Sthanam Lakshmi Kavitha, Saxena Neha, Mistari Vijay Krushna, Roy Tanusri, Jadhav Sameer Ralph, Sen Shamik
ACS Biomater Sci Eng. 2019 Jan 14;5(1):180-192. doi: 10.1021/acsbiomaterials.8b00313. Epub 2018 Sep 13.
Differentiation of stem cells into neurogenic lineage is of great interest for treatment of neurodegenerative diseases. While the role of chemical cues in regulating stem cell fate is well appreciated, the identification of physical cues has revolutionized the field of tissue engineering leading to development of scaffolds encoding one or more physical cues for inducing stem cell differentiation. In this study, using human mesenchymal stem cells (hMSCs) and mouse embryonic stem cells (mESCs), we have tested if stiffness and topography can be collectively tuned for inducing neuronal differentiation by culturing these cells on polyacrylamide hydrogels of varying stiffness (5, 10, and 20 kPa) containing rectangular grooves (10, 15, and 25 μm in width). While hMSCs maximally elongate and express neuronal markers on soft 5 kPa gels containing 10/15 μm grooves, single mESCs are unable to sense topographical features when cultured directly on grooved gels. However, this inability to sense topography is rescued by priming mESCs initially on soft 1 kPa flat gels and then replating these cells onto the grooved gels. Compared to direct culture on the grooved gels, this sequential adaptation increases both viability as well as neuronal differentiation. However, this two-step process does not enhance neuronal marker expression in hMSCs. In addition to highlighting important differences between hMSCs and mESCs in their responsiveness to physical cues, our study suggests that conditioning on soft substrates is essential for inducing topography-mediated neuronal differentiation in mESCs.
干细胞向神经源性谱系的分化对于神经退行性疾病的治疗具有重大意义。虽然化学信号在调节干细胞命运中的作用已得到充分认识，但物理信号的识别彻底改变了组织工程领域，导致开发出编码一种或多种物理信号以诱导干细胞分化的支架。在本研究中，我们使用人间充质干细胞（hMSCs）和小鼠胚胎干细胞（mESCs），通过将这些细胞培养在具有不同硬度（5、10和20 kPa）且含有矩形凹槽（宽度为10、15和25μm）的聚丙烯酰胺水凝胶上，测试了硬度和拓扑结构是否可以共同调节以诱导神经元分化。虽然hMSCs在含有10/15μm凹槽的柔软5 kPa凝胶上最大程度地伸长并表达神经元标记物，但单个mESCs直接培养在有凹槽的凝胶上时无法感知拓扑特征。然而，通过先将mESCs接种在柔软的1 kPa平板凝胶上，然后将这些细胞重新接种到有凹槽的凝胶上，可以挽救这种无法感知拓扑结构的情况。与直接培养在有凹槽的凝胶上相比，这种顺序适应增加了细胞活力以及神经元分化。然而，这个两步过程并没有增强hMSCs中神经元标记物的表达。除了突出hMSCs和mESCs在对物理信号反应方面的重要差异外，我们的研究表明，在柔软底物上进行预处理对于诱导mESCs中拓扑结构介导的神经元分化至关重要。
