cultured in hPL was compared to the mRNA profile of hMuStem cells cultured in HS. The profiles were generated in triplicates using the 3'DGE-Seq technology.

目的：被称为MuStem细胞的肌肉来源干细胞群体，是肌肉疾病细胞治疗的极具潜力的候选细胞制剂。为验证该制剂能否真正成为治疗产品并符合未来临床应用的准入要求，需预先证明采用符合药品生产质量管理规范（Good Manufacturing Practices，GMPs）的细胞制备工艺可实现其疗效。本研究旨在评估两种无异种动物源血液衍生物——人血清（human serum, HS）与人血小板裂解液（human platelet lysate, hPL）——作为替代物的应用效果，以取代此前存在争议但一直沿用的胎牛血清（fetal bovine serum, FBS），用于人MuStem细胞（human MuStem, hMuStem）的分离与扩增。
方法：本研究采用商业化制备的HS与hPL，对分离并体外扩增的hMuStem细胞开展对照研究，对比基于FBS的培养基体系下获得的细胞增殖速率、集落形成能力、肌源性分化潜能及多向分化能力，以评估两种血液衍生物的影响。同时，通过流式细胞术与高通量3'数字基因表达RNA测序分析各组细胞的表型与全局基因表达谱，以明确受试人类营养组分可能存在的差异化影响。
结果：相较于基于FBS的培养基，添加HS与hPL的培养基可有效支持hMuStem细胞的长期增殖，并提升其集落形成能力，且不会显著改变细胞的表达谱，同时可减少生长因子的添加量。体外分化实验联合转化生长因子β1（transforming growth factor β1, TGF-β1）去除实验显示，当采用hPL培养基培养时，hMuStem细胞的肌源性分化潜能与融合能力均有所降低，且该过程不依赖TGF-β1。采用hPL来源的3D水凝胶或纤维蛋白原缺失型hPL进行实验证实，不含肝素的hPL衍生物仍可维持良好的肌源性分化潜能。此外，将hPL培养基替换为HS或纤维蛋白原缺失型hPL后，细胞的肌源性降低表型可快速逆转。
结论：综上，本研究的原创性结果表明，HS与hPL可作为FBS的高效且适用的替代物，用于符合GMPs要求的hMuStem细胞批次制备。本研究采用3'DGE-Seq技术完成了三次生物学重复的mRNA测序，对比了hPL与HS培养基培养的hMuStem细胞的mRNA表达谱。