A dual cardiomyocyte reporter model derived from human pluripotent stem cells.
Ontology highlight
ABSTRACT:
SUBMITTER: Jiang Y
PROVIDER: S-EPMC8164304 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) raise many possibilities for cardiac research but they exhibit an immature phenotype, which influences experimental outcomes. The aim of our research is to investigate the effects of a topographical gradient substrate on the morphology and function of commercially available hiPSC-CM. The lateral dimensions the microgrooves on the substrate varied from 8 to 100 μm space between the 8 μm grooves on one axis and from ∼5 nm to ∼1 μm in depth on the other axis. Cells were seeded homogeneously across the substrate and according to the manufacturers protocols. At days 4 and 10, measures of eccentricity, elongation, orientation, sarcomere length (SL), and contractility of the hiPSC-CM were taken. Only the deepest and widest region (8-30 μm wide and 0.85-1 μm deep) showed a significantly higher percentage of hiPSC-CM with an increased eccentricity (31.3 ± 6.4%), elongation (10.4 ± 4.3%), and orientation (<10°) (32.1 ± 2.7%) when compared with the control (flat substrate) (15.8 ± 5.0%, 3.4 ± 2.7%, and 10.6 ± 1.1%, respectively). Additionally, during stimulus-induced contraction, the relaxation phase of the twitch was prolonged (400 ms) compared to nonelongated cells (200 ms). These findings support the potential use of dual microgradient substrates to investigate substrate topographies that stimulate migration and/or maturation of hiPSC-CM.
| S-EPMC5155309 | biostudies-literature
Project description:Induced pluripotent stem cell (iPSC)-based disease modeling has a great potential for uncovering the mechanisms of pathogenesis, especially in the case of neurodegenerative diseases where disease-susceptible cells can usually not be obtained from patients. So far, the iPSC-based modeling of neurodegenerative diseases has mainly focused on neurons because the protocols for generating astrocytes from iPSCs have not been fully established. The growing evidence of astrocytes' contribution to neurodegenerative diseases has underscored the lack of iPSC-derived astrocyte models. In the present study, we established a protocol to efficiently generate iPSC-derived astrocytes (iPasts), which were further characterized by RNA and protein expression profiles as well as functional assays. iPasts exhibited calcium dynamics and glutamate uptake activity comparable to human primary astrocytes. Moreover, when co-cultured with neurons, iPasts enhanced neuronal synaptic maturation. Our protocol can be used for modeling astrocyte-related disease phenotypes in vitro and further exploring the contribution of astrocytes to neurodegenerative diseases.
| S-EPMC7763297 | biostudies-literature
Project description:Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) exhibit a fetal phenotype that limits in vitro and therapeutic applications. Strategies to promote cardiomyocyte maturation have focused interventions on differentiated hPSC-CMs, but this study tests priming of early cardiac progenitor cells (CPCs) with polyinosinic-polycytidylic acid (pIC) to accelerate cardiomyocyte maturation. CPCs were differentiated from hPSCs using a monolayer differentiation protocol with defined small molecule Wnt temporal modulation, and pIC was added during the formation of early CPCs. pIC priming did not alter the expression of cell surface markers for CPCs (>80% KDR+/PDGFRα+), expression of common cardiac transcription factors, or final purity of differentiated hPSC-CMs (∼90%). However, CPC differentiation in basal medium revealed that pIC priming resulted in hPSC-CMs with enhanced maturity manifested by increased cell size, greater contractility, faster electrical upstrokes, increased oxidative metabolism, and more mature sarcomeric structure and composition. To investigate the mechanisms of CPC priming, RNAseq revealed that cardiac progenitor-stage pIC modulated early Notch signaling and cardiomyogenic transcriptional programs. Chromatin immunoprecipitation of CPCs showed that pIC treatment increased deposition of the H3K9ac activating epigenetic mark at core promoters of cardiac myofilament genes and the Notch ligand, JAG1. Inhibition of Notch signaling blocked the effects of pIC on differentiation and cardiomyocyte maturation. Furthermore, primed CPCs showed more robust formation of hPSC-CMs grafts when transplanted to the NSGW mouse kidney capsule. Overall, epigenetic modulation of CPCs with pIC accelerates cardiomyocyte maturation enabling basic research applications and potential therapeutic uses. Stem Cells 2019;37:910-923.
