Zhu H, Sun T, Wang Y, Wang T, Ma C, Wang C, Liu C, Guo Y
School of Laboratory Medicine Bengbu Medical College, Bengbu 233000, China.
School of Life Sciences, Bengbu Medical College, Bengbu 233000, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2021 Jun 20;41(6):820-827. doi: 10.12122/j.issn.1673-4254.2021.06.03.
To establish an efficient protocol for directed differentiation of miniature-swine induced pluripotent stem cells (iPSCs) into GABAergic progenitors in a chemically defined system.
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.
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.
The miniature-swine iPSCsderived GABA progenitors may serve as promising donor cells for neural grafting for treatment of neurodegenerative diseases.
在化学成分明确的体系中建立一种将小型猪诱导多能干细胞（iPSCs）定向分化为γ-氨基丁酸能祖细胞的高效方案。
我们采用两阶段方案诱导猪iPSCs分化。在第一阶段，悬浮培养3天后源自猪iPSCs的胚状体（EBs）在神经诱导培养基（含SB431542、DMH1和FGF2）中诱导至第12天，以分化为原始神经上皮细胞（NECs）。在第二阶段，原始NECs在神经诱导培养基（含Pur和B27）中诱导以获得神经玫瑰花结，并在第21天进一步分化为γ-氨基丁酸能神经元祖细胞。用CM-DiI标记后，将祖细胞立体定向移植到6-羟基多巴胺损伤的帕金森病（PD）模型大鼠的黑质（SN）中，并观察其在体内的细胞存活、迁移和分化情况。
猪iPSCs可在饲养细胞层上稳定传代，并表达多能干细胞标志物OCT4、Nanog、SSEA1和TRA-160。核型分析表明不存在其他物种细胞的污染。在诱导分化的第12天，细胞形成含有呈神经玫瑰花结形式的NECs的贴壁集落，其表达神经上皮标志物PAX6、SOX2和Nestin以及神经突标志物βⅢ微管蛋白（Tuj1）。诱导21天后，NECs分化为高表达NKX2.1和FOXG1的γ-氨基丁酸能神经祖细胞。移植8周后，iPSCs-iGABA祖细胞在PD大鼠的纹状体中存活，并分化为γ-氨基丁酸能神经元和TH+神经元，显著改善了大鼠的运动障碍。
小型猪iPSCs来源的γ-氨基丁酸能祖细胞可能作为神经移植治疗神经退行性疾病的有前景的供体细胞。
