title: A simple method for differentiation of H9 cells into neuroectoderm.
abstract: Human embryonic stem cells (ESCs) can form neuroectoderm (NE), providing a platform for in vitro dissection of NE formation. However, human ESCs can differentiate into all three germ layers. It thus is crucial to develop efficient methods for differentiation of human ESCs into NE cells. Both plating cell density and localized cell density (LCD) affect NE differentiation. Here, we developed a cell cluster-based NE differentiation method, in which both plating cell density and LCD are under control. Using our new method, high plating cell densities promote expression of PAX6, a NE marker protein. Two SMAD signaling blockers, SB431542 and NOGGIN, downregulate OCT4 and upregulate PAX6, while does not affect mRNA expression of GATA2 after 5 d of differentiation. Moreover, IB analysis showed a time-dependent upregulation of PAX6 and beta-III-tubulin together with a downregulation of OCT4 during the neural differentiation. Coexpression of both TH and beta-III-tubulin in the H9-derived cells was also detected, proving the NE cells have an ability to differentiate into one of the specific neurons. Together, we established a simple method for generating NE cells from H9 cells, which might contribute to develop high efficient method for neural differentiation.
authors.affiliation: Institute of Stem Cell and Tissue Engineering & Department of Histology and Embryology, School of Basic Medical Sciences, Anhui Medical University, Hefei, Anhui 230032, China; School of Nursing, Anhui Medical University, Hefei, Anhui 230032, China.
authors.forename: Annuo
authors.initials: A
authors.lastname: Liu
authors.affiliation: Institute of Stem Cell and Tissue Engineering & Department of Histology and Embryology, School of Basic Medical Sciences, Anhui Medical University, Hefei, Anhui 230032, China.
authors.forename: Dijuan
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Institute of Clinical Pharmacology, Anhui Medical University, Hefei, Anhui 230032, China.
authors.forename: Lihua
authors.initials: L
authors.lastname: Liu
authors.affiliation: School of Nursing, Anhui Medical University, Hefei, Anhui 230032, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Gong
authors.affiliation: Institute of Stem Cell and Tissue Engineering & Department of Histology and Embryology, School of Basic Medical Sciences, Anhui Medical University, Hefei, Anhui 230032, China; Central Laboratory of Molecular and Cellular Biology, School of Basic Medical Sciences, Anhui Medical University, Hefei, Anhui 230032, China. Electronic address: chaol1974@ahmu.edu.cn.
authors.forename: Chao
authors.initials: C
authors.lastname: Liu
mesh_terms: D002452:Cell Count / Q000379:methods; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology*; D006801:Humans; D054258:Neural Plate / Q000166:cytology*; D009474:Neurons / Q000166:cytology*
keywords: Cell cluster; H9; Localized cell density; Neuroectoderm; Reaggregation