Immunomodulation by systemic administration of human-induced pluripotent stem cell-derived mesenchymal stromal cells to enhance the therapeutic efficacy of cell-based therapy for treatment of myocardial infarction

Figure 1. Flow chart of the experiment.

Figure 2. Intravenous pre-transplantation systemic administration of hiPSC-MSCs improved left ventricular function. To assess left ventricular (LV) function, echocardiographic measurement was performed of left ventricular ejection fraction (LVEF) (A), fractional shortening (FS) (B), LV end-systolic dimension (LVESD) (C), and LV end-diastolic dimension (LVEDD) (D). Intravenous pre-transplantation of hiPSC-MSCs could further improve LVEF, increase FS and decrease LVESD in the MSC-hiPSC-CM group and MSC-hiPSC-MSC group compared with the S-hiPSC-CM group and S-hiPSC-MSC group. No change was observed in LVEDD; invasive hemodynamic study was performed to determine the maximal positive pressure derivative (+dP/dtmax) (E), and the slope of end systolic pressure-volume relationship (ESPVR) (F) 4 weeks following induction of myocardial infarction (MI) in different groups of animals. Intravenous pre-transplantation of hiPSC-MSCs could further improve +dP/dtmax and ESPVR in MSC-hiPSC-CM group and MSC-hiPSC-MSC group compared with S-hiPSC-CM group and S-hiPSC-MSC group.

Figure 3. Intravenous pre-transplantation systemic administration of hiPSC-MSCs improved survival of transplanted cells in mice with MI. To evaluate the survival of intramyocardial transplanted hiPSC-cardiomyocytes (CMs) or hiPSC-MSCs, DiR labelled hiPSC-CMs or hiPSC-MSCs were detected on the infarcted hearts using an IVIS spectrum in vivo imaging system 7 and 28 days after intramyocardial transplantation (A). The quantity of intramyocardial transplanted hiPSC-MSCs and hiPSC-CMs in DiR signal images was indicated by the radiant efficiency of fluorescent intensity. The DiR signal was stronger in the MSC-hiPSC-CM group and MSC-hiPSC-MSC group compared with the S-hiPSC-CM group and S-hiPSC-MSC group respectively (C-D). Immunofluorescent staining of intramyocardial transplanted cells in the peri-infarct area was performed 4 weeks after induction of MI (B). The quantity of intramyocardial transplanted hiPSC-CMs and hiPSC-MSCs on immunofluorescent images was represented by the number of anti-human Troponin I positive cells and anti-human CD105 positive cells, respectively. Intramyocardially transplanted CMs were represented by human Troponin I positive cells (green). Intramyocardially…

Figure 4. Intravenous pre-transplantation systemic administration of hiPSC-MSCs increased Tregs and decreased the infiltration of macrophages and apoptosis in the peri-infarct area. Hematoxylin and eosin (H&E) staining was performed to show the peri-infarct regions of the LV and to measure the cardiomyocyte cross sectional area in the peri-infarct area. To measure intramyocardial inflammation in the peri-infarct area, immunofluorescent staining of Tregs, macrophages and apoptotic cells in the peri-infarct area was performed 4 weeks after MI induction. Tregs were represented by CD4 (green) and Foxp3 (red) positive cells and cell nuclei were counterstained by DAPI (blue). Macrophages were represented by CD68 positive cells (green) and cell nuclei were counterstained by DAPI (blue). Apoptotic cells were represented by TdT-mediated dUTP Nick-End Labeling (TUNEL) positive cells (green) and cell nuclei were counterstained by DAPI (blue). Positive cells were counted under fluorescent microscopy and expressed as count per mm2. (A). Pre-transplantation systemic administration of hiPSC-MSCs significantly reduced the cross sectional area of native CMs in MSC-hiPSC-CM and MSC-hiPSC-MSC groups compared…

Figure 5. Intravenous pre-transplantation systemic administration of hiPSC-MSCs increased Tregs and decreased NK cells in the spleen. To identify splenic Tregs, splenocytes were stained with anti-mouse CD4-FITC and anti-mouse Foxp3-APC and analyzed by flow cytometry. “Fluorescence Minus One” (FMO) controls were obtained by omitting anti-mouse CD4-FITC antibody or anti-mouse Foxp3-APC antibody from the Tregs staining. Analysis of FMO controls for Tregs showed that omitting anti-mouse CD4-FITC antibody or anti-mouse Foxp3-APC antibody did not significantly change the frequency of positive cells for the other marker, confirming the validity of the gating strategy (A). To determine splenic NK cells, splenocytes were stained with anti-mouse CD49b-FITC and anti-mouse NK1.1-APC antibodies and analyzed by flow cytometry. FMO controls were obtained by omitting anti-mouse CD49b-FITC antibody or anti-mouse NK1.1-APC antibody from the NK cell staining. Analysis of FMO controls for NK cells showed that omitting anti-mouse CD49b-FITC antibody or anti-mouse NK1.1-APC antibody did not significantly change the frequency of positive cells for the other marker, confirming the validity of the gating strategy…

Figure 6. Cytokine profiles changes after co-culture for 7 days with CD4 positive splenocytes and hiPSC-MSCs. To demonstrate altered cytokine profiles, the supernatant level of cytokines was measured at day 7 (A). interferon (IFN)-γ (B) and tumor necrosis factor (TNF)-α (C), and interleukin (IL)-17A (D) in the supernatant reduced significantly after co-culture with hiPSC-MSCs for 7 days, compared with the cytokine level in the supernatant of the CD4 positive cell population alone. The cytokine level of IL-2 (E), IL-6 (F) and IL-10 (G) remained unchanged.