HUMSCs And Their Exosomes Restore The Spermatogenic Of Non-Human Primate Animals NOA Model

Part I Isolation and identification of human umbilical cordmesenchymal stem cells and their exosomes[Purpose]Umbilical cord mesenchymal stem cells were isolated from the umbilical cord afterdelivery of the fetus, and the obtained mesenchymal stem cells were identified bydirectional differentiation and surface labeling, umbilical cord mesenchymal stem cellswere cultured without serum, collected the serum-free culture supernatant, andultracentrifugation was performed. The exosomes were obtained by the method, and theobtained exosomes were identified by electron microscopy, surface labeling, andnanoparticle tracking analysis (NTA)[Methods[1. Under sterile conditions, take about 15cm of the umbilical cord of the full-termhealthy fetus, and the umbilical cord mesenchymal stem cells are separated by tissueadherence method2. Passage and purification of the obtained cells, and identification by cell morphcell immunofluorescence, cell surface labeling, osteogenesis and adipogenesisdifferentiation3 The umbilical cord mesenchymal stem cells cultured by the serum-free mediumand the culture supernatant is collected, and the exosomes derived from the umbilical cordmesenchymal stem cells are obtained by ultracentrifugation.4. The obtained exosomes were identified by electron microscopy, surface markerdetection and particle size analysis(NTA)[Results]1. Observed under the microscope, the obtained umbilical cord mesenchymal stemcells were uniform in size and similar in shape and they showed a spiral shape. Theexpression rates of HUMSCs positive markers CD44, CD73, CD90 and CD1OSwere>90%detected by flow cytometry, the negative markers CD34, CD45, HLA-DR4and after adipogenesis differentiation, oil red o staining was positive, immunofluorescenceresults showed that CD45, CD90, CD105 and Cp29 were expressed in cells2. The obtained exosomes derived from HUMSCs were spherical double-layeredmembranes under electron microscope, with uniform size, regular shape and uniformdistribution. The expression rates of positive surface markers CD9, CD63 and CD8I byflow cytometry were 70%, 50%o, 90%o, particle number to particle diameter ratio is normaldistribution measured by NTA, the average particle size is 135.5nmConclusions1.The tissue adherence method is a simple and stable method for obtaining umbilicalcord mesenchymal stem cells, and the HUMSCs with higher purity can be obtained by thetissue adherence method;2.The exosome with high purity and stable qualily can be obtained by ultracentrifugation of serum-free cell culture supernatant.Part II Establishment of non-human primate animals azoospermiamodel by busulfan[Purpose]A stable model of non-obstructive azoospermia in macaques was establighed by intravenous injectionof busulfan, providing an ideal amimal model for further study of non-oostnuctive azoospermia in human[Methods]1. Randomly selected 12 adult male macaques, and divided into control group and experimental group2. Collecting and storing venous blood of the experimental group macaque,subcutaneous injection of Granulocyte Colony Stimulating Factor(G-CSF) and intravenous injection of busulfan 6mg/kg;3. After the injection of busulfan, collect the venous blood and injected G-CSF subcutaneously again,4. Record the sperm count, sperm vitality, and the structure of the seminiferous tubulesof the experimental group macaques every two weeks, and compared with the control group,observed the effects of busulfan on the spermatogenic function of macaques[Results]1. After 4 weeks of injection of busulfan, the mortality rate of macaques in theexperimental group was 02. There was no significant change in the body weight of the macaques in the experimental group, and there was no abnormality in blood routine and biochemical results compared with the control group,3. In the experimental group, the sperm count of the mature monkeys was 0.The testicular tissue sections showed obvious destruction of the seminiferous tubule structure and there was a significant"cavitation-like"change The number of spermatogenic epithelial cells and the number of cells were significantly reduced[Conclusions]1. Busulfan 6mg/kg intravenous injection can successfully establish a non-obstructive macaque azoospermia model;2. Re-injecting storage blood and subcutaneous injection of G-CSF can significantlyreduce the effect of busulfan on the function of rhesus hematopoietic system and reducemortality.Part III HUMSCs and their exosomes restore the spermatogenic ofnon-human primate animals NOA model[Purpose]To investigate the effects of umbilical cord mesenchymal stem cells(HUMSCs)andtheir exosomes on the spermatogenic function of non-obstructive azoospermia macaqueModels[Methods]1. A single intravenous injection of busulfan 6mg/kg to establish 9 non-obstructiveazoospermia macaque models, and confirmed the successful modeling2. Randomly divided the macaques into three groups: HUMSCs group, exosome group.and stroke-physiological saline solution( SPSS) group,3. The HUMSCs group was transplanted into the human umbilical cord mesenchymalstem cell suspension in the seminiferous tubule; the exosome group was injected with theexosome suspension in the testicular seminiferous tubule, the SPSS group was injected withstroke-physiological saline solution in the testicular seminiferous tubule.4 After 11 weeks, analyze the sperm count, sperm viability, and the structure ofseminiferous tubules, compare between the groups. Evaluate the function of HUMSCs andtheir exosomes of recovery of spermatogenic function in the non-obstructive azoospermiamacaque model[Results]1. The sperm counts in the spermatozoa of the HUMSCs group and the exosome groupwere significantly increased, and the viability was significantly increased compared withthe SPSS group, and the HUMSCs group was superior to the exosome group,2. The testicular tissue sections of the macaque in HUMSCs group and the exosomegroup showed that the cells in the seminiferous tubules were closely arranged, the numberof cell layers increased, and spermatogenic cells were observed at all levels, while the SPSSgroup showed obvious"cavitating structure[Conclusions]1. Transplantation of umbilical cord mesenchymal stem cells and their exosomes inseminiferous tubules can restore the spermatogenic function of non-obstructiveazoospermia macaque models in varying degrees2. HUMSCs have better recovery effect on spermatogenic function of macaque model than exosomes.