Cardiomyogenic Potential Research Articles

The developmental origin of postnatal cardiac regeneration

Recent studies have shown that postnatal mammalian hearts undergo cardiomyocyte refreshment. However, evidence is lacking for the cell origin of these postnatal cardiomyogenesis. We first confirmed that myocardial injury triggered the expression of embryonic cardiogenesis genes at the external part of the heart. To determine whether cardiac lineage-specific progenitor cells exist in the postnatal mammalian heart and have cardiomyogenic potential, we created coronary artery ligation on the Nkx2.5 enhancer-eGFP transgenic mice. The number of Nkx2.5 enhancer-eGFP cells was increasing after myocardial injury and peaked at 2 weeks post injury. The activated eGFP+ cells expressed cardiac precursor markers. The eGFP signaling was not expressed in mature cardiomyocytes, hematopoietic cells or fibroblasts. These activated Nkx2.5 enhancer-eGFP cells would differentiate into striated cardiomyocytes in vitro. We further confirmed that cardiac progenitor cells contributed directly to postnatal cardiomyogenesis by lineage tracing using inducible Cre system. To trace the developmental origin of the activated Nkx2.5 enhancer-eGFP cells, we created different lineage-Cre/Nkx2.5 enhancer-eGFP/ ROSA26 reporter triple transgenic mice. The post-injury Nkx2.5 enhancer-eGFP+ cells originated from the embryonic epicardial cells, but did not originate from the pre-existing cardiomyocytes, endothelial cells, cardiac neural crest cells, and perinatal/ postnatal epicardial cells. Taken together, our data confirmed that cardiac-lineage progenitor cells, which originated from embryonic epicardium-derived cells, contributed to postnatal mammamian cardiomyogenesis. The discovery of cardiomyogenic cell population in the postnatal heart enables the future cell therapy for cardiac regeneration.

Cardiomyogenic potential of human amniotic fluid derived stem cells

Cardiomyogenic potential of human amniotic fluid derived stem cells

Abstract 252: Endocardial Cushion-derived Cardiovascular Progenitor Cells

Introduction: Regenerative therapies utilizing cardiac progenitor cells (CPCs) derived from human embryonic stem cells (hESC) have the potential to regenerate damaged myocardium post-myocardial infarctions. Previous studies have identified multipotent progenitors localized in the endocardial cushions of the developing mouse heart. Recent data in mice have also shown that the loss of a single transcription factor, Scl, enabled robust cardiomyogenic potential in these progenitor cells. Further analyses have delineated that these progenitors can be identified by co-expression of CD31 and PDGFRα. Recapitulation of this phenotypic progenitor cell from hESCs may have potential for clinical regenerative therapies. Hypothesis: We hypothesize that CD31 + /PDGFRα + cells sorted from wild type mouse and human fetal heart tissue possess nascent cardiomyogenic potential. In addition, we believe that close mimicry of the in vivo developmental process of cushion formation would generate a hESC-derived CPC expressing CD31 and PDGFRα. Methods: CD31 + /PDGFRα + cells sorted from wild type mouse heart and human fetal heart are plated in cardiac differentiation medium. Through a stage-specific differentiation protocol, hESCs are first differentiated into mesodermal progenitor cells, which then are sorted for the endothelial marker CD31. Under cardiac culture conditions, these CD31 + cells express the CPC marker PDGFRα. The CD31 + /PDGFRα + cells are then isolated and characterized through quantitative PCR, immunostaining, and functional assays. Results: Wild type mouse heart CD31 + /PDGFRα + cells, when isolated at E14.5 and plated in cardiac differentiation medium, develop beating colonies. In addition, CD31 + /PDGFRα + cells are present in human fetal hearts. Moreover, hESC-derived CD31 + /PDGFRα + cells, when cultured in cardiac medium, show increased expression of cardiomyogenic markers. Conclusions: We conclude that endogenous hESC-derived CD31 + /PDGFRα + cells have cardiogenic potential through in vitro differentiation, exhibited by their expression of cardiomyocyte characteristics and ability to beat. These cells may represent putative endocardial cushion-derived CPCs.

