猪诱导多能干细胞可定向分化为前脑GABA能神经元前体

Directed differentiation of porcine induced pluripotent stem cells into forebrain GABAergic neuron progenitors

目的探讨猪诱导多能干细胞(iPSCs)体外定向分化为GABA能神经元前体的方法体系。方法猪iPSCs诱导分化为GABA能神经元前体遵循两个阶段,第1阶段,猪iPSCs悬浮培养,第3天时形成类胚体,采用神经诱导培养基NIM(SB431542、DMH1、FGF2)继续诱导,第12天分化为原始神经上皮细胞。第2阶段,使用含Pur、B27的NIM培养基悬浮培养形成神经球,至第21天时形成GABA能神经元前体。CM-DiI标记后,定向移植帕金森(PD)模型大鼠黑质纹状体,检测其在宿主脑内存活、迁移及分化状况。结果猪iPSCs在饲养层细胞上稳定传代,表达多能性标记OCT4、Nanog、SSEA1和TRA-160,并且核型分析显示没有其他物种来源细胞污染。第12天经诱导分化获得原始神经上皮细胞能够形成玫瑰花环结构,并表达其表面标记物(PAX6、SOX2和Nestin)与神经微管蛋白标志物Tuj1。第21天诱导细胞高表达GABA能神经元前体的表面特异性抗原NKX2.1和前脑标志物FOXG1。移植8周后,体内可分化为GABA能神经元与多巴胺能神经元,明显改善PD大鼠运动行为。结论结合无血清培养基筛选法逐步定向诱导猪iPSCs高效分化为前脑GABA能神经元前体,移植后能够显著改善PD大鼠的运动功能障碍,为诱导GABA能神经元前体移植治疗神经损伤疾病奠定基础。

Objective To establish an efficient protocol for directed differentiation of miniature-swine induced pluripotent stem cells(iPSCs)into GABAergic progenitors in a chemically defined system.Methods We adopted a two-stage protocol for inducing the differentiation of porcine iPSCs.In the first stage,embryoid bodies(EBs)derived from porcine iPSCs after 3 days of suspension culture were induced in neural induction medium(containing SB431542,DMH1 and FGF2)till day 12 to differentiate into primitive neuroepithelia cells(NECs).In the second stage,the primitive NECs were induced in neural induction medium(containing Pur and B27)to obtain neural rosettes,which further differentiated into GABAergic neuron progenitors on day 21.After labeling with CM-DiI,the progenitor cells were stereotactically transplanted into the substantia nigra(SN)of 6-OHDA-lesioned PD model rats,and the cell survival,migration and differentiation in vivo were observed.Results Porcine iPSCs could be passaged stably on the feeder cell layer and expressed the pluripotent stem cell markers OCT4,Nanog,SSEA1and TRA-160.Karyotype analysis demonstrated the absence of contamination by cells from other species.On day 12 of induced differentiation,the cells formed adherent colonies containing NECs in the form of neural rosettes,which expressed the neuroepithelial markers PAX6,SOX2 and Nestin and the neurite marker beta III Tubulin(Tuj1).After induction for 21 days,the NECs differentiated into GABAergic neural progenitors highly expressing NKX2.1 and FOXG1.Eight weeks after transplantation,the iPSCs-iGABA progeniters survived in the striatum of the PD rats,where they differentiate into GABAergic neurons and TH+neurons and significantly improved dyskinesia of the rats.Conclusion The miniature-swine iPSCs derived GABA progenitors may serve as promising donor cells for neural grafting for treatment of neurodegenerative diseases.