Human Cardiac-Derived Stem/Progenitor Cells Fine-Tune Monocyte-Derived Descendants Activities toward Cardiac Repair

Figure 1. Human cardiac-derived stem/progenitor cells (hCPC) have the capacity to recruit CD14+CD16− monocytes. (A) Expression of informative markers on CD14+CD16− monocytes isolated from peripheral blood mononuclear cells (PBMC) as determined by flow cytometry (representative dot plot in the upper panel). Results are presented as percentage of positive cells (lower left panel) and geometric mean (lower right panel). (B) Migration of monocytes in response to hCPC- or interferonγ (IFNγ)-hCPC conditioned media or to RPMI medium was determined by transwell migration assay. The number of migrating cells was determined using flow cytometry counting beads. (C) The levels of CCL2 and CX3CL1 produced by hCPC- and IFNγ-hCPC as determined by specific enzyme-linked immunosorbent assay. (D) CD14+CD16− monocytes migration in response to hCPC and IFNγ-hCPC conditioned medium in the presence of 20 nM INCB 3284 dimesylate CCR-2 antagonist, 5 µg/mL polyclonal anti-MCP-1, or anti-IgG isotype control was determined as in (B). Data on the graphs represent the mean ± SEM from five independent experiments conducted with PBMC isolated from five different donors against the same hCPC, and are representative of…

Figure 2. CD14+CD16− monocytes develop an anti-inflammatory profile in the presence of allogeneic human cardiac-derived stem/progenitor cells (hCPC). CD14+CD16− monocytes were cultured for 5 days in the absence (medium) or the presence of hCPC (hCPC) or interferonγ (IFNγ)-hCPC (IFNγ-hCPC) at a ratio of 5:1. (A) Expression of informative markers presented as percentage of positive cells (upper panel) and geometric mean (lower panel) was determined by flow cytometry. (B) Phagocytic activity was evaluated using Red pHrodo Zymozan A BioPaticles phagocytosis assay. Results are presented as percentage of pHrodo-positive cells. (C) TNFα and IL10 secretion by monocytes populations as determined by specific enzyme-linked immunosorbent assay. Data on the graphs represent the mean ± SEM from five independent experiments conducted with peripheral blood mononuclear cells isolated from five different donors against the same hCPC and are representative of data obtained with two other hCPC from different donors. Each data point represents the mean of experimental triplicates. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns: not significant.

Figure 3. Allogeneic human cardiac-derived stem/progenitor cells (hCPC) impact CD14+CD16− monocytes differentiation/polarization programs. CD14+CD16− monocytes were differentiated/polarized with a cocktail of macrophage colony-stimulating factor (M-CSF)/interferonγ (IFNγ)/lipopolysaccharide (LPS) (A) or M-CSF/IL-4/IL-13 (B) in the absence (medium) or the presence of hCPC (hCPC) or IFNγ-hCPC (IFNγ-hCPC) for 5 days. Then by flow cytometry, we determined (A) expression of relevant markers of activated-M1 presented as percentage of positive cells (upper panel) and geometric mean (lower panel). (B) Expression of relevant markers of M2a presented as percentage of positive cells (upper panel) and geometric mean (lower panel). Data on the graphs represent the mean ± SEM from five independent experiments conducted with peripheral blood mononuclear cells (PBMC) isolated from five different donors against the same hCPC and are representative of data obtained with two other hCPC from different donors. Each data point represents the mean of experimental triplicates. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns: not significant.

Figure 4. Human cardiac-derived stem/progenitor cells (hCPC) promote IL10 production, influence phagocytic activity, and previse immuno-regulatory capacity of CD14+CD16– derived macrophages. CD14+CD16− monocytes were differentiated/polarized/activated as in Figure 3 in the presence (A,B) or absence (C,D) of cell-cell contact. (A) TNFα and IL10 secretion by CD14+CD16− monocyte-derived activated-M1 (left panel) and CD14+CD16− monocyte-derived M2a (right panel) in the presence or absence of hCPC, as evaluated by specific enzyme-linked immunosorbent assay. (B) Phagocytic activity of CD14+CD16− monocyte-derived activated-M1 (left panel) and CD14+CD16− monocyte-derived M2a (right panel) in the presence or absence of hCPC was determined by Red pHrodo Zymozan A BioPaticles phagocytosis assay. hCPC or interferonγ (IFNγ)-hCPC were compared to medium. (C) Modulation of PHA-induced T cells proliferation by activated-M1 or (D) M2a in the absence or the presence of hCPC was determined by tailored immunomodulation assays as described under Section “Materials and Methods.” T cells proliferation under different experimental conditions was evaluated by measuring the loss of carboxyfluorescein succinimidyl…

