Abstract
Clinical trials using adult stem cells to regenerate damaged heart tissue continue to this day1,2, despite ongoing questions of efficacy and a lack of mechanistic understanding of the underlying biological effect3. The rationale for these cell therapy trials is derived from animal studies that show a modest but reproducible improvement in cardiac function in models of cardiac ischaemic injury4,5. Here we examine the mechanistic basis for cell therapy in mice after ischaemia–reperfusion injury, and find that—although heart function is enhanced—it is not associated with the production of new cardiomyocytes. Cell therapy improved heart function through an acute sterile immune response characterized by the temporal and regional induction of CCR2+ and CX3CR1+ macrophages. Intracardiac injection of two distinct types of adult stem cells, cells killed by freezing and thawing or a chemical inducer of the innate immune response all induced a similar regional accumulation of CCR2+ and CX3CR1+ macrophages, and provided functional rejuvenation to the heart after ischaemia–reperfusion injury. This selective macrophage response altered the activity of cardiac fibroblasts, reduced the extracellular matrix content in the border zone and enhanced the mechanical properties of the injured area. The functional benefit of cardiac cell therapy is thus due to an acute inflammatory-based wound-healing response that rejuvenates the infarcted area of the heart.
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Data availability
All raw data generated or analysed in this study are available from the corresponding author upon reasonable request. Original source data used to generate graphs in each of the figures and Extended Data figures are available as Microsoft Excel data sheet files from the corresponding author.
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Acknowledgements
This work was supported by grants from the National Institutes of Health to J.D.M., S.S. and M.N. J.D.M. was supported by the Howard Hughes Medical Institute and the American Heart Association (19MERIT34370048). R.J.V. was supported by a National Research Service Award from the NIH (F32 HL128083) and a Career Development Award from the American Heart Association (19CDA34670044). All flow cytometric data were acquired using equipment maintained by the Research Flow Cytometry Core in the Division of Rheumatology at Cincinnati Children’s Hospital Medical Center.
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J.D.M. and R.J.V. conceived the study. R.J.V., M.M., M.A.S., H.K., A.K.J., J.A.S., A.J.Y. and V.H. performed experiments and generated all the data shown in the manuscript. S.S. provided oversight and technical help along with J.A.S. in measuring myocardial scar mechanical properties. M.N. provided theoretical assessment of the project and advice in experimental design. J.D.M. and R.J.V interpreted the data and wrote the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Characterization of cells used in the injection studies and initial neutrophil response to injections.
a, Flow cytometry analysis of MNCs isolated from Rosa26-mTomato mice for intracardiac injection. Singlet cells were selected by forward and side scatter properties followed by mTomato-positivity. mTomato+ cells were then assessed for surface expression of CD3e, CD11b, Ly6c, CD45R, Ly6G or Ter119 via antibodies. b, Flow cytometry plots for CPCs immunolabelled with antibodies against mesenchymal (Sca1), endothelial (CD31) or haematopoietic (CD45) lineage markers as indicated in the plots. An unstained negative control (unst.) plot is also shown. Gating was determined versus unstained negative controls. Similar results in a, b were obtained from at least three independent cell preparations. c, Quantification via flow cytometry of total neutrophil levels in dissociated hearts from MNC-, zymosan- or saline-injected male and female C57Bl/6J mice, three days after injection. As a comparison, data from n = 5 C57Bl/6J mice isolated one day after I–R injury are also shown when neutrophil levels are high. Data are from n = 4 (MNC and zymosan) or n = 2 (saline) mice. Numerical data are summarized as box-and-whisker plots, indicating the median value (black bar inside box), 25th and 75th percentiles (bottom and top of box, respectively), and minimum and maximum values (bottom and top whisker, respectively).
