Monica B Setien1, Kylie R Smith1, Kaleb Howard2, Kathleen Williams3, Steve T Suhr4, Erin K Purcell5. 1. Department of Biomedical Engineering, Michigan State University, East Lansing, MI, USA. 2. Neuroscience Program, Lyman Briggs College, Michigan State University, East Lansing, MI, USA; Miller School of Medicine, University of Miami, Miami, FL, USA. 3. Department of Electrical Engineering and Computer Science, Michigan State University, East Lansing, MI, USA. 4. Biomilab, LCC, Lansing, MI, USA. 5. Department of Biomedical Engineering, Michigan State University, East Lansing, MI, USA; Neuroscience Program, Lyman Briggs College, Michigan State University, East Lansing, MI, USA; Department of Electrical Engineering and Computer Science, Michigan State University, East Lansing, MI, USA. Electronic address: epurcell@egr.msu.edu.
Abstract
BACKGROUND: 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. NEW METHOD: 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. RESULTS: 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. CONCLUSION: Substrate coatings can be selected to enrich differentiated riPSCs for distinctive neuronal morphologies.
BACKGROUND: 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. NEW METHOD: 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. RESULTS: 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. CONCLUSION: Substrate coatings can be selected to enrich differentiated riPSCs for distinctive neuronal morphologies.
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