Efficient neural differentiation of mouse pluripotent stem cells in a serum-free medium and development of a novel strategy for enrichment of neural cells

• Published in: International journal of developmental neuroscience 2017-10, Vol.61 (1), p.112-124
• Main Authors: Verma, Isha, Rashid, Zubin, Sikdar, Sujit K., Seshagiri, Polani B.
• Format: Article
• Language: English
• Subjects: Action Potentials - drug effects; Action Potentials - physiology; Animals; Astrocytes; Cell culture; Cell Differentiation - drug effects; Cell Differentiation - physiology; Cells, Cultured; Culture Media, Serum-Free - pharmacology; Differentiation; Dopamine receptors; Electrophysiology; Embryo, Mammalian - cytology; Embryo, Mammalian - drug effects; Embryoid body; Enrichment; Flow Cytometry; Gene Expression Regulation, Developmental - drug effects; Gene Expression Regulation, Developmental - physiology; Glutamate Decarboxylase - metabolism; Glutamatergic transmission; Knockout serum replacement; Mice; Nerve Tissue Proteins - metabolism; Neural stem cells; Neurogenesis; Neurogenesis - drug effects; Neurons; Neurons - drug effects; Neurons - physiology; Neurosciences; Neurotransmitter Agents - metabolism; Oligodendrocytes; Organ Culture Techniques; Patch-Clamp Techniques; Pluripotency; Pluripotent stem cell; Pluripotent Stem Cells - drug effects; RNA, Messenger - metabolism; Serotonin; Serum-free medium; Stem cell transplantation; Stem cells; Time Factors; Transcription Factors - metabolism; Tubulin - metabolism; γ-Aminobutyric acid
• ISSN: 0736-5748; 1873-474X
• DOI: 10.1016/j.ijdevneu.2017.06.009

•PSCs undergo predominant neural differentiation in the KSR medium.•Different neuronal subtypes and glial cells are generated.•Differentiated neurons exhibit functional electrophysiological profiles.•EB scooping results in the enrichment of PSC-derived neural cells. Pluripotent stem cells (PSCs) offer an excellent model to study neural development and function. Although various protocols have been developed to direct the differentiation of PSCs into desired neural cell types, many of them suffer from limitations including low efficiency, long duration of culture, and the use of expensive, labile, and undefined growth supplements. In this study, we achieved efficient differentiation of mouse PSCs to neural lineage, in the absence of exogenous molecules, by employing a serum-free culture medium containing knockout serum replacement (KSR). Embryoid bodies (EBs) cultured in this medium predominantly produced neural cells which included neural progenitors (15–18%), immature neurons (8–24%), mature neurons (10–26%), astrocytes (27–61%), and oligodendrocytes (∼1%). Different neuronal subtypes including glutamatergic, GABAergic, cholinergic, serotonergic, and dopaminergic neurons were generated. Importantly, neurons generated in the KSR medium were electrically active. Further, the EB scooping strategy, involving the removal of the EB core region from the peripheral EB outgrowth, resulted in the enrichment of PSC-derived neural cells. Taken together, this study provides the evidence that the KSR medium is ideal for the rapid and efficient generation of neural cells, including functional neurons, from PSCs without the requirement of any other additional molecule.