Patrick van Vliet

MicroRNA-1 and -499 Regulate Differentiation and Proliferation in Human-Derived Cardiomyocyte Progenitor Cells

Objective—To improve regeneration of the injured myocardium, it is necessary to enhance the intrinsic capacity of the heart to regenerate itself and/or replace the damaged tissue by cell transplantation. Cardiomyocyte progenitor cells (CMPCs) are a promising cell population, easily expanded and efficiently differentiated into beating cardiomyocytes. Recently, several studies have demonstrated that microRNAs (miRNAs) are important for stem cell maintenance and differentiation via translational repression. We hypothesize that miRNAs are also involved in proliferation/differentiation of the human CMPCs in vitro. Methods and Results—Human fetal CMPCs were isolated, cultured, and efficiently differentiated into beating cardiomyocytes. miRNA expression profiling demonstrated that muscle-specific miR-1 and miR-499 were highly upregulated in differentiated cells. Transient transfection of miR-1 and -499 in CMPC reduced proliferation rate by 25% and 15%, respectively, and enhanced differentiation into cardiomyocytes in human CMPCs and embryonic stem cells, likely via the repression of histone deacetylase 4 or Sox6. Histone deacetylase 4 and Sox6 protein levels were reduced, and small interference RNA (siRNA)-mediated knockdown of Sox6 strongly induced myogenic differentiation. Conclusion—miRNAs regulate the proliferation of human CMPC and their differentiation into cardiomyocytes. By modulating miR-1 and -499 expression levels, human CMPC function can be altered and differentiation directed, thereby enhancing cardiomyogenic differentiation.

Human cardiomyocyte progenitor cells differentiate into functional mature cardiomyocytes: an in vitro model for studying human cardiac physiology and pathophysiology.

To date, there is no suitable in vitro model to study human adult cardiac cell biology. Although embryonic stem cells are able to differentiate into cardiomyocytes in vitro, the efficiency of this process is very low. Other methods to differentiate progenitor cells into beating cardiomyocytes rely on coculturing with rat neonatal cardiomyocytes, making it difficult to study human cardiomyocyte differentiation and (patho)physiology. Here we have developed a method for efficient isolation and expansion of human cardiomyocyte progenitor cells (CMPCs) from cardiac surgical waste or alternatively from fetal heart tissue. Furthermore, we provide a detailed in vitro protocol for efficient differentiation of CMPCs into cardiomyocytes with great efficiency (80–90% of differentiation). Once CMPCs are rapidly dividing (∼1 month after isolation), differentiation can be achieved in 3–4 weeks.

TGF-β1 induces efficient differentiation of human cardiomyocyte progenitor cells into functional cardiomyocytes in vitro

The adult mammalian heart has limited regenerative capacity and was generally considered to contain no dividing cells. Recently, however, a resident population of progenitor cells has been identified, which could represent a new source of cardiomyocytes. Here, we describe the efficient isolation and propagation of human cardiomyocyte progenitor cells (hCMPCs) from fetal heart and patient biopsies. Establishment of hCMPC cultures was remarkably reproducible, with over 70% of adult atrial biopsies resulting in robustly expanding cell populations. Following the addition of transforming growth factor beta, almost all cells differentiated into spontaneously beating myocytes with characteristic cross striations. hCMPC-derived cardiomyocytes showed gap-junctional communication and action potentials of maturing cardiomyocytes. These are the first cells isolated from human heart that proliferate and form functional cardiomyocytes without requiring coculture with neonatal myocytes. Their scalability and homogeneity are unique and provide an excellent basis for developing physiological, pharmacological, and toxicological assays on human heart cells in vitro.

The role of stem cells in cardiac regeneration.

