Reparative macrophage transplantation for myocardial repair: a refinement of bone marrow mononuclear cell-based therapy

Fig. 1. Production of M2-like macrophages by M-CSF + IL-4 treatment. Mouse BM-MNCs were isolated and treated with different M2-like macrophage induction protocols (M-CSF plus combination of polarizing cytokines) for 7 days. Freshly isolated BM-MNCs and M0 macrophages (treated with M-CSF alone) were used as controls. Bar charts presented as mean ± SEM. a Gating strategy for flow cytometry procedures. Cells double positive for F4/80 and CD206 within the CD11b+ population were defined as M2-like macrophages by flow cytometry. Representative dot plots from samples of freshly isolated BM-MNCs and M-CSF + IL-4 treated BM-MNCs are presented. b Quantification of M2-like macrophage induction. Percentages of M2-like macrophages among total cultured cells were measured by flow cytometry at the end of the induction protocols. *p < 0.05 vs. all other groups; †p < 0.05 vs. BM-MNC; §p < 0.05 vs. M0; #p < 0.05 vs. IL-4 IL-10 TGF-β1. N = 4–7 independent samples. c Representative flow cytometry images for each induction protocol. Representative dot plots from M0; IL-10; IL-4 + IL-10; IL-4 + TGF-β1; IL-4 + IL-10 + TGF-β1 protocols are presented. d Quantification of M2-like macrophage yield. For each…

Fig. 2. Optimization of M-CSF + IL-4 treatment. The M-CSF + IL-4 treatment was further analyzed in terms of duration, purity and treatment sequence. Bar charts presented as mean ± SEM. a Representative images for the timeline of M2-polarization by M-CSF + IL-4 treatment. BM-MNCs were treated with M-CSF + IL-4 and analyzed for M2-like macrophage markers by flow cytometry at days 4, 5, 6 and 7. Freshly isolated BM-MNCs (day 0) served as controls. b M2-like macrophage polarization timeline by M-CSF + IL-4 treatment. Percentages of M2-like macrophages among total cultured cell numbers were calculated from flow cytometry (refer to a). *both days 6 and 7 significant p < 0.05 vs. all days 0, 4 and 5, †p < 0.05 vs. day 0; §p < 0.05 vs. day 4. N = 4–6 independent samples. c M1-like macrophage production by M-CSF + IL-4 treatment. BM-MNCs were treated with M-CSF + IL-4 and analyzed for M1-like macrophage markers (CD80+) by flow cytometry at days 0, 4 and 7. M1-like macrophages were obtained by treatment of BM-MNCs with M-CSF, LPS and IFN-γ and served as positive controls. *p < 0.05. N = 4 independent samples. d Comparison against the sequential polarization approach. BM-MNC were either cultured…

Fig. 3. Pro-reparative, anti-inflammatory phenotype of M2(IL-4) macrophages. Gene expression and cytokine secretion profiles of M2-like macrophages obtained after 6 days of M-CSF + IL-4 treatment [M2(IL-4) macrophages] and freshly isolated BM-MNCs were compared. Bar charts presented as mean ± SEM. a Upregulation of M2-like macrophage markers and myocardial repair-related genes in M2(IL-4) macrophages. Fold change in mRNA levels were assessed by real-time qPCR using GAPDH as a reference gene. Expression in BM-MNCs was normalized to 1, which denotes no change in mRNA relative expression normalized to GAPDH. Data pooled from 6 to 12 independent biological samples and three technical replicates. *p < 0.05. b Pro-reparative, anti-inflammatory secretome of M2(IL-4) macrophages. Cytokines levels were assessed by ELISA after 24 h of culture. Data pooled from three independent biological samples and two technical replicates. *p < 0.05

Fig. 4. Enhanced post-MI cardiac recovery by M2(IL-4) macrophage transplantation. After induction of MI by left coronary artery ligation in mice, 5 × 105 BM-MNCs or 5 × 105 M2(IL-4) macrophages or PBS alone were administered intramyocardially to the border zone surrounding the infarct. Bar charts presented as mean ± SEM. a Representative echocardiography images at day 28 after treatment. M-mode short axis echocardiography images highlighting changes in anterior wall motion in relation to treatment. b Improved cardiac function and reduced ventricular dimension by M2(IL-4) macrophage transplantation. LVDs (left ventricular dimension at systole), LVDd (left ventricular dimension at diastole) and LVFS (left ventricular fractional shortening) were measured using transthoracic echocardiography (see a). *p < 0.05 vs. both BM-MNC and PBS groups; §p < 0.05 vs. PBS group. N = 9 mice in PBS and BM-MNC groups and N = 11 mice in M2(IL-4) group. c Increased scar thickness and reduced infarct size by M2(IL-4) macrophage transplantation. Hearts were collected at day 28 after treatment and subjected to picrosirius red staining. Representative images of each group are presented. Thickness of infarct left…

