Isolation, Culture and Cryopreservaion of Neural Stem Cell
Nestin is a member of the class Ⅵ intermediated filament proteins and a well-known stem cell marker in the neural lineage. The nestin gene consists of four exons and three introns. It has been known that the nestin transgene containing the second intron is sufficient to direct tissue specific expression of the nestin gene in developing embryos. In this study, we investigated whether the second intron of the rat nestin gene is sufficient to confer tissue-specific expression of the gene in neural stem cells in vitro and in vivo system. To study the specificity of the second intron of the nestin gene, eGFP expression was determined in transgenic mice carrying the nestin 2nd intron-eGFP transgene. eGFP expression was high in developing embryos but not in the adult mice. Neural stem cell were isolated from the forebrain of E12.5 transgenic mice and sorted by FACS based on GFP expression. Both GFP (+) and (-) cells could generate neurospheres but the differentiation potential into oligodendrocyte was reduced in eGFP(-) neurospheres. To determine differentiation ability neurosphere from single cell, we selected by clonal selection and cultured for 10 day. Single cell was formed neurosphere and expressed GFP or not. Neural stem cells (NSCs) derived from fetal and adult central nervous system has been expanded, differentiated, and fount to be multipotent and self-renewing. In this study, we investigated whether the NSCs from cryopreserved fetal brain tissue or cultured neurospheres maintain the differentiation potential compared with fresh(not frozen) tissue derived NSCs. Neural stem cell was prepared from gestation 10 to 15 weeks human fetal brain. Cultured neural stem cell was expressed nestin, a well-known stem cell marker in the neural lineage and positive neuronal marker(β-tubulin-III) and glial marker(Glial Fibrillary Acidic Protein, GFAP) after differentiation. To compared with not frozen and frozen tissue or neurospheres, tissue or neurosphere was cryopreserved in liquid nitrogen for 2 weeks. The cell derived from frozen tissue or neurospheres was expressed nestin and neuronal marker(β-tubulin-III) and glial marker(Glial Fibrillary Acidic Protein, GFAP) differentiation markers after differentiation. The results indicated that cryopreserved fetal brain tissue or neurospheres maintains NSCs fate, so tissue and neurospheres could store in liquid nitrogen. The Cryopreservation of tissue or neurosphere, expand the chances for use of the NSCs could therapeutic approach.
Nestin, 신경줄기세포, 신경구, 냉동보존, 세포치료제, neural stem cell, neurosphere, Cryopreservation
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