Abstract 136: Distinct Temporal Requirements for Scl to Repress Cardiomyogenesis in Endothelial-derived Cells in Hemogenic Tissues and the Heart

Identification of precursors with the capacity to generate cardiomyocytes is critical for advancing cardiac regenerative medicine. By analyzing knockout embryos for the bHLH factor Scl, we demonstrated that endothelial cells in hematopoietic tissues and the heart possess latent cardiomyogenic capacity. Furthermore, analysis of tamoxifen-inducible Rosa26-Cre ERT2 Scl fl/fl embryos suggested that the time window during which Scl is required for cardiac repression extends later in the heart versus the yolk sac. However, the cell types in which Scl acts remained elusive. We then deleted Scl in a cell-type specific manner in early mesoderm using Mesp1-Cre and in endothelial cells using Tie2-Cre. Lineage tracing in Mesp1-Cre Rosa26-YFP embryos demonstrated that at E9.5, a large majority of hematopoietic and endothelial cells in the yolk sac and heart were labeled. Moreover, deletion of Scl in Mesp1-Cre Scl fl/fl embryos phenocopied the germline knockout, essentially abrogating hematopoiesis and promoting the emergence of CD31 + PDGFRα + cardiomyogenic precursors and ectopic expression of the cardiomyocyte genes Myl7 and Tnnt2 in yolk sac vasculature. In contrast, deletion of Scl after endothelium had been specified in Tie2-Cre Scl fl/fl embryos did not grossly affect yolk sac hematopoiesis, nor did it induce ectopic cardiomyogenesis in hemogenic tissues. However, endothelial-derived cells in the hearts of Tie2-Cre Scl fl/fl embryos evidenced profound expansion of CD31 + PDGFRα + cardiogenic precursors at E11.5 and E13.5, as well as displayed dramatic upregulation of Myl7 and Tnnt2 , showing that the requirement for Scl to repress the cardiomyogenic program extends longer in endothelial derivatives in the heart than in the yolk sac. These data demonstrate that endocardial-derived cells in the heart retain latent cardiomyogenic potential until mid-gestation and nominate Scl as a critical regulator of endocardial fate.

Spheroid Formation and Enhanced Cardiomyogenic Potential of Adipose-Derived Stem Cells Grown on Chitosan

Mesenchymal stem cells may differentiate into cardiomyocytes and participate in local tissue repair after heart injury. In the current study, rat adipose-derived adult stem cells (ASCs) grown on chitosan membranes were observed to form cell spheroids after 3 days. The cell seeding density and surface modification of chitosan with Arg-Gly-Asp–containing peptide had an influence on the sizes of ASC spheroids. In the absence of induction, these spheroids showed an increased level of cardiac marker gene expression (Gata4, Nkx2-5, Myh6, and Tnnt2) more than 20-fold versus cells on the tissue culture polystyrene (TCPS) dish. Induction by 5-azacytidine or p38 MAP kinase inhibitor (SB202190) did not further increase the cardiac marker gene expression of these spheroids. Moreover, the enhanced cardiomyogenic potential of the spheroids was highly associated with the chitosan substrates. When ASC spheroids were plated onto TCPS with either basal or cardiac induction medium for 9 days, the spheroids spread into a monolayer and the positive effect on cardiomyogenic marker gene expression disappeared. The possible role of calcium ion and the up-regulation of adhesion molecule P-selectin and chemokine receptor Cxcr4 were demonstrated in ASC spheroids. Applying these spheroids to the chronic myocardial infarction animal model showed better functional recovery versus single cells after 12 weeks. Taken together, this study suggested that the ASC spheroids on chitosan may form as a result of calcium ion signaling, and the transplantation of these spheroids may offer a simple method to enhance the efficiency of stem cell–based therapy in myocardial infarction.

