Kidney organoid reproducibility across multiple human iPSC lines and diminished off target cells after transplantation revealed by single cell transcriptomics
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PROVIDER: PRJNA562165 | ENA |
REPOSITORIES: ENA
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Project description:Human iPSC-derived kidney organoids have the potential to revolutionize discovery, but assessing their consistency and reproducibility across iPSC lines, and reducing the generation of off-target cells remain an open challenge. Here, we used single cell RNA-Seq (scRNA-Seq) to profile 450,118 cells to show that organoid composition and development are comparable to human fetal and adult kidneys. Although cell classes were largely reproducible across iPSC lines, time points, protocols, and replicates, cell proportions were variable between different iPSC lines. Off-target cell proportions were the most variable. Prolonged in vitro culture did not alter cell types, but organoid transplantation under the mouse kidney capsule diminished off-target cells. Our work shows how scRNA-seq can help score organoids for reproducibility, faithfulness and quality, that kidney organoids derived from different iPSC lines are comparable surrogates for human kidney, and that transplantation enhances their formation by diminishing off-target cells.
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Project description:The kidney differentiation protocol takes induced pluripotent stem cells through to kidney organoid via directed differentiation in approximately 25 days. The cells are grown in a monolayer in a dish for seven days and are subjected to growth factors before being pelleted on day seven. The organoids then continue to differentiate as a 3D structure, with at least 8 distinct kidney cell types identifiable around day 18. Here we investigate the reproducibility of the kidney differentiation protocol using RNA sequencing from day 18 organoids. Organoid differentiations were performed in separate batches, and within one of the batches, from three separate vials. The resulting RNA-seq data was analysed to identify genes with highly variable expression between batches and vials.
2018-10-31 | GSE89044 | GEO
Project description:Transcriptomic profiles of 6 commercially-available human patient-derived gastrointestinal organoid lines were obtained and compared to transcriptomic profile of a commercially available human iPSC-induced colon organoid line. Transcriptomic profile of iPSC-derived human colon organoid line was compared after culture in either Corning growth-factor-reduced Matrigel (Corning 356231) or MilliporeSigma growth-factor-reduced ECMGel (E6909)
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Project description:Matrigel, a mouse tumor extracellular matrix (ECM) protein mixture, is an indispensable component of most organoid tissue culture. However, it has limited the utility of organoids for drug development and regenerative medicine due to its tumor-derived origin, batch-to22 batch variation, high cost, and safety issues. Here, we demonstrate that gastrointestinal (GI) tissue-derived ECM hydrogels are a suitable substitute for Matrigel in GI organoid culture. We found that the development and function of GI organoids grown in GI ECM hydrogels are comparable or often superior to those in Matrigel. In addition, GI ECM hydrogels enabled long-term subculture and transplantation of GI organoids by providing GI tissue-mimetic microenvironments. Tissue-specific and age-related ECM profiles of GI ECM hydrogels that affect organoid development were also elucidated through proteomic analysis. Together, our results suggest that ECM hydrogels derived from decellularized GI tissues are an effective alternative to the current gold standard, Matrigel, and produce organoids suitable for GI disease modeling, drug development, and tissue regeneration.
2022-01-31 | PXD023705 | Pride
Project description:Reproducibility in molecular and cellular studies is fundamental to scientific discovery. To establish the reproducibility of a well-defined long term neuronal differentiation protocol, we repeated the cellular and molecular comparison of the same two iPSC lines across five distinct laboratories. Despite uncovering acceptable variability within individual laboratories, we detect poor cross-site reproducibility of the differential gene expression signature between these two lines. Factor analysis identifies the laboratory as the largest source of variation along with several variation-inflating confounds such as passaging effects and progenitor storage. Single cell transcriptomics shows substantial cellular heterogeneity underlying inter-laboratory variability and being responsible for biases in differential gene expression inference. Factor analysis-based normalization of the combined dataset can remove the nuisance technical effects, enabling the execution of robust hypothesis generating studies. Our study shows that multi-center collaborations can expose systematic biases and identify critical factors to be standardized when publishing novel protocols, contributing to increased cross-site reproducibility.
