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.
Keywords: Cell culture substrates; Electrophysiology; Induced pluripotent stem cells; Neural substrate; Rat neural progenitor cells.
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Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation.Organogenesis. 2014;10(4):365-77. doi: 10.1080/15476278.2015.1011921. Organogenesis. 2014. PMID: 25629202 Free PMC article.
Highly efficient methods to obtain homogeneous dorsal neural progenitor cells from human and mouse embryonic stem cells and induced pluripotent stem cells.Stem Cell Res Ther. 2018 Mar 15;9(1):67. doi: 10.1186/s13287-018-0812-6. Stem Cell Res Ther. 2018. PMID: 29544541 Free PMC article.
Human induced pluripotent stem cell-derived neural stem cells survive, migrate, differentiate, and improve neurologic function in a rat model of middle cerebral artery occlusion.Stem Cell Res Ther. 2013 Jun 14;4(3):73. doi: 10.1186/scrt224. Stem Cell Res Ther. 2013. PMID: 23769173 Free PMC article.
Reverse engineering human neurodegenerative disease using pluripotent stem cell technology.Brain Res. 2016 May 1;1638(Pt A):30-41. doi: 10.1016/j.brainres.2015.09.023. Epub 2015 Sep 28. Brain Res. 2016. PMID: 26423934 Free PMC article. Review.
Applications of induced pluripotent stem cell technologies in spinal cord injury.J Neurochem. 2017 Jun;141(6):848-860. doi: 10.1111/jnc.13986. Epub 2017 Apr 5. J Neurochem. 2017. PMID: 28199003 Review.