Generation Of Functional Cardiomyocytes Research Articles

Generation of Functional Cardiomyocytes from Human Gastric Fibroblast-Derived Induced Pluripotent Stem Cells

Coronary artery diseases are major problems of the world. Coronary artery disease patients frequently suffer from peptic ulcers when they receive aspirin treatment. For diagnostic and therapeutic purposes, the implementation of panendoscopy (PES) with biopsy is necessary. Some biopsy samples are wasted after the assay is completed. In the present study, we established a protocol for human gastric fibroblast isolation and induced pluripotent stem cell (iPSC) generation from gastric fibroblasts via PES with biopsy. We showed that these iPSCs can be differentiated into functional cardiomyocytes in vitro. To our knowledge, this is the first study to generate iPSCs from gastric fibroblasts in vitro.

Small-molecule-based generation of functional cardiomyocytes from human umbilical cord-derived induced pluripotent stem cells.

The purpose of this study was to investigate the cardiac-differentiation potential of induced pluripotent stem cells (iPSCs) generated from human umbilical cord-derived mesenchymal cells. Spontaneous beating colonies were observed at day 7 after the sequential addition of CHIR99021 and IWP-4. The combined use of CHIR99021 and IWP-4 downregulated the expression of pluripotency markers while upregulating cardiac transcription factors and cardiomyocyte-specific markers. The derived cardiomyocytes demonstrated typical sarcomeric structures and action-potential features; most importantly, the derived cells exhibited responsiveness to β-adrenergic and muscarinic stimulations. The analyses of molecular, structural, and functional properties revealed that the derived cardiomyocytes were similar to cardiomyocytes derived from BJ foreskin fibroblast cells. In summary, our results demonstrate that functional cardiomyocytes can be generated from human umbilical cord-derived cells. The methodology described here has potential as a means for the production of functional cardiomyocytes from discarded human umbilical cord tissue.

Impact of Biomaterials on Differentiation and Reprogramming Approaches for the Generation of Functional Cardiomyocytes.

The irreversible loss of functional cardiomyocytes (CMs) after myocardial infarction (MI) represents one major barrier to heart regeneration and functional recovery. The combination of different cell sources and different biomaterials have been investigated to generate CMs by differentiation or reprogramming approaches although at low efficiency. This critical review article discusses the role of biomaterial platforms integrating biochemical instructive cues as a tool for the effective generation of functional CMs. The report firstly introduces MI and the main cardiac regenerative medicine strategies under investigation. Then, it describes the main stem cell populations and indirect and direct reprogramming approaches for cardiac regenerative medicine. A third section discusses the main techniques for the characterization of stem cell differentiation and fibroblast reprogramming into CMs. Another section describes the main biomaterials investigated for stem cell differentiation and fibroblast reprogramming into CMs. Finally, a critical analysis of the scientific literature is presented for an efficient generation of functional CMs. The authors underline the need for biomimetic, reproducible and scalable biomaterial platforms and their integration with external physical stimuli in controlled culture microenvironments for the generation of functional CMs.

Generation of functional cardiomyocytes from rat embryonic and induced pluripotent stem cells using feeder-free expansion and differentiation in suspension culture.

The possibility to generate cardiomyocytes from pluripotent stem cells in vitro has enormous significance for basic research, disease modeling, drug development and heart repair. The concept of heart muscle reconstruction has been studied and optimized in the rat model using rat primary cardiovascular cells or xenogeneic pluripotent stem cell derived-cardiomyocytes for years. However, the lack of rat pluripotent stem cells (rPSCs) and their cardiovascular derivatives prevented the establishment of an authentic clinically relevant syngeneic or allogeneic rat heart regeneration model. In this study, we comparatively explored the potential of recently available rat embryonic stem cells (rESCs) and induced pluripotent stem cells (riPSCs) as a source for cardiomyocytes (CMs). We developed feeder cell-free culture conditions facilitating the expansion of undifferentiated rPSCs and initiated cardiac differentiation by embryoid body (EB)-formation in agarose microwell arrays, which substituted the robust but labor-intensive hanging drop (HD) method. Ascorbic acid was identified as an efficient enhancer of cardiac differentiation in both rPSC types by significantly increasing the number of beating EBs (3.6 ± 1.6-fold for rESCs and 17.6 ± 3.2-fold for riPSCs). These optimizations resulted in a differentiation efficiency of up to 20% cTnTpos rPSC-derived CMs. CMs showed spontaneous contractions, expressed cardiac markers and had typical morphological features. Electrophysiology of riPSC-CMs revealed different cardiac subtypes and physiological responses to cardio-active drugs. In conclusion, we describe rPSCs as a robust source of CMs, which is a prerequisite for detailed preclinical studies of myocardial reconstruction in a physiologically and immunologically relevant small animal model.

