Anke M. Smits

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.

Human cardiomyocyte progenitor cell transplantation preserves long-term function of the infarcted mouse myocardium

AIMS Recent clinical studies revealed that positive results of cell transplantation on cardiac function are limited to the short- and mid-term restoration phase following myocardial infarction (MI), emphasizing the need for long-term follow-up. These transient effects may depend on the transplanted cell-type or its differentiation state. We have identified a population of cardiomyocyte progenitor cells (CMPCs) capable of differentiating efficiently into beating cardiomyocytes, endothelial cells, and smooth muscle cells in vitro. We investigated whether CMPCs or pre-differentiated CMPC-derived cardiomyocytes (CMPC-CM) are able to restore the injured myocardium after MI in mice. METHODS AND RESULTS MI was induced in immunodeficient mice and was followed by intra-myocardial injection of CMPCs, CMPC-CM, or vehicle. Cardiac function was measured longitudinally up to 3 months post-MI using 9.4 Tesla magnetic resonance imaging. The fate of the human cells was determined by immunohistochemistry. Transplantation of CMPCs or CMPC-CM resulted in a higher ejection fraction and reduced the extent of left ventricular remodelling up to 3 months after MI when compared with vehicle-injected animals. CMPCs and CMPC-CM generated new cardiac tissue consisting of human cardiomyocytes and blood vessels. Fusion of human nuclei with murine nuclei was not observed. CONCLUSION CMPCs differentiated into the same cell types in situ as can be obtained in vitro. This excludes the need for in vitro pre-differentiation, making CMPCs a promising source for (autologous) cell-based therapy.

Progenitor cells isolated from the human heart: a potential cell source for regenerative therapy

Background. In recent years, resident cardiac progenitor cells have been identified in, and isolated from the rodent heart. These cells show the potential to form cardiomyocytes, smooth muscle cells, and endothelial cells in vitro and in vivo and could potentially be used as a source for cardiac repair. However, previously described cardiac progenitor cell populations show immature development and need co-culture with neonatal rat cardiomyocytes in order to differentiate in vitro. Here we describe the localisation, isolation, characterisation, and differentiation of cardiomyocyte progenitor cells (CMPCs) isolated from the human heart.Methods. hCMPCs were identified in human hearts based on Sca-1 expression. These cells were isolated, and FACS, RT-PCR and immunocytochemistry were used to determine their baseline characteristics. Cardiomyogenic differentiation was induced by stimulation with 5-azacytidine.Results. hCMPCs were localised within the atria, atrioventricular region, and epicardial layer of the foetal and adult human heart. In vitro, hCMPCs could be induced to differentiate into cardiomyocytes and formed spontaneously beating aggregates, without the need for co-culture with neonatal cardiomyocytes.Conclusion. The human heart harbours a pool of resident cardiomyocyte progenitor cells, which can be expanded and differentiated in vitro. These cells may provide a suitable source for cardiac regeneration cell therapy. (Neth Heart J 2008;16: 163-9.)

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.

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.

Endothelial cells are activated during hypoxia via endoglin/ALK-1/SMAD1/5 signaling in vivo and in vitro.

Endoglin (ENG) promotes angiogenesis by enhancing activation of TGF-beta type I receptors ALK-1 and ALK-5. ALK-1 phosphorylates transcription factors SMAD1/5, which bind to BMP-responsive elements (BRE), whereas ALK-5 phosphorylates SMAD3, which binds to CAGA elements. Expression of ENG is increased during myocardial infarction (MI). We investigated which ENG signaling pathway is activated in endothelial cells during hypoxia. Expression of ENG, ALK-1, ALK-5, and phosphorylated SMAD1/3/5 by immunostaining and immunoblotting in a mouse model of myocardial infarction (MI) and in hypoxic human aortic endothelial cells (HAECs) was evaluated. Activation of BRE and CAGA was measured by luciferase assays in cells transfected with plasmids expressing ENG or ALK-1 and the number of cells was quantified. mRNA expression of the target genes of TGF-beta signaling, ID1 and BCL-X, was quantified by real-time RT-PCR. Expression of ENG, ALK-1 and phosphorylated SMAD1/5, but not ALK-5 or phosphorylated SMAD3, was significantly increased in hypoxic endothelial cells in vivo and in vitro. Overexpression of both ENG and ALK-1 significantly increased BRE but not CAGA activity, expression of ID1 and BCL-X and the number of HAECs at hypoxia. ENG/ALK-1 signaling is one of the factors that regulate endothelial cell activity during adaptive cardiac angiogenesis.

Low oxygen tension positively influences cardiomyocyte progenitor cell function.

Previously we observed that cardiomyocyte progenitor cells (hCMPCs) isolated from the human heart differentiate spontaneously into cardiomyocytes and vascular cells when transplanted after myocardial infarction (MI) in the ischemic heart. After MI, deprivation of oxygen is the first major change in the cardiac environment. How cells handle hypoxia is highly cell type dependent. The effect of hypoxia on cardiac stem or progenitor cells remains to be elucidated. Here, we show for the first time that short‐ and long‐term hypoxia have different effects on hCMPCs. Short‐term hypoxia increased the migratory and invasive capacities of hCMPCs likely via mesenchymal transformation. Although long‐term exposure to low oxygen levels did not induce differentiation of hCMPCs into mature cardiomyocytes or endothelial cells, it did increase their proliferation, stimulated the secretome of the cells which was shifted to a more anti‐inflammatory profile and dampened the migration by altering matrix metalloproteinase (MMP) modulators. Interestingly, hypoxia greatly induced the expression of the extracellular matrix modulator thrombospondin‐2 (TSP‐2). Knockdown of TSP‐2 resulted in increased proliferation, migration and MMP activity. In conclusion, short exposure to hypoxia increases migratory and invasive capacities of hCMPCs and prolonged exposure induces proliferation, an angiogenic secretion profile and dampens migration, likely controlled by TSP‐2.

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.

Cardiomyogenic differentiation-independent improvement of cardiac function by human cardiomyocyte progenitor cell injection in ischaemic mouse hearts

We previously showed that human cardiomyocyte progenitor cells (hCMPCs) injected after myocardial infarction (MI) had differentiated into cardiomyocytes in vivo 3 months after MI. Here, we investigated the short‐term (2 weeks) effects of hCMPCs on the infarcted mouse myocardium. MI was induced in immunocompromised (NOD/scid) mice, immediately followed by intramyocardial injection of hCMPCs labelled with enhanced green fluorescent protein (hCMPC group) or vehicle only (control group). Sham‐operated mice served as reference. Cardiac performance was measured 2 and 14 days after MI by magnetic resonance imaging at 9.4 T. Left ventricular (LV) pressure–volume measurements were performed at day 15 followed by extensive immunohistological analysis. Animals injected with hCMPCs demonstrated a higher LV ejection fraction, lower LV end‐systolic volume and smaller relaxation time constant than control animals 14 days after MI. hCMPCs engrafted in the infarcted myocardium, did not differentiate into cardiomyocytes, but increased vascular density and proliferation rate in the infarcted and border zone area of the hCMPC group. Injected hCMPCs engraft into murine infarcted myocardium where they improve LV systolic function and attenuate the ventricular remodelling process 2 weeks after MI. Since no cardiac differentiation of hCMPCs was evident after 2 weeks, the observed beneficial effects were most likely mediated by paracrine factors, targeting amongst others vascular homeostasis. These results demonstrate that hCMPCs can be applied to repair infarcted myocardium without the need to undergo differentiation into cardiomyocytes.