Extracellular matrix (ECM) provides both structural support and dynamic microenvironment for cells regulating their behavior and fate. As a critical component of stem cell niche ECM maintains stem cells and activates their proliferation and differentiation under specific stimuli. Mesenchymal stem/stromal cells (MSCs) regulate tissue-specific stem cell functions locating in their immediate microenvironment and producing various bioactive factors, including ECM components. We evaluated the ability of MSC-produced ECM to restore stem and progenitor cell microenvironment in vitro and analyzed the possible mechanisms of its effects. Human MSC cell sheets were decellularized by different agents (detergents, enzymes, and apoptosis inductors) to select the optimized combination (CHAPS and DNAse I) based on the conservation of decellularized ECM (dECM) structure and effectiveness of DNA removal. Prepared dECM was non-immunogenic, supported MSC proliferation and formation of larger colonies in colony-forming unit-assay. Decellularized ECM effectively promoted MSC trilineage differentiation (adipogenic, osteogenic, and chondrogenic) compared to plastic or plastic covered by selected ECM components (collagen, fibronectin, laminin). Interestingly, dECM produced by human fibroblasts could not enhance MSC differentiation like MSC-produced dECM, indicating cell-specific functionality of dECM. We demonstrated the significant integrin contribution in dECM-cell interaction by blocking the stimulatory effects of dECM with RGD peptide and suggested the involvement of key intracellular signaling pathways activation (pERK/ERK and pFAK/FAK axes, pYAP/YAP and beta-catenin) in the observed processes based on the results of inhibitory analysis. Taken together, we suppose that MSC-produced dECM may mimic stem cell niche components in vitro and maintain multipotent progenitor cells to insure their effective response to external differentiating stimuli upon activation. The obtained data provide more insights into the possible role of MSC-produced ECM in stem and progenitor cell regulation within their niches. Our results are also useful for the developing of dECM-based cell-free products for regenerative medicine.

细胞外基质（ECM）为细胞调节其行为和命运提供了结构支持和动态微环境。作为干细胞生态位的重要组成部分，ECM可以维持干细胞并在特定刺激下激活其增殖和分化。间充质干/基质细胞（MSC）调节组织特异性干细胞功能，这些功能位于其直接的微环境中并产生各种生物活性因子，包括ECM成分。我们评估了MSC产生的ECM恢复干细胞和祖细胞微环境的能力。体外并分析了其影响的可能机制。基于去细胞ECM（dECM）结构的保守性和DNA去除的有效性，通过不同的试剂（去污剂，酶和凋亡诱导剂）使人MSC细胞表层脱细胞，以选择优化的组合（CHAPS和DNAse I）。制备的dECM具有非免疫原性，在集落形成单位测定中支持MSC增殖和形成更大的集落。与塑料或被选定的ECM成分（胶原蛋白，纤连蛋白，层粘连蛋白）覆盖的塑料相比，脱细胞的ECM可有效地促进MSC三系分化（脂肪形成，成骨和软骨形成）。有趣的是，由人类成纤维细胞产生的dECM不能像MSC产生的dECM一样增强MSC分化，表明dECM具有细胞特异性功能。我们通过阻滞dECM与RGD肽的刺激作用，证明了整联蛋白在dECM细胞相互作用中的重要作用，并提出了关键细胞内信号通路激活（pERK / ERK和pFAK / FAK轴，pYAP / YAP和β-catenin）的参与基于抑制分析的结果观察到的过程。两者合计，我们认为MSC生产的dECM可能模仿干细胞的生态位成分体外并维持多能祖细胞，以确保它们在激活后对外部分化刺激产生有效反应。获得的数据提供了更多有关MSC产生的ECM在其壁ni内干细胞和祖细胞调节中可能发挥作用的见解。我们的结果对于开发基于dECM的再生医学无细胞产品也很有用。