15 research outputs found
Modelling renal defects in Bardet-Biedl syndrome patients using human iPS cells
Bardet-Biedl syndrome (BBS) is a ciliopathy with pleiotropic effects on multiple tissues, including the kidney. Here we have compared renal differentiation of iPS cells from healthy and BBS donors. High content image analysis of WT1-expressing kidney progenitors showed that cell proliferation, differentiation and cell shape were similar in healthy, BBS1, BBS2, and BBS10 mutant lines. We then examined three patient lines with BBS10 mutations in a 3D kidney organoid system. The line with the most deleterious mutation, with low BBS10 expression, expressed kidney marker genes but failed to generate 3D organoids. The other two patient lines expressed near normal levels of BBS10 mRNA and generated multiple kidney lineages within organoids when examined at day 20 of organoid differentiation. However, on prolonged culture (day 27) the proximal tubule compartment degenerated. Introducing wild type BBS10 into the most severely affected patient line restored organoid formation, whereas CRISPR-mediated generation of a truncating BBS10 mutation in a healthy line resulted in failure to generate organoids. Our findings provide a basis for further mechanistic studies of the role of BBS10 in the kidney
Imaging-Based Screen Identifies Laminin 411 as a Physiologically Relevant Niche Factor with Importance for i-Hep Applications.
Use of hepatocytes derived from induced pluripotent stem cells (i-Heps) is limited by their functional differences in comparison with primary cells. Extracellular niche factors likely play a critical role in bridging this gap. Using image-based characterization (high content analysis; HCA) of freshly isolated hepatocytes from 17 human donors, we devised and validated an algorithm (Hepatocyte Likeness Index; HLI) for comparing the hepatic properties of cells against a physiological gold standard. The HLI was then applied in a targeted screen of extracellular niche factors to identify substrates driving i-Heps closer to the standard. Laminin 411, the top hit, was validated in two additional induced pluripotent stem cell (iPSC) lines, primary tissue, and an in vitro model of α1-antitrypsin deficiency. Cumulatively, these data provide a reference method to control and screen for i-Hep differentiation, identify Laminin 411 as a key niche protein, and underscore the importance of combining substrates, soluble factors, and HCA when developing iPSC applications
Modelling renal defects in Bardet-Biedl syndrome patients using human iPS cells
Bardet-Biedl syndrome (BBS) is a ciliopathy with pleiotropic effects on multiple tissues, including the kidney. Here we have compared renal differentiation of iPS cells from healthy and BBS donors. High content image analysis of WT1-expressing kidney progenitors showed that cell proliferation, differentiation and cell shape were similar in healthy, BBS1, BBS2, and BBS10 mutant lines. We then examined three patient lines with BBS10 mutations in a 3D kidney organoid system. The line with the most deleterious mutation, with low BBS10 expression, expressed kidney marker genes but failed to generate 3D organoids. The other two patient lines expressed near normal levels of BBS10 mRNA and generated multiple kidney lineages within organoids when examined at day 20 of organoid differentiation. However, on prolonged culture (day 27) the proximal tubule compartment degenerated. Introducing wild type BBS10 into the most severely affected patient line restored organoid formation, whereas CRISPR-mediated generation of a truncating BBS10 mutation in a healthy line resulted in failure to generate organoids. Our findings provide a basis for further mechanistic studies of the role of BBS10 in the kidney
Phenotypic plasticity in the pancreas: new triggers, new players:Cell Differentiation and DevelopmentCell Differentiation and Development
Differentiation of beta-like cells from human induced pluripotent stem cell-derived pancreatic progenitor organoids
Human induced pluripotent stem cells (hiPSCs) and organoids are important for modeling human development and disease in vitro. In this study, we describe a protocol to differentiate hiPSC toward pancreatic progenitor (PP) organoids and beta-like cells. We detail the expansion and seeding of hiPSC, PP differentiation, organoid expansion, and the differentiation of PP into beta cells. Upon differentiation, organoids contained beta, delta, and alpha cells. For complete details on the use and execution of this protocol, please refer to Cujba et al. (2022)
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Precise identification of cell states altered in disease using healthy single-cell references.
