CCR2 macrophage response determines the functional outcome following cardiomyocyte transplantation.

BACKGROUND: The immune response is a crucial factor for mediating the benefit of cardiac cell therapies. Our previous research showed that cardiomyocyte transplantation alters the cardiac immune response and, when combined with short-term pharmacological CCR2 inhibition, resulted in diminished functional benefit. However, the specific role of innate immune cells, especially CCR2 macrophages on the outcome of cardiomyocyte transplantation, is unclear. METHODS: We compared the cellular, molecular, and functional outcome following cardiomyocyte transplantation in wildtype and T cell- and B cell-deficient Rag2del mice. The cardiac inflammatory response was assessed using flow cytometry. Gene expression profile was assessed using single-cell and bulk RNA sequencing. Cardiac function and morphology were determined using magnetic resonance tomography and immunohistochemistry respectively. RESULTS: Compared to wildtype mice, Rag2del mice show an increased innate immune response at steady state and disparate macrophage response after MI. Subsequent single-cell analyses after MI showed differences in macrophage development and a lower prevalence of CCR2 expressing macrophages. Cardiomyocyte transplantation increased NK cells and monocytes, while reducing CCR2-MHC-IIlo macrophages. Consequently, it led to increased mRNA levels of genes involved in extracellular remodelling, poor graft survival, and no functional improvement. Using machine learning-based feature selection, Mfge8 and Ccl7 were identified as the primary targets underlying these effects in the heart. CONCLUSIONS: Our results demonstrate that the improved functional outcome following cardiomyocyte transplantation is dependent on a specific CCR2 macrophage response. This work highlights the need to study the role of the immune response for cardiomyocyte cell therapy for successful clinical translation.

BPIFB4 and its longevity-associated haplotype protect from cardiac ischemia in humans and mice.

Long-living individuals (LLIs) escape age-related cardiovascular complications until the very last stage of life. Previous studies have shown that a Longevity-Associated Variant (LAV) of the BPI Fold Containing Family B Member 4 (BPIFB4) gene correlates with an extraordinarily prolonged life span. Moreover, delivery of the LAV-BPIFB4 gene exerted therapeutic action in murine models of atherosclerosis, limb ischemia, diabetic cardiomyopathy, and aging. We hypothesize that downregulation of BPIFB4 expression marks the severity of coronary artery disease (CAD) in human subjects, and supplementation of the LAV-BPIFB4 protects the heart from ischemia. In an elderly cohort with acute myocardial infarction (MI), patients with three-vessel CAD were characterized by lower levels of the natural logarithm (Ln) of peripheral blood BPIFB4 (p = 0.0077). The inverse association between Ln BPIFB4 and three-vessel CAD was confirmed by logistic regression adjusting for confounders (Odds Ratio = 0.81, p = 0.0054). Moreover, in infarcted mice, a single administration of LAV-BPIFB4 rescued cardiac function and vascularization. In vitro studies showed that LAV-BPIFB4 protein supplementation exerted chronotropic and inotropic actions on induced pluripotent stem cell (iPSC)-derived cardiomyocytes. In addition, LAV-BPIFB4 inhibited the pro-fibrotic phenotype in human cardiac fibroblasts. These findings provide a strong rationale and proof of concept evidence for treating CAD with the longevity BPIFB4 gene/protein.

The Effects of Hypoxic Preconditioned Murine Mesenchymal Stem Cells on Post-Infarct Arrhythmias in the Mouse Model.

