Generation of H1 PAX6WT/EGFP reporter cells to purify PAX6 positive neural stem/progenitor cells
- Department of Pathophysiology, Key Lab for Shock and Microcirculation Research of Guangdong, Southern Medical University, Guangzhou, 510515 (China)
- Key Laboratory of Regenerative Biology and Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530 (China)
- Guangzhou Biocare Institute of Cancer, Building D, Guangzhou International Business Incubator, No. 3, Juquan Road, Guangzhou Science Park, Guangzhou, 510663 (China)
Highlights: • We develop a talen-based gene targeting system to purify Pax6{sup +} neural progenitor/stem cells by inserting reporter gene into endogenous pax6 gene locus of human pluripotent stem cells. • The neural progenitor cell we purified possesses neural pluripotency and could be expanded and specified to post-mitotic neurons in vitro efficiently. • With this reporter cell line, we also screened out 1 NPC-specific microRNA, hsa-miR-99a-5p, and 3 ESCs-enriched miRNAs, hsa-miR-302c-5p, hsa-miR-512–3p and hsa-miR-518 b. Neural conversion from human pluripotent cells (hPSCs) is a potential therapy to neurological disease in the future. However, this is still limited by efficiency and stability of existed protocols used for neural induction from hPSCs. To overcome this obstacle, we developed a reporter system to screen PAX6{sup +} neural progenitor/stem cells using transcription activator like effector nuclease (TALEN). We found that knock-in 2 A-EGFP cassette into PAX6 exon of human embryonic stem cells H1 with TALEN-based homology recombination could establish PAX6{sup WT/EGFP} H1 reporter cell line fast and efficiently. This reporter cell line could differentiate into PAX6 and EGFP double positive neural progenitor/stem cells (NPCs/NSCs) after neural induction. Those PAX6{sup WT/EGFP} NPCs could be purified, expanded and specified to post-mitotic neurons in vitro efficiently. With this reporter cell line, we also screened out 1 NPC-specific microRNA, hsa-miR-99a-5p, and 3 ESCs-enriched miRNAs, hsa-miR-302c-5p, hsa-miR-512–3p and hsa-miR-518 b. In conclusion, the TALEN-based neural stem cell screening system is safe and efficient and could help researcher to acquire adequate and pure neural progenitor cells for further application.
- OSTI ID:
- 23136960
- Journal Information:
- Biochemical and Biophysical Research Communications, Vol. 502, Issue 4; Other Information: Copyright (c) 2018 Elsevier Inc. All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); ISSN 0006-291X
- Country of Publication:
- United States
- Language:
- English
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