Cultivation and identification of neural GABAergic precursor cells derived from medial ganglionic eminence of embryonic mice
10.3760/cma.j.issn.1671-8925.2017.05.002
- VernacularTitle:胎鼠内侧神经节隆起区γ-氨基丁酸能前体细胞的培养及鉴定
- Author:
Yifei LI
1
;
Zhenyu LI
;
Wenjing CHEN
;
Chen CHEN
;
Ning LIU
;
Ruxiang XU
Author Information
1. 南方医科大学
- Keywords:
Medial ganglionic eminence;
Precursor cell;
Interneuron
- From:
Chinese Journal of Neuromedicine
2017;16(5):439-444
- CountryChina
- Language:Chinese
-
Abstract:
Objective To explore a method for isolation,cultivation and identification of GABAergic precursor cells derived from medial ganglionic eminence (MGE-NPCs) of embryonic mice.Methods The MGE brain tissues of pregnant mice of 14.5 d were isolated under stereomicroscope;and the cells from these tissues were cultured to third passage or above in serum-free medium with SHH signal path stimulator.(1) Fluorescence immunocytochemistry was used to detect the expressions of neural stem cells (NSCs) markers nestin,sex determining region Y-box protein 2 (SOX2),MGE transcription factor NK2 homeobox 1 (NKX2-1) and intemeuron progenitor marker LIM homeobox 6 (LHX6) to identify the NSCs maintenance ability.(2) Proliferation potential of MGE-NPCs was detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and bromodeoxyuridine (BrdU) labeling one and 3 d after culture in vitro.(3) The expressions of neuronal marker neuronal nuclear antigen (NeuN),and interneuron markers gamma-aminobutyric acid (GABA),glutamic acid decarboxylase(GAD67),and parvalbumin (PV) were examined by immunocytofluorescent 5 d after induced culture with GABAergic induced medium.(4) The differentiations of MGE-NPCs into GABA-,GAD67-and PV-positive cells in vivo one month after transplantation into the mice were detected by frozen section immunofluorescence staining.Results The neurospheres with self-renewal and proliferation capacity were obtained from the MGE of embryonic mice.Immunofluorescent staining showed that nestin,SOX2,Nkx2.1 and LHX6 positively expressed in the MGE-NPCs.The results of MTT assay revealed that the optical density (OD) one d after culture was significantly less than that 3 d after culture (0.392±0.032 vs.0.811±0.017,P＜0.05).BrdU labeling indicated that the ratio of proliferated MGE-NPCs one d after culture was significantly less than that 3 d after culture (45.086±7.122 vs.61.786±10.540,P＜0.05).The MGE-NPCs could differentiate into NeuN-,GABA-,GAD67-and PV-positive inhibitory interneurons 5 d after differentiation culture and one month after transplantion into mice.Conclusion The MGE-NPCs cultured in vitro still have NSCs characteristics of neuronal precursors and remain the capacity of differentiated intemeuron.