Extended Data Fig. 2 Basal cardiac structure and function after cell or zymosan injection in uninjured mice.
a, Schematic of all experiments performed in this figure, in which C57Bl/6J eight-week-old male and female mice received intracardiac injection of mTomato-labelled MNCs, Alexa 594-labelled zymosan or sterile saline and were assessed by echocardiography after two weeks. b–f, Echocardiography-measured fractional shortening (FS) percentage (b), heart rate (HR) as beats per minute (bpm) under isoflurane anaesthesia (c), left ventricular posterior wall thickness in diastole (LVPWT;d) in millimetres (d), left ventricular end-systolic volume (LVESV) in microlitres (e) and left ventricular end-diastolic volume (LVEDV) in microlitres (f). All values in b–f were unchanged with injection of MNCs or zymosan versus saline. All numerical data are summarized as box-and-whisker plots, indicating the median value (black bar inside box), 25th and 75th percentiles (bottom and top of box, respectively), and minimum and maximum values (bottom and top whisker, respectively). The number of mice for each group in b–f is indicated below each respective box-and-whisker plot.
Extended Data Fig. 3 Characterization of inflammatory and functional effects of the intracardiac injection protocol.
a, Schematic of experiments performed in b–e. Eight-week-old C57Bl/6J (uninjured) male and female mice received intracardiac injection of sterile saline, or a sham procedure in which the heart was exposed by thoracotomy but no intracardiac injections were done. Naive mice without surgery served as an additional control. b–e, Quantification via flow cytometry of immune cells from enzymatically dissociated hearts from the above groups of mice. Total CD11b+ myeloid cells (b), CD11b+Ly6Chigh (c) or CD11b+Ly6Clow (d) monocytes, and CD11b+F4/80+ macrophages (e) from n = 4 mice per group are shown, normalized to the starting weight of dissociated tissue. *P < 0.05 versus naive and #P < 0.05 versus sham by one-way ANOVA with Tukey’s post hoc test (exact P values are shown in the panel). f, Schematic of experiments performed in g–i in which eight-week-old male and female C57Bl/6J mice received I–R injury followed by either intracardiac injection of saline or thoracotomy (sham) after one week, and then were assessed by echocardiography two weeks later. g–i, Fractional shortening (g), left ventricular end-systolic volume (h) and heart rate under isoflurane anaesthesia (i), as measured by echocardiography in the groups indicated. The number of mice for each group is indicated in each graph in g–i. All numerical data in this figure are summarized as box-and-whisker plots, indicating the median value (black bar inside box), 25th and 75th percentiles (bottom and top of box, respectively), and minimum and maximum values (bottom and top whisker, respectively).
Extended Data Fig. 4 Additional echocardiographic parameters and effect of treatment with MNCs killed by freezing and thawing, after I–R injury.
a–c, Echocardiography to measure left ventricular end-systolic volume in microlitres (a), left ventricular end-diastolic volume in microlitres (b) or heart rate under isoflurane anaesthesia (c) in mice that received an intracardiac injection of MNCs, CPCs, zymosan or sterile saline, three weeks after I–R. These data were collected in the same group of mice shown in Fig. 3b, d. Data in a are significantly different, as assessed by one-way ANOVA with Dunnett’s post hoc test (exact P values are shown in the panels). d, Schematic of experiment in which mice received an intracardiac injection of mTomato+ MNCs or MNCs killed by freezing and thawing one week after I–R, and then two weeks later the cardiac ventricular fractional shortening percentage was measured by echocardiography. Exact P values are shown in the panel versus I–R and saline, which involved one-way ANOVA with Dunnett’s post hoc test to examine significance. The sham, I–R and saline, and I–R and MNC groups shown here in d are the same data as also shown in Fig. 3, as these studies were performed in parallel. All numerical data are summarized as box-and-whisker plots, indicating the median value (black bar inside box), 25th and 75th percentiles (bottom and top of box, respectively), and minimum and maximum values (bottom and top whisker, respectively). The number of mice for each group is indicated below the respective plot.
Extended Data Fig. 5 Characterization of cell types labelled from Ccr2-RFP × Cx3cr1-GFP knock-in mice.
a, Schematic of experiments in which hearts from male and female double-heterozygous Ccr2-RFP × Cx3cr1-GFP knock-in mice (n = 3) were isolated and analysed by flow cytometry at 4 months of age. b, Representative flow cytometry plots with the gating strategy shown, in which singlet cells were first selected by forward and side scatter properties followed by gating on endogenous GFP and RFP fluorescence for CX3CR1+CCR2− (GFP+RFP−) or CX3CR1+CCR2+ (GFP+RFP+) cells. c–f, Cells within the GFP+RFP− or GFP+RFP+ gates as shown in b were then assessed for surface marker expression via antibodies. CD64+CD169+ macrophages (c), CD11b+CD11c+ dendritic cells (d) or CD11b+Ly6G+ neutrophils (e) are shown as a percentage of all GFP+RFP− or GFP+RFP+ cells. f, Ly6c positivity in the total GFP+RFP− or GFP+RFP+ populations is also shown. All data are represented as the mean ± s.e.m. from n = 3 mice.
