Krijn R. Vrijsen

Cardiomyocyte progenitor cell-derived exosomes stimulate migration of endothelial cells

•  Cell therapy •  The paracrine hypothesis •  Exosomes •  Collection CMPC secreted exosomes •  Conditioned medium and exosomes in in vitro scratch wound assay •  Exosomal signalling via MMP and EMMPRIN •  Discussion

MicroRNA-155 prevents necrotic cell death in human cardiomyocyte progenitor cells via targeting RIP1.

To improve regeneration of the injured myocardium, cardiomyocyte progenitor cells (CMPCs) have been put forward as a potential cell source for transplantation therapy. Although cell transplantation therapy displayed promising results, many issues need to be addressed before fully appreciating their impact. One of the hurdles is poor graft‐cell survival upon injection, thereby limiting potential beneficial effects. Here, we attempt to improve CMPCs survival by increasing microRNA‐155 (miR‐155) levels, potentially to improve engraftment upon transplantation. Using quantitative PCR, we observed a 4‐fold increase of miR‐155 when CMPCs were exposed to hydrogen‐peroxide stimulation. Flow cytometric analysis of cell viability, apoptosis and necrosis showed that necrosis is the main cause of cell death. Overexpressing miR‐155 in CMPCs revealed that miR‐155 attenuated necrotic cell death by 40 ± 2.3%via targeting receptor interacting protein 1 (RIP1). In addition, inhibiting RIP1, either by pre‐incubating the cells with a RIP1 specific inhibitor, Necrostatin‐1 or siRNA mediated knockdown, reduced necrosis by 38 ± 2.5% and 33 ± 1.9%, respectively. Interestingly, analysing gene expression using a PCR‐array showed that increased miR‐155 levels did not change cell survival and apoptotic related gene expression. By targeting RIP1, miR‐155 repressed necrotic cell death of CMPCs, independent of activation of Akt pro‐survival pathway. MiR‐155 provides the opportunity to block necrosis, a conventionally thought non‐regulated process, and might be a potential novel approach to improve cell engraftment for cell therapy.

Exosomes from Cardiomyocyte Progenitor Cells and Mesenchymal Stem Cells Stimulate Angiogenesis Via EMMPRIN

To date, cellular transplantation therapy has not yet fulfilled its high expectations for cardiac repair. A major limiting factor is lack of long-term engraftment of the transplanted cells. Interestingly, transplanted cells can positively affect their environment via secreted paracrine factors, among which are extracellular vesicles, including exosomes: small bi-lipid-layered vesicles containing proteins, mRNAs, and miRNAs. An exosome-based therapy will therefore relay a plethora of effects, without some of the limiting factors of cell therapy. Since cardiomyocyte progenitor cells (CMPC) and mesenchymal stem cells (MSC) induce vessel formation and are frequently investigated for cardiac-related therapies, the pro-angiogenic properties of CMPC and MSC-derived exosome-like vesicles are investigated. Both cell types secrete exosome-like vesicles, which are efficiently taken up by endothelial cells. Endothelial cell migration and vessel formation are stimulated by these exosomes in in vitro models, mediated via ERK/Akt-signaling. Additionally, these exosomes stimulated blood vessel formation into matrigel plugs. Analysis of pro-angiogenic factors revealed high levels of extracellular matrix metalloproteinase inducer (EMMPRIN). Knockdown of EMMPRIN on CMPCs leads to a diminished pro-angiogenic effect, both in vitro and in vivo. Therefore, CMPC and MSC exosomes have powerful pro-angiogenic effects, and this effect is largely mediated via the presence of EMMPRIN on exosomes.

Transendocardial cell injection is not superior to intracoronary infusion in a porcine model of ischaemic cardiomyopathy: a study on delivery efficiency

