Human iPS-derived pre-epicardial cells direct cardiomyocyte aggregation expansion and organization in vitro.
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ABSTRACT:
SUBMITTER: Tan JJ
PROVIDER: S-EPMC8370973 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Epicardial cells (EpiCs) are necessary for myocardium formation, yet little is known about crosstalk between EpiCs and cardiomyocytes (CMs) during development and the potential impact of EpiCs on CM maturation. To investigate the effects of EpiCs on CM commitment and maturation, we differentiated human pluripotent stem cells (hPSCs) to cardiac progenitor cells (CPCs) and EpiCs, and cocultured EpiCs and CPCs for two weeks. When EpiCs were allowed to form epicardial-derived cells, we observed increased expression of cTnI in developing CMs. In the presence of the TGFβ inhibitor A83-01, EpiCs remained in the epicardial state and induced CM proliferation, increased MLC2v expression, and led to less organized sarcomeres. These effects were not observed if CPCs were treated with EpiC-conditioned medium or if CPCs were indirectly cocultured with EpiCs. Finally, single cell RNA sequencing identified that EpiC-CPC coculture had bi-directional effects on transcriptional programs in EpiCs and CMs, and biased EpiC lineages from a SFRP2-enriched population to a DLK1- or C3-enriched population. This work suggests important crosstalk between EpiCs and CMs during differentiation which can be used to influence cell fate and improve the ability to generate cardiac cells and tissues for in vitro models and development of cardiac cellular therapies.
| S-EPMC8766908 | biostudies-literature
Project description:The purpose of this study was to fabricate pulsatile tubular cardiac tissue using cell sheet based-tissue engineering. First, we fabricated human induced pluripotent stem cell (hiPSc)-derived cardiomyocyte sheets and normal human dermal fibroblast (NHDF) sheets which are harvested from temperature responsive culture dishes only by lowering the temperature. Then tubular cardiac tissues are formed by wrapping one hiPSc-derived cardiomyocyte sheet and three NHDF sheets around an octagonal column, and both ends of the tubular tissue were covered with fibrin and collagen gel. The octagonal column with the tubular tissue was connected to an in vitro circulation system in a culture box. After four-day culture, the cardiac tissue survived and pulsated spontaneously in the circulation system. Furthermore, the analysis with a Millar catheter inserted into the cardiac tubes revealed significant inner pressure changes generated by their beating. In addition, the tubular cardiac tissue pulsated in response to the electrical stimulation. Although histological analyses demonstrated that cardiac troponin T-positive cells stratified the inner surface of the tubular tissues, gene expression analyses showed an immature state of these cardiomyocytes. Thus, cell sheet-based tissue engineering realized human pulsatile tubular cardiac tissue fabrication and we believe that these tubular cardiac tissues should contribute to future drug screening and regenerative therapy for heart diseases.
| S-EPMC6813561 | biostudies-literature
Project description:The epicardium and its derivatives provide trophic and structural support for the developing and adult heart. Here we tested the ability of human embryonic stem cell (hESC)-derived epicardium to augment the structure and function of engineered heart tissue in vitro and to improve efficacy of hESC-cardiomyocyte grafts in infarcted athymic rat hearts. Epicardial cells markedly enhanced the contractility, myofibril structure and calcium handling of human engineered heart tissues, while reducing passive stiffness compared with mesenchymal stromal cells. Transplanted epicardial cells formed persistent fibroblast grafts in infarcted hearts. Cotransplantation of hESC-derived epicardial cells and cardiomyocytes doubled graft cardiomyocyte proliferation rates in vivo, resulting in 2.6-fold greater cardiac graft size and simultaneously augmenting graft and host vascularization. Notably, cotransplantation improved systolic function compared with hearts receiving either cardiomyocytes alone, epicardial cells alone or vehicle. The ability of epicardial cells to enhance cardiac graft size and function makes them a promising adjuvant therapeutic for cardiac repair.
