The Feasibility Study Of Human Adipose-derived Stem Cells And Induced Pluripotent Stem Cell-derived Neural Stem Cells On Their Treatment In Stroke Mouse Model

Both human adipose stem cells(h ADSCs)and human induced pluripotent stem cell-derived neural stem cells(hi PSC-NSCs)are considered to be the ideal seed cells for the treatment of stroke,howere,there are few reports on the effects and mechanisms of h ADSCs and hi PSC-derived NSCs in repairing ischemic stroke,To provide new theoretical basis and technical support for the treatment of central nervous system diseases(CNS)using stem cells and regenerative medicine,the aim of this topic is to explore which stem cell is better to repair cerebral infarction and study its regulatory mechanism.This subject is divided into two parts,in vitro and in vivo experiments.In vitro experiments,the research contents include the isolation,culture and identification of h ADSCs,human i PSCs culture,induction and differentiation of NSCs,and the identification on the expression of Nestin and Sox2 of NSC markers by using immunofluorescence staining technique.In vivo experiment,the h ADSCs and hi PSC-derived NSCs which were labeled with Enhanced Green Fluorescence Protein expressing FG12 lentivirus and injected into MCAO mouse infarct area by using the improved method.Then we observed the effects of the two different kinds of stem cells on the repairation of neurological impairment caused by ischemic brain injury in mouse,to score the neurological function by using the 5 grade score method,and to detect the change of spatial learning and memory ability by using Morris Test in mouse,and to investigate the migration,differentiation and function integration of h ADSCs and hi PSC-NSCs in the brain,and to explore the mechanism of the two different kinds of stem cells in the treatment of brain injury.The paper contains three chapters:In vitro experimentChapter?: The isolation,culture and phenotype identification of human adipose stem cellsObjective: To acquire a mature and stable method of isolating and culturing h ADSCs in vitro,and to explore its biological characteristics.Methods: Primary culture of the h ADSCs was obtained by sticking-wall culture method,and the culture was enlarged,and the cell-flow technique was used to identify the surface markers of h ADSCs in the third-fifth generation.Results: After two weeks of the primary culture of hADSCs,the cells were observed by microscope,and the cells were demonstrating a typical fibroblast-like and shuttle-shaped morphology.By using immunofluorescence assay and the surface markers of h ADSCs,such as CD44,CD45,CD19,CD73,CD105,CD11 b,CD34 and HLA-DR,to identify h ADSCs.By using flow cytometry technique to identify immune phenotype of the h ADSCs,the results was that h ADSCs highly express mesenchymal stem cells surface markers such as CD44(100%),CD73(100%),CD90(100%),CD105(99.5%)and CD73CD105(99.5%),whereas,h ADSCs hardly expressed hematopoietic stem cells surface markers such as CD34,CD45,CD19,CD11 b and HLA-DR(all <3%).The results of this experiment showed that the separated and cultured h ADSCs had the same characteristics of mesenchymal stem cells,and the relative purity was higher than 97%.In vitro experiments,the differentiation of h ADSCs into three-line was induced by different methods,which showed the potential of multi-directional differentiation.By using Oil Red O staining to identify the cultured HADSCs could be induced into adipose cells,and by using Alizarin staining to identify the cultured h ADSCs could be induced into osteoblasts,and by using Alcian blue staining to identify cultured h ADSCs could be induced into chondrocytes.Conclusion: The topic establishes the mature and stable human adipose stem cell culture system in vitro.The separated and cultured h ADSCs not only has the ability of multi-directional differentiation potential but also has the ability of strong amplification in vitro and the characteristics of mesenchymal stem cells.At present,in the regenerative medicine,HADSCs is considered to be an ideal seed cells on the treatment in stroke.Chapter ?: hi PSCs were cultivated and induced into the NSCs.Objective: To obtain a stable passages of the hi PSCs and hi PSCs-derived NSC,and to explore the biological characteristics,and to repair ischemic brain damage by transplantation therapy.Methods: The experimental group adopted the mature and stable culture and differentiation system,we cultured human i PSCs cells and induced them to directionally differentiate into NSC,then by using immunofluorescence staining technique to identify the expression of the cell markers Nestin and Sox2.At the same time,the induction medium of non-recombinant human basic fibroblast growth factor(BFGF)was used to induce hi PSCs-NSC to differentiate into neurons and astrocytes and the expression of TUJ1 and GFAP was further identified by immunofluorescence staining.Results: we successfully obtained the induced and