Figure 5. CD14+CD16− monocytes-derived immature cells (iDC) acquire a macrophage-like profile in the presence of allogeneic human cardiac-derived stem/progenitor cells (hCPC). CD14+CD16− monocytes were differentiated into iDC with a GM-CSF/IL-4 cocktail in the absence (medium) or presence of hCPC (hCPC) or interferonγ (IFNγ)-hCPC (IFNγ-hCPC) at a ratio of 5:1. Co-cultures were conducted in the presence (A,B) or absence of cell–cell contact (C). (A) Expression of relevant iDC markers (left panel), of dendritic cell maturation markers (middle panel), and of FCγR (right panel) as determined by flow cytometry. Results are presented as the percentage of positive cells (upper panel) and geometric mean (lower panel). (B) IL10 and TNFα secretion by CD14+CD16− monocyte-derived iDC in the absence (medium) or the presence of hCPC (hCPC) or IFNγ-hCPC (IFNγ-hCPC) determined by specific enzyme-linked immunosorbent assay. (C) Modulation of PHA-induced T cells proliferation by CD14+CD16− monocyte-derived iDC in the absence (medium) or the presence of hCPC (hCPC) or IFNγ-hCPC (IFNγ-hCPC). T cells proliferation was analyzed by flow cytometry as described in Figure 4. Results are presented as division index…

Figure 6. Human cardiac-derived stem/progenitor cells (hCPC) confer macrophage-like profile and impair the capacity of mature DC (mDC) to induce allogeneic T cells proliferation. CD14+CD16− monocytes were differentiated into immature cells as in Figure 5 then their maturation to mDC was induced with lipopolysaccharide (LPS). (A) Expression of relevant markers (left panel), of dendritic cell maturation markers (middle panel), and of FCγR (right panel) by CD14+CD16− monocyte-derived mDC in the absence (medium) or the presence of hCPC (hCPC) or IFNγ-hCPC (IFNγ-hCPC) as determined by flow cytometry. Results are presented as the percentage of positive cells (upper panel) and geometric mean (lower panel). (B) CD14+CD16− monocyte-derived mDC in the absence (medium) or the presence of hCPC (hCPC) or IFNγ-hCPC (IFNγ-hCPC) were used to stimulate the proliferation of allogeneic carboxyfluorescein succinimidyl ester (CFSE)-labeled peripheral blood lymphocyte (PBL) at a PBL/DC ratio of 5:1 as described under Section “Materials and Methods.” The percentage of CD4+ and CD8+ proliferating T-cells was determined by flow cytometry as described in Figure 4. Results are presented as representative dot plots…

Figure 7. mDChCPC and mDCIFNγ-hCPC produce IL10 rather than TNFα and immune-modulate an ongoing immune response. (A) The levels of IL10 and TNFα were quantified by specific enzyme-linked immunosorbent assay from supernatants of co-cultures conducted as in Figure 6. (B) Modulation of PHA-induced T cells proliferation by mature DC (mDC) in the absence (medium) or the presence of hCPC (hCPC) or IFNγ-hCPC (IFNγ-hCPC) was determined by immunomodulation assays described under Section “Materials and Methods.” T cells proliferation was analyzed by flow cytometry as in Figure 4. Results are presented as division index compared to PHA or medium (lower panel) and representative histograms in the upper panel. Data on the graphs represent the mean ± SEM from four independent experiments conducted with Peripheral blood mononuclear cells (PBMC) isolated from four different donors and are representative of data obtained with two other hCPC from different donors. Each data point represents the mean of experimental triplicates. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns: not significant.