Rho kinase inhibitor Y27632 promotes survival of human induced pluripotent stem cells during differentiation into functional midbrain dopaminergic progenitor cells in vitro
10.12122/j.issn.1673-4254.2024.02.05
- VernacularTitle:Rho激酶抑制剂Y27632促进人诱导多能干细胞来源原始神经上皮细胞向多巴胺能神经前体细胞的转化
- Author:
Yangyang LI
1
;
Jiajia XU
;
Chengcheng JIANG
;
Zilong CHEN
;
Ying CHEN
;
Mengjiao YING
;
Ao WANG
;
Caiyun MA
;
Chunjing WANG
;
Yu GUO
;
Changqing LIU
Author Information
1. 蚌埠医科大学安徽省神经再生技术与医用新材料工程研究中心,安徽 蚌埠 233000
- Keywords:
human induced pluripotent stem cells;
Y27632;
primitive neuroepithelial cells;
dopaminergic neural progenitor cells
- From:
Journal of Southern Medical University
2024;44(2):236-243
- CountryChina
- Language:Chinese
-
Abstract:
Objective To improve the efficiency of induced differentiation of primitive neural epithelial cells derived from human induced pluripotent stem cells(hiPSCs-NECs)into functional midbrain dopaminergic progenitor cells(DAPs).Methods HiPSCs were cultured in mTeSRTM medium containing DMH1(10 μmol/L),SB431542(10 μmol/L),SHH(200 ng/mL),FGF8(100 ng/mL),purmorphamine(2 μmol/L),CHIR99021(3 μmol/L),and N2(1%)for 12 days to induce their differentiation into primitive neuroepithelial cells(NECs).The hiPSCs-NECs were digested with collagenase IV and then cultured in neurobasal medium supplemented with 1%N2,2%B27-A,BDNF(10 ng/mL),GDNF(10 ng/mL),AA,TGF-β,cAMP,and 1%GlutaMax in the presence of different concentrations of Rho kinase inhibitor Y27632,and the culture medium was changed the next day to remove Y27632.Continuous induction was performed until day 28 to obtain DAPs.Results Human iPSCs expressed the pluripotency markers OCT4,SOX2,Nanog,and SSEA1 and were positive for alkaline phosphatase staining.The hiPSCs-NECs were obtained on day 13 in the form of neural rosettes expressing neuroepithelial markers SOX2,nestin,and PAX6.In digested hiPSCs-NECs,the addition of 5 μmol/L Y27632 significantly promoted survival of the adherent cells,increased cell viability and the proportion of S-phase cells(P<0.01),and reduced the rate of apoptotic cells(P<0.05).On day 28 of induction,the obtained cells highly expressed the specific markers of DAPS(TH,FOXA2,NURR1,and Tuj1).Conclusion Treatment with Y27632(5 μmol/L)for 24 h significantly promotes the survival of human iPSCs-NECs during their differentiation into DPAs without affecting the cell differentiation,which indirectly enhances the efficiency of cell differentiation.
