Intramyocardial injected human umbilical cord-derived mesenchymal stem cells (HucMSCs) contribute to the recovery of cardiac function and the migration of CD4+ T cells into the infarcted heart via CCL5/CCR5 signaling

Fig. 1. The safety evaluation of HucMSCs in vivo. A Representative images of subcutaneous tumor formation between HucMSC and A549 groups on day 60. B The survival curves of HucMSC and A549 groups were observed for 120 days. *p < 0.05 versus A549 group. C Representative H&E immunohistochemical photos of the lung, liver, kidney and spleen on day 28. Scale bar = 100 µm. D The AST, ALT, Cr, LDH, CK, CK-MB levels in serum were measured in different groups on day 7 or 28. ns, no significant difference versus MI-N.S group. MI, myocardiac infarct; N.S, normal saline; HucMSCs, Human umbilical cord-derived mesenchymal stem cells; AST, aspartate transaminase; ALT, alanine aminotransferase; Cr, Creatine; LDH, Lactate dehydrogenase, CK, Creatine kinase; CK-MB, Creatine kinase isoenzyme

Fig. 2. HucMSCs administration improved cardiac function of mice post MI injury. A The flow diagram of the experiment design. The two groups: MI-N.S and MI-HucMSC. After MI injury was established, mice were immediately injected N.S or HucMSCs by three points into the peri-infarcted heart zone. B Representative echocardiographic images (M-mode) in two groups on day 7 following MI surgery. C Statistical results of cardiac function on day 7 between MI-N.S and MI-HucMSC groups (n = 13 per group). D Representative echocardiographic images (M-mode) in two groups on day 28. E Statistical results of cardiac function on day 28 between MI-N.S and MI-HucMSC groups (n = 13 per group) following MI surgery. *p < 0.05 versus MI-N.S group. LVEF left ventricular ejection fraction; LVFS left ventricular fractional shortening; LVEDV/LVESV left ventricular end diastolic/systolic volume; LVEDD/LVESD left ventricular end diastolic/systolic diameter

Fig. 3. Retention of HucMSCs in vivo. A The fluorescence intensity of DiR-labeled HucMSCs were detected in multiple organs including heart, lung, liver, spleen and kidney on day 7 after MI. B Compared with MI-N.S group, Alu-sx was positively expressed in MI-HucMSC group by genomic PCR. Neg con negative control; Pos con positive control; PCR ploymerase chain reaction. C Representative immunofluorescence images of GFP-HucMSC hearts on day 7. GFP green fluorescence protein. D Representative immunofluorescence images of MI-HucMSC hearts stained with human mitochondrion on day 28. Green, human mitochondrion; Blue, DAPI. Scale bar = 100 µm

Fig. 4. HucMSCs treatment contributed to the angiogenesis, attenuated myocardial fibrosis and hypertrophy in hearts after MI. A Representative immunofluorescence images of hearts stained with CD31 between MI-N.S and MI-HucMSC groups on both day 7 and 28. Green, CD31; Blue, DAPI. Scale bar = 100 µm. B Statistical results of the CD31 density (per field) of MI-N.S and MI-HucMSC groups both on day 7 and 28. C The mRNA expression of VEGF-α in infarcted hearts of two groups on day 7. D Representative Masson’s Trichrome-stained histological sections of hearts between MI-N.S and MI-HucMSC groups. Scale bar = 1000 µm. E The fibrosis area of heart tissues was measured using ImageJ software. F The mRNA levels of COL I, COL II, MMP-2 and TIMP2 in infarcted hearts of two groups. G Representative immunofluorescence images of hearts stained with WGA between MI-N.S and MI-HucMSC groups. Green, WGA; Blue, DAPI. Scale bar = 50 µm. H The cross-sectional areas of heart sections were estimated using ImageJ software. I The mRNA levels of ANP, BNP, MYH7 and MYH6 in infarcted hearts of two groups. *p < 0.05, **p < 0.01, ***p < 0.001 versus MI-N.S group. VEGF vascular endothelial growth factor; COL collage; MMP…

Fig. 5. Increase of immune cells in the infarcted heart after HucMSCs treatment on day 7. A Representative immunofluorescence images of hearts stained with human mitochondrion and CD3/CD4 in MI-HucMSC groups. Green, HucMSCs; Red, CD3; Blue, DAPI. Scale bar = 50 µm. B Representative immunofluorescence images of hearts stained with CD3 and CD4 between MI-N.S and MI-HucMSC groups. Green, CD3; Red, CD4; Blue, DAPI. Scale bar = 75 µm. C Representative immunofluorescence images of hearts stained with CD4 and Ki67 between MI-N.S and MI-HucMSC groups. Red, CD4; Green, Ki67; Blue, DAPI. Yellow arrow indicates CD4+Ki67+ double positive cells. Scale bar = 75 µm. D Representative immunofluorescence images of hearts stained with CD4 and FoxP3 between MI-N.S and MI-HucMSC groups. Green, CD4; Red, FoxP3; Blue, DAPI. Yellow arrow indicates CD4+FoxP3+ double positive cells. Scale bar = 50 µm. E Statistical results of percent (%) of CD3+ or CD4+ T cells between MI-N.S and MI-HucMSC groups. F Statistical results of cell counts of CD4+Ki67+ T cells between MI-N.S and MI-HucMSC groups. G Statistical results of cell counts of CD4+FoxP3+ Tregs between MI-N.S and MI-HucMSC groups. H The flow diagram of the…

Fig. 6. HucMSCs promoted CD4+ T cells migration into the injured heart via high levels of CCL5 post MI. A Statistical results of the T cells associated cytokines and chemokines in the heart between MI-N.S and MI-HucMSC groups on day 7. B The radar plots showed differences in cytokines between MI-N.S and MI-HucMSC groups on day 7. C The protein levels of CCL2 and CCL5 in heart tissues of two groups on day 7. *p < 0.05 and ***p < 0.001 versus MI-N.S group. D In vitro, statistical results of migrated CD4+ T cells number via different treatments. ##p < 0.01 versus Control group, &&&p < 0.001 versus MI-N.S group, *p < 0.05 versus MI-HucMSC group

Fig. 7. The CCR5 antagonist inhibited the cardioprotective effect of HucMSCs administration post MI injury. A The flow diagram of the experiment design. The three groups: MI-N.S, MI-HucMSC and MI-HucMSC-CCR5 antagonist. After MI injury was established, mice were immediately injected N.S or HucMSCs by three points into the peri-infarcted heart zone. Then, the MI-HucMSC-CCR5 antagonist group was intraperitoneally injected 20 mg/kg/d CCR5 antagonist. B Representative echocardiographic images (M-mode) in three groups on day 7 following MI surgery. C Statistical results of cardiac function on day 7 among MI-N.S (n = 6), MI-HucMSC (n = 6) and MI-HucMSC-CCR5 antagonist (n = 6) groups. *p < 0.05 versus MI-N.S group; #p < 0.05 versus MI-HucMSC group

Fig. 8. Schematic diagram of the role of HucMSCs in MI. Intramyocardial injection of HucMSCs upregulated the CD4+FoxP3+ Tregs and contributed to the migration of CD4+ T cells into the injured heart via CCL5/CCR5 pathway. MI myocardiac infarct; N.S normal saline; HucMSCs Human umbilical cord-derived mesenchymal stem cells; Tregs regulatory T cells; CCL C–C Motif Chemokine Ligand; CCR C–C Motif Chemokine receptor