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Project description:We report a fully human UC-derived organoid system that integrates ECM hydrogel, MSCs and reprogrammed hepatocytes, achieving the treatment of acute hepatic failure in case of extreme failure.To explore the mechanism of our organoid in hepatic failure, we performed single cell RNA sequencing of transplanted organoids as well as the original organoid prior to transplantation, we have demonstrated that MSCs were primarily responsible for inhibiting the secretion of inflammatory-inducing cytokines and promoting the secretion of inflammation-inhibiting cytokines by recruiting immune cells migrating into organoid to triggering immune response.
2021-12-31 | GSE165944 | GEO
Project description:Reproducibility in molecular and cellular studies is fundamental to scientific discovery. To establish the reproducibility of a well-defined long term neuronal differentiation protocol, we repeated the cellular and molecular comparison of the same two iPSC lines across five distinct laboratories. Despite uncovering acceptable variability within individual laboratories, we detect poor cross-site reproducibility of the differential gene expression signature between these two lines. Factor analysis identifies the laboratory as the largest source of variation along with several variation-inflating confounds such as passaging effects and progenitor storage. Single cell transcriptomics shows substantial cellular heterogeneity underlying inter-laboratory variability and being responsible for biases in differential gene expression inference. Factor analysis-based normalization of the combined dataset can remove the nuisance technical effects, enabling the execution of robust hypothesis generating studies. Our study shows that multi-center collaborations can expose systematic biases and identify critical factors to be standardized when publishing novel protocols, contributing to increased cross-site reproducibility.
2018-08-20 | GSE118734 | GEO
Project description:Organoid technology provides a revolutionary paradigm towards therapy, yet to be applied in humans mainly because of the reproducibility and scalability challenges. Here, we overcome these limitations by evolving scalable organ bud production platform entirely from human induced pluripotent stem cells (iPSC). By conducting massive ‘reverse’ screen experiments, we identified effective triple progenitor populations for generating liver buds in a highly reproducible manner: hepatic endoderm, endothelial and septum mesenchyme progenitors. Furthermore, we achieved human scalability by developing an omni-well-array culture platform for mass-producing homogenous and miniaturized liver buds on a clinically relevant large scale (>108-cell scale). Vascularized and functional liver tissues generated entirely from iPSC significantly improved subsequent hepatic functionalization potentiated by stage-matched developmental progenitor interactions, enabling functional rescue against acute liver failure via transplantation. Overall, our study provides a stringent manufacture platform for multi-cellular organoid supply, thus facilitating clinical and pharmaceutical applications especially for the treatment of liver diseases through multi-industrial collaborations.
2018-01-10 | GSE108937 | GEO
Project description:The kidney organoid differentiation protocol takes induced pluripotent stem cells through to kidney organoid via directed differentiation in approximately 25 days. The cells are grown in a monolayer in a dish for seven days and are subjected to growth factors before being pelleted on day seven. The organoids then continue to differentiate as a 3D structure, with at least 8 distinct kidney cell types identifiable around day 18. Here the EPCAM+ epithelial fraction was isolated from day 25 kidney organoids using MACS-enrichment and RNA-sequencing libraries generated.
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Project description:Human induced pluripotent stem cell (hiPSC)-derived cardiovascular cells are promising cell source for cell therapy to repair the heart. Cardiac microtissue consisted of cardiomyocytes and fibroblast cells exhibited much better physiological functions. How different cardiovascular cell types interact and evolve in 3D microenvironment is unknown. In this study, we performed single-cell transcriptome profiling of hiPSC-derived mini-cardiac organoid consisted of cardiomyocytes, endothelial cells and smooth muscle cells. Our analysis showed that cardiac fibroblasts emerged spontaneously in 3D microenvironment which in turn facilitated the maturation of cardiomyocytes. HiPSC-derived cardiomyocytes, endothelial cells and smooth muscle cells assembled into mini-cardiac organoid in collagen-matrigel after 2 weeks. Single-cell study uncovered significant cell fate shift and improvement in cardiomyocyte maturation status upon-multilineage co-culture. Ligand-receptor analysis identified DLK1-Notch signaling to be one of the most upregulated pathways in the fibroblast population. Modulate the activity of DLK1-Notch signaling affected the assembly of the mini-cardiac organoid and the expression of immune regulatory genes. Interestingly, transplantation of trilineage mini-cardiac organoid into a rat model of myocardial infarction leads to significant improvement of cardiac function. Collectively, our single-cell analysis of mini-cardiac organoid provided rich information about cell fate dynamics and multilineage cross-talks occurred in the 3D microenvironment, which bring new insight on the molecular mechanism that promotes cardiomyocyte maturation and heart repair.
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