Small Molecules Compound VCR Induce Rat Fibroblasts To Reprogram Into Neural Progenitor Cells Under Hypoxia And Its Therapeutic Effect On Parkinson’s Disease

Objective:Neural progenitor cells that are capable of self-renewing and differentiate into neural cell lineage hold great promise for potential cell therapy.However,the source of neural progenitor cells has become the key to clinical application because they are too difficult to obtain directly.Here we describe an efficient protocol for neural progrnitor cells generated from rat embryonic fibroblastsby a chemical cocktail VCR under hypoxic conditions（5%O₂）.To study the repair mechanism of transplanted chemical induced neural progrnitor cells（ci RNPCs）to improve the motor function of Parkinson’s rats.Methods:The reprogramming of rat fibroblasts into ci RNPCs mainly undergoes two major stages.Firstly,the rat fibroblasts was cultured in KSR medium containing VCR（V:VPA;C:CHIR99021;R:Repsox）and 10,000U/m L LIF for 15 days,under a physiological hypoxic condition（37℃,5%CO₂,5%O₂）.The formation of dense cell colonies was observed for intermediate cells.Secondly,the induced intermediate cells were digested with 0.25%trypsin,seeded in a low adhesion plate,and cultured in NEM medium under normoxia.The expression of markers was identified by immunofluorescent staining and Western Blotting.Transcriptome and epigenome analyses were performed with cells at different stages of process.The rat model of Parkinson’s disease with lateral damage was prepared by injecting 6-OHDA into the right substantia nigra（SN）at two coordinates by brain stereotaxic apparatus.After 2 weeks,APO-induced rotation test was used to screen the successfully modeled Parkinson’s rat model.After labeling with the red dye CM-Di I,ci RNPCs were directionally transplanted into the substantia nigra-striatal region of the Parkinson’s rat brain.The motor coordination ability of rats in control group,PD model group and transplantation group was compared through APO-induced rotation test,open field test,rotarod test and water maze test.Eight weeks after the cells were transplanted,the brains of the rats in each group were taken for HE staining to observe the number and morphological changes of the cells at the injury site;the expression of tyrosine hydroxylase（TH）in each group was detected by immunohistochemistry.Twenty-six weeks after cell transplantation,the survival,migration and differentiation of ci RNPCs in the host brain were observed by immunofluorescence.Results:After 5～10 days of hypoxia induction,a clear trend of cell aggregation has been observed,compact cell colonies were observed in REFs treated with VCR for 15 days under hypoxic condition.Approximately 30 colonies emerged from 1×10~5 cells,and most colonies were positive for AP staining.Moreover,when these cells were cultured further in suspension,free-floating neurospheres formed and stained positive for neural progenitor cells（Nestin,Sox2 and Pax6）.These ci RNPCs could differentiate into glial cells and neurons,and express neuron marker Tuj1 and astrocyte marker GFAP in vitro.Compared with the PD model group,there were significant differences in the APO-induced rotation test,open field test,rotarod test and water maze test of the rats in the transplantation group（p<0.01）,which indicated that the motor function of the Parkinson’s rats was significantly improved after transplantation of cells.Eight weeks after cell transplantation,HE staining showed that compared with the control group,the number of cells in the injured area of the PD model group was significantly reduced,and the arrangement was disordered.Compared with the PD model group,the number of cells in the transplantation group increased and the cells were arranged more regularly.The results of TH immunohistochemistry showed that the expression of TH in the injured area of the rats in the PD model group was significantly lower than that in the control group,while the expression of TH in the transplantation group was significantly higher than that in the PD model group.Twenty-six weeks after cell transplantation,the immunofluorescence results were observed,and CM-Di I-positive cells were found,which also expressed neuron markerβ-Ⅲ-tubulin,dopamine neuron marker TH,GABAergic neuron marker GABA,excitatory postsynaptic membrane marker GAD67,presynaptic membrane marker Synapsin,postsynaptic membrane marker PSD95 and astrocyte marker GFAP.Conclusion:1.Our research demonstrates that rat fibroblasts can be directly transformed into neural progenitor cells by small molecule compounds VCR without introducing exogenous factors,which can be used an attractive donor material for the neural grafting in neurodegenerative diseases.2.The transplanted ci RNPCs can survive for at least 26 weeks in the Parkinson’s rat brain microenvironment,simultaneously undergo long-distance migration,and differentiate into different functional neurons,producing synaptic connections with autologous neurons and functioning.