Stem cell therapy is a new strategy for chronic ischaemic heart disease in patients. However, no consensus exists on the most optimal delivery strategy. This randomized study was designed to assess cell delivery efficiency of three clinically relevant strategies: intracoronary (IC) and transendocardial (TE) using electromechanical mapping guidance (NOGA) compared to surgical delivery in a chronic pig model of ischaemic cardiomyopathy. Twenty‐four animals underwent delivery of 107 autologous Indium‐oxine‐labelled bone marrow‐derived mesenchymal stem cells (MSC) 4 weeks after infarction and were randomized to one of three groups (n = 8 each group): IC, TE or surgical delivery (reference group). Primary endpoint was defined as percentage (%) of injected dose per organ and assessed by in vivo gamma‐emission counting. In addition, troponin and coronary flow were assessed before and after MSC injection. Blinded endpoint analysis showed no significant difference in efficiency after surgical (16 ± 4%), IC (11 ± 1%) and TE (11 ± 3%) (P = 0.52) injections. IC showed less variability in efficiency compared with TE and surgical injection. Overall, TE injection showed less distribution of MSC to visceral organs compared with other modalities. Troponin rise and IC flow did not differ between the percutaneous groups. This randomized study showed no significant difference in cell delivery efficiency to the myocardium in a clinically relevant ischaemic large animal model between IC and TE delivery. In addition, no differences in safety profile were observed. These results are important in view of the choice of percutaneous cell delivery modality in future clinical stem cell trials.

MiR-155 inhibits cell migration of human cardiomyocyte progenitor cells (hCMPCs) via targeting of MMP-16.

Undesired cell migration after targeted cell transplantation potentially limits beneficial effects for cardiac regeneration. MicroRNAs are known to be involved in several cellular processes, including cell migration. Here, we attempt to reduce human cardiomyocyte progenitor cell (hCMPC) migration via increasing microRNA‐155 (miR‐155) levels, and investigate the underlying mechanism. Human cardiomyocyte progenitor cells (hCMPCs) were transfected with pre‐miR‐155, anti‐miR‐155 or control‐miR (ctrl‐miR), followed by scratch‐ and transwell‐ assays. These functional assays displayed that miR‐155 over‐expression efficiently inhibited cell migration by 38 ± 3.6% and 59 ± 3.7% respectively. Conditioned medium from miR‐155 transfected cells was collected and zymography analysis showed a significant decrease in MMP‐2 and MMP‐9 activities. The predicted 3′‐UTR of MMP‐16, an activator of MMP‐2 and ‐9, was cloned into the pMIR‐REPORT vector and luciferase assays were performed. Introduction of miR‐155 significantly reduced luciferase activity which could be abolished by cotransfection with anti‐miR‐155 or target site mutagenesis. By using MMP‐16 siRNA to reduce MMP‐16 levels or by using an MMP‐16 blocking antibody, hCMPC migration could be blocked as well. By directly targeting MMP‐16, miR‐155 efficiently inhibits cell migration via a reduction in MMP‐2 and ‐9 activities. Our study shows that miR‐155 might be used to improve local retention of hCMPCs after intramyocardial delivery.

microRNA-1 enhances the angiogenic differentiation of human cardiomyocyte progenitor cells

Instigated by the discovery of adult cardiac progenitor cells, cell replacement therapy has become a promising option for myocardial repair in the past decade. We have previously shown that human-derived cardiomyocyte progenitor cells (hCMPCs) can differentiate into cardiomyocyte-, endothelial-, and smooth muscle-like cells in vitro, and in vivo after transplantation in a mouse model of myocardial infarction, resulting in preservation of cardiac function. However, to allow successful repopulation of the injured myocardium, it is of key importance to restore myocardial perfusion by the formation of new vasculature. Several studies have shown that microRNAs regulate vascular differentiation of different stem/progenitor cells. Here, we show that miR-1 is upregulated in hCMPCs during angiogenic differentiation. Upregulation of miR-1 enhanced the formation of vascular tubes on Matrigel and within a collagen matrix, and also increased hCMPC motility, as shown by planar and transwell migration assays. By western blot, qRT-PCR and luciferase reporter assays, miR-1 was found to directly target and inhibit the expression of sprouty-related EVH1 domain-containing protein 1 (Spred1). Knocking down Spred1 phenocopies the functional effect seen for miR-1 upregulation. Using a systems biology approach, we found that in hCMPCs, miR-1 is proposed to control a network of genes predominantly involved in angiogenesis-related processes, including the Spred1 pathway. Our data shows that by upregulation of miR-1, the angiogenic differentiation of hCMPCs can be enhanced, which may be used as a new therapeutic approach to improve the efficiency of cell-based therapy for cardiac regeneration by enhancing the formation of new vasculature.

Non-surgical stem cell delivery strategies and in vivo cell tracking to injured myocardium.