Characterization of Cardiac-derived Myelo-monocytoid Progenitor Cells

Cells migrating from heart tissue biopsies during ex vivo culture have been associated with cardiomyogenic and angiogenic potential. We have characterized cells shed by murine cardiac explants cultured in MesenCult MSC medium. A relatively uniform population with a distinguishable morphology emerged from the early cellular outgrowth. This population expressed monocyte/macrophage and hematopoietic markers (CD11b+, CD14+, CD45+) but neither mesenchymal (CD90–, CD105–) nor endothelial (CD31–) markers. It was maintained in culture with a stable phenotype for more than 10 months. When cultured in differentiation medium, cells expressed cardiac sarcomeric α-actinin. They spontaneously formed sphere clusters (“cardiospheres”). This process was enhanced by TNFα. Spheres from neonatal mice exhibited spontaneous beating. To assess whether “contaminating” myocardial tissue fragments would explain CS beating, neonatal Z/EG transgenic mice were injected with an adeno-associated virus (AAV) vector expressing Cre-recombinase from a cardiac-specific promoter (Ncx1), resulting in the excision of a lacZ gene and activatation of the expression of the second reporter gene, enhanced green fluorescent protein (EGFP). Cardiac explants from Z/EG mice receiving AAV-9.Ncx1-CRE expressed EGFP, whereas the cellular outgrowth did not, demonstrating that the presence of cardiomyocytes in the outgrowth was not required for the generation of cardiac progenitor cells. Whether the myelo-monocytoid population originated from a retained hematologic component of explants or cardiac-resident leukocytes remains unclear. These results are in line with recent data on monocyte-derived multipotent cells (MOMC) with cardiomyogenic potential. They suggest that the cardiac-derived myelo-monocytoid cell, although not considered a classical adult progenitor cell, may have regenerative potential.

Study of cardiomyogenic potential of human Amniotic Fluid Stem Cells

It has been shown that Amniotic fluid stem cells (AFSCs) have characteristics intermediate between pluripotent embryonic and lineage-restricted adult stem cells, and are non-tumorigenic and low immunogenic. Moreover, they are obtained without destroying human embryos, so that preventing most of the ethical and social controversy. Human AFSCs express some genes specific of both embryonic (OCT3/4, NANOG, c-MYC) and primordial germ stem cells (Fragilis, Stella, c-KIT). We have demonstrated that hAFSCs form in vitro embryoid bodies (EBs) and express markers of three germ layers. Studies reported the ability of hAFSCs to differentiate in vitro into adipocytes and osteocytes, but only few data are available on their cardiomyogenic potential. Aim of this study is to analyze hAFSCs differentiation through the cardiac pathway. Embryonic Bodies (EBs) were obtained from hAFSCs cultured in presence of ascorbic acid and 5-aza-2’-deoxycytidine. Cardiomyogenic potential of hAFSCs and EBs was explored by WB and immunoflorescent analyses of specific markers. Simultaneous quantitative detection and cellular localization analysis of cardiomyogenic markers were conducted with an ImageStream multispectral imaging flow cytometer (Amnis-Seattle, WA) equipped with IDEAS statistical software. We evidenced that both AFSCs and EBs at early stage express Nkx2.5, a transcription factor expressed by cardiomyocytes precursor cells. Moreover, ImageStream imaging cytometer analysis evidenced that EBs formation was accompanied by an up-regulation of Nkx2.5 expression (36.54±1.83% and 64.68±3.23% positive cells in hAFSC and EBs respectively, p<.005) and by a significant nuclear translocation (12.98±0.64% and 37.98±1.9% nuclear positive cells in hAFSC and EBs respectively, p<.005). Microscopical analysis evidenced inside the EBs cells positive for the presence of cardiac α-Myosin heavy chain protein structurally organized in oriented filaments. Moreover, we detected cells expressing Connexin43. These results evidenced that EBs obtained from hAFSCs cultured in permissive conditions terminally differentiate into cardiomyocytes and suggest them as possible model to study the cardiac differentiation.

