Reprogramming Of Mouse Embryonic Fibroblasts Into Induced Oligodendrocyte Precursor Cells Via Indirect Lineage Conversion

Indirect lineage conversion provides a safe,efficient,and new approach for regenerative medicine by reprogramming differentiated somatic cells into lineage-specific precursor cells via forced expression of transcription factors.The myelin surrounding the axon of nerve cell is essential for proper signal conduction in the nerve system and maintenance of brain homeostasis,loss or dysfunction of myelin contributes to a variety of neurological diseases,such as multiple sclerosis(MS)and congenital leukodystrophies.Transplantation of oligodendrocyte precursor cells(OPCs)is a promising therapeutic strategy for those demyelinating diseases.After the brain tissue is injured,OPCs will be differentiated into OLs which can remyelinate the axon,but this kind of repair ability is very limited because there are only 5% to 8% OPCs,which are maintained a state of slow proliferation or quiescence in adult brain.In view of this problem,mouse embryonic fibroblasts(MEFs)——NIH/3T3 cells were reprogrammed into induced oligodendrocyte progenitor cells(iOPCs)through overexpression of three kinds of neuro-related transcription factors including Olig2,Nkx6.2,and Sox10.Those generated cells are similar to primary OPCs in morphology and expression of marker genes and have the ability to differentiate into OLs and astrocytes in vitro,which lays a foundation for the cell therapy of the demyelinating diseases,construction of disease modeling and development of new drug.Part 1: Isolation,culture,and induced differentiation of primary OPCs from Kunming mouseMouse OPCs is a kind of cells which is easy to be damaged and difficult to survive,and it is very sensitive to trypsin.In order to improve the isolation efficiency of mouse OPCs in vitro and utilize those cells to evaluate the quality of iOPCs,effects of different digestion methods and age of embryo or mouse on the growth status,total cell number,and cell survival rate of OPCs were explored in order to obtain suitable parameters for isolation and culture of OPCs.The results showed that:(1)The number of OPCs obtained after being treated with 0.125 % trypsin and 0.25 % chicken serum for 15 min was the highest,which significantly higher than that of other treatment groups(P<0.05)and control group(P<0.01).(2)Compared with newborn mouse,OPCs could be easily isolated from fetus at the gestational age of 18.5 days.Those OPCs appeared clear delamination after being cultured only for 7 days to 9 days,which shortened the time of culture in vitro.(3)The cell viability of OPCs obtained from whether fetus at the gestational age of 18.5 days or newborn mouse at the age of 1 day with improved purification method was significantly higher than that with traditional purification method(P<0.01),and the purity of generated OPCs was up to above 97 %.(4)Those isolated OPCs could be differentiated into OLs in serum-free medium supplemented with 40 ng/mL T3,and be differentiate into astrocytes in medium added with 10 % fetal bovine serum.The purity of OLs or astrocytes was more than 95 %.Therefore,mouse OPCs with typical morphology,good growth,and in vitro differentiation ability could be obtained by improved digestion and purification scheme from fetus at the gestational age of 18.5 days.Part 2: Optimization for transfection of plasmid DNA encoding Olig 2 in NIH/3T3 cells with different vectorsIn order to improve the transfection efficiency of plasmid DNA encoding Olig 2 in NIH/3T3 cells and lay the foundation for the preparation of iOPCs,the non-viral vectors(Lipofectamine 2000,Lipofectamine LTX)at different concentration(2.5 ?g/mL,5 ?g/mL,and 7.5 ?g/mL)combined with plasmid DNA at different concentration(1 ?g/mL,2 ?g/mL,3 ?g/mL,4 ?g/mL,and 5 ?g/mL),and magnetic nanoparticle transfection vector 1:1 bound to plastid DNA Poly-MAG were used to transfect the NIH/3T3 cells.Green fluorescent protein(GFP)gene acted as marker gene.Transfection parameters with higher efficiency and lower cytotoxicity were screened through comparing the transfection efficiency and cytotoxicity in different treatment groups.The results showed that no significantly difference was observed among 7.5 ?g/mL Lipofectamine 2000 combined with 3 ?g/mL plasmid DNA treatment group,7.5 ?g/mL Lipofectamine LTX combined with 2 ?g/mL plasmid DNA treatment group and 2 ?g/mL Poly-MAG combined with 2 ?g/mL plasmid DNA treatment group,which has the highest transfection efficiency among the other treatment groups with the same kinds of transfection reagent,respectively(P>0.05).However,the cytotoxicity of 2 ?g/mL Poly-MAG was lower(12.53±1.97 %)when the transfection efficiency of three kinds of transfection agents was the highest respectively.Poly-MAG could be rapidly enriched in the cell surface under the presence of external magnetic field and the Olig2 gene would be stably expressed.Therefore,2 ?g/mL Olig 2 NIH/3T3 carried by 2 ?g/mL Poly-MAG is suitable to transfect the NIH/3T3 cells.Part 3: Study on the feasibility of reprogramming NIH/3T3 cells into iOPCs via indirect lineage conversionIn order to establish the system of indirect lineage conversion to convert NIH/3T3 cells to iOPCs,overexpressions of transcription factors Olig 2,Nkx 6.2 and Sox 10 were mediated with Poly-MAG,the generated cells were identified based on the observation of morphology,immunocytochemical stain,real-time RCR(RT-PCR)and Western Blot detection,which lays a foundation for mechanism on regeneration of myelin,drug screening,and cell therapy,and so on.The results showed that:(1)iOPCs had the typical OPCs-like morphological characteristics,which grow in circular or elliptical shape with strong refraction.Most of them had two protrusions,and a few of them had three protrusions,they were very similar to mouse primary OPCs;(2)iOPCs expressed OPCs-specific antigen-A2B5,the ratio of A2B5 positive cells was 68.37±2.05 %;(3)Compared with the control group,the expressions of PDGFR?,S100?,NG2,and Olig2 genes of iOPCs were significantly up-regulated(P<0.01);(4)iOPCs could be induced to differentiate into MBP positive OLs or GFAP positive astrocytes in vitro,the rate of positive cells was 72.67±2.52 % or 75.36±1.98 %,respectively;(5)Compared with primary OPCs,the A2B5 protein levels of iOPCs were not significantly different(P>0.05),which indicated that cells overexpressing three neuronal transcription factors are similar to mouse primary OPCs,and the OPCs-specific protein A2B5 were expression.Therefore,differentiated NIH/3T3 cells could be reprogrammed into iOPCs via overexpression of transcription factors including Olig 2,Nkx 6.2,and Sox 10 mediated with magnetic transfection vector.Conclusions: OPCs with good morphology,rapid proliferation and differentiation ability could be efficiently isolated after the brain cortex of Kunming mouse fetus at the gestational age of 18.5 days was digested with 0.125 % trypsin combined with 0.25 % chicken serum,mixed cells were cultured in vivo for 9 days,and followed by be purified with the improved process.NIH/3T3 cells could be higher efficiently transfected with 2 ?g/mL plasmid DNA encoding Olig 2 mediated with 2 ?g/mL Poly-MAG and the cytotoxicity caused by this process was lower.NIH/3T3 cells could be successfully reprogrammed into iOPCs via forced expression of three kinds of neuro-related transcription factors including Olig 2,Nkx 6.2,and Sox 10 mediated with Poly-MAG,which could provide abundant cells for the study of mechanism on loss and regeneration of myelin,treatment of demyelination-related diseases.