| S-EPMC6824587 | biostudies-literature
Project description:BackgroundCertain nonmammalian species such as zebrafish have an elevated capacity for innate heart regeneration. Understanding how heart regeneration occurs in these contexts can help illuminate cellular and molecular events that can be targets for heart failure prevention or treatment. The epicardium, a mesothelial tissue layer that encompasses the heart, is a dynamic structure that is essential for cardiac regeneration in zebrafish. The extent to which different cell subpopulations or states facilitate heart regeneration requires research attention.MethodsTo dissect epicardial cell states and associated proregenerative functions, we performed single-cell RNA sequencing and identified 7 epicardial cell clusters in adult zebrafish, 3 of which displayed enhanced cell numbers during regeneration. We identified paralogs of hapln1 as factors associated with the extracellular matrix and largely expressed in cluster 1. We assessed HAPLN1 expression in published single-cell RNA sequencing data sets from different stages and injury states of murine and human hearts, and we performed molecular genetics to determine the requirements for hapln1-expressing cells and functions of each hapln1 paralog.ResultsA particular cluster of epicardial cells had the strongest association with regeneration and was marked by expression of hapln1a and hapln1b. The hapln1 paralogs are expressed in epicardial cells that enclose dedifferentiated and proliferating cardiomyocytes during regeneration. Induced genetic depletion of hapln1-expressing cells or genetic inactivation of hapln1b altered deposition of the key extracellular matrix component hyaluronic acid, disrupted cardiomyocyte proliferation, and inhibited heart regeneration. We also found that hapln1-expressing epicardial cells first emerge at the juvenile stage, when they associate with and are required for focused cardiomyocyte expansion events that direct maturation of the ventricular wall.ConclusionsOur findings identify a subset of epicardial cells that emerge in postembryonic zebrafish and sponsor regions of active cardiomyogenesis during cardiac growth and regeneration. We provide evidence that, as the heart achieves its mature structure, these cells facilitate hyaluronic acid deposition to support formation of the compact muscle layer of the ventricle. They are also required, along with the function of hapln1b paralog, in the production and organization of hyaluronic acid-containing matrix in cardiac injury sites, enabling normal cardiomyocyte proliferation and muscle regeneration.
| S-EPMC9308751 | biostudies-literature
Project description:One of the major challenges in cell-based cardiac regenerative medicine is the in vitro construction of three-dimensional (3D) tissues consisting of induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) and a blood vascular network supplying nutrients and oxygen throughout the tissue after implantation. We have successfully built a vascularized iPSC-CM 3D-tissue using our validated cell manipulation technique. In order to evaluate an availability of the 3D-tissue as a biomaterial, functional morphology of the tissues was examined by light and transmission electron microscopy through their implantation into the rat infarcted heart. Before implantation, the tissues showed distinctive myofibrils within iPSC-CMs and capillary-like endothelial tubes, but their profiles were still like immature. In contrast, engraftment of the tissues to the rat heart led the iPSC-CMs and endothelial tubes into organization of cell organelles and junctional apparatuses and prompt development of capillary network harboring host blood supply, respectively. A number of capillaries in the implanted tissues were derived from host vascular bed, whereas the others were likely to be composed by fusion of host and implanted endothelial cells. Thus, our vascularized iPSC-CM 3D-tissues may be a useful regenerative paradigm which will require additional expanded and long-term studies.
| S-EPMC5651879 | biostudies-other
Project description:Epicardial cells (EpiCs) form the epicardium, the outer layer of the heart surrounding the myocardium. During development and in response to injury EpiCs undergo an epithelial to mesenchymal transition (EMT) migrating inward to create fibroblasts and smooth muscle cells in the myocardium. EpiCs have been shown to be necessary for proper myocardium formation, yet little is known about crosstalk between EpiCs and cardiomyocyte (CMs) and the potential impact of EpiCs on CM maturation. Here, we differentiated human pluripotent stem cells (hPSCs) to both cardiac progenitor cells (CPCs) and EpiCs, and cocultured EpiCs and CPCs in the presence of A83-01, to inhibit TGFβ signaling and EMT, for two weeks.
