Reparative macrophages play an important role in cardiac repair post-myocardial infarction (MI). Bone marrow mononuclear cells (BM-MNCs) have been investigated as a donor for cell therapy but with limited clinical success. These cells, however, may be utilized as a source for reparative macrophages. This translational study aimed to establish a robust in vitro protocol to produce functional reparative macrophages from BM-MNCs and to establish pre-clinical evidence of the efficacy of reparative macrophage transplantation for the treatment of MI. Mouse BM-MNCs were treated with M-CSF plus IL-4, IL-10, TGF-β1 or combinations of these in vitro. The concomitant administration of M-CSF and IL-4 produced the highest rate and largest number of CD11b + F4/80 + CD206 + reparative macrophages. Expression and secretion of tissue repair-related factors including IGF-1, TGF-β1, VEGF and IL1-ra were remarkably enhanced in reparative macrophages compared to BM-MNCs. These cells were transplanted in a mouse MI model, resulting in evident improvement in cardiac function recovery, compared to BM-MNC transplantation. Histological studies showed that reparative macrophage transplantation enhanced myocardial tissue repair including augmented microvascular formation, reduced cardiomyocyte hypertrophy and attenuated interstitial fibrosis. Moreover, survival of reparative macrophages in the heart post-transplantation was increased compared to BM-MNCs. Reparative macrophage transplantation also increased host-derived reparative macrophages in part through TGF-β secretion. In conclusion, concomitant M-CSF + IL-4 treatment effectively produced reparative macrophages from BM-MNCs in vitro. Transplantation of produced reparative macrophage achieved a superior therapeutic efficacy, compared to BM-MNC transplantation, through the enhanced quantity and quality of donor cell engraftment. Further development of this advanced cell-based therapy is warranted. Electronic supplementary material The online version of this article (10.1007/s00395-019-0742-1) contains supplementary material, which is available to authorized users.

Post-operative adhesions are a leading cause of abdominal surgery-associated morbidity. Exposed fibrin clots on the damaged peritoneum, in which the mesothelial barrier is disrupted, readily adhere to surrounding tissues, resulting in adhesion formation. Here we show that resident F4/80HighCD206− peritoneal macrophages promptly accumulate on the lesion and form a ‘macrophage barrier’ to shield fibrin clots in place of the lost mesothelium in mice. Depletion of this macrophage subset or blockage of CD11b impairs the macrophage barrier and exacerbates adhesions. The macrophage barrier is usually insufficient to fully preclude the adhesion formation; however, it could be augmented by IL-4-based treatment or adoptive transfer of this macrophage subset, resulting in robust prevention of adhesions. By contrast, monocyte-derived recruited peritoneal macrophages are not involved in the macrophage barrier. These results highlight a previously unidentified cell barrier function of a specific macrophage subset, also proposing an innovative approach to prevent post-operative adhesions.

Stem cell therapy utilizing bone marrow mononuclear cells (BMC’s) is a potential strategy to treat heart failure patients with improvement in symptom profile and cardiac function. We describe a rationale for concurrent BMC and left ventricular assist device therapy in selected heart failure patients. This combination therapy has demonstrated improved myocardial perfusion and cardiac function in patients with advanced ischemic cardiomyopathy. Moreover, preclinical data support improved cell retention with left ventricular unloading. The beneficial effects of BMC’s are likely through a paracrine mechanism initiating a ‘cardiac-repair’ process. Combination therapy of BMC’s and a left ventricular assist device may exhibit a synergistic effect with improved engraftment of BMC’s through left ventricular unloading.

Mesenchymal stromal cell (MSC) transplantation has been investigated as an advanced treatment of heart failure; however, further improvement of the therapeutic efficacy and mechanistic understanding are needed. Our previous study has reported that epicardial placement of fibrin sealant films incorporating rat amniotic membrane-derived (AM)-MSCs (MSC-dressings) could address limitations of traditional transplantation methods. To progress this finding toward clinical translation, this current study aimed to examine the efficacy of MSC-dressings using human AM-MSCs (hAM-MSCs) and the underpinning mechanism for myocardial repair. Echocardiography demonstrated that cardiac function and structure were improved in a rat ischemic cardiomyopathy model after hAM-MSC-dressing therapy. hAM-MSCs survived well in the rat heart, enhanced myocardial expression of reparative genes, and attenuated adverse remodeling. Copy number analysis by qPCR revealed that upregulated reparative genes originated from endogenous rat cells rather than hAM-MSCs. These results suggest hAM-MSC-dressing therapy stimulates a secondary release of paracrine factors from endogenous cells improving myocardial repair ("secondary paracrine effect"), and cardiac M2-like macrophages were identified as a potential cell source of repair. We demonstrated hAM-MSCs increased M2-like macrophages through not only enhancing M2 polarization but also augmenting their proliferation and migration capabilities via PGE 2 , CCL2, and TGF-b1, resulting in enhanced cardiac function after injury.