Survival, Integration, and Differentiation of Unrestricted Somatic Stem Cells in the Heart

Unrestricted somatic stem cells (USSCs) derived from human umbilical cord blood represent an attractive cell source to reconstitute the damaged heart. We have analyzed the cardiomyogenic potential and investigated the fate of USSCs after transplantation into rat heart in vivo. USSCs demonstrated cardiomyogenic differentiation properties characterized by the spontaneously beating activity and the robust expression of cardiac α-actinin and troponin T (cTnT) at protein and mRNA level after cocultivation with neonatal rat cardiomyocytes. To study the fate in vivo, eGFP⁺ USSCs were injected transcoronarily into immunosuppressed rats via a catheter-based technique. Nearly 80% USSCs were retained within the myocardium without altering cardiac hemodynamics. After 7 days, 20% of the transplanted cells survived in the host myocardium and showed elongated morphology with weak expression of cardiac-specific markers, while some eGFP⁺ USSCs were found to integrate into the vascular wall. After 21 days, only a small fraction of USSCs were found in the myocardium (0.13%); however, the remaining cells clearly exhibited a sarcomeric structure similar to mature cardiomyocytes. Identical results were also obtained in nude rats. In addition, we found some cells stained positively for activated caspase 3 paralleled by the massive infiltration of CD11b⁺ cells into the myocardium. In summary, USSCs can differentiate into beating cardiomyocytes by cocultivation in vitro. After coronary transplantation in vivo, however, long-term survival of differentiated USSCs was rather low despite a high initial fraction of trapped cells.

TGFβ-Dependent Epithelial-to-Mesenchymal Transition Is Required to Generate Cardiospheres from Human Adult Heart Biopsies

Autologous cardiac progenitor cells (CPCs) isolated as cardiospheres (CSps) represent a promising candidate for cardiac regenerative therapy. A better understanding of the origin and mechanisms underlying human CSps formation and maturation is undoubtedly required to enhance their cardiomyogenic potential. Epithelial-to-mesenchymal transition (EMT) is a key morphogenetic process that is implicated in the acquisition of stem cell-like properties in different adult tissues, and it is activated in the epicardium after ischemic injury to the heart. We investigated whether EMT is involved in the formation and differentiation of human CSps, revealing that an up-regulation of the expression of EMT-related genes accompanies CSps formation that is relative to primary explant-derived cells and CSp-derived cells grown in a monolayer. EMT and CSps formation is enhanced in the presence of transforming growth factor β1 (TGFβ1) and drastically blocked by the type I TGFβ-receptor inhibitor SB431452, indicating that TGFβ-dependent EMT is essential for the formation of these niche-like 3D-multicellular clusters. Since TGFβ is activated in the myocardium in response to injury, our data suggest that CSps formation mimics an adaptive mechanism that could potentially be enhanced to increase in vivo or ex vivo regenerative potential of adult CPCs.

Abstract 42: In Vivo Differentiation of Epigenetically Reprogrammed Human Endothelial Progenitor Cells into Cardiomyocytes Enhances Functional and Anatomical Postinfarct Myocardial Repair

Currently, bone marrow derived endothelial progenitor cells (human CD34+ cells, EPC) are being used clinically to improve vascularization in patients with ischemic heart disease. While it is generally accepted that CD34+ cells predominantly work through a paracrine mechanism, there exists no convincing evidence that these cells trans-differentiate into functional cardiomyocytes (CMC). Since ischemic heart disease leads to substantial loss of CMC, improving cardiomyogenic plasticity of an existing autologous cell therapy is of obvious import. EPC and CMC both differentiate from a common mesodermal progenitor however; during EC-specific lineage differentiation, CMC specific genes are epigenetically silenced. We hypothesized that reprogramming of CD34+ cells using small molecules targeting key epigenetic repressive marks may recapitulate their cardiomyogenic potential. Human CD34+ EPCs were treated with inhibitors of histone deacetylases (valproic acid) for 24 hours followed by an additional 24 hours with the DNA methyltransferase inhibitor (5-Azacytidine). This forty-eight hour treatment led to the reactivation of pluripotency associated and CMC specific mRNA expression while EC specific gene expression was maintained. Intra-myocardial transplantation of a sub-therapeutic dose of reprogrammed CD34+ cells in an acute myocardial infarction mouse model showed significant improvement in LV function compared to the same number of control CD34+ cells that are therapeutically equivalent to no treatment at all. This was histologically supported by de novo CMC differentiation. In addition to increased cardiomyogenic plasticity, drug treatment also enhanced the inherent therapeutic capacity of the CD34+ cells as shown by reduced fibrosis, increased capillary density, increased proliferation, increased cell survival and increased secretion of angiogenic factors. Taken together, our results suggest that epigenetically reprogrammed CD34+ cells are “super-CD34+ cells” that have an enhanced paracrine effect, display a more plastic phenotype and improve post-infarct cardiac repair by both neo-cardiomyogenesis and neovascularization.