differentiated NSC.After 21 days of adding serum-free induction media,a typical nerve Rosette structure could be observed under a microscope.By using immunofluorescence staining,cell balls were positively expressing Nestin and Sox2.After the adherent cells were cultured for 20 days,the cells formed a distinct neural network structure.By using immunofluorescence staining to identify the cell markers,the results showed highly express Nestin,Sox2.After the cells were cultured for 1 week with no serum non-recombinant human basic fibroblast growth factor(BFGF)media,the cell balls were differentiated and formed a large number of neural network structure.The expression of cell markers including Nestin,SOX2,TUJ1 and GFAP were identified by immunofluorescence staining,and the results showed that the hi PSCs-derived NSC could be induced and differentiated into neuron and astrocytes.Conclusion: we have successfully established a mature and stable culture and differentiation system of the hi PSCs-derived NSC,the acquired NSC with the nature of neural stem cells,has a strong potential to differentiate into the nervous system,and in the current translational medicine,hi PSCs-NSC is also considered to be the ideal seed cells for the treatment in stroke.In vivo experimentChapter ?: The evaluation of the therapeutic efficacy of Human adipose-derived stem cells and Induced pluripotent stem cell-derived neural stem cells on their treatment in stroke mouse modelObjective: To establish a mature and stable ischemic stroke(MCAO)model and to evaluate the effects of two different kinds of stem cells on the injury repairation of nerve function in animals,and to provide basic support for the clinical application and the transformation in regenerative medicine.Methods: The SPF C57/BL6 female mice at the age of 4-6 weeks and with the weight of 16-18 g were selected to establish a mouse model for stroke.we used the score of neurological function and the living tissue experiment to verify the success of the mouse MCAO model established by line suppository.We randomly assigned the experimental animals to four groups: Hi PSCs-NSC injection group,HADSCs injection group,PBS group and Sham group.The specific method is that each day,we use the animal behavioral score to evaluate the model established for all animals within 14 days.7 days after the success of the model,the h ADSCs(106 cells/each animal)and hi PSC-NSCs(106cells/each animal)labeled with Enhanced Green Fluorescence Protein expressing FG12 lentivirus were injected into MCAO mouse infarct area in situ way with a slowly,slightly multi-point.Then we injected the same volume of PBS into the control group in the same way,but the Sham group did not inject any substance.Next,the 5-level scoring method(Zea Longa 5-grade scale)was used to evaluate the recovery of nerve function in mouse.35 days after cell transplantation,the recovery of learning and memory ability of mouse was evaluated by the Morris Test,and the infarct area of moue brain was evaluated by TTC staining.Results: 96% Mice were survived,and the successful MCAO mice with a neural function score were between 2 and 3,then TTC staining were performed and showed that the infarct location and the infarct area were the same size.Hi PSC-NSCs and h ADSCs were injected into the mouse brain tissue and it is possible for them to survive for a long time,and the results of immunofluorescence staining showed that the transplanted cells expressed nerve cells fluorescent markers such as GFAP?Iba1 in the transplanted area and the brain injury area after 35 days.So,the transplanted cells could migrate to ischemic area and differentiate into nerve cells;the scores of injection cell groups were significantly lower than those of MCAO-PBS-injected cells.The scores between injecting cells group and MCAO-PBS group were significantly different(P<0.05),however,the scores between MCAO-hi PSCs-NSC-EGFP group and MCAO-h ADSC-EGFP group had no significant difference.In addition,the experimental results of the Morris Test proved that both the hi PSCs-NSC and HADSCs improved the learning and memory abilities of mice,the spatial learning and memory ability of cells injection group was better than that before cells transplantation,the same time node cells injection group was better than MCAO-PBS Group(P<0.05);whereas,compared MCAO-hi PSC-NSC-EGFP group with MCAO-h ADSC-EGFP group,there is no significant difference in learning and memory function restoration(P>0.05).Conclusion: The MCAO model is a kind of mice cerebral ischemia model with high repeatability and good stability.Hi PSC-NSCs and h ADSCs were separately injected into the brain of mice in situ way,which could directly migrate to cerebral ischemia and differentiate into nerve cells and could survive long-term.Both hi PSC-NSCs and h ADSCs were significantly promoted neurological function recovery after cerebral ischemia injury in mice,and there was no significant difference in therapeutic efficacy.