Jan Willem Buikema

Cardiac Stem Cell Treatment in Myocardial Infarction: A Systematic Review and Meta-Analysis of Preclinical Studies

RATIONALE Cardiac stem cells (CSC) therapy has been clinically introduced for cardiac repair after myocardial infarction (MI). To date, there has been no systematic overview and meta-analysis of studies using CSC therapy for MI. OBJECTIVE Here, we used meta-analysis to establish the overall effect of CSCs in preclinical studies and assessed translational differences between and within large and small animals in the CSC therapy field. In addition, we explored the effect of CSC type and other clinically relevant parameters on functional outcome to better predict and design future (pre)clinical studies using CSCs for MI. METHODS AND RESULTS A systematic search was performed, yielding 80 studies. We determined the overall effect of CSC therapy on left ventricular ejection fraction and performed meta-regression to investigate clinically relevant parameters. We also assessed the quality of included studies and possible bias. The overall effect observed in CSC-treated animals was 10.7% (95% confidence interval 9.4-12.1; P<0.001) improvement in ejection fraction compared with placebo controls. Interestingly, CSC therapy had a greater effect in small animals compared with large animals (P<0.001). Meta-regression indicated that cell type was a significant predictor for ejection fraction improvement in small animals. Minor publication bias was observed in small animal studies. CONCLUSIONS CSC treatment resulted in significant improvement of ejection fraction in preclinical animal models of MI compared with placebo. There was a reduction in the magnitude of effect in large compared with small animal models. Although different CSC types have overlapping culture characteristics, we observed a significant difference in their effect in post-MI animal studies.Rationale: Cardiac stem cells (CSC) therapy has been clinically introduced for cardiac repair after myocardial infarction (MI). To date, there has been no systematic overview and meta-analysis of studies using CSC therapy for MI. Objective: Here, we used meta-analysis to establish the overall effect of CSCs in preclinical studies and assessed translational differences between and within large and small animals in the CSC therapy field. In addition, we explored the effect of CSC type and other clinically relevant parameters on functional outcome to better predict and design future (pre)clinical studies using CSCs for MI. Methods and Results: A systematic search was performed, yielding 80 studies. We determined the overall effect of CSC therapy on left ventricular ejection fraction and performed meta-regression to investigate clinically relevant parameters. We also assessed the quality of included studies and possible bias. The overall effect observed in CSC-treated animals was 10.7% (95% confidence interval 9.4–12.1; P <0.001) improvement in ejection fraction compared with placebo controls. Interestingly, CSC therapy had a greater effect in small animals compared with large animals ( P <0.001). Meta-regression indicated that cell type was a significant predictor for ejection fraction improvement in small animals. Minor publication bias was observed in small animal studies. Conclusions: CSC treatment resulted in significant improvement of ejection fraction in preclinical animal models of MI compared with placebo. There was a reduction in the magnitude of effect in large compared with small animal models. Although different CSC types have overlapping culture characteristics, we observed a significant difference in their effect in post-MI animal studies. # Novelty and Significance {#article-title-50}

Wnt/β-catenin signaling directs the regional expansion of first and second heart field-derived ventricular cardiomyocytes

In mammals, cardiac development proceeds from the formation of the linear heart tube, through complex looping and septation, all the while increasing in mass to provide the oxygen delivery demands of embryonic growth. The developing heart must orchestrate regional differences in cardiomyocyte proliferation to control cardiac morphogenesis. During ventricular wall formation, the compact myocardium proliferates more vigorously than the trabecular myocardium, but the mechanisms controlling such regional differences among cardiomyocyte populations are not understood. Control of definitive cardiomyocyte proliferation is of great importance for application to regenerative cell-based therapies. We have used murine and human pluripotent stem cell systems to demonstrate that, during in vitro cellular differentiation, early ventricular cardiac myocytes display a robust proliferative response to β-catenin-mediated signaling and conversely accelerate differentiation in response to inhibition of this pathway. Using gain- and loss-of-function murine genetic models, we show that β-catenin controls ventricular myocyte proliferation during development and the perinatal period. We further demonstrate that the differential activation of the Wnt/β-catenin signaling pathway accounts for the observed differences in the proliferation rates of the compact versus the trabecular myocardium during normal cardiac development. Collectively, these results provide a mechanistic explanation for the differences in localized proliferation rates of cardiac myocytes and point to a practical method for the generation of the large numbers of stem cell-derived cardiac myocytes necessary for clinical applications.