Co-culture with neonatal cardiomyocytes enhances the proliferation of iPSC-derived cardiomyocytes via FAK/JNK signaling.

BackgroundWe previously reported that the pluripotent stem cells can differentiate into cardiomyocytes (CMs) by co-culture with neonatal CMs (NCMs) in vitro. However, the involving mechanism is not clear.MethodsMouse induced pluripotent stem cells (iPSCs) were cultured in hanging drops to form embryoid bodies (EBs) and to induce myocardial differentiation. Co-culture of EBs and NCMs was established in a transwell insert system, while EBs grown alone in the wells were used as controls.ResultsCo-culture with NCMs markedly increased the generation of functional CMs from iPSCs. The focal adhesion kinase (FAK) phosphorylation, and c-Jun N-terminal kinase (JNK) phosphorylation in co-culture were higher than that in EBs grown alone. Treating FAK small interfering RNA (FAK siRNA) or specific inhibitor for JNK (SP600125) to iPSCs significantly reduced the phosphorylation of JNK and the expressions of Mef2c and Bcl-2. The expressions of cTnT and MLC-2V were also decreased. Our results revealed that co-culture with NCMs significantly enhance the differentiation ability of iPSCs by increasing Mef2c and Bcl-2 expressions concomitantly with a marked augment on cell proliferation through JNK signaling pathways.ConclusionsThese findings indicated that co-culture of EBs with NCMs induces genes expressed in a mature pattern and stimulates the proliferation of iPSC-derived CMs (iPS-CMs) by activating FAK/JNK signaling.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-016-0112-2) contains supplementary material, which is available to authorized users.

Generation of Functional Cardiomyocytes from Efficiently Generated Human iPSCs and a Novel Method of Measuring Contractility.

Human induced pluripotent stem cells (iPSCs) derived cardiomyocytes (iCMCs) would provide an unlimited cell source for regenerative medicine and drug discoveries. The objective of our study is to generate functional cardiomyocytes from human iPSCs and to develop a novel method of measuring contractility of CMCs. In a series of experiments, adult human skin fibroblasts (HSF) and human umbilical vein endothelial cells (HUVECs) were treated with a combination of pluripotent gene DNA and mRNA under specific conditions. The iPSC colonies were identified and differentiated into various cell lineages, including CMCs. The contractile activity of CMCs was measured by a novel method of frame-by-frame cross correlation (particle image velocimetry-PIV) analysis. Our treatment regimen transformed 4% of HSFs into iPSC colonies at passage 0, a significantly improved efficiency compared with use of either DNA or mRNA alone. The iPSCs were capable of differentiating both in vitro and in vivo into endodermal, ectodermal and mesodermal cells, including CMCs with >88% of cells being positive for troponin T (CTT) and Gata4 by flow cytometry. We report a highly efficient combination of DNA and mRNA to generate iPSCs and functional iCMCs from adult human cells. We also report a novel approach to measure contractility of iCMCs.

In vitro cardiomyogenic differentiation of human dental pulp stem cells. The role of 5-azacytidine

Cellular cardiomyoplasty has been introduced as a potential therapy for treating heart failure and has generated significant interest in identifying various cell types capable of restoring the injured myocardium. Autologous human dental pulp stem cells (hDPSCs) show the ability to differentiate into various cell type such as osteoblasts1, chondroblasts, and adipoblasts. Aim of this study is to investigate the aptitude of hDPSCs to differentiate towards a cardiomyogenic phenotype in vitro. In this study we developed an efficient protocol for the generation of functional cardiomyocytes. Second passaged cells were treated with 5μM of 5-azacytidine2, a well-known demethylating agent, for 48h. Initial exposition of cultured hDPSCs to 5-azacytidine leads to development of embryoid bodies (EBs) expressing specific transcripts markers of the three germ layers as : ectoderm (MAP2), mesoderm (MSX1), and endoderm (PAX6) and various precardiac markers including MYH6, MESP. EBs subsequently plated onto gelatin-coated tissue culture dishes express cardiac markers as Nkx2.5 and connexin 43 determinated by PCR and immunofluorescence analysis at confocal microscopy, indicating that EBs can differentiate into functional cardiomyocytes. These results demonstrate that hDPSCs can be an easily source of stem cells able to undergo versus the cardiomyiogenic lineage.