| S-EPMC6599574 | biostudies-literature
Project description:BackgroundThe Δ160E mutation in TNNT2, which encodes troponin T, is a rare pathogenic variant identified in patients with hypertrophic cardiomyopathy and is associated with poor prognosis. Thus, a convenient human model recapitulating the pathological phenotype caused by TNNT2 Δ160E is required for therapeutic development.MethodsWe identified a heterozygous in-frame deletion mutation (c.478_480del, p.Δ160E) in TNNT2 in a patient with familial hypertrophic cardiomyopathy showing progressive left ventricular systolic dysfunction, leading to advanced heart failure. To investigate the pathological phenotype caused by Δ160E, we generated a set of isogenic induced pluripotent stem cells carrying the heterozygous Δ160E, homozygously corrected or homozygously introduced Δ160E using genome editing and differentiated them into cardiomyocytes (Hetero-Δ160E-, wild type-, and Homo-Δ160E-induced pluripotent stem cells [iPSC]-derived cardiomyocytes [iPSC-CMs]).ResultsHetero-Δ160E-iPSC-CMs exhibited prolonged calcium decay, relaxation impairment, and hypertrophy compared to wild type-iPSC-CMs. Notably, these phenotypes were further exacerbated in Homo-Δ160E-iPSC-CMs. Overexpression of R-GECO-fused Δ160E mutant troponin T prolonged decay time and time to peak of the myofilament-localized calcium transient in iPSC-CMs, indicating that sarcomeric calcium retention with Δ160E may affect intracellular calcium concentration. High-content imaging analysis detected remarkable nuclear translocation of NFATc1, especially in Homo-Δ160E-iPSC-CMs, indicating that the Δ160E mutation promotes hypertrophic signaling pathway in a dose-dependent manner. Increased phosphorylation of CaMKIIδ (calcium/calmodulin-dependent protein kinase IIδ) and phospholamban at Thr17 was observed in Homo- and Hetero-Δ160E-iPSC-CMs. Epigallocatechin-3-gallate, a calcium desensitizing compound, shortened prolonged calcium decay and relaxation duration in Δ160E-iPSC-CMs.ConclusionsIsogenic iPSC-CMs recapitulate the prolonged calcium decay, relaxation impairment, and subsequent calcium-regulated signaling pathways caused by the TNNT2 Δ160E mutation and can serve as a human model for therapeutic development to prevent hypertrophic cardiomyopathy pathology.
| S-EPMC9584061 | biostudies-literature
Project description:Human induced pluripotent stem cells (hiPSCs) can differentiate into neurons and glia via neural progenitor cells and are widely used for neurogenic studies. However, directly visualizing the transition status during the neural differentiation of live cells is difficult. Here, targeting NEUROG2 (NGN2) and TUBB3 as markers of neurogenic cells and neurons, respectively, we established TUBB3EGFP/NGN2TagRFP dual-reporter hiPSCs using CRISPR-Cas9 technology. We induced the differentiation of cortical neurons from dual-reporter hiPSCs, successfully visualizing cell-fate conversion in two-dimensional (2D) culture and 3D organoids. The reporter cells were used to monitor drug effects to enhance neural induction, responses to gene knockdown, transplantation to the embryonic mouse brain, and live imaging at single-cell resolution. Notably, the earliest REELIN-positive neurons showed a distinctive migration pattern, and their production was accelerated by HES1-function loss. Together, these results demonstrate the potential for dual-reporter hiPSCs in therapeutic neural regeneration strategies and studies on human cortical development.
| S-EPMC9481873 | biostudies-literature
Project description:Dilated cardiomyopathy (DCM) is the most common type of cardiomyopathy that account for the majority of heart failure cases. The present study aimed to reveal the underlying molecular mechanisms of DCM and provide potential biomarkers for detection of this condition. The public dataset of GSE35108 was downloaded, and 4 normal induced pluripotent stem cell (iPSC)-derived cardiomyocytes (N samples) and 4 DCM iPSC-derived cardiomyocytes (DCM samples) were utilized. Raw data were preprocessed, followed by identification of differentially expressed genes (DEGs) between N and DCM samples. Crucial functions and pathway enrichment analysis of DEGs were investigated, and protein-protein interaction (PPI) network analysis was conducted. Furthermore, a module network was extracted from the PPI network, followed by enrichment analysis. A set of 363 DEGs were identified, including 253 upregulated and 110 downregulated genes. Several biological processes (BPs), such as blood vessel development and vasculature development (FLT1 and MMP2), cell adhesion (CDH1, ITGB6, COL6A3, COL6A1 and LAMC2) and extracellular matrix (ECM)-receptor interaction pathway (CDH1, ITGB6, COL6A3, COL6A1 and LAMC2), were significantly enriched by these DEGs. Among them, MMP2, CDH1 and FLT1 were hub nodes in the PPI network, while COL6A3, COL6A1, LAMC2 and ITGB6 were highlighted in module 3 network. In addition, PENK and APLNR were two crucial nodes in module 2, which were linked to each other. In conclusion, several potential biomarkers for DCM were identified, such as MMP2, FLT1, CDH1, ITGB6, COL6A3, COL6A1, LAMC2, PENK and APLNR. These genes may serve significant roles in DCM via involvement of various BPs, such as blood vessel and vasculature development and cell adhesion, and the ECM-receptor interaction pathway.