2021-10-12 | GSE168956 | GEO
Project description:Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13(high)CD133(+) cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4? and ?-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development.
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Project description:Atrial fibrillation (AF) is the most common arrhythmia in the United States, affecting approximately 1 in 10 adults, and its prevalence is expected to rise as the population ages. Treatment options for AF are limited; moreover, the development of new treatments is hindered by limited (1) knowledge regarding human atrial electrophysiological endpoints (e.g., conduction velocity [CV]) and (2) accurate experimental models. Here, we measured the CV and refractory period, and subsequently calculated the conduction wavelength, in vivo (four subjects with AF and four controls), and ex vivo (atrial slices from human hearts). Then, we created an in vitro model of human atrial conduction using induced pluripotent stem (iPS) cells. This model consisted of iPS-derived human atrial cardiomyocytes plated onto a micropatterned linear 1D spiral design of Matrigel. The CV (34-41 cm/s) of the in vitro model was nearly five times faster than 2D controls (7-9 cm/s) and similar to in vivo (40-64 cm/s) and ex vivo (28-51 cm/s) measurements. Our iPS-derived in vitro model recapitulates key features of in vivo atrial conduction and may be a useful methodology to enhance our understanding of AF and model patient-specific disease.
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Project description:Mesenchymal stem cell (MSC)-based therapy has shown great promises in various animal disease models. However, this therapeutic potency has not been well claimed when applied to human clinical trials. This is due to both the availability of MSCs at the time of administration and lack of viable expansion strategies. MSCs are very susceptible to in vitro culture environment and tend to adapt the microenvironment which could lead to cellular senescence and aging. Therefore, extended in vitro expansion induces loss of MSC functionality and its clinical relevance. To combat this effect, this work assessed a novel cyclical aggregation as a means of expanding MSCs to maintain stem cell functionality. The cyclical aggregation consists of an aggregation phase and an expansion phase by replating the dissociated MSC aggregates onto planar tissue culture surfaces. The results indicate that cyclical aggregation maintains proliferative capability, stem cell proteins, and clonogenicity, and prevents the acquisition of senescence. To determine why aggregation was responsible for this phenomenon, the integrated stress response pathway was probed with salubrial and GSK-2606414. Treatment with salubrial had no significant effect, while GSK-2606414 mitigated the effects of aggregation leading to in vitro aging. This method holds the potential to increase the clinical relevance of MSC therapeutic effects from small model systems (such as rats and mice) to humans, and may open the potential of patient-derived MSCs for treatment thereby removing the need for immunosuppression.
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Project description:Morphology and function of the nervous system is maintained via well-coordinated processes both in central and peripheral nervous tissues, which govern the homeostasis of organs/tissues. Impairments of the nervous system induce neuronal disorders such as peripheral neuropathy or cardiac arrhythmia. Although further investigation is warranted to reveal the molecular mechanisms of progression in such diseases, appropriate model systems mimicking the patient-specific communication between neurons and organs are not established yet. In this study, we reconstructed the neuronal network in vitro either between neurons of the human induced pluripotent stem (iPS) cell derived peripheral nervous system (PNS) and central nervous system (CNS), or between PNS neurons and cardiac cells in a morphologically and functionally compartmentalized manner. Networks were constructed in photolithographically microfabricated devices with two culture compartments connected by 20 microtunnels. We confirmed that PNS and CNS neurons connected via synapses and formed a network. Additionally, calcium-imaging experiments showed that the bundles originating from the PNS neurons were functionally active and responded reproducibly to external stimuli. Next, we confirmed that CNS neurons showed an increase in calcium activity during electrical stimulation of networked bundles from PNS neurons in order to demonstrate the formation of functional cell-cell interactions. We also confirmed the formation of synapses between PNS neurons and mature cardiac cells. These results indicate that compartmentalized culture devices are promising tools for reconstructing network-wide connections between PNS neurons and various organs, and might help to understand patient-specific molecular and functional mechanisms under normal and pathological conditions.
| S-EPMC4744060 | biostudies-literature