Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion

Mesenchymal stem/stromal cells (MSCs) exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM) prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower) to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25) human telomerase reverse transcriptase (hTERT) transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs) and decidua basalis (DMSCs), respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies.

间充质干细胞/基质细胞（Mesenchymal stem/stromal cells, MSCs）在离体扩增过程中会出现不良表型改变，这限制了基础研究与细胞治疗所需的大批量高质量原代MSCs的制备。原代MSCs在由原代MSCs制备的脱细胞细胞外基质（decellularized extracellular matrix, dECM）上扩增时，可保留包括增殖能力与分化潜能在内的多项理想MSC特性。然而，需使用低传代代次原代MSCs（第3代及以内）制备dECM的要求，极大限制了该技术的实用性与影响力。本研究发现，在由高传代（第25代）人端粒酶逆转录酶（human telomerase reverse transcriptase, hTERT）转导的永生化MSC细胞系制备的dECM上扩增的原代MSCs，同样表现出增殖能力与成骨分化能力的提升。本研究采用两株hTERT转导的胎盘来源MSC细胞系：CMSC29与DMSC23[分别源自胎盘绒毛膜绒毛（placental chorionic villi, CMSCs）与基蜕膜（decidua basalis, DMSCs）]，用于制备dECM包被的培养底物。这些dECM底物在结构与生化特性上存在差异。在dECM-DMSC23上培养的原代DMSCs，经14天离体扩增后细胞数量较对照组提升3倍，且成骨分化能力显著增强；在dECM-DMSC23上培养的原代CMSCs则表现出细胞数量提升2倍、成骨分化能力增强的结果。本研究得出结论：源自胎盘不同部位的永生化MSC细胞系所制备的dECM，在维持原代MSCs核心特性的同时，对原代MSCs扩增的支持能力存在差异。此外，本研究首次证实可利用高传代细胞制备dECM以促进原代MSCs的扩增，这一进展极大提升了基于dECM的技术的可行性与应用前景。