Study On The Functional Differences Of Different Cell Subgroups Of Human Oligodendrocyte Precursor Cells And The Safety Evaluation Of Transplantation In Young Animals

Preoligodendrocytes(Pre-OLs)are an important cell source for myelination in the central nervous system.In premature infants,brain tissue ischemia and hypoxia often lead to the destruction of Pre-OLs in the white matter of the brain,which leads to disorders of myelination and affects the development of the nervous system.Therefore,timely repair of damaged myelin sheath and restoration of remyelination are very important for the growth and development of newborns.At present,the main way to treat demyelinating diseases is still medical treatment,but the effect is not good,and the increasingly mature cell transplantation therapy is expected to become the gospel for patients with demyelinating diseases.In the human body,Pre-OLs are differentiated from human oligodendrocyte precursor cells(hOPCs)with stronger proliferation and migration capabilities.Therefore,hOPCs are a potential source of cells for cell transplantation to treat human demyelinating diseases.Studies have shown that during the development of hOPCs,they can roughly go through three stages:early,middle and late hOPCs.These different stages of hOPCs may include different cell subgroups.For example,chondroitin sulfate proteoglycan 4 positive cells(NG2+)are the main cell subsets of early hOPCs,while α-N-acetylneuraminic acid α-2,8-sialyltransferase 1 positive cells(A2B5+)are One of the importantsubgroups of mid-term hOPCs.The hOPCs used in this study were obtained according to our team’s existing method of preparing human-source OPCs.Previous research results have confirmed the myelination effect of our team’s hOPCs,but it is not clear whether there are different cell subgroups in the hOPCs,and whether there are functional differences between different cell subgroups.In addition,the safety of cell transplantation therapy is also very important.There are essential differences between newborns and adults,such as the development of major organs,tissues,and nervous system,so newborns may have different adverse reactions from adults after cell transplantation.More importantly,OPCs are derived from glial progenitor cells,and glioma is currently the most common primary nervous system tumor.Therefore,we cannot ignore whether the transplantation of OPCs leads to tumor formation.In short,the preclinical safety research of cell transplantation for the treatment of neonatal demyelinating diseases has extremely important significance.In summary,in this study,we first used flow cytometry and single-cell sequencing to preliminarily study the cell subpopulations of hOPCs.Finally,NG2+cell subgroups and A2B5+cell subgroups were obtained through cell sorting,and the functional differences between the two groups of cells were studied.Then,hOPCs were transplanted into the lateral ventricle of young Sprague-Dawley(SD)rats to study their biological safety,and the ability to form myelin was verified by transplanting congenital demyelinated shiverer mice.Part ⅠThe functional difference of human oligodendrocyte precursor cell subgroupsAIM:To compare the remyelination effects of hOPCs at different stages by studying the differences between NG2+and A2B5+cells.STUDY DESIGN:Using cell sorting technology,we obtained NG2+/-,A2B5+/-cells.Further research was then conducted via in vitro cell proliferation and migration assays,single-cell sequencing,mRNA sequencing,and cell transplantation into shiverer mice.RESMLTS:The results showed that the myelinating ability of A2B5+cells was stronger than that of NG2+cells,while the proliferation and migration ability was weaker than that of NG2+cells.In addition,the migration,proliferation,and myelination capacities of the negative cell population were stronger than those of the positive cell population.CONCLUSIONS:These results indicate that early hOPCs rich in NG2+ cells are not suitable for cell transplantation to treat demyelinating diseases.In addition,among the four groups of cells,the negative cell group has the best final remyelination effect,so the negative group is also worthy of further study in the future.Part ⅡStudy on the safety human oligodendrocyte precursor cells transplanted into young animalsAIM:We aimed to evaluate the safety of hOPCs developed in our laboratory and transplanted into the lateral ventricles of young animals.STUDY DESIGN:Several acute and chronic toxicity experiments were conducted in which different doses of hOPCs were transplanted into the lateral ventricles of Sprague-Dawley rats of different ages.The toxicity,biodistribution,and tumor formation ability of the injected hOPCs were examined by evaluating the rats’ vital signs,developmental indicators,neural reflexes,as well as by hematology,immunology,and pathology.RESMLTS:Overall,our results show that transplanted hOPCs into young mice are non-toxic to their organ function or immune system.The transplanted cells engrafted in the brain and did not appear in other organs,nor did they cause tissue proliferation or tumor formation.In terms of efficacy,the transplanted hOPCs were able to form myelin in the corpus callosum,alleviate the trembling phenotype of shiverer mice,and promote normal development.CONCLUSIONS:The transplantation of hOPCs is safe;they can effectively form myelin in the brain,thereby providing a theoretical basis for the future clinical transplantation of hOPCs.