After myocardial infarction, injured cardiomyocytes are replaced by fibrotic tissue promoting the development of heart failure. Cell transplantation has emerged as a potential therapy and stem cells may be an important and powerful cellular source. Embryonic stem cells can differentiate into true cardiomyocytes, making them in principle an unlimited source of transplantable cells for cardiac repair, although immunological and ethical constraints exist. Somatic stem cells are an attractive option to explore for transplantation as they are autologous, but their differentiation potential is more restricted than embryonic stem cells. Currently, the major sources of somatic cells used for basic research and in clinical trials originate from the bone marrow. The differentiation capacity of different populations of bone marrow‐derived stem cells into cardiomyocytes has been studied intensively. The results are rather confusing and difficult to compare, since different isolation and identification methods have been used to determine the cell population studied. To date, only mesenchymal stem cells seem to form cardiomyocytes, and only a small percentage of this population will do so in vitro or in vivo. A newly identified cell population isolated from cardiac tissue, called cardiac progenitor cells, holds great potential for cardiac regeneration. Here we discuss the potential of the different cell populations and their usefulness in stem cell based therapy to repair the damaged heart.

Early cardiac development: a view from stem cells to embryos

From the 1920s, early cardiac development has been studied in chick and, later, in mouse embryos in order to understand the first cell fate decisions that drive specification and determination of the endocardium, myocardium, and epicardium. More recently, mouse and human embryonic stem cells (ESCs) have demonstrated faithful recapitulation of early cardiogenesis and have contributed significantly to this research over the past few decades. Derived almost 15 years ago, human ESCs have provided a unique developmental model for understanding the genetic and epigenetic regulation of early human cardiogenesis. Here, we review the biological concepts underlying cell fate decisions during early cardiogenesis in model organisms and ESCs. We draw upon both pioneering and recent studies and highlight the continued role for inxa0vitro stem cells in cardiac developmental biology.

Isolation and expansion of resident cardiac progenitor cells.

After myocardial infarction, loss of viable cardiomyocytes severely impairs cardiac function. Recently, stem cell transplantation has been put forward as a promising approach to repair the damaged heart. Although several clinical trials have already been performed, the dominant beneficial effects are probably due to neoangiogenesis and arteriogenesis. However, replacement of cardiomyocytes is vital to improve cardiac function in the long term. Stem cells and progenitor cells, with the capacity to differentiate into cardiomyocytes, have been described in both embryonic and adult tissues. Upon stimulation, cardiac progenitor cells proliferate and differentiate into cardiomyocytes, vascular smooth muscle cells, and endothelial cells. Currently however, high proliferation rates and differentiation of cardiac progenitor cells beyond the fetal stage have not yet been achieved. Full differentiation into adult cardiomyocytes in vitro and in vivo might be important for efficient integration with the host environment and therefore more research is needed to study factors that influence proliferation and differentiation. Here we will discuss the isolation of cardiac progenitor cells, their potential to differentiate into various cell types needed for cardiac repair, the possible mechanisms behind these events, and how these cells may be implemented in future clinical settings.

Increased Expression of the Transforming Growth Factor-β Signaling Pathway, Endoglin, and Early Growth Response-1 in Stable Plaques

Background and Purpose— Unstable atherosclerotic plaques are characterized by increased macrophages and reduced smooth muscle cells (SMCs) and collagen content. Endoglin, an accessory transforming growth factor-&bgr; (TGF&bgr;) receptor, is a modulator of TGF&bgr; signaling recently found to be expressed on SMCs in atherosclerotic plaques. Its function in plaque SMCs and plaque development is unknown. Early growth response-1 (EGR-1), a transcription factor downstream of TGF&bgr;, stimulates SMC proliferation and collagen synthesis. In atherosclerotic lesions, it is mainly expressed by SMCs. Therefore, we studied the TGF&bgr;, endoglin, and EGR-1 pathway in advanced atherosclerotic plaques in relation to plaque phenotype. Methods— Human carotid atherosclerotic plaques (n=103) were collected from patients undergoing carotid endarterectomy. Histologically, plaques were analyzed for plaque characteristics, ie, collagen, macrophage and SMC content, and intraplaque thrombus. Intraplaque endoglin, pSmad (indicative for TGF&bgr; signaling), EGR-1, and TGF&bgr; levels were analyzed using Western blots and enzyme-linked immunosorbent assays, respectively. Results— Higher endoglin and EGR-1 protein levels correlated positively with increased plaque collagen levels, increased smooth muscle cell content, and decreased intraplaque thrombi as well as TGF&bgr; signaling (pSmad). Although EGR-1 overexpression in vitro stimulated collagen synthesis, inhibiting endoglin resulted in lower EGR-1 levels, decreased SMC proliferation, and decreased collagen content. Conclusions— TGF&bgr; in human atherosclerotic plaques is active and signals through the TGF&bgr;/Smad pathway. For the first time, we show a strong association between endoglin and EGR-1, increased collagen and SMCs expression, decreased levels of intraplaque thrombosis, and a stable plaque phenotype.

