Zoomed image of the black square in (E) shows (F) DAB\stained MBP, (G) locality of GFP + oligodendrocytes, and (H) co\localization of GFP + oligodendrocytes and MBP. both mouse and human NSCs (Kim with defined culture conditions is sufficient to generate homogeneous, self\renewing and bipotent iOPCs from fully differentiated somatic cells through passing the aggregate stage and demonstrates its functionality in rat SCI models. Our strategy reduces host genome modifications by minimizing the use of transcription factors to a single factor and facilitates future therapeutic applications for demyelinating conditions, including SCI. Results Generation of iOPCs from adult mouse fibroblasts by (Mitchell and cultured in defined OPC induction medium. The transduced cells underwent morphological changes into spindle\shaped cells 14C21?days after induction (Fig?1B and C), whereas uninfected cells did not change during the entire process (Fig?1D). Next, we mechanically isolated the cells exhibiting spindle\shaped morphology and replated them in OPC medium supplemented Rabbit Polyclonal to Integrin beta5 with platelet\derived growth factor AA (PDGFCAA), an essential mitogen for OPCs (Noble transgene in the host genome of iOPC clones by genomic polymerase chain reaction (PCR) (Fig?EV1D). Exogenous expression of mRNA was dramatically silenced in TAK-242 S enantiomer both clones at passage 5 (P5), as examined by quantitative reverse transcriptionCPCR (qRTCPCR) (Fig?EV1E). Furthermore, the iOPCs maintained a normal mouse chromosome karyotype (2induction (Fig?EV1F). These results support that expression with our defined culture condition is sufficient to convert the cell fate of adult mouse fibroblasts into expandable iOPCs. Open in a separate window Figure 1 Generation and characterization of differentiation into mature oligodendrocytes (OPC\AGs, OPC aggregates).BCG Morphology of induction. (E) Appearance of OPC\AGs within 35?days after infection. (F) OPC\like cells outgrew from OPC\AGs on gelatin\coated plates. (G) Zoomed image of the white square in (F), which shows the bipolar morphology of OPC\like cells. Scale bars, 250?m.H Immunofluorescence images of iOPC\C1 and iOPC\C2 stained with OPC\specific markers, A2B5, PDGFR, NG2, and Olig2, in OPC medium. Cells were co\stained TAK-242 S enantiomer with A2B5 and PDGFR (left\most columns), A2B5 and NG2 (left\middle columns), NG2 and PDGFR (right\middle columns), and Olig2 and DAPI (right\most column). Cells were counterstained with DAPI. Scale bars, 75?m.I Morphology TAK-242 S enantiomer of iOPC clones at early (passage 3) and late passages (passage 31). Scale bars, 125?m.J Growth curves and mean doubling time (mDT) of iOPC clones at passage 3 (P3) and passage 31 (P31). Each point refers to the cell numbers of two iOPC clones every 24?h. Data are presented as the means??SD (and and transgene in the iOPC clones. Silencing of the exogenous transgene expression in iOPC clones. Gene expression was normalized to after 2?weeks of differentiation showing (G) O4\immunostained oligodendrocytes and GFAP\stained astrocytes derived from iOPC\C1. (H) Zoomed image of the white square in (G). Scale bars, 75?m.I The differentiation efficiency of iOPCs into O4+ oligodendrocytes and GFAP + astrocytes. Data are presented as the means??SD (Cspg4,and distinct from the fibroblast (Fig?3C). The hierarchical clustering, 3D PCA analysis, and the distance map showed that iOPCs TAK-242 S enantiomer and wtOPCs are tightly correlated (Fig?3D and Appendix Fig S1A and B). To validate the microarray data, we examined the mRNA expression of OPC\specific genes including and by qRTCPCR. Consistent with the microarray result, the expression level of OPC\specific genes was up\regulated in the iOPC clones relative to the fibroblasts (Fig?3E). Together, these results revealed a high degree of similarity in molecular identity between iOPCs and wtOPCs. Open in a separate window Figure 3 Global gene expression profiles of TAK-242 S enantiomer Cspg4and in iOPCs relative to fibroblasts. Graphs represent log2\fold changes after normalization to functionality of the iOPCs, we transplanted GFP\labeled iOPCs into adult rat SCI models ((Fig?4E and F). GFP+ iOPCs were distributed in the vicinity of the myelinated nerve fibers in the white matter (Fig?4G and H). This result suggests that myelination was.
Zoomed image of the black square in (E) shows (F) DAB\stained MBP, (G) locality of GFP + oligodendrocytes, and (H) co\localization of GFP + oligodendrocytes and MBP