Differentiation Of Human Oligodendrocyte Precursor Cells In Vitro And Myelination In Vivo

Background:White matter injury(WMI)is the most common form of brain injury in premature infants.At present,there is no effective therapy,leading to many sequelae such as cerebral palsy.Oligodendrocyte precursor cells(OPCs)are the only natural myelinating precursor cells in the central nervous system(CNS).OPCs are specifically damaged during WMI,causing oligodendrocytes(OLs)fails to maturation and myelination,Eventually,it leads to extensive demyelination in the white matter area,which causes neurological dysfunctions.Cell transplantation may be the hope of curing WMI,and OPCs are the ideal cell source..Our laboratory has successfully induced OPCs from human fetal brain-derived neural stem cells(NSCs)rapidly and steadily,but the ability of differentiation of OPCs into myelin sheath remains to be verified.Objective:To explore the functionalities of human OPCs induced by our laboratory,including the ability to differentiate into mature OLs in vitro and the ability to myelinate in vivo.Methods:The purity of human NSCs-induced OPCs was identified by immunofluorescence staining(IF)with specific markers of OPCs,such as PDGFR-a,A2B5,NG2,SOX10,etc.,and GFAP for astrocyte(AST)and Tuj-1 for neuron.In vitro differentiation experiment,the differentiation of OPCs was induced by adding different growth factor combinations into culture medium.The experimental groups included BM group,IGF-1 group,NT3 group,PDGF-AA group,IGF-1+NT3(IN)group,IGF-1+NT3+PDGF-AA(INP)group,IGF-1+NT3+EGF(INE)group,FBS group and OPCDQ gjroup(OPC differentiation medium of Sciencell company).GalC,MBP and GFAP markers were used for IF to identify the type and degree of differentiation of OPCs.In vivo myelination experiment,congenital demyelinating shiverer mice were used as animal models.The shiverer mouse(shi-/-)with complete MBP gene deletion was used as the negative control group and the treatment group,and the normal mouse(shi+/+)was used as the positive control.There were 5 mice in each group.The mice in the treatment group were stereotactically transplanted with OPCs 24 hours after birth,and the control group was treated with PBS instead of OPCs.At the age of 12 weeks,the formation of myelin sheath was assessed by transmission electron microscopy.Results:Human NSCs were induced to produce high-purity OPCs.The positive rate of each index was identified by IF:A2B5 was 88.21±2.40%,SOX10 was 90.41±1.72%,PDGFR-a was 85.12±0.23%,and NG2 was 88.47±0.32%.Few cells expressed GFAP and Tuj-1.In vitro differentiation experiment of OPCs,some cells in the OPCDQ group were observed to have small soma,increased processes and branches,or even a grid-like shape,which became more and more complex with the prolongation of culture time;some cells had large nuclei,less processes and relatively simple morphology.The IF results showed that 49.35±0.27%of cells expressed GalC,30.14±0.21%of cells expressed GFAP,and MBP staining was negative.The cells in the FBS group grew rapidly and showed spindle-like astrocyte morphology.The other groups had similar culture conditions.At 14 days of cell culture,there was no obvious OLs morphology,and the condition was poor,so the culture could not be maintained.In the experiment of OPCs transplanted into shiverer mice,the negative control group occasionally had thin and disordered myelin sheaths;the positive control group had the largest number of myelin sheaths with uniform morphology,clear boundary and close arrangement.Compared with negative group,the treatment group had significantly more myelin sheaths,a few of which were nearly normal,and the thickness was increased,but the myelin sheath was loose and separated,and most of the myelin sheath was only 1-2 layers.Conclusion:Human OPCs can differentiate into GalC+ immature oligodendrocytes in vitro,but fails to maturation,it may be related to the lack of a certain differentiation environment.And human OPCs can be myelinated in vivo,suggesting that transplanted human OPCs can survive,differentiate and mature.This study successfully demonstrated the human OPCs induced by our laboratory are functional.