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

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. Overall design: 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.

研究目的：被称为MuStem细胞的肌肉源性干细胞群体，是肌肉疾病细胞治疗的极具潜力的候选方案。为验证该制剂能否真正作为治疗产品，并符合未来临床应用的准入要求，目前需预先证明采用符合药品生产质量管理规范（Good Manufacturing Practices, GMPs）制备的细胞具有有效性。本研究旨在评估两种无异种血液衍生物——人血清（Human Serum, HS）和人血小板裂解液（human platelet lysate, hPL）——作为替代物的应用潜力，以替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（human MuStem, hMuStem）细胞的分离与扩增。
研究方法：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，以基于FBS培养基的实验结果为参照，评估其对细胞增殖速率、克隆形成能力、肌源性分化潜能及多向分化能力的影响。此外，通过流式细胞术与高通量3’数字基因表达RNA测序（3’ DGE RNA-seq），检测各组细胞的表型与全局基因表达谱，以明确所测试的人类营养成分可能产生的差异化影响。
研究结果：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，并提升其克隆形成能力，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。体外分化实验结合转化生长因子β1（Transforming Growth Factor β1, TGF-β1）缺失实验表明，当采用hPL培养基培养时，hMuStem细胞的肌源性分化水平及融合能力更低，且该调控过程不依赖TGF-β1。使用hPL来源的3D水凝胶或纤维蛋白原缺失型hPL的实验证实，不含肝素的hPL衍生物仍可维持显著的肌源性分化潜能。此外，当将hPL培养基替换为HS培养基或纤维蛋白原缺失型hPL培养基后，这种减弱的肌源性能力可快速恢复。
研究结论：综上，本研究的原创性结果表明，HS与hPL可作为FBS的高效且合适的替代物，用于符合GMPs要求的hMuStem细胞批量制备。
研究整体设计：将在hPL培养基中培养的hMuStem细胞的mRNA表达谱，与在HS培养基中培养的hMuStem细胞的mRNA表达谱进行对比。所有表达谱均采用3’数字基因表达测序（3'DGE-Seq）技术完成，设置三次生物学重复。