Transplanted miR-219-overexpressing oligodendrocyte precursor cells promoted remyelination and improved functional recovery in a chronic demyelinated model

Abstract

Oligodendrocyte precursor cells (OPCs) have the ability to repair demyelinated lesions by maturing into myelin-producing oligodendrocytes. Recent evidence suggests that miR-219 helps regulate the differentiation of OPCs into oligodendrocytes. We performed oligodendrocyte differentiation studies using miR-219-overexpressing mouse embryonic stem cells (miR219-mESCs). The self-renewal and multiple differentiation properties of miR219-mESCs were analyzed by the expression of the stage-specific cell markers Nanog, Oct4, nestin, musashi1, GFAP, Tuj1 and O4. MiR-219 accelerated the differentiation of mESC-derived neural precursor cells (NPCs) into OPCs. We further transplanted OPCs derived from miR219-mESCs (miR219-OPCs) into cuprizone-induced chronically demyelinated mice to observe remyelination, which resulted in well-contained oligodendrocyte grafts that migrated along the corpus callosum and matured to express myelin basic protein (MBP). Ultrastructural studies further confirmed the presence of new myelin sheaths. Improved cognitive function in these mice was confirmed by behavioral tests. Importantly, the transplanted miR219-OPCs induced the proliferation of endogenous NPCs. In conclusion, these data demonstrate that miR-219 rapidly transforms mESCs into oligodendrocyte lineage cells and that the transplantation of miR219-OPCs not only promotes remyelination and improves cognitive function but also enhances the proliferation of host endogenous NPCs following chronic demyelination. These results support the potential of a therapeutic role for miR-219 in demyelinating diseases.

Figure 1. MiR-219-overexpressed mESCs are pluripotent.

(A) A schematic protocol for directed differentiation of miR219-mESCs. Mouse ESCs were infected by miR-219 lentivirus at day 0. Twenty-four hours later, infected cells were selected under puromycin and grown in mESC medium without bFGF for 5 days, then switched to DIM medium supplemented with Pur and RA for 8 days to generate EBs. To induce NPCs, EBs were plated onto laminin/poly-ornithine-coated plates and cultured in DMEM/F12 supplemented with bFGF, RA, Pur and N2 for 7 days. On day 21, the medium was replaced with DMEM/F12 medium with 1% FBS but without bFGF, RA or Pur. (B) Relative miR-219 levels were detected by RT-PCR. The values shown are mean ± s.e.m. (n = 3), ***P < 0.001. (C–H) Undifferentiated miR219-mESC colonies express the pluripotency markers Nanog and Oct4. (I–N) miR219-mESC-derived GFP-positive NPCs overlap with nestin and musashi1 (Msi1). (O–W) In vitro terminal differentiation of miR219-mESCs into neurons, astrocytes, and oligodendrocytes was identified by Tuj1, GFAP and Galc. From C to N, scar bars = 50 μm (in Fig. 1N). From O to W, scale bars = 50 μm (in Fig. 1W). (X)) The mRNA levels of Nanog, Oct4, Galc, Tuj1, GFAP, Msi1 and nestin were detected by RT-PCR.

Figure 2. MiR-219 accelerates mESC differentiation into an oligodendrocyte lineage.

(A) A schematic protocol for the directed differentiation of miR219-mESCs into an oligodendrocyte linage. To generate OPCs, on day 15, NPCs were derived from mESCs grown for up to 28 days in DMEM/F12 supplemented with 10 ng/mL bFGF, 10 ng/mL PDGF-AA, 1% N2, and 2% KSR. Fluorescence immunocytochemistry imaging revealed that most of the GFP-positive cells in the scramble group were also A2B5 positive (B–D); a few were NG2 (E–G) and O4 (H–J) positive at 23 days in vitro. In the miR-219 group, GFP-positive cells expressed weak A2B5 (K–M) and strong NG2 (N–P) and O4 (Q–S) fluorescence intensity. Nuclei were stained with DAPI (blue). For oligodendrocyte maturation, miR219-OPCs were cultured in DMEM/F12 with 10% FBS for 7 days. Images show that OPCs differentiated into MBP- (T–U) and CNP- (V–W) positive mature oligodendrocytes. The percentages of A2B5-, NG2- and O4-positive cells in GFP-positive cells were counted (X). The data are presented as the mean ± s.e.m. *P < 0.05, **P < 0.001; n = 3 for each group. Scale bars: S = 50 μm; W = 20 μm.

Figure 3. MiR219-OPCs are associated with spinal axons in co-culture.

