Department of Biomedical Engineering, Michigan State University, East Lansing, MI, USA.
Neuroscience Program, Lyman Briggs College, Michigan State University, East Lansing, MI, USA; Miller School of Medicine, University of Miami, Miami, FL, USA.
J Neurosci Methods. 2020 May 15;338:108693. doi: 10.1016/j.jneumeth.2020.108693. Epub 2020 Mar 19.
Induced pluripotent stem cells (iPSCs) may be an advantageous source of neuronal cells to repair damage due to neurological disorders or trauma. Additionally, they are promising candidates to develop models to study underlying mechanisms of neurodegenerative diseases. While successful neural differentiation of iPSCs has been reported in mice, protocols detailing the generation of neural cells from rat iPSCs are relatively limited, and their optimization by manipulating cell culture methods has remained unexplored.
Here, we describe and compare the effects of four distinct, commonly used substrates on the neuronal differentiation of rat iPSC (riPSC) derived-neural progenitor cells. Our approach is to use substrate coating as a method to enrich differentiated riPSCs for neuronal subtypes with the desired morphology and maturity. We use a combination of electrophysiology, immunofluorescence staining, and Sholl analysis to characterize the cells generated on each substrate over a nine-day time course.
The surface coating presented by the cell culture substrate influences the polarity and arborization of differentiating neurons. Polyornithine-laminin coating promoted neuronal arborization and maturation, while Geltrex favored bipolar cells which displayed indicators of functional immaturity. Poly-d-lysine substrate was associated with limited neurite outgrowth and arborization. Gelatin was the least favorable substrate for the growth and differentiation of our cells. Comparison with Existing Method: Rat-derived neural progenitor cells have been previously derived; however, our methods to use substrate coatings to influence morphological and electrical maturity have not been explored previously.
Substrate coatings can be selected to enrich differentiated riPSCs for distinctive neuronal morphologies.
诱导多能干细胞（iPSCs）可能是修复神经紊乱或创伤导致的损伤的有利的神经元细胞来源。此外，它们是开发模型以研究神经退行性疾病潜在机制的有前途的候选物。虽然已经在小鼠中成功地进行了 iPSC 的神经分化，但详细描述从大鼠 iPSC 生成神经细胞的方案相对有限，并且通过操纵细胞培养方法对其进行优化仍未得到探索。
在这里，我们描述并比较了四种不同的、常用的基质对大鼠 iPSC（riPSC）衍生的神经祖细胞的神经分化的影响。我们的方法是使用基质涂层作为一种方法，使分化的 riPSC 富集具有所需形态和成熟度的神经元亚型。我们使用电生理学、免疫荧光染色和 Sholl 分析相结合的方法，在 9 天的时间过程中对每种基质上生成的细胞进行特征描述。
细胞培养基质呈现的表面涂层会影响分化神经元的极性和分支。多聚鸟氨酸-层粘连蛋白涂层促进神经元的分支和成熟，而 Geltrex 则有利于表现出功能不成熟迹象的双极细胞。多聚-d-赖氨酸基质与有限的神经突生长和分支有关。明胶是最不利于我们细胞生长和分化的基质。与现有方法的比较：以前已经衍生出大鼠来源的神经祖细胞；然而，我们使用基质涂层来影响形态和电成熟的方法以前没有被探索过。
可以选择基质涂层来富集具有独特神经元形态的分化 riPSC。