Pan-epicardial lineage tracing reveals that epicardium derived cells give rise to myofibroblasts and perivascular cells during zebrafish heart regeneration

Myocardial infarction (MI) leads to a severe loss of cardiomyocytes, which in mammals are replaced by scar tissue. Epicardial derived cells (EPDCs) have been reported to differentiate into cardiomyocytes during development, and proposed to have cardiomyogenic potential in the adult heart. However, mouse MI models reveal little if any contribution of EPDCs to myocardium. In contrast to adult mammals, teleosts possess a high myocardial regenerative capacity. To test if this advantage relates to the properties of their epicardium, we studied the fate of EPDCs in cryoinjured zebrafish hearts. To avoid the limitations of genetic labelling, which might trace only a subpopulation of EPDCs, we used cell transplantation to track all EPDCs during regeneration. EPDCs migrated to the injured myocardium, where they differentiated into myofibroblasts and perivascular fibroblasts. However, we did not detect any differentiation of EPDCs nor any other non-cardiomyocyte population into cardiomyocytes, even in a context of impaired cardiomyocyte proliferation. Our results support a model in which the epicardium promotes myocardial regeneration by forming a cellular scaffold, and suggests that it might induce cardiomyocyte proliferation and contribute to neoangiogenesis in a paracrine manner.

Ascorbic acid rescues cardiomyocyte development in Fgfr1−/− murine embryonic stem cells

Fibroblast growth factor receptor 1 (Fgfr1) gene knockout impairs cardiomyocyte differentiation in murine embryonic stem cells (mESC). Here, various chemical compounds able to enhance cardiomyocyte differentiation in mESC [including dimethylsulfoxide, ascorbic acid (vitC), free radicals and reactive oxygen species] were tested for their ability to rescue the cardiomyogenic potential of Fgfr1−/− mESC. Among them, only the reduced form of vitC, l-ascorbic acid, was able to recover beating cell differentiation in Fgfr1−/− mESC. The appearance of contracting cells was paralleled by the expression of early and late cardiac gene markers, thus suggesting their identity as cardiomyocytes. In the attempt to elucidate the mechanism of action of vitC on Fgfr1−/− mESC, we analyzed several parameters related to the intracellular redox state, such as reactive oxygen species content, Nox4 expression, and superoxide dismutase activity. The results did not show any relationship between the antioxidant capacity of vitC and cardiomyocyte differentiation in Fgfr1−/− mESC. No correlation was found also for the ability of vitC to modulate the expression of pluripotency genes. Then, we tested the hypothesis that vitC was acting as a prolyl hydroxylase cofactor by maintaining iron in a reduced state. We first analyze hypoxia inducible factor (HIF)-1α mRNA and protein levels that were found to be slightly upregulated in Fgfr1−/− cells. We treated mESC with Fe2+ or the HIF inhibitor CAY10585 during the first phases of the differentiation process and, similar to vitC, the two compounds were able to rescue cardiomyocyte formation in Fgfr1−/− mESC, thus implicating HIF-1α modulation in Fgfr1-dependent cardiomyogenesis.

Enhanced Angiogenic and Cardiomyocyte Differentiation Capacity of Epigenetically Reprogrammed Mouse and Human Endothelial Progenitor Cells Augments Their Efficacy for Ischemic Myocardial Repair

