Differentiation From Bone Mesenchymal Stem Cells Into Enteric Neurons In Vitro And The Primary Study Of Mechanism

Hirschsprung's disease (HD) is common digestive tract malformation in childrenwith an incidence rate of 1/2000～1/5000. The pathogenesis of HD is currently agreedthat embryonic neural crest cells are blocked in the digestive tract transition becauseof a number of reasons, so rectal and colon wall is absent of ganglion cells. HDmanifestes that spastic contraction of aganglionic segment and the secondarymegacolon due to expansion of the proximal intestine. To date, surgery is still themost effective treatment. Although surgical methods improve continuously andminimally invasive surgery makes great progress, postoperative complications are stillplagued patients and doctors. Cell transplantation, one of the top researches in recentyears, is expected to become another effective and safe treatment manner for HD.Bone mesenchymal stem cells (BMSC) can be easily isolated, cultured andpurified in vitro and multi-directional differentiation potential is one of the mostimportant biological characteristics. Now there are more and more reports aboutneural differentiation of BMSC. Many studies indicate that BMSC can differentiateinto nerve cells under certain conditions in vitro and transplant into the brain or spinalcord to treat the injury of the nervous system, which makes it possible that BMSCtreat intestinal nervous diseases and avoids the absence of neural stem cell and ethicalproblem. However, the mechanism of differentiation of BMSC into nerve cells is notclear, which may be concerned with the combined effect of agents that activated thedifferentiation-related gene expression. More common withβ-mercaptoethanol(BME), dimethyl sulfoxide (DMSO), retinoic and other chemical substances are oftenused as inducers. In addition, some growth factors and neurotrophic factors havegradually be taken seriously. On the one hand, neurotrophic factors may be used asinduced factors under a certain culture condition to induce BMSC to the nerve cell;On the other hand, neurotrophic factors can protect the nerve and promote neuritegrowth. Neural cell apoptosis may occur without the support of neurotrophic factors. In this experiment, we investigated the conditions of differentiation from BMSCto nerve cells, as well as neurotrophic factor expression changes, such as glial cellline-derived neurotrophic factor (GDNF) and rearranged during transfection (RET).The results showed that bFGF and EGF can not only induce neural differentiation ofBMSC, but also increase the expression of GDNF and RET significantly. GDNF mayplay an important role in the process of differentiation of BMSC into nerve cells. Inthe following experiment, GDNF and fetal gut condition medium (FGCM) inducedifferentiation of BMSC, and the induced cells expressed not only neuronal markerNSE and the enteric nervous marker nNOS, but also intestinal neurotransmitters VIP.This showed that differentiated enteric neurons in vitro not only have themorphological characteristics, but also a certain degree of function and it's possiblethat the induced cells could be transplanted to aganglionic segment in vivo. In order toimprove the differentiation rate of BMSC into enteric neurons, the experiment aboutBMSC expressing GDNF was carried out. The results showed that recombinantgenetic engineered BMSC can successfully express GDNF in vitro. The geneticengineered BMSC in FGCM can differentiate into enteric neurons and induction ofdifferentiation is higher than that of exogenous GDNF group.However, we have no idea whether the induced cells have the function of normalganglion and whether cell transplantation can improve the function of aganglionsegment. It still needs further study in vivo. PartⅠIsolation, cultivation, purification and identification ofrat bone mesenchymal stem cells in vitroObjective: To isolate and observe bone mesenchymal stem cells (BMSC) fromSprague-Dawley (SD) rats in vitro and purify the cells.Methods: Femur bone marrow of rats were harvested and all the cells wereplanted and were cultured in DMEM with 10% FBS. BMSC were purified afterseveral passages. They were identified by flow cytometry of CD90 and CD45.Results: BMSC grew clones after adherence. The cells became fusion afterseveral passages. BMSC at passage 4 were BMSC marker CD90 positive (93.4%) andhematopoietic marker CD45 negative on flow cytometry.Conclusions: BMSC can be successfully cultured in vitro. The cells can bepurified only through repeated adherence.PartⅡDifferentiation from bone mesenchymal stem cells intonerve