| S-EPMC5585721 | biostudies-literature
Project description:Tendon regeneration is difficult because detailed knowledge about tendon progenitor cells (TPCs), which produce tenocytes to repair tendon tissue, has not been revealed. Mohawk homeobox (MKX) is a marker of TPCs or tenocytes, but a human pluripotent stem cell (hPSC)-based reporter system that visualizes MKX+ cells has not been developed. Here, we established an hPSC-derived MKX-tdTomato reporter cell line and tested the induction ratio of MKX-tdTomato+ cells using our stepwise/xeno-free differentiation protocol. MKX-tdTomato+ cells were generated with high efficiency and expressed tendon-specific markers, including MKX, SCX, TNMD, and COL1A1. Our MKX-tdTomato hPSC line would be a useful tool for studying the development or regeneration of tendon tissue.
| S-EPMC9652995 | biostudies-literature
Project description:Reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs) has revolutionized the complex scientific field of disease modelling and personalized therapy. Cardiac differentiation of human iPSCs into cardiomyocytes (hiPSC-CMs) has been used in a wide range of healthy and disease models by deriving CMs from different somatic cells. Unfortunately, hiPSC-CMs have to be improved because existing protocols are not completely able to obtain mature CMs recapitulating physiological properties of human adult cardiac cells. Therefore, improvements and advances able to standardize differentiation conditions are needed. Lately, evidences of an epigenetic memory retained by the somatic cells used for deriving hiPSC-CMs has led to evaluation of different somatic sources in order to obtain more mature hiPSC-derived CMs.
| S-EPMC7013592 | biostudies-literature
Project description:Avoiding immune rejection after allogeneic induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) transplantation is a concern. However, mesenchymal stem cells (MSCs) can suppress immune rejection. To determine whether MSC co-transplantation can reduce immune rejection after allogeneic iPSC-CM transplantation, the latter cell type, harbouring a luciferase transgene, was subcutaneously transplanted alone or together with syngeneic MSCs into BALB/c mice. Bioluminescence imaging revealed that MSC co-transplantation significantly improved graft survival (day 7: iPSC-CMs alone 34 ± 5%; iPSC-CMs with MSCs, 61 ± 7%; P = 0.008). MSC co-transplantation increased CD4 + CD25 + FOXP3 + regulatory T cell numbers, apoptotic CD8-positive T cells, and IL-10 and TGF-beta expression at the implantation site. Analysis using a regulatory T cell depletion model indicated that enhanced regulatory T cell populations in the iPSC-CM with MSC group partially contributed to the extended iPSC-CM survival. Further, MSCs affected activated lymphocytes directly through cell-cell contact, which reduced the CD8/CD4 ratio, the proportion of Th1-positive cells among CD4-positive cells, and the secretion of several inflammation-related cytokines. Syngeneic MSC co-transplantation might thus control allogeneic iPSC-CM rejection by mediating immune tolerance via regulatory T cells and cell-cell contact with activated lymphocytes; this approach has promise for cardiomyogenesis-based therapy using allogeneic iPSC-CMs for severe heart failure.
| S-EPMC7067786 | biostudies-literature
Project description:During spaceflight, the cardiovascular system undergoes remarkable adaptation to microgravity and faces the risk of cardiac remodeling. Therefore, the effects and mechanisms of microgravity on cardiac morphology, physiology, metabolism, and cellular biology need to be further investigated. Since China started constructing the China Space Station (CSS) in 2021, we have taken advantage of the Shenzhou-13 capsule to send human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) to the Tianhe core module of the CSS. In this study, hPSC-CMs subjected to space microgravity showed decreased beating rate and abnormal intracellular calcium cycling. Metabolomic and transcriptomic analyses revealed a battery of metabolic remodeling of hPSC-CMs in spaceflight, especially thiamine metabolism. The microgravity condition blocked the thiamine intake in hPSC-CMs. The decline of thiamine utilization under microgravity or by its antagonistic analog amprolium affected the process of the tricarboxylic acid cycle. It decreased ATP production, which led to cytoskeletal remodeling and calcium homeostasis imbalance in hPSC-CMs. More importantly, in vitro and in vivo studies suggest that thiamine supplementation could reverse the adaptive changes induced by simulated microgravity. This study represents the first astrobiological study on the China Space Station and lays a solid foundation for further aerospace biomedical research. These data indicate that intervention of thiamine-modified metabolic reprogramming in human cardiomyocytes during spaceflight might be a feasible countermeasure against microgravity.
| S-EPMC10999445 | biostudies-literature