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Updated: May 17, 2025
Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing
Published on: June 9, 2017
Investigation of the morphological, cellular, biochemical, and molecular modifications in the BG01V human embryonic
1Department of Cell Biology, ICMR-National Institute of Nutrition, Tarnaka, Hyderabad, India; Department of Biotechnology & Bioinformatics, School of life Sciences, University of Hyderabad, Hyderabad, India.
Abstract:
Changes in the morphology, metabolic activity, intracellular calcium (Ca2 +) transients, expression of topoisomerase-2β (Topo-2β), and senescence of human embryonic stem cells (hESCs)-derived neuronal cells on basic hESC culture media and neuronal differentiation medium at different time intervals is not clear. Hence, we aimed to investigate the morphological, cellular, biochemical, and molecular alterations in the BG01V hESC-derived neuronal cells on basic hESC culture media and neuronal differentiation media at different time intervals.
Materials And Methods:
BG01V hESC-derived neuronal cells grown on basic hESC culture media and neuronal differentiation media were evaluated for morphological changes by microscopy, metabolic activity by MTT assay, cell viability by Trypan Blue exclusion assay, cellular activity by estimating the Ca2+ deposits, cellular senescence by senescence-associated beta-galactosidase (SA-β-gal) activity, and level of Topo-2β using Western blotting at different time intervals.
Results:
Contrasting to the BG01V hESCs grown on basic hESC culture media, a notable increase in the neuronal cell-like structures, neuritic outgrowth, and expression of nestin protein on neural induction was observed. Higher levels of Ca2+ deposits, metabolic activity, SA-β-gal activity, and Topo-2β expression in BG01V hESC-derived neuronal cells grown on neuronal differentiation media on day 12 compared to hESCs grown on basic hESC culture media including other days were noted.
Conclusion:
This study suggests the increase of calcium salts reflecting the calcium activity at distinct phases of neuronal differentiation, ranging from neural induction to neurite extension. The metabolic and SA-β-gal activity of BG01V hESC-derived neuronal cells may suggest the ongoing biological aging process. Upregulation and activation of Topo-2β upon differentiation induction at the mid-phase suggest the activation of inducible gene loci and downregulation of Topo-2β at a later stage.