cells in vitroObjective: To investigate neural differentiation of rat bone mesenchymal stemcells (BMSC) and expression change of neural markers.Methods: At passage 4, BMSC were induced by basic fibroblast growth factor(bFGF, 10ng/ml), epidermal growth factor (EGF, 10ng/ml) and both of them inDMEM with 10% FBS for 7 days, respectively. The expression of neural specificenolase (NSE) and glial fibrillary acidic protein (GFAP) were detected byimmunocytochemistry. The expression of glial cell line-derived neurotrophic factor(GDNF) and rearranged during transfection (RET) mRNA was detected by RT-PCR.Results: After 7 days of induction, a certain number of cells showed expression of GFAP and NSE by immunocytochemistry method in experimental group, while thecells showed no expression of GFAP and NSE in control group. In bFGF group andbFGF+ EGF group, the positive rate of NSE was significantly higher than EGF group,while expression of GFAP in EGF group was obviously higher. BMSC expressed lowlevel GDNF mRNA and no RET mRNA, however, after induction, GDNF mRNAhighly increased and RET mRNA expressed, especially in bFGF+ EGF group.Conclusions: bFGF and EGF can not only induce neural differentiation of BMSC, butalso increase the expression of GDNF and RET significantly. GDNF may play animportant role in the process of differentiation of BMSC into nerve cells. The cellsinduced by bFGF are mainly neurons, while the induced cells by EGF are gliacytes.PartⅢDifferentiation from BMSC into enteric neurons in vitroand comparison of two different methodsObjective: To investigate the conditions and feasibility of differentiation frombone mesenchymal stem cells (BMSC) into enteric neurons in vitro and compare theefficiency of two methods.Methods: BMSC were cultured in DMEM supplemented with 10% fetal bovineserum (FBS) and induced by two methods after passage 4. The first one: BMSC wereinduced by 10ng/ml glial cell line-derived neurotrophic factor (GDNF) and fetal gutcondition medium (FGCM) for 7 days. The second one: The total mRNA wasextracted from newborn intestine by one-step method. GDNF cDNA was acquired byRT-PCR. The vector of PIRES2-EGFP-GDNF was constructed and identified bydouble enzyme digestion. Its sequence was analyzed by sequencing. The transgeneticexpression results were identified with immunofluorescence detection andimmunocytochemical stain. The transgentic cells were induced with diluted FGCM. The expression of neural specific enolase (NSE), vasoactive intestinal peptide (VIP)and nitric oxide synthase (nNOS) was detected by immunohistochemistry.Results: After 7 days of induction, a certain number of cells showed expressionof NSE, VIP and nNOS by immunohistochemistry method in both experimental group,while the cells showed no expression of VIP and NSE in control group. The inducedneurons form BMSC expressing GDNF were VIP and nNOS positive higher thanexogenous GDNF-induced cells.Conclusions: GDNF and FGCM can induce differentiation of BMSC intoenteric neurons. The efficiency of differentiation of GDNF-modified-BMSC intoenteric neurons is higher than that of exogenous GDNF-induced cells.PartⅣNeurotrophic factor expression changes of BMSC afterdifferentiation into enteric neurons and the study of mechanismObjective: To investigate the expression changes of glial cell line-derivedneurotrophic factor (GDNF), the receptor RET and C-kit of bone mesenchymal stemcells (BMSC) after differentiation into enteric neurons.Methods: At passage 4, BMSC were induced by 10ng/mlGDNF and fetal gutcondition medium (FGCM) for 7 days. The expression of neural specific enolase(NSE), vasoactive intestinal peptide (VIP) and nitric oxide synthase (nNOS) wasdetected by immunocytochemistry. The expression change of GDNF, RET and c-kitmRNA was detected by RT-PCR and protein was detected by western blot.Results: After 7 days of induction, a certain number of cells showed expressionof NSE, VIP and nNOS by immunohistochemistry method in experimental group,while the cells showed no expression of VIP and NSE in control group. BMSCexpressed low GDNF and no RET or c-kit mRNA, while after induction, the expression of GDNF mRNA was highly increased RET and C-kit mRNA expressed.GDNF protein was expressed.Conclusions: GDNF and FGCM can not only induce differentiation of BMSCinto enteric neurons, but also increase the expression of GDNF, RET and c-kit mRNA.GDNF-RET singaling pathway may play an important role in enteric neuraldifferentiation. C-kit may play a role at a certain stage of development of entericnervous system and be blocked after receiving a signal of mature enteric neurons.