Prospective identification and culture of rat enteric neural stem cells (ENSCs)

Abstract

Hirschprung's disease (HD), a very common congenital abnormality in children, occurs mainly due to the congenital developmental defect of the enteric nervous system. The absence of enteric ganglia from the distal gut due to deletion in gut colonization by neural crest progenitor cells may lead to HD. The capacity to identify and isolate the enteric neuronal precursor cells from developing and mature tissues would enable the development of cell replacement therapies for HD. However, a mature method to culture these cells is a challenge. The present study aimed to propose a method to culture enteric neural stem cells (ENSCs) from the DsRed transgenic fetal rat gut. The culture medium used contained 15 % chicken embryo extract, basic fibroblast growth factor, and epidermal growth factor. ENSCs were cultured from embryonic day 18 in DsRed transgenic rat. Under inverted microscope and fluorescence staining, ENSCs proliferated to form small cell clusters on the second day of culture. The neurospheres-like structure were suspended in the medium, and there were some filaments between the adherent cells from day 3 to day 6 of the culture. The neurospheres were formed by ENSCs on day 8 of the culture. Network-like connections were formed between the adherent cells and differentiated cells after adding 10 % FBS. The differentiated cells were positive for neurofilament and glial fibrillary acidic protein antibodies. The present study established a method to isolate and culture ENSCs from E18 DsRed transgenic rats in the terminal stage of embryonic development. This study would offer a way to obtain plenty of cells for the future research on the transplantation of HD.

Exposure to GDNF Enhances the Ability of Enteric Neural Progenitors to Generate an Enteric Nervous System

“…Of the numerous studies examining isolation and in vitro culture of rodent and human ENS progenitors, most use only bFGF and EGF growth factors in the culture media, usually in the presence of differing combinations of N2 and B27 supplements and/or chick embryo extract ( Belkind-Gerson et al., 2015 , Binder et al., 2015 , Cheng et al., 2015 , Dettmann et al., 2014 , Gao et al., 2016 , Hotta et al., 2013 , Lindley et al., 2008 , Metzger et al., 2009a ). Few studies have included GDNF in the culture medium ( Becker et al., 2012 , Schriemer et al., 2016 ), and to date no study has made direct comparisons between established ENCC culture conditions and GDNF treatment.…”

Recent advances in regenerative medicine to treat enteric neuropathies: use of human cells

“…Formation of NLBs from dissociated gut cells is now a common method to isolate and study enteric neural progenitors . However, a recent study showed that not all NLBs of cells that form in cultures of dissociated gut cells contain neural stem cells and not all NLBs give rise to neural derivatives under differentiation conditions .…”

“…method to isolate and study enteric neural progenitors. [22][23][24][25][26][27][28] However, a recent study showed that not all NLBs of cells that form in cultures of dissociated gut cells contain neural stem cells and not all NLBs give rise to neural derivatives under differentiation conditions. 29 Furthermore, NLBs generated from the outer layers of small intestine of adult mice contain more neural crest-derived cells than NLBs generated from the large intestine.…”