Heart failure is a major public health problem, and inflammation is involved in its pathogenesis. Inflammatory Ly6C hi monocytes accumulate in mouse hearts after pressure overload and are detrimental to the heart; however, the types of cells that drive inflammatory cell recruitment remain uncertain. Here, we showed that a distinct subset of mouse cardiac fibroblasts became activated by pressure overload and recruited Ly6C hi monocytes to the heart. Single-cell sequencing analysis revealed that a subset of cardiac fibroblasts highly expressed genes transcriptionally activated by the transcription factor NF-B, as well as C-C motif chemokine ligand 2 (Ccl2) mRNA, which encodes a major factor in Ly6C hi monocyte recruitment. The deletion of the NF-B activator IKK in activated cardiac fibroblasts attenuated Ly6C hi monocyte recruitment and preserved cardiac function in mice subjected to pressure overload. Pseudotime analysis indicated two single-branch trajectories from quiescent fibroblasts into inflammatory fibroblasts and myofibroblasts. Our results provide insight into the mechanisms underlying cardiac inflammation and fibroblast-mediated inflammatory responses that could be therapeutically targeted to treat heart failure.

Background: Reparative (alternatively activated or M2-like) macrophages play an important role in post-myocardial infarction (MI) cardiac repair. Transplantation of bone marrow mononuclear cells (BM-MNCs) is an emerging therapy for MI while its therapeutic efficacy in previous clinical trials is not satisfactory. We hypothesized that induced differentiation/polarisation of BM-MNCs to reparative macrophages before transplantation may enhance the effect of BM-MNC transplantation. Purpose: This study aimed to develop a robust in vitro protocol to produce reparative macrophages from BM-MNCs and to establish the pre-clinical proof of concept data for reparative macrophage transplantation for the treatment of MI. Methods & Results: Mouse BM-MNCs were treated with M-CSF plus IL-4, IL-10, TGF-β1 or combinations of these in vitro . The concomitant M-CSF+IL-4 protocol produced the highest rate and number of CD11b + F4/80 + CD206 + macrophages. Expression and secretion of tissue repair-related factors of the produced cells, including IGF-1, TGF-β1, VEGF and IL1-ra, were more extensive compared to BM-MNCs. Then, reparative macrophages, BM-MNCs or PBS only were intramyocardially injected in a mouse MI model. At 4 weeks after treatment, echocardiography demonstrated that reparative macrophage transplantation markedly improved cardiac function (left ventricular ejection fraction; 57.2±1.6%, n=11) compared to both BM-MNC transplantation (48.4±1.3%, n=9) and control group (44.4±2.0%, n=9). Histological studies showed that infarct size was the smallest after reparative macrophage transplantation in association with the greatest tissue repair in the peri-infarct myocardium, including augmented microvascular formation, reduced cardiomyocyte hypertrophy and reduced pathological interstitial fibrosis. It was also found that reparative macrophage transplantation increased host-derived reparative macrophages through TGF-β1 secretion. Conclusion: M-CSF+IL-4 treatment was effective in producing reparative macrophages from BM-MNCs in vitro . Addition of this pre-treatment improved the therapeutic effect of BM-MNC transplantation. Further pre-clinical and clinical development of this advanced cell therapy is warranted.