Ventricular arrhythmias associated with myocardial infarction (MI) have a significant impact on mortality in patients following heart attack. Therefore, targeted reduction of arrhythmia represents a therapeutic approach for the prevention and treatment of severe events after infarction. Recent research transplanting mesenchymal stem cells (MSC) showed their potential in MI therapy. Our study aimed to investigate the effects of MSC injection on post-infarction arrhythmia. We used our murine double infarction model, which we previously established, to more closely mimic the clinical situation and intramyocardially injected hypoxic pre-conditioned murine MSC to the infarction border. Thereafter, various types of arrhythmias were recorded and analyzed. We observed a homogenous distribution of all types of arrhythmias after the first infarction, without any significant differences between the groups. Yet, MSC therapy after double infarction led to a highly significant reduction in simple and complex arrhythmias. Moreover, RNA-sequencing of samples from stem cell treated mice after re-infarction demonstrated a significant decline in most arrhythmias with reduced inflammatory pathways. Additionally, following stem-cell therapy we found numerous highly expressed genes to be either linked to lowering the risk of heart failure, cardiomyopathy or sudden cardiac death. Moreover, genes known to be associated with arrhythmogenesis and key mutations underlying arrhythmias were downregulated. In summary, our stem-cell therapy led to a reduction in cardiac arrhythmias after MI and showed a downregulation of already established inflammatory pathways. Furthermore, our study reveals gene regulation pathways that have a potentially direct influence on arrhythmogenesis after myocardial infarction.

Monitoring the maturation of the sarcomere network: a super-resolution microscopy-based approach.

The in vitro generation of human cardiomyocytes derived from induced pluripotent stem cells (iPSC) is of great importance for cardiac disease modeling, drug-testing applications and for regenerative medicine. Despite the development of various cultivation strategies, a sufficiently high degree of maturation is still a decisive limiting factor for the successful application of these cardiac cells. The maturation process includes, among others, the proper formation of sarcomere structures, mediating the contraction of cardiomyocytes. To precisely monitor the maturation of the contractile machinery, we have established an imaging-based strategy that allows quantitative evaluation of important parameters, defining the quality of the sarcomere network. iPSC-derived cardiomyocytes were subjected to different culture conditions to improve sarcomere formation, including prolonged cultivation time and micro patterned surfaces. Fluorescent images of α-actinin were acquired using super-resolution microscopy. Subsequently, we determined cell morphology, sarcomere density, filament alignment, z-Disc thickness and sarcomere length of iPSC-derived cardiomyocytes. Cells from adult and neonatal heart tissue served as control. Our image analysis revealed a profound effect on sarcomere content and filament orientation when iPSC-derived cardiomyocytes were cultured on structured, line-shaped surfaces. Similarly, prolonged cultivation time had a beneficial effect on the structural maturation, leading to a more adult-like phenotype. Automatic evaluation of the sarcomere filaments by machine learning validated our data. Moreover, we successfully transferred this approach to skeletal muscle cells, showing an improved sarcomere formation cells over different differentiation periods. Overall, our image-based workflow can be used as a straight-forward tool to quantitatively estimate the structural maturation of contractile cells. As such, it can support the establishment of novel differentiation protocols to enhance sarcomere formation and maturity.

Combined Gemcitabine and Immune-Checkpoint Inhibition Conquers Anti-PD-L1 Resistance in Low-Immunogenic Mismatch Repair-Deficient Tumors.

Tumors arising in the context of Lynch Syndrome or constitutional mismatch repair deficiency are hypermutated and have a good response towards immune-checkpoint inhibitors (ICIs), including α-PD-L1 antibodies. However, in most cases, resistance mechanisms evolve. To improve outcomes and prevent resistance development, combination approaches are warranted. Herein, we applied a combined regimen with an α-PD-L1 antibody and gemcitabine in a preclinical tumor model to activate endogenous antitumor immune responses. Mlh1-/- mice with established gastrointestinal tumors received the α-PD-L1 antibody (clone 6E11; 2.5 mg/kg bw, i.v., q2wx3) and gemcitabine (100 mg/kg bw, i.p., q4wx3) in mono- or combination therapy. Survival and tumor growth were recorded. Immunological changes in the blood were routinely examined via multi-color flow cytometry and complemented by ex vivo frameshift mutation analysis to identify alterations in Mlh1-/--tumor-associated target genes. The combined therapy of α-PD-L1 and gemcitabine prolonged median overall survival of Mlh1-/- mice from four weeks in the untreated control group to 12 weeks, accompanied by therapy-induced tumor growth inhibition, as measured by [18F]-FDG PET/CT. Plasma cytokine levels of IL13, TNFα, and MIP1ß were increased and also higher than in mice receiving either monotherapy. Circulating splenic and intratumoral myeloid-derived suppressor cells (MDSCs), as well as M2 macrophages, were markedly reduced. Besides, residual tumor specimens from combi-treated mice had increased numbers of infiltrating cytotoxic T-cells. Frameshift mutations in APC, Tmem60, and Casc3 were no longer detectable upon treatment, likely because of the successful eradication of single mutated cell clones. By contrast, novel mutations appeared. Collectively, we herein confirm the safe application of combined chemo-immunotherapy by long-term tumor growth control to prevent the development of resistance mechanisms.