Induction of Pluripotent Stem Cells from a Cynomolgus Monkey Using a Polycistronic Simian Immunodeficiency Virus–Based Vector, Differentiation Toward Functional Cardiomyocytes, and Generation of Stably Expressing Reporter Lines

Induced pluripotent stem cells (iPSCs) represent a novel cell source for regenerative therapies. Many emerging iPSC-based therapeutic concepts will require preclinical evaluation in suitable large animal models. Among the large animal species frequently used in preclinical efficacy and safety studies, macaques show the highest similarities to humans at physiological, cellular, and molecular levels. We have generated iPSCs from cynomolgus monkeys (Macaca fascicularis) as a segue to regenerative therapy model development in this species. Because typical human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors show poor transduction of simian cells, a simian immunodeficiency virus (SIV)-based vector was chosen for efficient transduction of cynomolgus skin fibroblasts. A corresponding polycistronic vector with codon-optimized reprogramming factors was constructed for reprogramming. Growth characteristics as well as cell and colony morphology of the resulting cynomolgus iPSCs (cyiPSCs) were demonstrated to be almost identical to cynomolgus embryonic stem cells (cyESCs), and cyiPSCs expressed typical pluripotency markers including OCT4, SOX2, and NANOG. Furthermore, differentiation in vivo and in vitro into derivatives of all three germ layers, as well as generation of functional cardiomyocytes, could be demonstrated. Finally, a highly efficient technique for generation of transgenic cyiPSC clones with stable reporter expression in undifferentiated cells as well as differentiated transgenic cyiPSC progeny was developed to enable cell tracking in recipient animals. In conclusion, our data indicate that cyiPSCs represent a valuable cell source for establishment of macaque-based allogeneic and autologous preclinical cell transplantation models for various fields of regenerative medicine.

Generation of functional cardiomyocytes from mouse induced pluripotent stem cells

Background Induced pluripotent stem (iPS) cells allow derivation of autologous differentiated cells for cell therapy. The purpose of this study was to compare the cardiac differentiation potential of mouse iPS cells with embryonic stem (ES) cells and demonstrate that they could produce functional cardiomyocytes. Methods iPS cells were prepared from mouse embryonic fibroblasts by lentiviral mediated expression of four transcription factors (Oct4/Sox2/Klf4/C-myc). To induce cardiac cell differentiation, iPS-S-6 or D3-ES cells were induced to form embryoid bodies (EBs) using a two-medium culture protocol, then plated onto gelatin-coated plates and maintained in DMEM. Results Following classification of the generation periods of contracting EBs into early (d8–d11), middle (d12–d15) and late (d16–20), iPS cells in the early period exhibited characteristics similar to ES cells. In iPS cells from the middle period group, the ratio of contracting EBs was significantly increased compared to ES cells, and the difference persisted in cells from the late period group (p < 0.05). The percentage of contracting EBs formed from iPS and ES cells were 44.8% and 33.3%, respectively. In addition, iPS cell-derived cardiomyocytes exhibited mRNA expression of cardiac mesoderm markers such as GATA4 and NKX2.5, and cardiomyocyte markers such as α1s, α1c, α-MHC, β-MHC, Cx40, TnI, TnT, ANF and Hey2. Single cardiomyocytes exhibited typical cross-striated myofibrillar organization, and electrophysiological studies revealed functional cardiac-specific voltage-gated Na +, Ca 2+ and K + channels. Conclusions These results demonstrate that functional cardiomyocytes can be generated from iPS cells, and suggest that these cells may be useful for the treatment of cardiovascular disease.