Concise Review: Engineering Myocardial Tissue: The Convergence of Stem Cells Biology and Tissue Engineering Technology

Advanced heart failure represents a leading public health problem in the developed world. The clinical syndrome results from the loss of viable and/or fully functional myocardial tissue. Designing new approaches to augment the number of functioning human cardiac muscle cells in the failing heart serve as the foundation of modern regenerative cardiovascular medicine. A number of clinical trials have been performed in an attempt to increase the number of functional myocardial cells by the transplantation of a diverse group of stem or progenitor cells. Although there are some encouraging suggestions of a small early therapeutic benefit, to date, no evidence for robust cell or tissue engraftment has been shown, emphasizing the need for new approaches. Clinically meaningful cardiac regeneration requires the identification of the optimum cardiogenic cell types and their assembly into mature myocardial tissue that is functionally and electrically coupled to the native myocardium. We here review recent advances in stem cell biology and tissue engineering and describe how the convergence of these two fields may yield novel approaches for cardiac regeneration. Stem Cells 2013;31:2587–2598

Engineering Myocardial Tissue: The Convergence of Stem Cells Biology and Tissue Engineering Technology

Advanced heart failure represents a leading public health problem in the developed world. The clinical syndrome results from the loss of viable and/or fully functional myocardial tissue. Designing new approaches to augment the number of functioning human cardiac muscle cells in the failing heart serve as the foundation of modern regenerative cardiovascular medicine. A number of clinical trials have been performed in an attempt to increase the number of functional myocardial cells by the transplantation of a diverse group of stem or progenitor cells. Although there are some encouraging suggestions of a small early therapeutic benefit, to date, no evidence for robust cell or tissue engraftment has been shown, emphasizing the need for new approaches. Clinically meaningful cardiac regeneration requires the identification of the optimum cardiogenic cell types and their assembly into mature myocardial tissue that is functionally and electrically coupled to the native myocardium. We here review recent advances in stem cell biology and tissue engineering and describe how the convergence of these two fields may yield novel approaches for cardiac regeneration. Stem Cells 2013;31:2587–2598

Stem cells in heart failure

Heart failure (HF) is a leading public health problem resulting in an increased risk of cardiovascular complications and mortality. Despite advances in medical therapy, the prevalence of HF is still increasing. The Framingham study demonstrated that the life time risk for developing HF is one in five for both men and women. Evidence-based treatment of chronic HF is mainly focused on suppressing the chronic neurohormonal activation, which may prevent the deterioration of heart function. However, despite many therapeutic strategies patients diagnosed with HF have a poor prognosis. Conventional medical strategies for the treatment of HF due to myocardial infarction (MI) do not attempt to correct the underlying cause (i.e. loss of viable or functional myocardial tissue), raising a need for strategies aimed at myocardial regeneration and repair. Therefore novel approaches to increase the number of functioning cardiac muscle cells in the failing heart might open new avenues for the treatment of patients with advanced HF. Over the past few years, several thousand patients have been treated with various forms of bone marrow stem cell therapy during acute MI. On the basis of early animals studies, cell-based therapy clinical trials were quickly initiated, with the hope that injected or circulating haematopoietic stem cells would lead to cardiac regeneration. Indeed, several studies have reported a modest but statistically significant improvement in left ventricular (LV) ejection fraction in patients’ post-MI. In a post hoc analysis of the REPAIR-AMI trial, for example, it was suggested that bone marrow stem cell therapy was most effective in patients with markedly depressed LV function, which may indicate that the possible beneficial effects of bone marrow cell therapy may be restricted to a specific group of patients. Similar results were observed in the HEBE trial, which showed overall no benefit of bone marrow stem cells after MI. However, in a sub-population of patients with a dilated LV at baseline, bone marrow stem cell therapy prevented further dilation compared with controls, which is in concert with other observations. A recent meta-analysis which included 811 participants with acute MI from 13 trials showed a 3% improvement in LV ejection fraction in the experimental group. Thus, intracoronary infusion of bone marrow derived stem cells appears to result in a modest but reproducible short-term improvement in LV systolic function in the setting of acute MI. In the current issue of the journal, Strauer et al. extend these findings to the chronic HF population. In an open label, nonrandomized, prospective study, 391 patients with an ischaemic aetiology of HF were included, of which 191 patients received an intracoronary bone marrow cell infusion. Post-treatment the authors reported an improvement in LV performance and a longterm decrease in mortality of patients treated with stem cells. These results suggest that bone marrow cells may have a positive effect on cardiac remodelling and improving function in patients with chronic HF. Although the study is the largest in its kind, it needs to be emphasized that the study was open label and was not randomized. Patients who refused to undergo bone marrow cell infusion were included in the control group. This may well lead to a significant bias and limits the value of the current trial. In addition, at baseline the intervention group had a 7% lower LV ejection fraction compared with the control group. Despite these reported improvements in LV systolic function, uncertainties remain. For example the preferred route of administration is still subject to debate. Furthermore, the mechanism, by which stem cells exert their potentially beneficial effects remain poorly understood. In the light of several experimental studies, there is little evidence to support the onset of cardiac muscle regeneration following bone marrow therapy. As a result, it has been proposed that the bone marrow derived stem cells may exert a paracrine effect to improve angiogenesis. This is supported by a study in a large animal model of acute MI that showed injection of bone marrow derived stem cells increased capillary density and improved collateral perfusion and regional cardiac function. Furthermore, it has been previously shown

