Study On Biological Properties Of Human Fetal Bone Marrow Derived Mesenchymal Stem Cells And Their Neuroprotective Effects For Rat Dopaminergic Neurons After GDNF Gene-modification

Parkinson's disease (PD) is a chronic progressive neurodegenerativedisease of the central nervous system (CNS) that the nigrostriataldopaminergic pathways are affected. The application of DA precursorL-dopa is the major treatment of PD, which is unable to hinder thedisease progress. Transplantation of genetic modified stem cells might bethe promising therapeutic option for PD. Abortive fetal bone marrowderived mesenchymal stem cells (fBMMSCs) were isolated and culturedin the experiment by their inherent ability of adherence and theirbiological properties were investigated. The fBMMSCs were served asvectors of glial cell line-derived neurotrophic factor (GDNF) and were transplanted into Parkinson's disease model of rat organotypicmesencepholic slices in vitro lesioned by 6-hydroxydopamine (6-OHDA)to determine the neuroprotection for dopaminergic neurons. Theassessment was performed that whether the fetal bone marrow derivedMSCs could be an alternative source for application in cell and genetherapy.PartⅠThe isolation, primary culture, and biological propertiesof fetal bone marrow derived mesenchymal stem cellsObjective: To perform the isolation and primary culture of fetalbone marrow derived mesenchymal stem cells and to study theirbiological properties. Methods: MSCs were isolated respectively fromthe femur bone marrow of 16 weeks old, 17 weeks old, 25 weeks oldabortive fetus using adherence method and cultured and expanded inL-DMEM with 10％fetal bovine serum (FBS). The fifth passagefBMMSCs were picked up for 1. analysis of expression of cell surfacephenotype: CD73, CD90, CD105, CD34,CD45, CD13,CD44, CD133,HLA-DR with flow cytometry. 2. drawing of fBMMSCs growth curve. 3.quantification of fBMMSCs proliferation with BrdU incorporation assaysand phase S detection. 4. examination of Bmi-1, Oct4, Nanog, Sox2,Nestin, Rex-1 message RNA expression in fBMMSCs with RT-PCR. 5. analysis of karyotype of fBMMSCs. 6. detection of the GDNF proteinwith immuofluorescence and ELISA assays after transduced respectivelywith lentiviral vector that contained a constitutively expressing greenfluorescent protein (GFP) gene or GDNF gene. 7. investigation of thecorrelation of Bmi-1, p16 gene and senescence of fBMMSCs withinduction of replicative senescence of fBMMSCs by successive passages,by means of comparing with Bmi-1, p16 mRNA expression in fBMMSCsat passage 5, passage 15, passage 30. Results: MSCs were successfullyisolated from the femur bone marrow of three abortive fetus which werepositive for CD73, CD90, CD105, CD13, CD44 and negative for CD34,CD45, CD133, HLA-DR. Growth curves depicted an initial lag phase ofday 2, followed by a log phase in which cells divided at exponential ratesafter 3 days. The log phase was followed by a plateau phase. The rates ofBrdU incorporation in fBMMSCs were over one third, and fBMMSCs inphase S were more than ten percent. The fBMMSCs expressed Bmi-1,Oct4, Nanog, and Sox2 mRNA and did not express Nestin and Rex-1mRNA. After transduced respectively with lentiviral vector thatcontained a constitutively expressing green fluorescent protein (GFP)gene or GDNF gene, GFP labeled fBMMSCs were detected positive withfluorescence microscope, the screened GDNF genetic modifiedfBMMSCs were also shown positive with Immuofluorescence stainingfor GDNF and the secretion of GDNF in cultured medium was detected with ELISA assays. With the increase of passage number, the mRNAexpression level of Bmi-1 gene gradually decreased, so did that of p16gene. Conclusion: Fetal MSCs can be successfully isolated from femurbone marrow and are easy to proliferate and to perform genemanipulation. The replicative senescence of fBMMSCs has correlationwith the mRNA expression level of Bmi-1.PartⅡThe neuroprotective effects of GDNF gene-modifiedhuman fetal bone marrow derived mesenchymal stem cells fordopaminergic neurons in organotypic slices of ratmesencephalon of Parkinson's modelObjective: To investigate the neuroprotective effects of GDNFgene-modified human fetal bone marrow derived MSCs for dopaminergicneurons in rat organotypic mesencephalic slices of Parkinson's model.Methods: The organotypic slices of mesencephalon were generated frompostnatal day 7 Wistar rats. After cultured for 8 days, organotypiccultures were exposed to 10μM 6-hydroxydopamine (6-OHDA) for 24hto induce in vitro Parkinson's model, and then were subdivided intocontrol group (without donor cells administration), GFP-fBMMSCsgroup (transplantation of GFP-fBMMSCs) and GDNF-fBMMSCs group(transplantation of GDNF-fBMMSCs) for experiments. LDH level in cultured medium was analyzed with spectrophotometric analyzer and DAlevels in the cultured medium and tissue extracts were detected withHPLC at different time. Immuofluorescence staining forTH-immunoreactive cells in mesencephalic slices was performed at week3 of post-transplantation. Results: At the end of first week ofpost-transplantation, LDH levels declined in all the three experimentgroups in comparison with pre-transplantation. In addition, the declineddegree in GDNF-fBMMSCs group was the greatest among all the threegroups, followed by that in GFP-fBMMSCs group and the lowest incontrol group, but the difference between each group was not significant(P＞0.05). The levels of DA in the cultured medium and tissue extractswere the highest in GDNF-fBMMSCs group, followed by that inGFP-fBMMSCs group and the lowest in control group. There wassignificant difference between each group (P＜0.05) except that nosignificant difference of the DA level in the cultured medium betweenGDNF-fBMMSCs group and GFP-fBMMSCs group (P＞0.05). At week3 of post-transplantation, the decline trend of LDH level was simiiar tothat at week 1 of post-transplantation. There was significant differencebetween that in cell transplantation groups (GFP-fBMMSCs group andGDNF-fBMMSCs group) and that in control group (P＜0.05), and nosignificant difference between that in cell transplantation groups (P＞0.05). The levels of DA in the cultured medium and tissue extracts and the numbers of TH-reactive cells in the organotypic mesencephalic sliceswere the highest in GDNF-fBMMSCs group, followed by that inGFP-fBMMSCs and the lowest in control group. Except that nosignificant difference of DA level in the cultured medium betweenGDNF-fBMMSCs group and GFP-fBMMSCs group (P＞0.05), therewas significant difference between GDNF-fBMMSCs group and the othertwo groups (P＜0.05) and no significant difference between the latter twogroups (P＞0.05). Conclusion: GDNF secretion from GDNFgene-modified fetal bone marrow derived MSCs possesses biologicalactivity. The neuroprotective effects for dopaminergic neurons areobserved after GDNF gene-modified fBMMSCs transplanted into ratmesencephalic slices of Parkinson's model. The transplantation ofnon-modified fBMMSCs also has partial neuroprotective effects fordopaminergic neuron in the early stage of transplantation.