Investigation of neural crest stem cell therapy and niche factors for Hirschsprung's disease

先天性巨结肠症，又称无神经节细胞症或巨结肠，是一种大肠(结肠)发育障碍，每5000名儿童中就有1人发生这种疾病。RET KO(Ret^&lt；[-/-]&gt；)小鼠是一种无肠道神经系统的先天性巨结肠模型小鼠。与Ret^&lt；[-/-]&gt；KO小鼠无神经节结肠是利用无血清培养再生神经球样小体(NLBS)的方法研究先天性巨结肠的再生医学策略。虽然有一些报道描述了NLBS在肠道中的产生，但无血清培养过程尚未见报道。P0-CRE/FLOXED-EGFP小鼠被认为是利用EGFP荧光靶向神经脊源干细胞(NCSCs)群体，并可以利用FACS细胞分选器分离NCSCs丰富的细胞群体。我们从转基因小鼠P0-CRE/FLOXP的肠道中获得了NLBS-EGFP，然后在无血清培养基中成功培养细胞。培养10天至3周后，无血清培养的EGFP阳性细胞形成神经球样结构，表达NCSC基因。多聚D-赖氨酸层粘连蛋白包被的玻片小室和含10%胎牛血清的培养液培养4天后，神经细胞、神经胶质细胞和肌成纤维细胞标志物蛋白均有表达。

Hirschsprung's disease, also called aganglionosis or megacolon, is a developmental disorder of large intestine (colon) which occurs in 1 in 5000 children. Ret KO (Ret^<[-/-]>) mouse were one of the Hirschsprung's disease model mouse which have no intestinal nerve systems. An object of this NLB co-culture study with Ret^<[-/-]>KO mouse aganglionic colon is to research regenerative medicine tactics for Hirschsprung's disease using de novo Neurosphere-like Bodies (NLBs) generation protocol with serum free culture to regenerate enteric nervous system.Although there are several report described Generation of NLBs in gut but serum free culture procedure have not reported.P0-Cre/floxed-EGFP mice are known to be used to target neural crest derived stem cells (NCSCs) population using EGFP fluorescent and could separate NCSCs rich cell population using FACS cell sorter.We generated Neurosphere-like Bodies (NLBs) from the E13.5 embryo gut of transgenic mice P0-Cre/floxp-EGFP and then cells were cultured in the serum-free medium successfully. After 10 days to 3 w cultures, serum-Free-cultured EGFP-positive cells formed Neurosphere-like structures that expressed NCSC genes. Neurons, glial cells, and myofibroblasts marker protein expression were confirmed after 4days culture of NLBs using poly-D-lysine-laminin coated slide chamber and 10% FBS contained medium.