Although bone marrow endothelial progenitor cell (EPC)-based therapies improve the symptoms in patients with ischemic heart disease, their limited plasticity and decreased function in patients with existing heart disease limit the full benefit of EPC therapy for cardiac regenerative medicine. We hypothesized that reprogramming mouse or human EPCs, or both, using small molecules targeting key epigenetic repressive marks would lead to a global increase in active gene transcription, induce their cardiomyogenic potential, and enhance their inherent angiogenic potential. Mouse Lin-Sca1(+)CD31(+) EPCs and human CD34(+) cells were treated with inhibitors of DNA methyltransferases (5-Azacytidine), histone deacetylases (valproic acid), and G9a histone dimethyltransferase. A 48-hour treatment led to global increase in active transcriptome, including the reactivation of pluripotency-associated and cardiomyocyte-specific mRNA expression, whereas endothelial cell-specific genes were significantly upregulated. When cultured under appropriate differentiation conditions, reprogrammed EPCs showed efficient differentiation into cardiomyocytes. Treatment with epigenetic-modifying agents show marked increase in histone acetylation on cardiomyocyte and pluripotent cell-specific gene promoters. Intramyocardial transplantation of reprogrammed mouse and human EPCs in an acute myocardial infarction mouse model showed significant improvement in ventricular functions, which was histologically supported by their de novo cardiomyocyte differentiation and increased capillary density and reduced fibrosis. Importantly, cell transplantation was safe and did not form teratomas. Taken together, our results suggest that epigenetically reprogrammed EPCs display a safe, more plastic phenotype and improve postinfarct cardiac repair by both neocardiomyogenesis and neovascularization.

Cardio-myogenic differentiation of human amniotic fluid colony derived stem cells in the form of embryonic body-like structure

To investigate cardio-myogenic differentiation potential of human amniotic fluid colony derived stem cells (HAFCDSC) in the form of embryonic body (EB)-like structure in vitro. HAFCDSC were isolated from second trimester amniotic fluid which was backup of amniocentesis specimens. The forth passage of HAFCDSC were cultured by hanging-drop preparation in complete medium for 5 days to form EB-like structures followed by inducing medium in regular tissue culture dishes for 2 weeks (experiment group). The EB-like structures cultured in complete medium were served as control group. Cardiomyocyte phenotypes were detected by RT-PCR and immunofluorescence staining. HAFCDSC could form EB-like structures 24 h after hanging drop culture, and the diameter of EB-like structures gradually increased with culture duration and the mean diameter of EB-like structures reached 237.3 ± 26.7 µm on 5(th) day. The formation rate of EB-like structures was 93.5%. RT-PCR analysis showed EB-like structures expressed stem cell related mRNA. Immunofluorescence staining demonstrated that some cardiomyocyte phenotypes such as Desmin, α-Actinin and Tn I were expressed by HAFCDSCs in EB-like structures after cardio-myogenic induction. Cardiomyocytes specific mRNA including Desmin, α-Actin, Tn I, Tn T, T-box and NK2.5 were also transcripted as detected by RT-PCR. No positive results was found in control group. HAFCDSC can form uniform EB-like structures after hanging drop culture for 5 days. HAFCDSC differentiate into cardiomyocyte-like cells through the form of EB-like structures after induction by 5-Aza.

Abstract 16730: Enhancing the Cardiomyogenic Potential of Human Mesenchymal Stem Cells by Modulation of miR Expression

Introduction: it has been shown that mesenchymal stem cells (MSC) can differentiate into cardiomyocytes (CMC) both in vitro and in vivo. However, the efficiency of cardiac differentiation is very limited. Recently, it has been suggested that miR1, 133 and 499 are involved in cardiac development and stem cell differentiation. The role of these miR in MSC differentiation is currently unknown. Accordingly, we tested if the overexpression of miR1, 133 and 499 may favour the differentiation of MSC into CMC. Methods: miR1, 133, and 499 were transiently overexpressed, one by one or in different combinations, in human MSC. Cardiac differentiation was evaluated by measuring the expression of the cardiac specific markers GATA4, Nkx2.5, Tbx5, MEF2c, MLC2v, Cx43, cTnT and α-MHC. Western Blot and immunocytochemistry (ICC) for cTnT and Cx43 were also performed. Results: miR499 alone increased the expression of the early cardiac genes GATA4, Tbx5, MLC2v. In particular, miR499 exerted more pronounced effects compared with miR1 and miR133. Concomitant overexpression of miR499 and miR133 further increased the expression of Tbx5 and MEF2c. Western Blot analysis showed that miR499 increased the expression of late cardiac genes when overexpressed alone or in combination with miR133. Finally, ICC staining confirmed that miR499 in association with miR133 increase Cx43 and cTnT protein expression. Finally, the expression of cardiac specific protein α-MHC was more pronounced in MSC overexpressing miR499 alone or in combination with miR133. Conclusions: concomitant overexpression of miR499 and miR133 successfully commit AMSC into CMC. Our data suggests that miR modulation can represent a promising approach to improve the efficiency of cardioregnerative therapy with multipotent stem cells.

