Abstract：
An in-vitro model of human bone marrow mesenchymal stem cells (hBM-MSCs) myogenic commitment by synergic effect of a differentiation media coupled with human primary skeletal myoblasts (hSkMs) co-culture was developed adopting both conventional static co-seeding and perfused culture systems. Static co-seeding provided a notable outcome in terms of gene expression with a significant increase of Desmin (141-fold) and Myosin heavy chain II (MYH2, 32-fold) at day 21, clearly detected also by semi-quantitative immunofluorescence. Under perfusion conditions, myogenic induction ability of hSkMs on hBM-MSCs was exerted by paracrine effect with an excellent gene overexpression and immunofluorescence detection of MYH2 protein; furthermore, due to the dynamic cell culture in separate wells, western blot data were acquired confirming a successful cell commitment at day 14. A significant increase of anti-inflammatory cytokine gene expression, including IL-10 and IL-4 (15-fold and 11-fold, respectively) at day 14, with respect to the pro-inflammatory cytokines IL-12A (7-fold at day 21) and IL-1β (1.4-fold at day 7) was also detected during dynamic culture, confirming the immunomodulatory activity of hBM-MSCs along with commitment events. The present study opens interesting perspectives on the use of dynamic culture based on perfusion as a versatile tool to study myogenic events and paracrine cross-talk compared to the simple co-seeding static culture.
摘要：
通过分化培养基与人原代成肌细胞(hSkMs)共培养的协同作用,开发了人骨髓间充质干细胞(hBM-MSCs)的体外模型,其采用常规静态共接种和灌注培养系统。静态共接种在基因表达方面提供了显著结果,在第21天时Desmin(141倍)和肌球蛋白重链II(MYH2,32倍)显著增加,这也通过半定量免疫荧光清楚地检测到。在灌注条件下，hSkMs对hBM-MSCs的成肌诱导能力是通过旁分泌效应发挥的，具有良好的基因过表达和MYH2蛋白的免疫荧光检测；由于在单独的孔中动态细胞培养，获得的westernblot数据证实了在第14天成功的细胞承诺。抗炎细胞因子基因表达显著增加，包括IL-10和IL-4（15倍和11倍，分别）在第14天，在动态培养过程中还检测到促炎细胞因子IL-12A（第21天的7倍）和IL-1β（第7天的1.4倍），确认hBM-MSC的免疫调节活性以及承诺事件。与简单的共播种静态培养相比，本研究为基于灌注的动态培养作为研究肌源性事件和旁分泌串扰的通用工具的使用开辟了有趣的观点。