Expanding mouse ventricular cardiomyocytes through GSK-3 inhibition.

Controlled proliferation of cardiomyocytes remains a major limitation in cell biology and one of the main underlying hurdles for true modern regenerative medicine. Here, a technique is described for robust expansion of early fetal‐derived mouse ventricular cardiomyocytes on a platform usable for high‐throughput molecular screening, tissue engineering and, potentially, in vivo translational experiments. This method provides a small‐molecule approach to control proliferation or differentiation of early beating cardiomyocytes through modulation of the Wnt/β‐catenin signaling pathway. Moreover, isolation and expansion of fetal cardiomyocytes takes less than 3 weeks, yields a relatively pure (∼70%) functional myogenic population, and is highly reproducible. Curr. Protoc. Cell Biol. 61:23.9.1‐23.9.10.

Untangling the Biology of Genetic Cardiomyopathies with Pluripotent Stem Cell Disease Models

Purpose of ReviewRecently, the discovery of strategies to reprogram somatic cells into induced pluripotent stem (iPS) cells has led to a major paradigm change in developmental and stem cell biology. The application of iPS cells and their cardiac progeny has opened novel directions to study cardiomyopathies at a cellular and molecular level. This review discusses approaches currently undertaken to unravel known inherited cardiomyopathies in a dish.Recent FindingsWith improved efficiency for mutation correction by genome editing, human iPS cells have now provided a platform to untangle the biology of cardiomyopathies. Multiple studies have derived pluripotent stem cells lines from patients with genetic heart diseases. The generation of cardiomyocytes from these cells lines has, for the first time, enable the study of cardiomyopathies using cardiomyocytes harboring patient-specific mutations and their corrected isogenic counterpart. The molecular analyses, functional assays, and drug tests of these lines have led to new molecular insights in the early pathophysiology of left ventricular non-compaction cardiomyopathy (LVNC), hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), arrhythmogenic right ventricular cardiomyopathy (ARVC), and others.SummaryThe advent of iPS cells offers an exceptional opportunity for creating disease-specific cellular models to investigate their underlying mechanisms and to optimize future therapy through drug and toxicity screening. Thus far, the iPS cell model has improved our understanding of the genetic and molecular pathophysiology of patients with various genetic cardiomyopathies. It is hoped that the new discoveries arising from using these novel platforms for cardiomyopathy research will lead to new diagnostic and therapeutic approaches to prevent and treat these diseases.