The Stuttering Progress of Cell Therapy for Heart Disease

Stem cell therapy has emerged as a potential therapeutic strategy for myocardial infarction (MI). Multiple cell types used to regenerate the injured heart have been tested in clinical trials. The results of studies of skeletal myoblasts (SKMs) have been resoundingly negative, and the bone marrow-derived-cell experience leaves much to be desired. A number of lessons arise from the large-scale bone marrow-derived-cell trials: (i) efficacy has been inconsistent and, overall, modest; however, unexpectedly meaningful benefits on clinical end points have been reported; (ii) cardiac engraftment of cells is disappointingly low, and delivery methods need to be optimized and combined with strategies to boost retention; (iii) the cardiomyogenic potential of bone marrow cells is low; however, functional benefit can be achieved through indirect pathways; and (iv) autologous cell therapy has severe limitations; highly standardized allogeneic cell products are attractive. Given the spotty trajectory of cell therapy to date, a more systematic approach to product development and preclinical optimization will facilitate more effective clinical translation.

Synthetic sulfonyl-hydrazone-1 positively regulates cardiomyogenic microRNA expression and cardiomyocyte differentiation of induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) are obtained from adult cells through overexpression of pluripotency factors. iPSCs share many features with embryonic stem cells (ESCs), circumventing ethical issues, and, noteworthy, match donor's genotype. iPSCs represent therefore a valuable tool for regenerative medicine. Cardiac differentiation of ESCs can be enhanced via microRNAs (miRNAs) and small chemical compounds, which probably act as chromatin remodelers. Cardiomyogenic potential of iPSCs is currently intensely investigated for cell therapy or in vitro drug screening and disease modeling. However, influences of small compounds on iPSC-related cardiomyogenesis have not yet been investigated in details. Here, we compared the effects of two small molecules, bis-peroxo-vanadium (bpV) and sulfonyl-hydrazone-1 (SHZ) at varying concentrations, during cardiac differentiation of murine iPSCs. SHZ (5 µM) enhanced specific marker expression and cardiomyocyte yield, without loss of cell viability. In contrast, bpV showed negligible effects on cardiac differentiation rate and appeared to induce Casp3-dependent apoptosis in differentiating iPSCs. Furthermore, SHZ-treated iPSCs were able to increase beating foci rate and upregulate early and late cardiomyogenic miRNA expression (miR-1, miR-133a, and miR-208a). Thus, our results demonstrate that small chemical compounds, such as SHZ, can constitute a novel and clinically feasible strategy to improve iPSC-derived cardiac differentiation.

In vitro cardiomyogenic potential of human amniotic fluid stem cells

Stem cell therapy for damaged cardiac tissue is currently limited by a number of factors, including inability to obtain sufficient cell numbers, the potential tumorigenicity of certain types of stem cells and the possible link between stem cell therapy and the development of malignant arrhythmias. In this study, we investigated whether human amniotic fluid-derived stem (hAFS) cells could be a potential source of cells for cardiac cell therapy, by testing the in vitro differentiation capabilities. Undifferentiated hAFS cells express several cardiac genes, including the transcription factor mef2, the gap junction connexin43, and H- and N-cadherin. A 24 h incubation with 5-aza-2'-deoxycytidine (5-AZA-dC) induced hAFS cell differentiation along the cardiac lineage. Evidence for this differentiation included morphological changes, upregulation of cardiac-specific genes (cardiac troponin I and cardiac troponin T) and redistribution of connexin43, as well as downregulation of the stem cell marker SRY-box 2 (sox2). When co-cultured with neonatal rat cardiomyocytes (NRCs), hAFS cells formed both mechanical and electrical connections with the NRCs. Dye transfer experiments showed that calcein dye could be transferred from NRCs to hAFS cells through cellular connections. The gap junction connexin43 likely involved in the communication between the two cell types, because 12-O-tetradecanoylphorbol 13-acetate (TPA) could partially block cellular crosstalk. We conclude that hAFS cells can be differentiated into a cardiomyocyte-like phenotype and can establish functional communication with NRCs. Thus, hAFS cells may potentially be used for cardiac cell therapy.

