Generation and Maturation of Human iPSC-derived 3D Organotypic Cardiac Microtissues in Long-Term Culture
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ABSTRACT:
PROVIDER: PRJNA863293 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR20697534.fastq.gz | Fastqsanger.gz | |||
| SRR20697535.fastq.gz | Fastqsanger.gz | |||
| SRR20697536.fastq.gz | Fastqsanger.gz | |||
| SRR20697537.fastq.gz | Fastqsanger.gz | |||
| SRR20697538.fastq.gz | Fastqsanger.gz |
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Project description:We have established a scaffold-free protocol to generate multicellular, beating and self-organized human organotypic cardiac microtissues (hOCMTs) in vitro from human induced pluripotent stem cells (hiPSCs) that can be cultured for long term. This is achieved by differentiation of hiPSC in 2D monolayer culture towards cardiovascular lineage, followed by further aggregation on low-attachment culture dishes in 3D. The generated hOCMTs containing multiple cell types that physiologically compose the heart, gradually self-organize and beat without external stimuli for more than 50 days. We have shown that 3D hOCMTs display improved cardiac specification, survival and maturation as compared to standard monolayer cardiac differentiation in 2D. We also confirmed the functionality of hOCMTs by metabolic flux analysis and their response to cardioactive and cardiotoxic drugs in long term culture. This study could help to develop more physiologically-relevant cardiac tissue models, and represent a powerful platform for future translational research in cardiovascular biology.
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Project description:Cardiovascular diseases remain the leading cause of death worldwide; hence there is an increasing focus on developing physiologically relevant in vitro cardiovascular tissue models suitable for studying personalized medicine and pre-clinical tests. Despite recent advances, models that reproduce both tissue complexity and maturation are still limited. We have established a scaffold-free protocol to generate multicellular, beating human cardiac microtissues in vitro from hiPSCs-namely human organotypic cardiac microtissues (hOCMTs)-that show some degree of self-organization and can be cultured for long term. This is achieved by the differentiation of hiPSC in 2D monolayer culture towards cardiovascular lineage, followed by further aggregation on low-attachment culture dishes in 3D. The generated hOCMTs contain multiple cell types that physiologically compose the heart and beat without external stimuli for more than 100 days. We have shown that 3D hOCMTs display improved cardiac specification, survival and metabolic maturation as compared to standard monolayer cardiac differentiation. We also confirmed the functionality of hOCMTs by their response to cardioactive drugs in long-term culture. Furthermore, we demonstrated that they could be used to study chemotherapy-induced cardiotoxicity. Due to showing a tendency for self-organization, cellular heterogeneity, and functionality in our 3D microtissues over extended culture time, we could also confirm these constructs as human cardiac organoids (hCOs). This study could help to develop more physiologically-relevant cardiac tissue models, and represent a powerful platform for future translational research in cardiovascular biology.
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Project description:Cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) are functionally immature, but this is improved by incorporation into engineered tissues or forced contraction. Here, we showed that tri-cellular combinations of hiPSC-derived CMs, cardiac fibroblasts (CFs), and cardiac endothelial cells also enhance maturation in easily constructed, scaffold-free, three-dimensional microtissues (MTs). hiPSC-CMs in MTs with CFs showed improved sarcomeric structures with T-tubules, enhanced contractility, and mitochondrial respiration and were electrophysiologically more mature than MTs without CFs. Interactions mediating maturation included coupling between hiPSC-CMs and CFs through connexin 43 (CX43) gap junctions and increased intracellular cyclic AMP (cAMP). Scaled production of thousands of hiPSC-MTs was highly reproducible across lines and differentiated cell batches. MTs containing healthy-control hiPSC-CMs but hiPSC-CFs from patients with arrhythmogenic cardiomyopathy strikingly recapitulated features of the disease. Our MT model is thus a simple and versatile platform for modeling multicellular cardiac diseases that will facilitate industry and academic engagement in high-throughput molecular screening.
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Project description:Human-iPSC-Derived Cardiac Stromal Cells Enhance Maturation in 3D Cardiac Microtissues and Reveal Non-cardiomyocyte Contributions to Heart Disease
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Project description:Human-iPSC-Derived Cardiac Stromal Cells Enhance Maturation in 3D Cardiac Microtissues and Reveal Non-cardiomyocyte Contributions to Heart Disease
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Project description:Organotypic human tissue models constitute promising systems to facilitate drug discovery and development. They allow to maintain native cellular phenotypes and functions, which enables long-term pharmacokinetic and toxicity studies, as well as phenotypic screening. To trace relevant phenotypic changes back to specific targets or signaling pathways, comprehensive proteomic profiling is the gold-standard. A multitude of proteomic workflows have been applied on 3D tissue models to quantify their molecular phenotypes; however, their impact on analytical results and biological conclusions has not been evaluated. Here, we compared the performance of twelve mass spectrometry-based global proteomic workflows that differed in the amount of cellular input, lysis protocols and quantification methods for the analysis of primary human liver spheroids. We find that the different protocols differ majorly in the total number and subcellular compartment bias of identified proteins, which is particularly relevant for the reliable quantification of transporters and drug metabolizing enzymes. However, using a model of non-alcoholic fatty liver disease, we show that critical disease pathways are robustly identified using a standardized high throughput-compatible workflow based on thermal lysis, even using only individual spheroids (1,500 cells) as input. The results increase the applicability of proteomic profiling to phenotypic screens in organotypic microtissues and provides a scalable platform for deep phenotyping from limited biological material.
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Project description:Single-cell RNA-seq: We used single-cell RNAseq to investigate the maturation of astrocytes within human cortical spheroids Bulk RNA-seq: Bulk sequencing from astrocytes and neurons purified (via immunopanning) from iPSC-derived coritical spheroids at varying in vitro differentiation states
2017-07-31 | GSE99951 | GEO
Project description:The generation of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offers an unlimited source of patient-specific human cardiomyocytes. However, the use of iPSC-CM in vitro-models to guide the clinic selection of particular drugs for individual patients remains a vision. A major limitation represents the immature phenotype of iPSC-CMs, which alters their sensitivity towards physiological and pathophysiological stimuli, cardioactive drugs or toxic substances, in comparison to adult cardiomyocytes (CMs). In this study, we aim to generate iPSC-CMs with an advanced maturation state by combining the use of MM with the alignment of the cells on nanopatterned surfaces (NP) and induction of an increased contractile workload by electrical stimulation (ES). Under the combined influence of MM, NP and ES, iPSC-CMs develop a more complex cellular structure, increased mitochondrial content and enhanced electrophysiological properties. Furthermore, the response of iPSC-CMs matured under influence of MM, NP and ES to isoprenaline as well as verapamil differs to less mature iPSC-CMs cultured in B27-medium. Taken together, our results reveal that the combination of MM, NP and ES strongly improves the maturation state of iPSC-CMs and that this advanced maturation state critically affects the cell behavior in functional studies as well as response to cardioactive drugs.
| EGAS00001007491 | EGA