Abstract
P19 embryonal carcinoma cells (EC-cells) provide a simple and robust culture system for studying neural development. Most protocols developed so far for directing neural differentiation of P19 cells depend on the use of culture medium supplemented with retinoic acid (RA) and serum, which has an undefined composition. Hence, such protocols are not suitable for many molecular studies. In this study, we achieved neural differentiation of P19 cells in a serum- and RA-free culture medium by employing the knockout serum replacement (KSR) supplement. In the KSR-containing medium, P19 cells underwent predominant differentiation into neural lineage and by day 12 of culture, neural cells were present in 100% of P19-derived embryoid bodies (EBs). This was consistently accompanied by the increased expression of various neural lineage-associated markers during the course of differentiation. P19-derived neural cells comprised of NES+ neural progenitors (~ 46%), TUBB3+ immature neurons (~ 6%), MAP2+ mature neurons (~ 2%), and GFAP+ astrocytes (~ 50%). A heterogeneous neuronal population consisting of glutamatergic, GABAergic, serotonergic, and dopaminergic neurons was generated. Taken together, our study shows that the KSR medium is suitable for the differentiation of P19 cells to neural lineage without requiring additional (serum and RA) supplements. This stem cell differentiation system could be utilized for gaining mechanistic insights into neural differentiation and for identifying potential neuroactive compounds.
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Acknowledgments
This work was supported by a grant from the Department of Biotechnology, Ministry of Science and Technology, India (Grant no. BT/PR5425/AAQ/1/498/2012). The authors thank the bio-imaging and FCM facilities of the Indian Institute of Science for providing help in conducting ICC and FCM experiments, respectively.
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I.V. and P.B.S. were involved in the conceptualization of the study, designing of experimental strategies, and preparation of the manuscript. I.V. conducted all the experiments and analyzed the data. Both authors read and approved the final manuscript for submission.
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Verma, I., Seshagiri, P.B. Directed differentiation of mouse P19 embryonal carcinoma cells to neural cells in a serum- and retinoic acid-free culture medium. In Vitro Cell.Dev.Biol.-Animal 54, 567–579 (2018). https://doi.org/10.1007/s11626-018-0275-1
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DOI: https://doi.org/10.1007/s11626-018-0275-1