Endothelial cells regulate cardiomyocyte development from embryonic stem cells

The molecules and environment that direct pluripotent stem cell differentiation into cardiomyocytes are largely unknown. Here, we determined a critical role of receptor tyrosine kinase, EphB4, in regulating cardiomyocyte generation from embryonic stem (ES) cells through endothelial cells. The number of spontaneous contracting cardiomyocytes, and the expression of cardiac-specific genes, including alpha-MHC and MLC-2V, was significantly decreased in EphB4-null ES cells. EphB4 was expressed in endothelial cells underneath contracting cardiomyocytes, but not in cardiomyocytes. Angiogenic inhibitors, including endostatin and angiostatin, inhibited endothelial cell differentiation and diminished cardiomyogenesis in ES cells. Generation of functional cardiomyocytes and the expression of cardiac-specific genes were significantly enhanced by co-culture of ES cells with human endothelial cells. Furthermore, the defects of cardiomyocyte differentiation in EphB4-deficient ES cells were rescued by human endothelial cells. For the first time, our study demonstrated that endothelial cells play an essential role in facilitating cardiomyocyte differentiation from pluripotent stem cells. EphB4 signaling is a critical component of the endothelial niche to regulate regeneration of cardiomyocytes.

Generation of Functional Cardiomyocytes From Adult Mouse Spermatogonial Stem Cells

Stem cell-based therapy is a promising approach for the treatment of heart failure. Adult stem cells with the pluripotency of embryonic stem cells (ESCs) would be an ideal cell source. Recently, we reported the successful establishment of multipotent adult germline stem cells (maGSCs) from mouse testis. These cultured maGSCs show phenotypic characteristics similar to ESCs and can spontaneously differentiate into cells from all 3 germ layers. In the present study, we used the hanging drop method to differentiate maGSCs into cardiomyocytes and analyzed their functional properties. Differentiation efficiency of beating cardiomyocytes from maGSCs was similar to that from ESCs. The maGSC-derived cardiomyocytes expressed cardiac-specific L-type Ca(2+) channels and responded to Ca(2+) channel-modulating drugs. Cx43 was expressed at cell-to-cell contacts in cardiac clusters, and fluorescence recovery after photobleaching assay showed the presence of functional gap junctions among cardiomyocytes. Action potential analyses demonstrated the presence of pacemaker-, ventricle-, atrial-, and Purkinje-like cardiomyocytes. Stimulation with isoproterenol resulted in a significant increase in beating frequency, whereas the addition of cadmium chloride abolished spontaneous electrical activity. Confocal microscopy analysis of intracellular Ca(2+) in maGSC-derived cardiomyocytes showed that calcium increased periodically throughout the cell in a homogenous fashion, pointing to a fine regulated Ca(2+) release from intracellular Ca(2+) stores. By using line-scan mode, we found rhythmic Ca(2+) transients. Furthermore, we transplanted maGSCs into normal hearts of mice and found that maGSCs were able to proliferate and differentiate. No tumor formation was found up to 1 month after cell transplantation. Taken together, we believe that maGSCs provide a new source of distinct types of cardiomyocytes for basic research and potential therapeutic application.

Generation and Characterization of Functional Cardiomyocytes from Rhesus Monkey Embryonic Stem Cells

Embryonic stem cells (ESCs) from mice and humans (hESCs) have been shown to be able to efficiently differentiate toward cardiomyocytes (CMs). Because murine ESCs and hESCs do not allow for establishment of pre-clinical allogeneic transplantation models, the aim of our study was to generate functional CMs from rhesus monkey ESCs (rESCs). Although formation of ectodermal and neuronal/glial cells appears to be the default pathway of the rESC line R366.4, we were able to change this commitment and to direct generation of endodermal/mesodermal cells and further differentiation toward CMs. Differentiation of rESCs resulted in an average of 18% of spontaneously contracting embryoid bodies (EBs) from rESCs. Semiquantitative reverse transcription-polymerase chain reaction analyses demonstrated expression of marker genes typical for endoderm, mesoderm, cardiac mesoderm, and CMs, including brachyury, goosecoid, Tbx-5, Tbx-20, Mesp1, Nkx2.5, GATA-4, FOG-2, Mlc2a, MLC2v, ANF, and alpha-MHC in rESC-derived CMs. Immunohistological and ultrastructural studies showed expression of CM-typical proteins, including sarcomeric actinin, troponin T, titin, connexin 43, and cross-striated muscle fibrils. Electrophysiological studies by means of multielectrode arrays revealed evidence of functionality, electrical coupling, and beta-adrenergic signaling of the generated CMs. This is the first study demonstrating generation of functional CMs derived from rESCs. In contrast to hESCs, rESCs allow for establishment of pre-clinical allogeneic transplantation models. Moreover, rESC-derived CMs represent a cell source for the development of high-throughput assays for cardiac safety pharmacology.