The Individual Effects of Cyclin-Dependent Kinase Inhibitors on Head and Neck Cancer Cells-A Systematic Analysis.

Cyclin-dependent kinase inhibitors (CDKi´s) display cytotoxic activity against different malignancies, including head and neck squamous cell carcinomas (HNSCC). By coordinating the DNA damage response, these substances may be combined with cytostatics to enhance cytotoxicity. Here, we investigated the influence of different CDKi´s (palbociclib, dinaciclib, THZ1) on two HNSCC cell lines in monotherapy and combination therapy with clinically-approved drugs (5-FU, Cisplatin, cetuximab). Apoptosis/necrosis, cell cycle, invasiveness, senescence, radiation-induced γ-H2AX DNA double-strand breaks, and effects on the actin filament were studied. Furthermore, the potential to increase tumor immunogenicity was assessed by analyzing Calreticulin translocation and immune relevant surface markers. Finally, an in vivo mouse model was used to analyze the effect of dinaciclib and Cisplatin combination therapy. Dinaciclib, palbociclib, and THZ1 displayed anti-neoplastic activity after low-dose treatment, while the two latter substances slightly enhanced radiosensitivity. Dinaciclib decelerated wound healing, decreased invasiveness, and induced MHC-I, accompanied by high amounts of surface-bound Calreticulin. Numbers of early and late apoptotic cells increased initially (24 h), while necrosis dominated afterward. Antitumoral effects of the selective CDKi palbociclib were weaker, but combinations with 5-FU potentiated effects of the monotherapy. Additionally, CDKi and CDKi/chemotherapy combinations induced MHC I, indicative of enhanced immunogenicity. The in vivo studies revealed a cell line-specific response with best tumor growth control in the combination approach. Global acting CDKi's should be further investigated as targeting agents for HNSCC, either individually or in combination with selected drugs. The ability of dinaciclib to increase the immunogenicity of tumor cells renders this substance a particularly interesting candidate for immune-based oncological treatment regimens.

Continuous Electrical Stimulation Affects Initial Growth and Proliferation of Adipose-Derived Stem Cells.

The aim of the study was to establish electrical stimulation parameters in order to improve cell growth and viability of human adipose-derived stem cells (hADSC) when compared to non-stimulated cells in vitro. hADSC were exposed to continuous electrical stimulation with 1.7 V AC/20 Hz. After 24, 72 h and 7 days, cell number, cellular surface coverage and cell proliferation were assessed. In addition, cell cycle analysis was carried out after 3 and 7 days. After 24 h, no significant alterations were observed for stimulated cells. At day 3, stimulated cells showed a 4.5-fold increase in cell numbers, a 2.7-fold increase in cellular surface coverage and a significantly increased proliferation. Via cell cycle analysis, a significant increase in the G2/M phase was monitored for stimulated cells. Contrastingly, after 7 days, the non-stimulated group exhibited a 11-fold increase in cell numbers and a 4-fold increase in cellular surface coverage as well as a significant increase in cell proliferation. Moreover, the stimulated cells displayed a shift to the G1 and sub-G1 phase, indicating for metabolic arrest and apoptosis initiation. In accordance, continuous electrical stimulation of hADSC led to a significantly increased cell growth and proliferation after 3 days. However, longer stimulation periods such as 7 days caused an opposite result indicating initiation of apoptosis.

Cardiomyocyte Transplantation after Myocardial Infarction Alters the Immune Response in the Heart.

