Abstract
Objective To establish and improve the method for culturing and purifying enteric neural progenitor cells (ENPCs) derived from murine embryos and seek optimal transfection for cell proliferation and differentiation experiments for further studies of the development and diseases of enteric nervous system. Methods Guts were collected from E16.5 mouse embryos and treated with collagenase.Single cell suspension was plated on medium and immunocytochemical identification performed after several exchanges of medium.Once ENPCs purity reached over 95%, mmu-miR-346 was transfected into ENPCs by lipo2000, RNAimax and lentivirus (LV) respectively.For assessing transfection efficiency, the gene expression of mmu-miR-346 was detected by real-time polymerase chain reaction (PCR). Ethynyldeoxyuridine (EdU) assay was performed for examining the effect of mmu-miR-346 on the proliferation of ENPCs.The differentiation of ENPCs was detected by the induction of differentiation medium. Results Neurospheres formed at Day 2.After 4 full exchanges of medium, immunocytochemical identification indicated (96.85±0.5357)% cells expressed Nestin and p75.The overexpression efficiency of LV was the highest among three methods.EdU showed mmu-miR-346 decreased the ratio of EdU(+ ) cells from (17.87±1.13)% to (6.73±0.43)%.TuJ1 immunofluorescence showed that mmu-miR-346 decreased the ratio of TuJ1(+ ) cells from (24.87±0.6)% to (11.90±0.85)%.Significant differences existed in cell proliferation and differentiation between transfected and control groups. Conclusions A primary method of culturing and purifying murin ENPCs may be improved by isolating and dissociating E16.5 murine gut.And an efficient lentivirus transfection is successfully selected for cell proliferation and differentiation assays.Thus it lays a foundation for understanding the development and diseases of enteric nervous system. Key words: Hirschsprung disease; Enteric nervous system; Transfection
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