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

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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.