Extended Data Fig. 6 Response of Cx3cr1 global gene-deleted mice to myocardial infarction injury.
a, Schematic of experiments in which eight-week-old male and female Cx3cr1-GFP heterozygous (het.) or Cx3cr1-GFP/GFP homozygous (knockout, KO) mice received myocardial infarction (MI) via permanent occlusion of the left coronary artery and were then followed out for 12 weeks. b, c, Representative confocal immunohistochemistry images from hearts of mice described in a at one day, three days or four weeks after myocardial infarction, showing endogenous GFP fluorescence from the Cx3cr1 knock-in allele. Immunohistochemistry for activated CD68 macrophages (white) is also shown in b. Micrographs are representative of n = 3 mice per time point. Scale bars, 100 μm. d, Quantification via flow cytometry of total monocyte, neutrophil and macrophage levels in dissociated whole hearts from Cx3cr1 heterozygous or knockout mice at three days after myocardial infarction. Data are from n = 11 (heterozygous) or n = 12 (knockout) mice. Numerical data are summarized as box-and-whisker plots, indicating the median value (black bar inside box), 25th and 75th percentiles (bottom and top of box, respectively), and minimum and maximum values (bottom and top whisker, respectively). e, Survival curve for Cx3cr1-GFP/GFP mice versus controls over 12 weeks (x axis shows days) after myocardial infarction injury. Control mice in this experiment included both Cx3cr1-GFP heterozygous and C57Bl/6J animals. The number (n) of mice for each group is indicated in the figure. *P = 0.0473 by two-sided Gehan–Breslow–Wilcoxon test.
Extended Data Fig. 7 Mechanical and structural improvements in cell-therapy- or zymosan-treated hearts after I–R.
a, Change in passive force generation over increasing stretch lengthening (per cent of L0) in isolated infarct strips from zymosan- or saline-injected hearts, analysed three weeks after surgical injury (injection of zymosan or saline occurred two weeks before collecting from mice). Exact P values are shown in the panel, which were determined by unpaired two-tailed t-test. The I–R and saline, and untreated control, data shown here are the same as in Fig. 4, as these studies were performed in parallel. Numerical data are summarized as box-and-whisker plots, indicating the median value (black bar inside box), 25th and 75th percentiles (bottom and top of box, respectively), and minimum and maximum values (bottom and top whisker, respectively), from the number (n) of mice indicated in the panels. b, Representative confocal micrographs from heart histological sections from Ccr2-RFP × Cx3cr1-GFP mice at ten days after I–R, showing the infarct border zone versus remote myocardium, and labelled with a biotin-conjugated CHP that detects immature or denatured collagen. CHP labelling was detected with a streptavidin-conjugated Alexa 647 secondary antibody (purple). c, Representative confocal micrographs of heart histological sections from the post-I–R border zone of Ccr2-RFP × Cx3cr1-GFP mice that received intracardiac injection of MNCs or zymosan at seven days after I–R, analysed after an additional three days. Endogenous RFP (red) or GFP (green) fluorescence shows CCR2+ or CX3CR1+ macrophages, respectively. Sections were treated with CHP (purple) as in b to visualize immature collagen and areas of active remodelling versus areas in which subtypes of macrophage differentially localize. Images in b, c are representative of n = 2 mice per group. Scale bars, 100 μm.
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Supplementary Tables
This file contains Supplementary Table 1 (Antibodies and Antibody Dilutions Used in this Study) and Supplementary Table 2 (Primers Used for RT-PCR Analysis in this Study).
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Vagnozzi, R.J., Maillet, M., Sargent, M.A. et al. An acute immune response underlies the benefit of cardiac stem cell therapy. Nature 577, 405–409 (2020). https://doi.org/10.1038/s41586-019-1802-2
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DOI: https://doi.org/10.1038/s41586-019-1802-2
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