Fig. 5. Enhanced donor cell survival after M2(IL-4) macrophage transplantation. Donor cell retention and localization was analyzed 28 days post-transplantation. Bar charts presented as mean ± SEM. a Quantitative donor cell retention. Quantitative cell retention of male BM-MNCs or M2(IL-4) macrophages in the female heart (LV wall excluding the ventricular septum) was carried out by analyzing genomic DNA levels of male-specific Sry gene within the left ventricular myocardium by qPCR. Sry signal was normalized for GAPDH expression and the corresponding number of cells was determined. Data presented as percentage of total transplanted cells. N = 5 hearts in each group; *p < 0.05. b Donor cell localization. Immunohistolabeling for CD206 and DAPI were performed using heart samples collected at day 28 after treatment. Donor cells were pre-labelled with PKH26 before transplantation. Representative images from the infarct area of the M2(IL-4) macrophage transplantation group are presented (lower lane). PKH26-positive donor cells were not detected in the BM-MNC group (upper lane). Scale bars = 50 μm

Fig. 6. Improved myocardial tissue repair after M2(IL-4) macrophage transplantation. The hearts were collected 28 days after treatment and analyzed for myocardial tissue repair by histological assessments. Bar charts presented as mean ± SEM. *p < 0.05 vs. both PBS and BM-MNC groups; N = 4 hearts in PBS and BM-MNC groups and N = 5 hearts in M2(IL-4) group. a Picrosirius red staining for quantification of interstitial fibrosis. Collagen volume fraction was measured in the infarct, infarct border zone and remote myocardium. Scale bars = 20 μm. b Isolectin-B4 (IB4) staining for quantification of microvascular formation in the border area. The number of IB4-positive capillaries was counted and presented as vessels/mm2. Scale bars = 50 μm. c Wheat germ agglutinin (WGA) and α-sarcomeric actinin (α-sarc) staining to measure cardiomyocyte size. The cross-sectional area of cardiomyocytes (α-sarc+, transversely cut, central nuclei) in border and remote areas was measured. At least 40 cardiomyocytes per area were assessed. Scale bars = 20 μm

Fig. 7. Upregulation of tissue repair-related genes in the myocardium after M2(IL-4) macrophage transplantation. Fold change in mRNA levels for myocardial repair-related factors were assessed in the left ventricular myocardium (excluding the ventricular septum) collected at day 28 after treatment using real-time qPCR with GAPDH as a reference gene. Expression in PBS treated hearts was normalized to 1, which denotes no change in mRNA relative expression normalized to GAPDH. Data pooled from five independent biological samples in each group and three technical replicates. Bar charts presented as mean ± SEM. *p < 0.05 vs. both PBS and BM-MNC groups

Fig. 8. Augmented host-derived M2-like macrophages by M2(IL-4) macrophage transplantation. The potential involvement of donor M2(IL-4) macrophages in the endogenous M2-like macrophage polarization process was investigated both in vivo and in vitro. Bar charts presented as mean ± SEM. a Increased M2-like macrophage accumulation after M2(IL-4) macrophage transplantation. Hearts were collected at 28 days after treatment and stained for M2-like (CD206+) macrophages. The number of CD206+ macrophages was calculated in the infarct, pre-infarct border, and remote myocardium. *p < 0.05 vs. both PBS and BM-MNC groups. N = 4 in PBS and BM-MNC groups; N = 5 hearts in M2(IL-4) group. Scale bars = 50 μm. b Role of TGF-β1 in M2-like polarization induced by M2(IL-4) macrophages. M0 macrophages (BM-MNCs treated with M-CSF for 5 days) were subjected to different types of conditioned medium (CM) with or without neutralization antibody to TGF-β for 24 h. The phenotype changes were analyzed by flow cytometry. ctrl ab control antibody, neut ab neutralization antibody. *p < 0.05 vs. M0, BM-MNC CM and M2(IL-4) CM + TGF-β neutralization antibody groups; †p < 0.05 vs. M0 and BM-MNC CM groups; N = 4 for all groups…