We investigated the influence of syngeneic cardiomyocyte transplantation after myocardial infarction (MI) on the immune response and cardiac function. Methods and Results: We show for the first time that the immune response is altered as a result of syngeneic neonatal cardiomyocyte transplantation after MI leading to improved cardiac pump function as observed by magnetic resonance imaging in C57BL/6J mice. Interestingly, there was no improvement in the capillary density as well as infarct area as observed by CD31 and Sirius Red staining, respectively. Flow cytometric analysis revealed a significantly different response of monocyte-derived macrophages and regulatory T cells after cell transplantation. Interestingly, the inhibition of monocyte infiltration accompanied by cardiomyocyte transplantation diminished the positive effect of cell transplantation alone. The number of CD68+ macrophages in the remote area of the heart observed after four weeks was also different between the groups. Transcriptome analysis showed several changes in the gene expression involving circadian regulation, mitochondrial metabolism and immune responses after cardiomyocyte transplantation. Conclusion: Our work shows that cardiomyocyte transplantation alters the immune response after myocardial infarction with the recruited monocytes playing a role in the beneficial effect of cell transplantation. It also paves the way for further optimization of the efficacy of cardiomyocyte transplantation and their successful translation in the clinic.

Dose-Independent Therapeutic Benefit of Bone Marrow Stem Cell Transplantation after MI in Mice.

Several cell populations derived from bone marrow (BM) have been shown to possess cardiac regenerative potential. Among these are freshly isolated CD133+ hematopoietic as well as culture-expanded mesenchymal stem cells. Alternatively, by purifying CD271+ cells from BM, mesenchymal progenitors can be enriched without an ex vivo cultivation. With regard to the limited available number of freshly isolated BM-derived stem cells, the effect of the dosage on the therapeutic efficiency is of particular interest. Therefore, in the present pre-clinical study, we investigated human BM-derived CD133+ and CD271+ stem cells for their cardiac regenerative potential three weeks post-myocardial infarction (MI) in a dose-dependent manner. The improvement of the hemodynamic function as well as cardiac remodeling showed no therapeutic difference after the transplantation of both 100,000 and 500,000 stem cells. Therefore, beneficial stem cell transplantation post-MI is widely independent of the cell dose and detrimental stem cell amplification in vitro can likely be avoided.

Quantitative Evaluation of the Sarcomere Network of Human hiPSC-Derived Cardiomyocytes Using Single-Molecule Localization Microscopy.

The maturation of iPSC-derived cardiomyocytes is still a critical point for their application in cardiovascular research as well as for their clinical use. Although multiple differentiation protocols have been established, researchers failed to generate fully mature cardiomyocytes in vitro possessing identical phenotype-related and functional properties as their native adult counterparts. Besides electrophysiological and metabolic changes, the establishment of a well structured sarcomere network is important for the development of a mature cardiac phenotype. Here, we present a super resolution-based approach to quantitatively evaluate the structural maturation of iPSC-derived cardiomyocytes. Fluorescence labelling of the α-actinin cytoskeleton and subsequent visualization by photoactivated localization microscopy allows the acquisition of highly resolved images for measuring sarcomere length and z-disc thickness. Our image analysis revealed that iPSC and neonatal cardiomyocyte share high similarity with respect to their sarcomere organization, however, contraction capacity was inferior in iPSC-derived cardiac cells, indicating an early maturation level. Moreover, we demonstrate that this imaging approach can be used as a tool to monitor cardiomyocyte integrity, helping to optimize iPSC differentiation as well as somatic cell direct-reprogramming strategies.

GLP: A requirement in cell therapies - perspectives for the cardiovascular field.

In biomedical research, enormous progress is being made and new candidates for putative medicinal products emerge. However, most published preclinical data are not conducted according to the standard Good Laboratory Practice (GLP). GLP is mandatory for preclinical analysis of Advanced Therapy Medicinal Products (ATMP) and thereby a prerequisite for planning and conduction of clinical trials. Not inconsiderable numbers of clinical trials are terminated earlier or fail - do inadequate testing strategies or missing specialized assays during the preclinical development contribute to this severe complex of problems? Unfortunately, there is also a lack of access to GLP testing results and OECD (Organisation for Economic Co-operation and Development) GLP guidelines are not yet adjusted to ATMP specialties. Ultimately, GLP offers possibilities to generate reliable and reproducible data. Therefore, this review elucidates different GLP aspects in drug development, speculates on reasons of putative low GLP acceptance in the scientific community and mentions solution proposals.

