南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (2): 175-182.doi: 10.12122/j.issn.1673-4254.2023.02.03

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人诱导多能干细胞体外定向分化为多巴胺能神经元祖细胞

徐佳佳，李洋洋，仲光尚，方祝玲，刘淳博，马彩云，王春景，郭 俣，刘长青   

  1. 蚌埠医学院生命科学学院，检验医学院，安徽 蚌埠 233000
  • 出版日期:2023-02-20 发布日期:2023-03-16

Directed differentiation of human induced pluripotent stem cells into midbrain dopaminergic neuron progenitors in vitro

XU Jiajia, LI Yangyang, ZHONG Guangshang, FANG Zhuling, LIU Chunbo, MA Caiyun, WANG Chunjing, GUO Yu, LIU Changqing   

  1. School of Life Sciences, School of Laboratory Medicine, Bengbu Medical College, Bengbu 233000, China
  • Online:2023-02-20 Published:2023-03-16

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摘要： 目的 探讨人诱导多能干细胞（hiPSCs）体外高效定向分化为中脑多巴胺能神经元祖细胞（DAPs）的方法体系。方法 人iPSCs诱导分化为DAPs遵循两个主要阶段，第1阶段是化学诱导产生中间态细胞，人iPSCs在含小分子化合物组合的神经诱导培养基中培养至第13天，其表观特征类似于原始神经上皮细胞。第2阶段，特异性诱导DAPs，将中间态细胞在神经分化培养基中诱导至第28天获得DAPs。CM-DiI染色后，定向移植帕金森（PD）大鼠模型右前脑内侧束（MFB），实验动物随机分为3组：健康对照组，模型组和DAPs移植组。移植后2周的大鼠全脑冷冻切片免疫荧光检测移植细胞在宿主脑内微环境中存活、迁移及分化状况，移植后8周，对细胞移植组PD大鼠进行行为学鉴定。结果 人iPSCs可在基质胶上稳定传代，具有正常的二倍体核型，表达多潜能性标记物OCT4、SOX2和Nanog，且碱性磷酸酶染色呈阳性。诱导分化至第13天获得原始神经上皮细胞形成玫瑰花结样结构，且能够表达其表面特征性标记物（SOX2、Nestin和PAX6，91.3%~92.8%）。诱导至第28天的DAPs高表达其特异性的标记物（TH、FOXA2、LMX1A和NURR1，93.3%~96.7%）。而且，DAPs移植PD大鼠脑内2周后，可分化为TH+，FOXA2+与Tuj1+神经元，移植后8周, 移植组较模型组相比，经水迷宫实验（P<0.0001）和阿扑吗啡（APO）诱导的旋转实验（P<0.0001）检测，PD大鼠的运动功能得到明显改善。结论 人iPSCs经多级诱导可高效分化为中脑DA能神经元祖细胞，具备体内和体外分化为功能神经元与胶质细胞的潜能，并可显著改善PD大鼠的运动功能障碍，为hiPSCs-DAPs移植治疗神经系统损伤疾病奠定细胞基础和实验依据。

关键词: 人诱导多能干细胞；多巴胺能神经元祖细胞；原始神经上皮细胞；细胞移植；帕金森模型大鼠

Abstract: Objective To establish an efficient protocol for directed differentiation of human induced pluripotent stem cells (hiPSCs) into functional midbrain dopaminergic progenitor cells (DAPs) in vitro. Methods hiPSCs were induced to differentiate into DAPs in two developmental stages. In the first stage (the first 13 days), hiPSCs were induced into intermediate cells morphologically similar to primitive neuroepithelial cells (NECs) in neural induction medium containing a combination of small molecule compounds. In the second stage, the intermediate cells were further induced in neural differentiation medium until day 28 to obtain DAPs. After CM-DiI staining, the induced DAPs were stereotactically transplanted into the right medial forebrain bundle (MFB) of rat models of Parkinson's disease (PD). Eight weeks after transplantation, the motor behaviors of PD rats was evaluated. Immunofluorescence assay of brain sections of the rats was performed at 2 weeks after transplantation to observe the survival, migration and differentiation of the transplanted cells in the host brain microenvironment. Results hiPSCs passaged stably on Matrigel showed a normal diploid karyotype, expressed the pluripotency markers OCT4, SOX2, and Nanog, and were positive for alkaline phosphatase. The primitive neuroepithelial cells obtained on day 13 formed dense cell colonies in the form of neural rosettes and expressed the neuroepithelial markers (SOX2, Nestin, and PAX6, 91.3%-92.8%). The DAPs on day 28 highly expressed the specific markers (TH, FOXA2, LMX1A and NURR1, 93.3-96.7%). In rat models of PD, the hiPSCs-DAPs survived and differentiated into TH+, FOXA2+ and Tuj1+ neurons at 2 weeks after transplantation. Eight weeks after transplantation, the motor function of PD rats was significantly improved as shown by water maze test (P<0.0001) and apomorphine-induced rotation test (P<0.0001) compared with rats receiving vehicle injection. Conclusion HiPSCs can be effectively induced to differentiate into DAPs capable of differentiating into functional neurons both in vivo and in vitro. In rat models of PD, the transplanted hiPSCs-DAPs can survive for more than 8 weeks in the MFB and differentiate into multiple functional neurocytes to ameliorate neurological deficits of the rats, suggesting the potential value of hiPSCs-DAPs transplantation for treatment of neurological diseases.

Key words: human induced pluripotent stem cells; dopaminergic progenitor cells; primitive neuro-epithelial cells; cell transplantation; Parkinson's disease; rat models