Translational Tissue Engineering Center, Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21287, USA.
J Cell Physiol. 2012 Nov;227(11):3701-8. doi: 10.1002/jcp.24078.
Mesenchymal condensation is a critical transitional stage that precedes cartilage formation during embryonic development. We hypothesized that "priming" hMSCs to recapitulate mesenchymal condensation events prior to inducing differentiation would enhance their subsequent chondrogenic properties. Our prior studies have suggested that exposing hMSCs to hypoxia (2% O(2)) induces condensation-like effects. We therefore assessed the effect of preconditioning for different time periods on the expression of condensation specific genes by growing hMSCs in expansion medium under different normoxic (20% O(2)) and hypoxic conditions for up to 2 weeks, and subsequently induced chondrogenesis of preconditioned hMSCs. The total cultivation time for each group was 4 weeks and the chondrogenic properties were assessed using gene expression, biochemical analysis, and histological staining. Our results demonstrated the benefits of preconditioning were both time- and oxygen-dependent. Condensation specific genes, SOX-9 and NCAM, were significantly up-regulated in hypoxic conditions at the end of 1 week. COL X and MMP13 expression was also lower than the normoxic samples at this time point. However, this group did not exhibit more efficient chondrogenesis after 4 weeks. Instead, hMSCs preconditioned for 1 week and subsequently differentiated, both under 20% O(2), resulted in the most efficient chondrogenesis. Interestingly, while hypoxia appears to positively enhance expression of chondrogenic genes, this did not produce an enhanced matrix accumulation. The results of this study emphasize the significance of considering the timing of specific cues in developing protocols for stem cell-based therapies and underscore the complexity in regulating stem cell differentiation and tissue formation.
间质凝聚是胚胎发育过程中软骨形成之前的一个关键过渡阶段。我们假设在诱导分化之前，使 hMSC 预先经历间质凝聚事件，会增强其随后的软骨生成特性。我们之前的研究表明，将 hMSC 暴露于低氧（2% O(2)) 环境中会诱导出类似于凝聚的效应。因此，我们评估了在不同时间点对 hMSC 进行预培养对凝聚特异性基因表达的影响，具体方法是在不同的常氧（20% O(2)) 和低氧条件下，将 hMSC 在扩增培养基中培养长达 2 周，随后诱导预培养 hMSC 的软骨生成。每个组的总培养时间为 4 周，并通过基因表达、生化分析和组织学染色评估其软骨生成特性。我们的结果表明，预培养的益处既依赖于时间又依赖于氧浓度。在第 1 周结束时，低氧条件下的 SOX-9 和 NCAM 等凝聚特异性基因表达显著上调。COL X 和 MMP13 的表达在此时也低于常氧样本。然而，这一组在 4 周后并没有表现出更高效的软骨生成。相反，在 20% O(2) 下预培养 1 周并随后分化的 hMSC 产生了最有效的软骨生成。有趣的是，虽然低氧似乎能正向增强软骨生成基因的表达，但这并没有产生更多的基质积累。本研究的结果强调了在开发基于干细胞的治疗方案时，考虑特定信号的时间的重要性，并突出了调节干细胞分化和组织形成的复杂性。