(A,B) NF-200 immunocytostaining shows neurites extending from spinal cord explants. High-magnification images of the box in A are shown in B. (C,D) Phase contrast micrographs show miR219-OPCs-derived cells exhibiting multipolar morphology and interacting with the neurites from spinal cord explants. The boxed area in C is presented in higher magnification in D. (E,F) After 2 weeks of co-culture, the processes of some GFP-positive oligodendrocytes have touched and elongated along the NF-200-positive neurites. The white arrows in image F and the high-magnification images of the box in image E indicate that GFP-positive oligodendrocytes made close contact with the neurites. Scale bars: A, C, E = 50 μm; B, D, F = 10 μm.

Figure 4. MiR-219 promotes the survival and differentiation of OPCs after transplantation in cuprizone-induced demyelinated mice.

(A) Representative images showing that surviving OPCs were identified at the injection site as GFP-positive cells 1 week after transplantation. (B) GFP-positive cells were co-labeled with the OPC marker NG2 in the corpus callosum 2 weeks after transplantation, representative image of n = 3. (C) At 4 weeks post-transplantation, a large fraction of GFP-positive cells in both the scramble and miR-219 groups co-expressed the oligodendrocyte marker MAG, representative image of n = 3. (D) MBP was observed when the OPC grafts survived for up to 8 weeks, representative image of n = 3. (E–G) Quantification of the number of NG2/GFP or MAG/GFP double positive cells, as well as the IOD of MBP/GFP double positive cells in the corpus callosum. The data are presented as the mean ± s.e.m. **P < 0.01, ***P < 0.001; Scale bars: A = 100 μm; B, C = 50 μm; D = 20 μm. IOD, integral optical density.

Figure 5. Enhanced remyelination in miR219-OPC-transplanted mice.

Toluidine blue staining of semi-thin resin sections of the corpus callosum in control mice (A,B), mice fed a cuprizone diet for 12 weeks followed by a normal diet for 8 weeks, treated with vehicle (C), engrafted with scramble-OPCs (D) and engrafted with miR219-OPCs (E). Dotted lines in A denote the borders of the corpus callosum and the striatum. The boxed image in A is shown in higher magnification in B. (F) The bar graph indicates the mean ± s.e.m of the number of myelinated axons (n = 4 per condition). Representative electron microscopic images of cross-sections of the corpus callosum of control mice (G), vehicle treated mice (H), scramble-OPCs transplanted mice (I) and miR219-OPC transplanted mice (J). The g ratio (axon diameter/fiber diameter) calculation (n = 4 per group, with 25–30 axons measured per group) significantly decreased in the scramble- and miR219-OPCs-transplanted groups, indicating improved myelin thickness (K). (L) Axon diameter was not significantly different in different samples (n = 4 per group, with 25–30 axons measured per group). All values are expressed as the mean ± s.e.m. **P < 0.01, ***P < 0.001 versus control group; ^#P < 0.05,^###P < 0.001 versus vehicle group; ^ΔP < 0.05 versus scramble group. Scale bars: A = 20 μm, B-E = 10 μm and G-J = 500 nm.

Figure 6. OPC transplantation enhances the proliferation of host SVZa cells in cuprizone-induced chronic demyelination lesions.

Mice were sacrificed and the cell types in the SVZa were assessed after transplantation 8 weeks. Semi-thin sections stained with toluidine blue (we converted the colour pictures into grey) show the cells in the SVZa in control (A), vehicle (B), scramble (C) and miR-219 groups of mice (D). Arrows in Figure A represent the various cell types: type E (red), type B (blue), type A (yellow) and type C (green). The bar graphs (K,L) indicate the total cell numbers and the different cell types in the SVZa (AOI, 600 μm × 450 μm). BrdU- (E–G) and nestin- (H–J) positive cells were also detected in the SVZa by immunofluorescence. Representative images are the same fields from three different conditions. The numbers of BrdU- and nestin-positive cells increased in the SVZa of miR219-OPC-transplanted mice, compared with vehicle treated mice (M) (AOI, 200 μm × 100 μm). All values are expressed as the mean ± s.e.m. (n = 4 per condition for semi-thin sections, n = 3 per condition for immunostaining). *P < 0.05, **P < 0.01 and ***P < 0.001. Scale bars: A–D = 10 μm and E-J = 100 μm.

Figure 7. OPC transplantation reverses cognitive deficits in cuprizone-induced demyelinated mice as shown by the step-through passive avoidance task and the MWM test.

(A) Step-through passive avoidance task. (B) Mean latency in the MWM hidden platform test. (C) Comparison of the time spent in the target quadrant on day 5 (where the platform was located during hidden platform training). (D) The number of crossings over the exact location of the former platform on day 5. The data are from 8 mice/group. All values are expressed as the mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001 versus control group; ^#p < 0.05, ^##p < 0.01 versus vehicle group, ^ΔP < 0.05 versus scramble group.