Heart failure is a major economic and public health problem. Despite the recent advances in drug therapy and coronary revascularization, the lost cardiomyocytes due to necrosis and apoptosis are not replaced by new myocardial tissue. Cell therapy is an interesting therapeutic option as it potentially improves contractility and restores regional ventricular function. Early clinical data demonstrated that cell transplantation, mainly delivered through non-surgical methods, is safe and feasible. However, several important issues need to be elucidated. This includes, next to determining the best cell type, the optimal delivery strategy, the biodistribution and the survival of implanted stem cells after transplantation. In this view, pre-clinical animal experiments are indispensable. Reporter genes, magnetic or radioactive labeling of stem cells have been developed to observe the fate and the distribution of transplanted cells using non-invasive imaging techniques. Several studies have demonstrated that these direct and non-direct labeling techniques may become an important tool in cell therapy. Integration of cell delivery and cell tracking will probably be a key for the success of cell therapy in patients. This review will provide a comprehensive overview on the various cell tracking and non-surgical cell delivery techniques, which are highly important in view of experimental and clinical studies.

Stem cell therapy for end-stage heart failure: indispensable role for the cell?

Purpose of reviewFor heart failure patients, the urgent need for heart transplantation exceeds the availability of donor hearts. Therefore, cell transplantation has emerged as an interesting and potential solution. This review will focus on the capability of different types of stem cells to regenerate the heart. Moreover, the mechanism for success will be addressed, focusing on the specific (and indispensable?) role of the cells. Recent findingsIn recent years, many types of stem cells have been described as a possible source for cell transplantation in failing hearts, with mixed outcomes. Cell transplantation is hampered by suboptimal delivery techniques, limited survival of cells, and reduced proliferation and differentiation rates in vivo. Interestingly, the number of injected cells that engrafted the heart successfully cannot explain the observed beneficial effects and, therefore, paracrine effects are suggested for the success in cell therapy. SummaryThis review summarizes the current types of stem or progenitor cells used in cardiac cell therapy and beneficial effects on heart function and morphology in preclinical studies. Currently, the observed effects suggest that paracrine effects might be responsible, thereby triggering mobilization and activation of resident (stem) cells, which challenges the classical concept and true regenerative capacity of cell therapy at this point.

Circulating Extracellular Vesicles Contain miRNAs and are Released as Early Biomarkers for Cardiac Injury

Plasma-circulating microRNAs have been implicated as novel early biomarkers for myocardial infarction (MI) due to their high specificity for cardiac injury. For swift clinical translation of this potential biomarker, it is important to understand their temporal and spatial characteristics upon MI. Therefore, we studied the temporal release, potential source, and transportation of circulating miRNAs in different models of ischemia reperfusion (I/R) injury. We demonstrated that extracellular vesicles are released from the ischemic myocardium upon I/R injury. Moreover, we provided evidence that cardiac and muscle-specific miRNAs are transported by extracellular vesicles and are rapidly detectable in plasma. Since these vesicles are enriched for the released miRNAs and their detection precedes traditional damage markers, they hold great potential as specific early biomarkers for MI.

Suppression of T cells by mesenchymal and cardiac progenitor cells is partly mediated via extracellular vesicles

Adverse remodeling after myocardial infarction (MI) is strongly influenced by T cells. Stem cell therapy after MI, using mesenchymal stem cells (MSC) or cardiomyocyte progenitor cells (CMPC), improved cardiac function, despite low cell retention and limited differentiation. As MSC secrete many factors affecting T cell proliferation and function, we hypothesized the immune response could be affected as one of the targets of stem cell therapy. Therefore, we studied the immunosuppressive properties of human BM-MSC and CMPC and their extracellular vesicles (EVs) in co-culture with activated T cells. Proliferation of T cells, measured by carboxyfluorescein succinimidyl ester dilution, was significantly reduced in the presence of BM-MSC and CMPC. The inflammatory cytokine panel of the T cells in co-culture, measured by Luminex assay, changed, with strong downregulation of IFN-gamma and TNF-alpha. The effect on proliferation was observed in both direct cell contact and transwell co-culture systems. Transfer of conditioned medium to unrelated T cells abrogated proliferation in these cells. EVs isolated from the conditioned medium of BM-MSC and CMPC prevented T cell proliferation in a dose-dependent fashion. Progenitor cells presence induces up- and downregulation of multiple previously unreported pathways in T cells. In conclusion, both BM-MSC and CMPC have a strong capacity for in vitro immunosuppression. This effect is mediated by paracrine factors, such as extracellular vesicles. Besides proliferation, many additional pathways are influenced by both BM-MSC and CMPC.