Kusuma Gina D, Brennecke Shaun P, O'Connor Andrea J, Kalionis Bill, Heath Daniel E
Pregnancy Research Centre, Department of Maternal-Fetal Medicine, Royal Women's Hospital, Parkville, Victoria, Australia.
Department of Chemical and Biomolecular Engineering, Particulate Fluids Processing Centre, The University of Melbourne, Parkville, Victoria, Australia.
PLoS One. 2017 Feb 2;12(2):e0171488. doi: 10.1371/journal.pone.0171488. eCollection 2017.
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/基质细胞（MSCs）在体外扩增过程中会出现不理想的表型变化，这限制了基础研究和细胞治疗所需的大量高质量原代MSCs的生产。当在由原代MSCs制备的脱细胞细胞外基质（dECM）上扩增时，原代MSCs保留了许多理想的MSC特性，包括增殖能力和分化潜能。然而，需要使用低传代次数的原代MSCs（第3代或更低）来制备dECM，这极大地限制了该技术的实用性和影响力。在此，我们报告，在由高传代次数（第25代）人端粒酶逆转录酶（hTERT）转导的永生化MSC细胞系制备的dECM上扩增的原代MSCs也表现出增殖增加和成骨分化增强。使用两种hTERT转导的胎盘来源的MSC细胞系CMSC29和DMSC23（分别来源于胎盘绒毛膜（CMSCs）和基蜕膜（DMSCs））来制备dECM包被的基质。这些dECM基质显示出结构和生化差异。与对照组相比，在dECM-DMSC23上培养的原代DMSCs在培养14天后细胞数量增加了三倍，成骨分化增强。在dECM-DMSC23上培养的原代CMSCs细胞数量增加了两倍，成骨分化增强。我们得出结论，源自胎盘不同部位的永生化MSC细胞系产生的dECM具有不同的支持原代MSC扩增增加的能力，同时保持重要的原代MSC特性。此外，这是首次证明使用高传代次数的细胞来制备可促进原代MSC扩增的dECM，这一进展大大提高了基于dECM的技术的可行性和适用性。
