Figure 1.
Cell surface marker profiles of EAEASCs and WtASCs.
A) Cells were analyzed by flow cytometry for MSC surface markers CD29, Sca1; hematopoietic markers CD34 and CD45; phagocytic lineage marker CD11b; and endothelial marker CD31. Gray filled: isotype control; gray line: WtASCs; black line: EAEASCs. B) Cell size based on forward scatter signal of flow cytometry (P = 0.26, t-test, n = 7).
Figure 2.
Analysis of differentiation of EAEASCs and WtASCs along osteogenic and adipogenic lineages.
A) Differentiated EAEASCs and WtASCs. Cells were incubated in adipogenic or osteogenic differentiation media for 21days and stained, respectively, with Oil Red O and Alizarin Red. For osteogenic differentiation, images were collected at 4× magnification. For adipogenic differentiation, images were obtained at 10× magnification. B) Quantification of osteogenesis and adipogenesis. For the quantitation of osteogenesis, the cells were de-stained with 10% cetylpyridinium chloride after stained with Alizarin Red. For the quantitation of adipogenesis, the cells were de-stained with isopropanol. Optical density (OD) was measured at 584 nm and normalized to protein content. The bar graph represents the ratio of normalized OD of differentiated cells and normalized OD of control cells. # means P<0.05 vs WtASCs (t-test, n = 3).
Figure 3.
Colony forming unit assays for EAEASCs and WtASCs.
A total of 100 cells were plated on 56.72 Nunc cell culture plates and incubated for 14 days. Cells were stained with 3% crystal violet, and colonies 2 mm or larger in diameter were counted. # means P<0.05 vs WtASCs (t-test, n = 5).
Figure 4.
Real-time PCR analysis of cytokine and chemokine profiles of EAEASCs and WtASCs.
Both cell types were cultured overnight, trypsinized, and analyzed by real-time PCR to determine the expression levels of various cytokines and chemokines TNFα, IL-6, MCP-1, MIP-1α, RANTES, KC, MIP-2α and VEGF. # means P<0.05 EAEASCs vs WtASCs (t-test, n = 3).
Figure 5.
Clinical scoring for disease onset and progression.
The clinical scores for the HBSS-treated (n = 20), EAEASC-treated (n = 20) and WtASC-treated (n = 20) EAE mouse groups were recorded daily for the duration of the study. WtASCs significantly reduced the clinical symptoms from PDI13 where the EAEASCs failed to mediate any therapeutic efficacy or improvement. # means P<0.01 vs HBSS-treated EAE group (post-hoc, n = 20); • means P<0.01 vs EAEASCs-treated EAE group (post-hoc, n = 20).
Figure 6.
Histologic and Immunohistochemical analysis on the spinal cord.
Spinal cords were collected at sacrifice from 3 mice per group. Each spinal cord was sectioned, mounted, and stained with luxol fast blue (LFB), H&E, and anti-GFAP for intact myelin, cell infiltration and astrocytes levels. Images were acquired at 4×, 40× and 10×magnification. The LFB stained sections (n = 3 mice; 3 sections/mouse) were quantified for the intensity of the blue staining using Aperio Software. The H&E stained sections (n = 3 mice; 3 sections/mouse) were quantified for the total cells per field using Fiji/Image J software. ## means P<0.01 EAEASCs-treated vs WtASCs-treated EAE group (post-hoc, n = 9); ** means P<0.01 EAEASCs-treated vs normal naïve mouse group (post-hoc, n = 9); ••means P<0.01 HBSS-treated vs WtASCs-treated EAE group (post-hoc, n = 9); ¥¥ means P<0.01 HBSS-treated vs normal naïve mouse group (post-hoc, n = 9).
Figure 7.
Lesion and cell infiltration analysis on the spinal cord.
Spinal cords were collected at sacrifice from 3 mice per group. Each spinal cord was sectioned, mounted, and stained with anti-CD3, CD11b, and CD45 followed by Hematoxylin counterstain for lesion size, number and cell infiltration analysis. # means P<0.05 vs EAEASC-treated EAE group (post-hoc, n = 9); & means P<0.05 vs HBSS-treated EAE group (post-hoc, n = 9).
Figure 8.
Pro-inflammatory cytokine protein levels in mouse serum.
Sera from 5 mice per group at sacrifice were pooled and analyzed by ELISA to detect levels of TNF-α, IL-12, and IL-17. # means P<0.05 vs HBSS-treated EAE group (post-hoc, n = 5); • means P<0.05 vs EAEASCs-treated EAE group (post-hoc, n = 5); & means P<0.05 vs normal naïve mouse group (post-hoc, n = 5).