Acknowledgements: We thank M. Morgan and R. Lindeboom for the critical reading of the manuscript, and R. Elmentaite, A. Missarova and all members of the Marioni and Teichmann laboratories for valuable discussions and feedback on this project. The PBMC studies included in this work were selected using the materials from the Chan–Zuckerberg Initiative workshop on ‘Assembling Tissue References’, which were kindly shared by L. Dratva. J.C.M. acknowledges core funding from Cancer Research UK (C9545/A29580) and the European Molecular Biology Laboratory. E.D., A.-M.C., A.J.O., K.B.M and S.A.T. acknowledge Wellcome Sanger core funding (WT206194).Funder: Core funding from the European Molecular Biology Laboratory.Funder: Core funding from Wellcome Sanger Institute (WT206194)Joint analysis of single-cell genomics data from diseased tissues and a healthy reference can reveal altered cell states. We investigate whether integrated collections of data from healthy individuals (cell atlases) are suitable references for disease-state identification and whether matched control samples are needed to minimize false discoveries. We demonstrate that using a reference atlas for latent space learning followed by differential analysis against matched controls leads to improved identification of disease-associated cells, especially with multiple perturbed cell types. Additionally, when an atlas is available, reducing control sample numbers does not increase false discovery rates. Jointly analyzing data from a COVID-19 cohort and a blood cell atlas, we improve detection of infection-related cell states linked to distinct clinical severities. Similarly, we studied disease states in pulmonary fibrosis using a healthy lung atlas, characterizing two distinct aberrant basal states. Our analysis provides guidelines for designing disease cohort studies and optimizing cell atlas use
Stem/progenitor cells in normal physiology and disease of the pancreas
Though embryonic pancreas progenitors are well characterised, the existence of stem/progenitor cells in the postnatal mammalian pancreas has been long debated, mainly due to contradicting results on regeneration after injury or disease in mice. Despite these controversies, sequencing advancements combined with lineage tracing and organoid technologies indicate that homeostatic and trigger-induced regenerative responses in mice could occur. The presence of putative progenitor cells in the adult pancreas has been proposed during homeostasis and upon different stress challenges such as inflammation, tissue damage and oncogenic stress. More recently, single cell transcriptomics has revealed a remarkable heterogeneity in all pancreas cell types, with some cells showing the signature of potential progenitors. In this review we provide an overview on embryonic and putative adult pancreas progenitors in homeostasis and disease, with special emphasis on in vitro culture systems and scRNA-seq technology as tools to address the progenitor nature of different pancreatic cells
An HNF1a truncation associated with maturity-onset diabetes of the young impairs pancreatic progenitor differentiation by antagonising HNF1b function
An HNF1α truncation associated with maturity-onset diabetes of the young impairs pancreatic progenitor differentiation by antagonizing HNF1β function
The HNF1α(p291fsinsC) truncation is the most common mutation associated with maturity-onset diabetes of the young 3 (MODY3). Although shown to impair HNF1α signaling, the mechanism by which HNF1α(p291fsinsC) causes MODY3 is not fully understood. Here we use MODY3 patient and CRISPR/Cas9-engineered human induced pluripotent stem cells (hiPSCs) grown as 3D organoids to investigate how HNF1α(p291fsinsC) affects hiPSC differentiation during pancreatic development. HNF1α(p291fsinsC) hiPSCs shows reduced pancreatic progenitor and β cell differentiation. Mechanistically, HNF1α(p291fsinsC) interacts with HNF1β and inhibits its function, and disrupting this interaction partially rescues HNF1β-dependent transcription. HNF1β overexpression in the HNF1α(p291fsinsC) patient organoid line increases PDX1(+) progenitors, while HNF1β overexpression in the HNF1α(p291fsinsC) patient iPSC line partially rescues β cell differentiation. Our study highlights the capability of pancreas progenitor-derived organoids to model disease in vitro. Additionally, it uncovers an HNF1β-mediated mechanism linked to HNF1α truncation that affects progenitor differentiation and could explain the clinical heterogeneity observed in MODY3 patients