CD271+ Human Mesenchymal Stem Cells Show Antiarrhythmic Effects in a Novel Murine Infarction Model.

BACKGROUND: Ventricular arrhythmias (VA) are a common cause of sudden death after myocardial infarction (MI). Therefore, developing new therapeutic methods for the prevention and treatment of VA is of prime importance. METHODS: Human bone marrow derived CD271+ mesenchymal stem cells (MSC) were tested for their antiarrhythmic effect. This was done through the development of a novel mouse model using an immunocompromised Rag2-/- γc-/- mouse strain subjected to myocardial "infarction-reinfarction". The mice underwent a first ischemia-reperfusion through the left anterior descending (LAD) artery closure for 45 minutes with a subsequent second permanent LAD ligation after seven days from the first infarct. RESULTS: This mouse model induced various types of VA detected with continuous electrocardiogram (ECG) monitoring via implanted telemetry device. The immediate intramyocardial delivery of CD271+ MSC after the first MI significantly reduced VA induced after the second MI. CONCLUSIONS: In addition to the clinical relevance, more closely reflecting patients who suffer from severe ischemic heart disease and related arrhythmias, our new mouse model bearing reinfarction warrants the time required for stem cell engraftment and for the first time enables us to analyze and verify significant antiarrhythmic effects of human CD271+ stem cells in vivo.

Angiogenic Potential of Bone Marrow Derived CD133+ and CD271+ Intramyocardial Stem Cell Trans- Plantation Post MI.

BACKGROUND: Bone marrow (BM)-derived stem cells with their various functions and characteristics have become a well-recognized source for the cell-based therapies. However, knowledge on their therapeutic potential and the shortage for a cross-link between distinct BM-derived stem cells, primed after the onset of myocardial infarction (MI), seems to be still rudimentary. Therefore, the post-examination of the therapeutic characteristics of such primed hematopoietic CD133+ and mesenchymal CD271+ stem cells was the object of the present study. METHODS AND RESULTS: The effects of respective CD133+ and CD271+ mononuclear cells alone as well as in the co-culture model have been explored with focus on their angiogenic potential. The phenotypic analysis revealed a small percentage of isolated cells expressing both surface markers. Moreover, target stem cells isolated with our standardized immunomagnetic isolation procedure did not show any negative alterations following BM storage in regard to cell numbers and/or quality. In vitro network formation relied predominantly on CD271+ stem cells when compared with single CD133+ culture. Interestingly, CD133+ cells contributed in the tube formation, only if they were cultivated in combination with CD271+ cells. Additional to the in vitro examination, therapeutic effects of the primed stem cells were investigated 48 h post MI in a murine model. Hence, we have found a lower expression of transforming growth factor ßeta 3 (TGFß3) as well as an increase of the proangiogenic factors after CD133+ cell treatment in contrast to CD271+ cell treatment. On the other hand, the CD271+ cell therapy led to a lower expression of the inflammatory cytokines. CONCLUSION: The interactions between CD271+ and CD133+ subpopulations the extent to which the combination may enhance cardiac regeneration has still not been investigated so far. We expect that the multiple characteristics and various regenerative effects of CD271+ cells alone as well as in combination with CD133+ will result in an improved therapeutic impact on ischemic heart disease.

Intramyocardial angiogenetic stem cells and epicardial erythropoietin save the acute ischemic heart.