Nkx2.5+ Cardiomyoblasts Contribute to Cardiomyogenesis in the Neonatal Heart

During normal lifespan, the mammalian heart undergoes limited renewal of cardiomyocytes. While the exact mechanism for this renewal remains unclear, two possibilities have been proposed: differentiated myocyte replication and progenitor/immature cell differentiation. This study aimed to characterize a population of cardiomyocyte precursors in the neonatal heart and to determine their requirement for cardiac development. By tracking the expression of an embryonic Nkx2.5 cardiac enhancer, we identified cardiomyoblasts capable of differentiation into striated cardiomyocytes in vitro. Genome-wide expression profile of neonatal Nkx2.5+ cardiomyoblasts showed the absence of sarcomeric gene and the presence of cardiac transcription factors. To determine the lineage contribution of the Nkx2.5+ cardiomyoblasts, we generated a doxycycline suppressible Cre transgenic mouse under the regulation of the Nkx2.5 enhancer and showed that neonatal Nkx2.5+ cardiomyoblasts mature into cardiomyocytes in vivo. Ablation of neonatal cardiomyoblasts resulted in ventricular hypertrophy and dilation, supporting a functional requirement of the Nkx2.5+ cardiomyoblasts. This study provides direct lineage tracing evidence that a cardiomyoblast population contributes to cardiogenesis in the neonatal heart. The cell population identified here may serve as a promising therapeutic for pediatric cardiac regeneration.

Stage-specific Effects of Bioactive Lipids on Human iPSC Cardiac Differentiation and Cardiomyocyte Proliferation

Bioactive lipids such as sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) regulate diverse processes including cell proliferation, differentiation, and migration. However, their roles in cardiac differentiation and cardiomyocyte proliferation have not been explored. Using a 96-well differentiation platform for generating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) we found that S1P and LPA can independently enhance cardiomyocyte generation when administered at an early stage of differentiation. We showed that the combined S1P and LPA treatment of undifferentiated hiPSCs resulted in increased nuclear accumulation of β-catenin, the canonical Wnt signaling pathway mediator, and synergized with CHIR99021, a glycogen synthase kinase 3 beta inhibitor, to enhance mesodermal induction and subsequent cardiac differentiation. At later stages of cardiac differentiation, the addition of S1P and LPA resulted in cell cycle initiation in hiPSC-CMs, an effect mediated through increased ERK signaling. Although the addition of S1P and LPA alone was insufficient to induce cell division, it was able to enhance β-catenin-mediated hiPSC-CM proliferation. In summary, we demonstrated a developmental stage-specific effect of bioactive lipids to enhance hiPSC-CM differentiation and proliferation via modulating the effect of canonical Wnt/β-catenin and ERK signaling. These findings may improve hiPSC-CM generation for cardiac disease modeling, precision medicine, and regenerative therapies.

Large-Scale Single-Cell RNA-Seq Reveals Molecular Signatures of Heterogeneous Populations of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells

Rationale: Human-induced pluripotent stem cell–derived endothelial cells (iPSC-ECs) have risen as a useful tool in cardiovascular research, offering a wide gamut of translational and clinical applications. However, inefficiency of the currently available iPSC-EC differentiation protocol and underlying heterogeneity of derived iPSC-ECs remain as major limitations of iPSC-EC technology. Objective: Here, we performed droplet-based single-cell RNA sequencing (scRNA-seq) of the human iPSCs after iPSC-EC differentiation. Droplet-based scRNA-seq enables analysis of thousands of cells in parallel, allowing comprehensive analysis of transcriptional heterogeneity. Methods and Results: Bona fide iPSC-EC cluster was identified by scRNA-seq, which expressed high levels of endothelial-specific genes. iPSC-ECs, sorted by CD144 antibody–conjugated magnetic sorting, exhibited standard endothelial morphology and function including tube formation, response to inflammatory signals, and production of NO. Nonendothelial cell populations resulting from the differentiation protocol were identified, which included immature cardiomyocytes, hepatic-like cells, and vascular smooth muscle cells. Furthermore, scRNA-seq analysis of purified iPSC-ECs revealed transcriptional heterogeneity with 4 major subpopulations, marked by robust enrichment of CLDN5, APLNR, GJA5, and ESM1 genes, respectively. Conclusions: Massively parallel, droplet-based scRNA-seq allowed meticulous analysis of thousands of human iPSCs subjected to iPSC-EC differentiation. Results showed inefficiency of the differentiation technique, which can be improved with further studies based on identification of molecular signatures that inhibit expansion of nonendothelial cell types. Subtypes of bona fide human iPSC-ECs were also identified, allowing us to sort for iPSC-ECs with specific biological function and identity.