Differential efficacy of human mesenchymal stem cells based on source of origin

Abstract

Mesenchymal stem cells (MSCs) are useful in tissue repair but also possess immunomodulatory properties. Murine and uncontrolled human trials suggest efficacy of MSCs in treating lupus. Autologous cells are preferable; however, recent studies suggest that lupus-derived MSCs lack efficacy in treating disease. Thus, the optimum derivation of MSCs for use in lupus is unknown. It is also unknown which in vitro assays of MSC function predict in vivo efficacy. The objectives for this study were to provide insight into the optimum source of MSCs and to identify in vitro assays that predict in vivo efficacy. We derived MSCs from four umbilical cords, four healthy bone marrows (BMs), and four lupus BMs. In diseased MRL/lpr mice, MSCs from healthy BM and umbilical cords significantly decreased renal disease, whereas lupus BM MSCs only delayed disease. Current in vitro assays did not differentiate efficacy of the different MSCs. However, differences in MSC efficacy were observed in B cell proliferation assays. Our results suggest that autologous MSCs from lupus patients are not effective in treating disease. Furthermore, standard in vitro assays for MSC licensing are not predictive of in vivo efficacy, whereas inhibiting B cell proliferation appears to differentiate effective MSCs from ineffective MSCs.

Figure 1. Survival curve, body and spleen weight, urinary albumin excretion, and serum dsDNA auto-antibody levels of MRL/lpr mice

A. Kaplan Meier survival curve of MRL/lpr mice receiving hMSCs. Mortality was recorded until the time of sacrifice (8 weeks post transplant) B. Body weight of MRL/lpr mice at time of sacrifice. C. Spleen weights of MRL/lpr mice at time of sacrifice. Results are expressed as the mean ±SEM. D. Urinary albumin excretion over time post MSC transpant. Results are expressed as the mean 24 hour albumin excretion (ug/mouse/day) ±SEM. E. Serum dsDNA auto-antibodies overtime post MSC transplant. Results are expressed as the mean ±SEM. PBS (n=8), UC-MSC (n=8), HBM-MSC (n=8), LBM-MSC (n=16). Statistical comparisons were performed using Log-rank test, 1way ANOVA with Tukey posttest, and 2way ANOVA with Bonferroni posttest. *p≤ 0.05; **p≤.01; ***p≤0.001.

Figure 2. Glomerular C3 intensity, IgG intensity, and overall glomerular pathology score in MRL/lpr mice

At time of sacrifice kidneys were embedded in OCT and paraffin for sectioning. OCT embedded kidney labeled with fluorescein-conjugated anti-mouse C3 or IgG. A. Representative images of C3 and IgG deposits (20x) in glomeruli. B. Graphical assessment of MFI for C3 and IgG deposition. C. H&E staining of paraffin embedded kidney were examined and graded for hypercellularity, mesangial expansion, necrosis, cresents, and membrane thickening. D. Scores are added together to yield overall renal pathology score. E. Interstitial inflammation scores. Results are expressed as the mean ±SEM. PBS (n=8), UC-MSC (n=8), HBM-MSC (n=8), LBM-MSC (n=9). Statistical comparisons were performed using 1way ANOVA with Tukey posttest. *p≤ 0.05; **p≤.01; ***p≤0.001.

Figure 3. Inflammatory markers and human gene expression in MRL/lpr mice

At time of sacrifice kidneys were isolated for qPCR analysis. A. Expression of mouse MCP1 and IFNγ expression in the kidney of MRL/lpr mice relative to GAPDH. B. Urinary MCP1 measured over time post MSC transplant by ELISA. C. Urinary IFNγ at 6 weeks post MSC transplant measured by ELISA. D. Human IDO1 and CFH expression relative to GAPDH from the kidney of MRL/lpr mice at time of sacrifice (8 weeks post transplant). Results are expressed as the mean ±SEM. PBS (n=4), UC-MSC (n=8), HBM-MSC (n=8), LBM-MSC (n=12). Statistical comparisons were performed using 1way ANOVA with Tukey posttest and 2way ANOVA with Bonferroni posttest. *p≤ 0.05; **p≤.01.

Figure 4. Percentages of inflammatory cells in the spleen and bone marrow of MRL/lpr mice

Flow cytometry analysis was performed on spleen and bone marrow isolated from mice injected with MSC at time of sacrifice. A. Percentages of CD3+CD8+, CD3+CD4+, and CD3+CD4+CD25+ T cells in the spleen. B. Percentages of TCRβ+, B220+, and CD138+ cells in the bone marrow. Results are expressed as the mean ±SEM. PBS (n=3), UC-MSC (n=4), HBM-MSC (n=5), LBM-MSC (n=15). Statistical comparisons were performed using Kruskal-Wallis test with Dunn’s multiple comparison posttest. *p≤ 0.05; **p≤.01; ***p≤0.001.

Figure 5. T cell and B cell proliferation in CFSE dilution assays co-cultured with different sources of MSC and IFNγ production of T cell proliferation assay

A. Representative plots of CD3+ T cell proliferation in CFSE dilution assay co-cultured with each source of MSC. B. Proliferation of CD3+ T cells incubated alone (n=5) or with varying ratios of UC-MSC (n=5), HBM-MSC (n=4), and LBM-MSC (n=5). Cultures were stimulated with 1µg/ml of anti-human CD3/CD28 antibodies. C. Supernatants were collected from PBMC co-culture assay, IFNγ and IL17A cytokine levels were analyzed by ELISA. D. Representative plots of CD19+ B cell proliferation in CFSE dilution assay co-cultured with each source of MSC. E. Proliferation of CD19+ B cells incubated alone (n=4) or 1:1 ratio with UC-MSC (n=5), HBM-MSC (n=4), and LBM-MSC (n=5). Cultures were stimulated with 2.5µg/ml CpG, 1µg/ml), 2.5µg/ml F(ab’)2 anti-human IgM/IgA/IgG, and 1000U/ml IL2. Results are expressed as the mean percentage of proliferating cells or pg/ml IFNγ ±SEM. Statistical comparisons were performed 2way ANOVA with Bonferroni posttest. **p≤.01; ***p≤0.001.

Figure 6. Flow cytometry and qPCR analysis of MSC markers after in vitro IFNγ licensing

UC-MSC (n=), HBM-MSC (n=), and LBM-MSC (n=) were cultured with 0, 5 10, or 50 ng/ml of IFNγ. A. SOCS1 expression relative to GAPDH in IFNγ licensed MSCs B. Percentage of HLA-ABC+ and B7-H1+ MSCs cultured with varying concentrations of IFNγ. C. IDO1, B7H1, CFH, and CCL9 expression relative to GAPDH in IFNγ licensed MSCs. n=3 for each MSC source. Results are expressed as the mean ±SEM. Statistical comparisons were performed using 2way ANOVA with Bonferroni posttest. *denotes comparison to UC-MSC; •denotes comparison to HBM-MSC. *p≤.05 **p≤.01; ***p≤0.001.