Introduction Mesenchymal stromal cell (MSC) transplantation is a promising treatment to promote myocardial repair. Among various sources, the amnion has an advantage in mass production of high-quality MSCs due to its large initial cell-yield and prenatal nature of isolated cells. In addition to the powerful tissue-repair potential, amnion-derived MSCs (AMSCs) exhibit a robust immunomodulative ability, enabling allogeneic transplantation without immunosuppressive reagents. We here report a novel bioengineering technique to deliver AMSCs for myocardial repair by epicardial placement of self-adhesive, bi-layered, AMSC-incorporating dressings (AMSC-dressing), which is fabricable on-site (Figure A). Methods and results AMSC-dressing was fabricated by spreading AMSC suspension on the inner layer of a fibrin sealant film, composed of fibrinogen and thrombin. Due to the resulting adhesive AMSC-fibrin complex, the AMSC-dressing firmly adhered to the heart surface without the need for suture or additional glue. The outer collagen layer of the film facilitated the easy handling and also protected the AMSC-fibrin complex from external damage. We applied a 1 cm2 dressing containing 0, 1, 2 or 4 millions of rat AMSCs to a rat ischemic cardiomyopathy model (4 weeks post coronary artery ligation). Intramyocardial (IM) injection of 4 millions of AMSCs and sham treatment were also conducted. Echocardiography and catheterization consistently demonstrated that AMSC-dressing therapy improved cardiac function and reduced heart dilatation in a dose-dependent manner compared to the sham control. Furthermore, this therapeutic effect exceeded that of IM injection (Figure B). Histological analyses revealed that AMSC-dressing therapy resulted in augmented myocardial tissue repair (increased neovascularization, attenuated pathological fibrosis and reduced cardiomyocyte hypertrophy) compared to IM injection and sham groups. These effects were associated with increased upregulation of a range of tissue repair-related genes including Il10, Cxcl12, Igf1, Timp1, Hif1a, Tgfb, Mmp2, Hgf, Fgf2 and Vegf. Of note, it was elucidated that both initial retention and subsequent survival of donor AMSCs were enhanced by the dressing technique compared to IM injection. In addition, in vitro studies demonstrated that culturing in a fibrin glue not only enhanced upregulation of tissue-repair genes of AMSCs but also improved their survival against environmental stress through activating the Akt/PI3K cell-survival pathway. Conclusion AMSC-dressing therapy enhanced both quantity and quality of donor cell engraftment, leading to the augmented therapeutic efficacy, compared to the current method. Furthermore, this technique is user-friendly and requires no specialized equipment at the treating hospital, highlighting its great potential to be a widely-adopted, standard treatment for heart failure. Further development of this advanced cell therapy towards clinical application is justified. Acknowledgement/Funding British Heart Foundation, Heart Research UK, Japan Agency for Medical Research and Development, Kaneka Corporation

Background Recent research has revealed that reparative (alternatively activated or M2-like) macrophages play an important role in post-myocardial infarction (MI) cardiac repair, proposing that augmentation of these cells will enhance recovery from MI. Transplantation of bone marrow mononuclear cells (BM-MNCs) is an emerging therapy for MI while its therapeutic efficacy in previous clinical trials is not satisfactory. Given that BM-MNCs are a natural source of macrophages, we hypothesized that induced differentiation/polarisation of BM-MNCs to reparative macrophages before transplantation may enhance the effect of BM-MNC transplantation. Purpose This study aimed to develop a robust in vitro protocol to produce reparative macrophages from BM-MNCs and to establish the pre-clinical proof of concept data for reparative macrophage transplantation for the treatment of MI. Methods and results Mouse BM-MNCs were treated with M-CSF plus IL-4, IL-10, TGF-β1 or combinations of these in vitro. The concomitant M-CSF+IL-4 protocol (both 20ng/ml) produced the highest rate (89.7±0.7%) and number (1.7-fold larger than the original cell number) of CD11b+F4/80+CD206+ macrophages. Expression and secretion of tissue repair-related factors of the produced cells, including IGF-1, TGF-β1, VEGF and IL1-ra, were more extensive compared to BM-MNCs. Then, 5x105 BM-MNC-derived reparative macrophages, 5x105 BM-MNCs, or saline only (control) were intramyocardially injected in a mouse MI model based on coronary artery ligation. At 4 weeks after treatment, echocardiography demonstrated that reparative macrophage transplantation markedly improved cardiac function (left ventricular ejection fraction; 57.2±1.6%, n=11) compared to both BM-MNC transplantation (48.4±1.3%, n=9) and control group (44.4±2.0%, n=9). Histological studies showed that infarct size was the smallest after reparative macrophage transplantation in association with the greatest tissue repair in the peri-infarct myocardium, including augmented microvascular formation, reduced cardiomyocyte hypertrophy and reduced pathological interstitial fibrosis. These were corresponded to amplified myocardial upregulation of tissue repair-related genes. Of note, survival of donor reparative macrophages in the heart post-transplantation was >10-fold greater compared to BM-MNCs. It was also found that reparative macrophage transplantation increased host-derived cardiac reparative macrophages. This might be a part of the mechanism by which reparative macrophage transplantation augmented myocardial repair, and our in vitro antibody neutralisation study indicated that TGF-β1 played a role in this donor macrophage-to-host macrophage pathway. Conclusion M-CSF+IL-4 treatment was effective in producing reparative macrophages from BM-MNCs in vitro. Addition of this pre-treatment improved the therapeutic effect of BM-MNC transplantation. Further pre-clinical and clinical development of this advanced cell therapy is warrantied. Acknowledgement/Funding British Heart Foundation (RG/15/3/31236); Heart Research UK (RG2618/12/13 and TRP06/15); St Barts Medical School London