Ischemic heart failure is the leading cause of mortality worldwide. An early boost of intracardiac regenerative key mechanisms and angiogenetic niche signaling in cardiac mesenchymal stem cells (MSCs) could improve myocardial infarction (MI) healing. Epicardial erythropoietin (EPO; 300 U kg-1) was compared with intraperitoneal and intramyocardial EPO treatments after acute MI in rats (n=156). Real-time PCR and confocal microscopy revealed that epicardial EPO treatment enhanced levels of intracardiac regenerative key indicators (SDF-1, CXCR4, CD34, Bcl-2, cyclin D1, Cdc2 and MMP2), induced transforming growth factor ß (TGF-ß)/WNT signaling in intramyocardial MSC niches through the direct activation of AKT and upregulation of upstream signals FOS and Fzd7, and augmented intracardiac mesenchymal proliferation 24 h after MI. Cardiac catheterization and tissue analysis showed superior cardiac functions, beneficial remodeling and increased capillary density 6 weeks after MI. Concomitant fluorescence-activated cell sorting, co-cultures with neonatal cardiomyocytes, angiogenesis assays, ELISA, western blotting and RAMAN spectroscopy demonstrated that EPO could promote cardiomyogenic differentiation that was specific of tissue origin and enhance paracrine angiogenetic activity in cardiac CD45-CD44+DDR2+ MSCs. Epicardial EPO delivery might be the optimal route for efficient upregulation of regenerative key signals after acute MI. Early EPO-mediated stimulation of mesenchymal proliferation, synergistic angiogenesis with cardiac MSCs and direct induction of TGF-ß/WNT signaling in intramyocardial cardiac MSCs could initiate an accelerated healing process that enhances cardiac recovery.

Vimentin-Induced Cardiac Mesenchymal Stem Cells Proliferate in the Acute Ischemic Myocardium.

In-depth knowledge of the mechanisms induced by early postischemic cardiac endogenous mesenchymal stem cells (MSCs) in the acutely ischemic heart could advance our understanding of cardiac regeneration. Herein, we aimed to identify, isolate, and initially characterize the origin, kinetics and fate of cardiac MSCs. This was facilitated by in vivo genetic cell fate mapping through green fluorescent protein (GFP) expression under the control of vimentin induction after acute myocardial infarction (MI). Following permanent ligation of the left anterior descending coronary artery in CreER+ mTom/mGFP+ mice, vimentin/GFP+ cells revealed ischemia-responsive activation, survival, and local enrichment inside the peri-infarction border zone. Fluorescence-activated cell sorting (FACS)-isolated vimentin/GFP+ cells could be strongly expanded in vitro with clonogenic precursor formation and revealed MSC-typical cell morphology. Flow-cytometric analyses demonstrated an increase in cardiac vimentin/GFP+ cells in the ischemic heart, from a 0.6% cardiac mononuclear cell (MNC) fraction at 24 h to 1.6% at 72 h following MI. Sca-1+CD45- cells within the vimentin/GFP+ subtype of this MNC fraction increased from 35.2% at 24 h to 74.6% at 72 h after MI. The cardiac postischemic vimentin/GFP+ MNC subtype showed multipotent adipogenic, chondrogenic, and osteogenic differentiation potential, which is distinctive for MSCs. In conclusion, we demonstrated a seemingly proliferative first response of vimentin- induced cardiac endogenous MSCs in the acutely ischemic heart. Genetically, GFP-targeted in vivo cell tracking, isolation, and in vitro expansion of this cardiac MSC subtype could help to clarify their reparative status in inflammation, fibrogenesis, cell turnover, tissue homeostasis, and myocardial regeneration.

Cardiac Mesenchymal Stem Cells Proliferate Early in the Ischemic Heart.

BACKGROUND/PURPOSE: Cardiac mesenchymal stem cells (MSCs) could stimulate cell-specific regenerative mechanisms after myocardial infarction (MI) depending on spatial origin, distribution, and niche regulation. We aimed at identifying and isolating tissue-specific cardiac MSCs that could contribute to regeneration. METHODS: Following permanent ligation of the left anterior descending coronary artery in rats (n = 16), early cardiac tissues and cardiac mononuclear cells (MNCs) were analyzed by immunohistology, confocal laser scanning microscopy, and flow cytometry, respectively. Early postischemic specific MSCs were purified by fluorescence-activated cell sorting, cultivated under standardized culture conditions, and tested for multipotent differentiation in functional identification kits. RESULTS: Cardiac MSC niches were detected intramyocardially in cell clusters after MI and characterized by positive expression for vimentin, CD29, CD44, CD90, CD105, PDGFRα, and DDR2. Following myocardial ischemia, proliferation was induced early and proliferation density was approximately 11% in intramyocardial MSC clusters of the peri-infarction border zone. Cluster sizes increased by 157 and 64% in the peri-infarction and noninfarcted areas of infarcted hearts compared with noninfarcted hearts 24 h following MI, respectively. Coincidentally, flow cytometry analyses illustrated postischemic moderate enrichments of CD45-CD44+ and CD45-DDR2+ cardiac MNCs. We enabled isolation of early postischemic culturable cardiac CD45-CD44+DDR2+ MSCs that demonstrated typical clonogenicity with colony-forming unit-fibroblast formation as well as adipogenic, chondrogenic, and osteogenic differentiation. CONCLUSIONS: MI triggered early proliferation in specific cardiac MSC niches that were organized in intramyocardial clusters. Following targeted isolation, early postischemic cardiac CD45-CD44+DDR2+ MSCs exhibited typical characteristics with multipotent differentiation capacity and clonogenic expansion.

