Y-27632 promotes differentiation of human embryonic stem cells into neuron-like cells

doi:10.3969/j.issn.2095-4344.0753

Abstract

Abstract:

BACKGROUND: Y-27632 is a Rho-associated coil-formed protein kinase inhibitor that can regulate the self-renewal of stem cells, promote clonal formation and cell survival, and regulate and protect neuronal cell growth and development. How to improve the differentiation efficiency of embryonic stem cells into neuron-like cells is highly important for nerve injury repair and nerve regeneration.
OBJECTIVE: To investigate the effect of Y-27632 on the differentiation efficiency and function of human embryonic stem cells into neuron-like cells.
METHODS: After resuscitation, human embryonic stem cells at passage 16 were subjected to morphological observation and staining identification. The embryoid bodies were prepared by suspension culture, and after 8 days of incubation, the cells were cultured in Sato medium containing different concentrations of Y-27632 (0, 5, 10, 20, 40 μmol/L) for 10 days and identification by staining. After induction for 18 days, the differentiation efficiency and neurite outgrowth were identified by immunofluorescence staining.
RESULTS AND CONCLUSION: The human embryonic stem cells co-expressed Oct4 and SSEA-3 stem cell specific markers. After suspension culture for 8 days and further adherent culture for 10 days, the cells could be differentiated into neuron-like cells with neurogenic morphology and expressing Tuj-1. Y-27632, especially at a concentration of 10 μmol/L, not only promoted cell proliferation (a significant increase in adherent cells an Tuj-1 positive cells), but also facilitated cell differentiation into neurons. Immunofluorescence staining findings showed that 10 μmol/L Y-27632 significantly increased the number of Tuj-1 positive cells and neurites and the length of neurites after 18 days of differentiation. These results indicate that Y-27632 not only promotes the differentiation of human embryonic stem cells into neuron-like cells, but also accelerates neurite outgrowth.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Embryonic Stem Cells, Cell Differentiation, Nerve Regeneration, Tissue Engineering

CLC Number:

R394.2

He Da-yan, Yang Yu-hong. Y-27632 promotes differentiation of human embryonic stem cells into neuron-like cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(13): 2063-2067.

Figures/Tables 1

2.1 人胚胎干细胞形态及鉴定人胚胎干细胞一直保持未分化状态，主要以集落形式贴壁生长，细胞排列紧密，克隆边缘清晰(图1A)。Oct4和SSEA-3是人胚胎干细胞未分化状态的重要检测指标，细胞传代后24 h，利用免疫荧光法对细胞行染色鉴定，胚胎干细胞基本都为双阳性，且与细胞核共定位(图1B-E)，说明胚胎干细胞保持未分化的状态。 2.2 人胚胎干细胞诱导分化后的细胞形态观察和荧光染色鉴定人胚胎干细胞经胰蛋白酶消化后，采用细菌培养皿进行悬浮分化培养，加入神经分化培养液，每天换液，培养8 d后，选取形态规则，细胞分布紧密的类胚体(图2A)，放入多聚赖氨酸预处理的培养皿中进行贴壁分化培养，加入Sato诱导培养基培养10 d后，利用免疫荧光法对细胞进行神经细胞特异蛋白Tuj-1染色鉴定，结果显示，有部分细胞具有类神经元形态，且表达Tuj-1(图2B)，说明胚胎干细胞能够向类神经元细胞分化。 2.3 Y-27632促进胚胎干细胞向类神经细胞元分化的免疫荧光染色鉴定类胚体细胞诱导分化过程中，加入不同浓度的Y-27632(0，5，10，20，40 μmol/L)，培养10 d后，进行免疫荧光染色并观察，Y-27632不仅能够促进细胞增殖(图3A)，同时促进细胞向类神经元细胞分化速率(图3B，C)，且在浓度为10 μmol/L时最为显著。 2.4 诱导后细胞形态观察和免疫荧光检测将类胚体培养18 d后，行免疫荧光染色观察，对照组和10 μmol/L Y-27632组的胚胎干细胞均能够向类神经元细胞分化，然而10 μmol/L Y-27632组的阳性细胞数明显多于对照组，且类神经元细胞突起更多、更长(图4)，说明Y-27632不仅促进胚胎干细胞向类神经元细胞分化，同时促进类神经元细胞突起的生长。 2.5 诱导后类神经元细胞突起数和长度检测类胚体诱导分化18 d后，对阳性表达Tuj-1的细胞进行突起数和突起长度统计，结果显示，10 μmol/L Y-27632处理后，显著增加了每一个细胞的突起数和突起长度(图5)，说明Y-27632促进类神经元细胞突起的生长。"

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