Hyperpolarization Induces Differentiation in Human Cardiomyocyte Progenitor Cells

In the past years, cardiovascular progenitor cells have been isolated from the human heart and characterized. These cells can differentiate into cardiomyocytes, smooth muscle cells and endothelial cells and are therefore of great value for investigation of the mechanisms that drive progenitor cell function and plasticity, drug testing and, potentially, therapeutical purposes. In this respect, most studies have focused on enhancing differentiation with chemicals or growth factors, or co-culture with other cell types. Although they have revealed important mechanisms, protocols need to be established that exclude the need for such factors when one considers using progenitor cells to repair the human heart. In this study we tested whether we could induce cardiomyogenic differentiation of human cardiomyocyte progenitor cells (CMPCs) by altering their membrane potential. We induced hyperpolarization in CMPCs by either co-culturing them with a Kir2.1-overexpressing cell line or by overnight culture in medium containing low potassium concentrations. Hyperpolarization led to increased intracellular calcium concentrations, activation of calcineurin signaling, increased cardiac-specific gene and protein expression levels and, ultimately, to the formation of spontaneously beating cardiomyocytes. Thus, hyperpolarization is sufficient to induce differentiation of CMPCs, thereby revealing a novel mechanism for cardiomyogenic differentiation of heart-derived progenitor cells.

Foetal and adult cardiomyocyte progenitor cells have different developmental potential.

In the past years, cardiovascular progenitor cells have been isolated from the human heart and characterized. Up to date, no studies have been reported in which the developmental potential of foetal and adult cardiovascular progenitors was tested simultaneously. However, intrinsic differences will likely affect interpretations regarding progenitor cell potential and application for regenerative medicine. Here we report a direct comparison between human foetal and adult heart‐derived cardiomyocyte progenitor cells (CMPCs). We show that foetal and adult CMPCs have distinct preferences to differentiate into mesodermal lineages. Under pro‐angiogenic conditions, foetal CMPCs form more endothelial but less smooth muscle cells than adult CMPCs. Foetal CMPCs can also develop towards adipocytes, whereas neither foetal nor adult CMPCs show significant osteogenic differentiation. Interestingly, although both cell types differentiate into heart muscle cells, adult CMPCs give rise to electrophysiologically more mature cardiomyocytes than foetal CMPCs. Taken together, foetal CMPCs are suitable for molecular cell biology and developmental studies. The potential of adult CMPCs to form mature cardiomyocytes and smooth muscle cells may be essential for cardiac repair after transplantation into the injured heart.

Human cardiomyocyte progenitor cells: a short history of nearly everything.

The high occurrence of cardiac disease in the Western world has driven clinicians and cardiovascular biologists to look for alternative strategies to treat patients. A challenging approach is the use of stem cells to repair the heart, in itself an inspiring thought. In the past 10 years, stem cells from different sources have been under intense investigation and, as a result, a multitude of studies have been published on the identification, isolation, and characterization, of cardiovascular progenitor cells and repair in different animal models. However, relatively few cardiovascular progenitor populations have been identified in human hearts, including, but not limited to, cardiosphere‐derived cells , cKit+ human cardiac stem cells , Isl1+ cardiovascular progenitors , and, in our lab, cardiomyocyte progenitor cells (CMPCs) . Here, we aim to provide a comprehensive summary of the past findings and present challenges for future therapeutic potential of CMPCs.