Mechanisms of stem cell based cardiac repair-gap junctional signaling promotes the cardiac lineage specification of mesenchymal stem cells.

Different subtypes of bone marrow-derived stem cells are characterized by varying functionality and activity after transplantation into the infarcted heart. Improvement of stem cell therapeutics requires deep knowledge about the mechanisms that mediate the benefits of stem cell treatment. Here, we demonstrated that co-transplantation of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) led to enhanced synergistic effects on cardiac remodeling. While HSCs were associated with blood vessel formation, MSCs were found to possess transdifferentiation capacity. This cardiomyogenic plasticity of MSCs was strongly promoted by a gap junction-dependent crosstalk between myocytes and stem cells. The inhibition of cell-cell coupling significantly reduced the expression of the cardiac specific transcription factors NKX2.5 and GATA4. Interestingly, we observed that small non-coding RNAs are exchanged between MSCs and cardiomyocytes in a GJ-dependent manner that might contribute to the transdifferentiation process of MSCs within a cardiac environment. Our results suggest that the predominant mechanism of HSCs contribution to cardiac regeneration is based on their ability to regulate angiogenesis. In contrast, transplanted MSCs have the capability for intercellular communication with surrounding cardiomyocytes, which triggers the intrinsic program of cardiogenic lineage specification of MSCs by providing cardiomyocyte-derived cues.

Cardiac Function Improvement and Bone Marrow Response -: Outcome Analysis of the Randomized PERFECT Phase III Clinical Trial of Intramyocardial CD133+ Application After Myocardial Infarction.

OBJECTIVE: The phase III clinical trial PERFECT was designed to assess clinical safety and efficacy of intramyocardial CD133+ bone marrow stem cell treatment combined with CABG for induction of cardiac repair. DESIGN: Multicentre, double-blinded, randomised placebo controlled trial. SETTING: The study was conducted across six centres in Germany October 2009 through March 2016 and stopped due slow recruitment after positive interim analysis in March 2015. PARTICIPANTS: Post-infarction patients with chronic ischemia and reduced LVEF (25-50%). INTERVENTIONS: Eighty-two patients were randomised to two groups receiving intramyocardial application of 5ml placebo or a suspension of 0.5-5×106 CD133+. OUTCOME: Primary endpoint was delta (∆) LVEF at 180days (d) compared to baseline measured in MRI. FINDINGS (PRESPECIFIED): Safety (n=77): 180d survival was 100%, MACE n=2, SAE n=49, without difference between placebo and CD133+. Efficacy (n=58): The LVEF improved from baseline LVEF 33.5% by +9.6% at 180d, p=0.001 (n=58). Treatment groups were not different in ∆LVEF (ANCOVA: Placebo +8.8% vs. CD133+ +10.4%, ∆CD133+vs placebo +2.6%, p=0.4). FINDINGS (POST HOC): Responders (R) classified by ∆LVEF≥5% after 180d were 60% of the patients (35/58) in both treatment groups. ∆LVEF in ANCOVA was +17.1% in (R) vs. non-responders (NR) (∆LVEF 0%, n=23). NR were characterized by a preoperative response signature in peripheral blood with reduced CD133+ EPC (RvsNR: p=0.005) and thrombocytes (p=0.004) in contrast to increased Erythropoeitin (p=0.02), and SH2B3 mRNA expression (p=0.073). Actuarial computed mean survival time was 76.9±3.32months (R) vs. +72.3±5.0months (NR), HR 0.3 [Cl 0.07-1.2]; p=0.067.Using a machine learning 20 biomarker response parameters were identified allowing preoperative discrimination with an accuracy of 80% (R) and 84% (NR) after 10-fold cross-validation. INTERPRETATION: The PERFECT trial analysis demonstrates that the regulation of induced cardiac repair is linked to the circulating pool of CD133+ EPC and thrombocytes, associated with SH2B3 gene expression. Based on these findings, responders to cardiac functional improvement may be identified by a peripheral blood biomarker signature. TRIAL REGISTRATION: ClinicalTrials.govNCT00950274.

