The Experimental Study Of Co-Culture In Vitro Of Interstitial Cells Of Cajal And Neuroepithelial Stem Cells In Rat

Aganglionic megacolon, i.e. Hirschsprung’s disease (HD), is a common and functional intestinal obstruction with an incidence of 1/3000-1/5000 live births. This disorder is characterized by severe constipation due to the absence of enteric ganglia and reduction of Interstitial cell of Cajal along a variable length of the intestine. Varying lengths of the distal colon are unable to relax, causing functional colonic obstruction over time. Symptoms range from neonatal intestinal obstruction to chronic progressive constipation in older children. HD was a multi-factors and multi-genes genetic disease caused by genetic and environmental agents. Abnormality of genes regulating gangliocytes’development such as RET、EDNRB、EDN3、GDNF maybe the inner causes, and ischemia、anoxia、toxin and inflammation maybe the environmental causes.The enteric nervous system (ENS) is a well-defined system of neurons and glial cells that regulates several aspects of gastrointestinal physiology, including motility and secretion. The ENS descends from migrating neural crest cells and is structured into different plexuses embedded in the gastrointestinal tract wall, including myenteric plexus and submucosal plexus. Disturbances appearing during this time affect the proper formation and/or the proper functioning of the ENS, thus leading to severe intestinal dysfunctions, e.g., Hirschsprung’s disease. Intersititial cells of Cajal (ICCs) of the gastrointestinal tract come from the mesenchymal cells of the embryonic layer in the primitive digestive tract, ICCs are the pacemaker cells of the slow wave movement of the intestinal wall, and participate in the transfer of the excitatory and inhibitory neurotransmitter through the contact with the ganglion cell axons in the intestinal wall. Abnormal deficiency or reduction of ICCs and NESCs maybe the immediate cause of HD.Based on the above concept, we addressed this issue to study the isolation and culture of ICCs and NESCs. In this research, NESCs derived from rat embryonic neural tube and ICCs derived from rat colon were co-cultivated in vitro which was designed to explore the interaction and value of the two kinds of cells in the occurrence, development and treatment of enteric nervous system diseases. This study can be divided into three parts.Part one:Isolation and culture of interstitial cells of Cajal from rat colon in vitroIn order to identify ICCs more accurately from cell morphology, we make much efforts in studying the isolation, culture ICCs from rat colon in vitro. With the enzymolysis method in combination with the density gradient centrifugation technology, the ICCs in the digestive tract can be separated and cultivated in vitro. The antibody of c-kit was used to identify the cultured ICCs. M199 medium contains serum and stem cell factor (SCF) was used for resuspending the cell suspension.After 24 hours, we changed the medium. After 3 days, the cell bodies of cultured ICCs seemed like spindles and stretched out a few axis-cylinders. After 5 days, longer axis-cylinders which form synapse each other were observed. Within 7 days in vitro, connections were formed with widespread snapes.Part two:Isolation and culture of neuroepithelial stem cells from neural tube in vitroIsolation and culture of NESCs are the base of realization of the stem cells. The NESCs were isolated from neural tubes of E11.5d Wistar rats. By microdissection and mechanical trituration, a cell suspension was obtained and resuspended in neurobasal medium contining B27. After 3 days, dozens of NESCs had proliferated to form neurosphere-like aggregates and a neurosphere consisted of hundreds of cells developed after cultured for 6 days. The generated neurospheres were characterized as Nestin positive. In the presence of serum, migrating cells from neurosphere differentiated into glial fibrillary acidic protein(GFAP)-positive astrocytes and microtubule associated protein 2(MAP2)-positive neurons.Part three:Co-culture of interstitial cells of Cajal and neuroepithelial stem cells in vitroNESCs and ICCs were co-cultivated in vitro which was designed to explore the interaction and value of the two kinds of cells in the occurrence, development. The third generation of NESCs were inoculated into the ICCs, which the medium is DMEM/F12 with 10% FBS. The control group is the single culture NESCs that is cultivated with DMEM/F12 containing 10% FBS. Protein gene product 9.5(PGP9.5) immunocytochemical staining revealed that the numbers of positive cells of the co-culture group was obviously more than that of the single culture of NESCs. After 7-day co-culture, NESCs were observed to differentiate into the neuronal nitric oxide synthase(nNOS)-positive neurons. The co-cultured ICCs survived well and connected with differentiated NESCs morphologically. Double immunofluorescence staining for c-kit and MAP2 showed that ICCs interconnected with neurons which differentiated from NESCs.ConclusionsIn summary, we successfully isolated and cultured NESCs derived from rat embryonic neural tube and ICCs derived from rat colon. By the co-culture of the two kinds of cells, we demonstrated that ICCs can promote the differentiation of NESCs into neurons, and interconnect with the differentiated neurons into a network morphologically, and then may participate in transmission of nerval signal. The findings may provide the theoretical basis for the treatment of nervous system diseases through the simultaneous transplantation of NESCs and ICCs.