Cardiogenol C can induce Mouse Hair Bulge Progenitor Cells to Transdifferentiate into Cardiomyocyte-like Cells

BackgroundHair bulge progenitor cells (HBPCs) are multipotent stem cells derived from the bulge region of mice vibrissal hairs. The purified HBPCs express CD34, K15 and K14 surface markers. It has been reported that HBPCs could be readily induced to transdifferentiate into adipocytes and osteocytes. However, the ability of HBPCs to transdifferentiate into cardiomyocytes has not yet been investigated.Methodology/Principal FindingsThe cardiomyogenic potential of HBPCs was investigated using a small cell-permeable molecule called Cardiogenol C. We established that Cardiogenol C could induce HBPCs to express transcription factors GATA4, Nkx2.5 and Tbx5, which are early specific markers for pre-cardiomyogenic cells. In prolonged cultures, the Cardiogenol C-treated HBPCs can also express muscle proteins, cardiac-specific troponin I and sarcomeric myosin heavy chain. However, we did not observe the ability of these cells to functionally contract. Hence, we called these cells cardiomyocyte-like cells rather than cardiomyocytes. We tried to remedy this deficiency by pre-treating HBPCs with Valproic acid first before exposing them to Cardiogenol C. This pretreatment inhibited, rather than improved, the effectiveness of Cardiogenol C in reprogramming the HBPCs. We used comparative proteomics to determine how Cardiogenol C worked by identifying proteins that were differentially expressed. We identified proteins that were involved in promoting cell differentiation, cardiomyocyte development and for the normal function of striated muscles. From those differentially expressed proteins, we further propose that Cardiogenol C might exert its effect by activating the Wnt signaling pathway through the suppression of Kremen1. In addition, by up-regulating the expression of chromatin remodeling proteins, SIK1 and Smarce1 would initiate cardiac differentiation.Conclusions/SignificanceIn conclusion, our CD34+/K15+ HBPCs could be induced to transdifferentiate into cardiomyocyte-like cells using a small molecule called Cardiogenol C. The process involves activation of the Wnt signaling pathway and altered expression of several key chromatin remodeling proteins. The finding is clinically significant as HBPCs offer a readily accessible and autologous source of progenitor cells for cell-based therapy of heart disease, which is one of major killers in developed countries.

Developmental patterns and characteristics of epicardial cell markers Tbx18 and Wt1 in murine embryonic heart

BackgroundAlthough recent studies have highlighted the role of epicardial cells during cardiac development and regeneration, their cardiomyogenic potential is still controversial due to the question of lineage tracing of epicardial cells. The present study therefore aimed to examine the the expression of Tbx18 and Wt1 in embryonic heart and to identify whether Tbx18 and Wt1 themselves expressed in the cardiomyocyte.MethodsMouse embryonic hearts were collected at different stages for immunofluorescence costaining with either Tbx18 and the cardiac transcription factor Nkx2.5 or Wilms tumor 1 (Wt1) and Nkx2.5.ResultsTbx18 and Wt1, but not Nkx2.5, were expressed in the proepicardium and epicardium. Tbx18 was expressed in cells within the heart from E10.5 to at least E14.5; these Tbx18-expressing cells were Nkx2.5 positive, except for a few cells that were Nkx2.5 negative at E14.5. Wt1 was expressed in cells within the heart from E12.5 to at least E14.5, but these Wt1-expressing cells were Nkx2.5 negative.ConclusionThe data obtained in this study demonstrate that Tbx18 is expressed in the cardiomyocytes from E10.5 to at least E14.5, and Wt1 is expressed within the heart from E12.5 to at least E14.5, but not in the cardiomyocyte. These findings may provide new insights on the role of the epicardial cells in cardiac regeneration.