GMP-conformant on-site manufacturing of a CD133+ stem cell product for cardiovascular regeneration.

BACKGROUND: CD133+ stem cells represent a promising subpopulation for innovative cell-based therapies in cardiovascular regeneration. Several clinical trials have shown remarkable beneficial effects following their intramyocardial transplantation. Yet, the purification of CD133+ stem cells is typically performed in centralized clean room facilities using semi-automatic manufacturing processes based on magnetic cell sorting (MACS®). However, this requires time-consuming and cost-intensive logistics. METHODS: CD133+ stem cells were purified from patient-derived sternal bone marrow using the recently developed automatic CliniMACS Prodigy® BM-133 System (Prodigy). The entire manufacturing process, as well as the subsequent quality control of the final cell product (CP), were realized on-site and in compliance with EU guidelines for Good Manufacturing Practice. The biological activity of automatically isolated CD133+ cells was evaluated and compared to manually isolated CD133+ cells via functional assays as well as immunofluorescence microscopy. In addition, the regenerative potential of purified stem cells was assessed 3 weeks after transplantation in immunodeficient mice which had been subjected to experimental myocardial infarction. RESULTS: We established for the first time an on-site manufacturing procedure for stem CPs intended for the treatment of ischemic heart diseases using an automatized system. On average, 0.88 × 106 viable CD133+ cells with a mean log10 depletion of 3.23 ± 0.19 of non-target cells were isolated. Furthermore, we demonstrated that these automatically isolated cells bear proliferation and differentiation capacities comparable to manually isolated cells in vitro. Moreover, the automatically generated CP shows equal cardiac regeneration potential in vivo. CONCLUSIONS: Our results indicate that the Prodigy is a powerful system for automatic manufacturing of a CD133+ CP within few hours. Compared to conventional manufacturing processes, future clinical application of this system offers multiple benefits including stable CP quality and on-site purification under reduced clean room requirements. This will allow saving of time, reduced logistics and diminished costs.

Applying 3D-FRAP microscopy to analyse gap junction-dependent shuttling of small antisense RNAs between cardiomyocytes.

Small antisense RNAs like miRNA and siRNA are of crucial importance in cardiac physiology, pathology and, moreover, can be applied as therapeutic agents for the treatment of cardiovascular diseases. Identification of novel strategies for miRNA/siRNA therapy requires a comprehensive understanding of the underlying mechanisms. Emerging data suggest that small RNAs are transferred between cells via gap junctions and provoke gene regulatory effects in the recipient cell. To elucidate the role of miRNA/siRNA as signalling molecules, suitable tools are required that will allow the analysis of these small RNAs at the cellular level. In the present study, we applied 3 dimensional fluorescence recovery after photo bleaching microscopy (3D-FRAP) to visualise and quantify the gap junctional exchange of small RNAs between neonatal cardiomyocytes in real time. Cardiomyocytes were transfected with labelled miRNA and subjected to FRAP microscopy. Interestingly, we observed recovery rates of 21% already after 13min, indicating strong intercellular shuttling of miRNA, which was significantly reduced when connexin43 was knocked down. Flow cytometry analysis confirmed our FRAP results. Furthermore, using an EGFP/siRNA reporter construct we demonstrated that the intercellular transfer does not affect proper functioning of small RNAs, leading to marker gene silencing in the recipient cell. Our results show that 3D-FRAP microscopy is a straightforward, non-invasive live cell imaging technique to evaluate the GJ-dependent shuttling of small RNAs with high spatio-temporal resolution. Moreover, the data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation where small RNAs act as signalling molecules within the intercellular network.