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

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.