Immunomodulatory effect of mesenchymal stromal cell overexpressing HLA-G1 in cell-based therapy for myocardial infarction

Fig. 1. Flow chart of the experiment

Fig. 2. Intravenous administration of HLA-G1 overexpressing hUCB-MSCs increased Treg cells and decreased NK cells. A Flow cytometry analysis of splenocytes was performed to evaluate the immunomodulatory effect of hUCB-MSCs. B and C Relative to the Sham group, AMI induction significantly increased splenic NK cells, but did not affect splenic Tregs in the AMI group. Furthermore, compared with the AMI group, intramyocardial transplantation of hiPSC-CMs did not affect splenic Tregs and NK cells in the AMI + CM group. However, the intravenous administration of hUCB-MSCs or HLA-G1 overexpressing hUCB-MSCs substantially increased splenic Treg cells and decreased splenic NK cells in the MSC + AMI + CM and HLA-G1 MSC + AMI + CM groups. HLA-G1 overexpressing hUCB-MSCs therapy further increased splenic Treg cells and decreased splenic NK cells in the HLA-G1 MSC + AMI + CM group, relative to the MSC + AMI + CM group. D Intravenous administration of hUCB-MSCs or HLA-G1 overexpressing hUCB-MSCs did not affect splenic CD4+ T cells. n = 6 mice/group. One-way ANOVA with Tukey post hoc test was performed for statistical analysis

Fig. 3. Cytokine profile of CD4+ splenocytes after 7 days of co-culture with hUCB-MSCs or HLA-G1 overexpressing hUCB-MSCs. A To assess the changes in cytokine profiles, the supernatant level of cytokines was measured at day 7. B Interferon (IFN)-γ, C tumor necrosis factor (TNF)-α, D interleukin (IL)-2, and E IL-17 A in the supernatant were reduced significantly after 7 days of co-culture with hUCB-MSCs, compared with the cytokine level in the supernatant of the CD4+ cell population alone. HLA-G1 overexpressing hUCB-MSCs indicated better immunomodulatory effects on these cytokines compared with hUCB-MSCs. The cytokine levels of F IL-10 and G IL-6 remained unchanged. n = 6 mice/group. One-way ANOVA with Tukey post hoc test was performed for statistical analysis

Fig. 4. Intravenous administration of HLA-G1 overexpressing hUCB-MSCs could increase cell engraftment. A To evaluate the cell engraftment of intramyocardially transplanted hiPSC-CMs, fluorescent imaging of DiR-labeled hiPSC-CM transplanted animals was performed on days 7 and 28. B and C Relative to the AMI + CM group, intravenous administration of hUCB-MSCs substantially increased engraftment of intramyocardially transplanted hiPSC-CMs in the MSC + AMI + CM group on days 7 and 28. Moreover, HLA-G1 overexpressing hUCB-MSCs therapy further increased the engraftment of transplanted hiPSC-MSCs in the HLA-G1 MSC + AMI + CM group, compared with the MSC + AMI + CM group on days 7 and 28 (B and C). n = 6 mice/group. One-way ANOVA with Tukey post hoc test was performed for statistical analysis

Fig. 5. Intravenous administration of HLA-G1 overexpressing hUCB-MSCs decreased infarct size and apoptosis. A Masson’s trichrome staining, H&E staining, and immunohistology staining of TUNEL were carried out to evaluate infarct size and apoptosis. B and C The H&E staining images showed the pathological changes in the peri-infarct area, whereas immunohistology staining indicated the rate of apoptosis in this region. The AMI group had a significant infarct area and an increased apoptosis rate. Furthermore, intramyocardial transplantation of hiPSC-CMs substantially decreased infarct size and apoptosis in the AMI + CM group, compared with the AMI group. Moreover, the intravenous administration of hUCB-MSCs or HLA-G1 overexpressing hUCB-MSCs significantly reduced infarct size and apoptosis in the MSC + AMI + CM and HLA-G1 MSC + AMI + CM groups, relative to the AMI and AMI + CM groups. In the HLA-G1 MSC + AMI + CM group, HLA-G1 overexpressing hUCB-MSCs therapy further reduced infarct size and apoptosis rate, compared with the MSC + AMI + CM group. n = 6 mice/group. One-way ANOVA with Tukey post hoc test was performed for statistical analysis

Fig. 6. Intravenous administration of HLA-G1 overexpressing hUCB-MSCs increased neovascularization. (A) Immunofluorescence staining of α-SMA and vWF was performed to assess neovascularization in the peri-infarct area. (B and C) In the AMI group, α-SMA- and vWF-positive vessels were reduced in the peri-infarct area. However, intramyocardial transplantation of hiPSC-CMs substantially increased neovascularization in the AMI + CM group, relative to the AMI group. Moreover, compared with the AMI and AMI + CM groups, intravenous administration of hUCB-MSCs or HLA-G1 overexpressing hUCB-MSCs increased angiogenesis in the MSC + AMI + CM and HLA-G1 MSC + AMI + CM groups. Compared with the MSC + AMI + CM group, the HLA-G1 MSC + AMI + CM group showed significantly increased angiogenesis after intravenous administration of HLA-G1 overexpressing hUCB-MSCs. n = 6 mice/group. One-way ANOVA with Tukey post hoc test was performed for statistical analysis

Fig. 7. Intravenous systemic administration of HLA-G1 overexpressing hUCB-MSCs improved heart function. A An Echocardiographic test was performed to assess the heart function. B Left ventricular ejection fraction (LVEF), C fractional shortening (FS), D LV end-systolic dimension (LVESD), and E LV end-diastolic dimension (LVEDD) were calculated in the parasternal long-axis view. B–E The AMI group indicated reduced LVEF and FS, whereas significantly increased LVESD and LVEDD, compared with the Sham group. However, relative to the AMI + CM group, intravenous administration of hUCB-MSCs or HLA-G1 overexpressing hUCB-MSCs increased LVEF and FS, while decreasing LVESD and LVEDD in the MSC + AMI + CM and HLA-G1 MSC + AMI + CM groups. The HLA-G1 overexpressing hUCB-MSCs therapy further improved the heart function in the HLA-G1 MSC + AMI + CM group, compared with the MSC + AMI + CM group. n = 6 mice/group. One-way ANOVA with Tukey post hoc test was performed for statistical analysis