Department of Chemical and Biomedical Engineering, Cleveland State University, Cleveland, Ohio.
Biotechnol Bioeng. 2018 Aug;115(8):2013-2026. doi: 10.1002/bit.26714. Epub 2018 Apr 27.
Endogenous adult cardiac regenerative machinery is not capable of replacing the lost cells following myocardial infarction, often leading to permanent alterations in structure-function-mechanical properties. Regenerative therapies based on delivering autologous stem cells within an appropriate 3D milieu could meet such demand, by enabling homing and directed differentiation of the transplanted cells into lost specialized cell populations. Since type I collagen is the predominant cardiac tissue matrix protein, we here optimized the 3D niche which could promote time-dependent evolution of cardiomyogenesis from human bone marrow-derived mesenchymal stem cells (BM-MSC). 3D collagen gel physical and mechanical characteristics were assessed using SEM and AFM, respectively, while the standalone and combined effects of collagen concentration, culture duration, and 5-azacytidine (aza) dose on the phenotype and genotype of MSC spheroids were quantified using immunofluorescence labeling and RT-PCR analysis. Increasing collagen concentration led to a significant increase in Young's modulus (p < 0.01) but simultaneous decrease in the mean pore size, resulting in stiffer gels. Spheroid formation significantly modulated MSC differentiation and genotype, mostly due to better cell-cell interactions. Among the aza dosages tested, 10 μM appears to be optimal, while 3 mg/ml gels resulted in significantly lower cell viability compared to 1 or 2 mg/ml gels. Stiffer gels (2 and 3 mg/ml) and exposure to 10 μM aza upregulated early and late cardiac marker expressions in a time-dependent fashion. On the other hand, cell-cell signaling within the MSC spheroids seem to have a strong role in influencing mature cardiac markers expression, since neither aza nor gel stiffness seem to significantly improve their expression. Western blot analysis suggested that canonical Wnt/β-catenin signaling pathway might be primarily mediating the observed benefits of aza on cardiac differentiation of MSC spheroids. In conclusion, 2 mg/ml collagen and 10 μM aza appears to offer optimal 3D microenvironment in terms of cell viability and time-dependent evolution of cardiomyogenesis from human BM-MSCs, with significant applications in cardiac tissue engineering and stem cell transplantation for regenerating lost cardiac tissue.
内源性成人心肌再生机制不能替代心肌梗死后丢失的细胞，这通常导致结构-功能-机械性能的永久性改变。基于在适当的 3D 环境中递送自体干细胞的再生疗法可以满足这种需求，使移植细胞归巢并定向分化为丢失的特化细胞群体。由于 I 型胶原是心脏组织基质蛋白的主要成分，我们在此优化了 3D 生态位，以促进人骨髓间充质干细胞（BM-MSC）向心肌发生的时间依赖性演变。使用 SEM 和 AFM 分别评估 3D 胶原凝胶的物理和机械特性，同时使用免疫荧光标记和 RT-PCR 分析定量评估胶原浓度、培养时间和 5-氮杂胞苷（aza）剂量对 MSC 球体表型和基因型的单独和组合作用。增加胶原浓度会导致杨氏模量显著增加（p<0.01），但同时平均孔径减小，导致凝胶变硬。球体形成显著调节 MSC 分化和基因型，主要是由于细胞间相互作用更好。在所测试的 aza 剂量中，10μM 似乎是最佳的，而 3mg/ml 凝胶的细胞活力明显低于 1 或 2mg/ml 凝胶。较硬的凝胶（2 和 3mg/ml）和暴露于 10μM aza 以时间依赖性方式上调早期和晚期心脏标志物的表达。另一方面，MSC 球体中的细胞-细胞信号似乎在影响成熟心脏标志物表达方面起着重要作用，因为 aza 或凝胶硬度都不能显著改善其表达。Western blot 分析表明，经典 Wnt/β-catenin 信号通路可能主要介导 aza 对 MSC 球体心肌分化的观察到的益处。总之，2mg/ml 胶原和 10μM aza 似乎在细胞活力和人 BM-MSCs 向心肌发生的时间依赖性演变方面提供了最佳的 3D 微环境，在心脏组织工程和干细胞移植以再生丢失的心脏组织方面具有重要应用。
