Project description:This study used scRNA-seq to characterise the transcriptome in 26 day-old iPSC-derived kidney organoids, treated with TGFB1, the EzH2 inhibitor GSK343, a combination of both or a vehicle control for 48 hours (days 24-26) before harvesting. 2 organoids per condition were pooled and dissociated using a cold-active protease. Nuclei were extracted and profiled using the 10X Genomics Single-cell 3' V3 kits. Libraries were sequenced using paired-end reads on an Illumina NextSeq 500. Initial processing was performed using CellRanger v3.1.0 (10X Genomics).
Project description:This study used snATAC-seq to profile Chromatin accessibility in 26 day-old iPSC-derived kidney organoids, treated with TGFB1, the EzH2 inhibitor GSK343, a combination of both or a vehicle control for 48 hours (days 24-26) before harvesting. 2 organoids per condition were pooled and dissociated using a cold-active protease. Nuclei were extracted and profiled using the 10X Genomics Single-cell ATAC reagent kit v1.1. Libraries were sequenced using paired-end reads on an Illumina NovaSeq 6000. Initial processing was performed using CellRanger ATAC v1.2.0 (10X Genomics).
Project description:scRNA-seq was used to characterise hiPSC-derived kidney organoids differentiated within fully synthetic self-assembling peptide hydrogels of variable mechanical strengths and compare these to organoids differentiated within the animal-derived matrix, Matrigel. Organoids were matured in the respective matrices until day 24 of differentiation and 6 organoids per support matrix were then pooled and dissociated using the cold-active protease from Bacillus licheniformis. Cells were processed on the 10x Genomics Chromium platform using the Single-Cell 3’ v3.1 protocol. The NextSeq500 (Illumina) was used to sequence the libraries generated and initial processing of the data was carried out using the 10X Genomic Cell Ranger v3.1.0 pipeline.
Project description:Experiment intended to obtain expression profiles of iPSC-derived human colon organoids compared to undifferentiated human iPSCs and a patient-derived colon organoid line
Project description:These files represent single cell RNA-Seq data generated on a 10x Chromium genomics platform from four biological replicates of iPSC-derived human kidney organoids, in two batches, differentiated according to our published protocol (Takasato et al., Nature Protocols 2016). The aggregated human organoid data contains populations representing endothelial cells, podocytes, stroma, nephron, and off-target populations with similarity to neurons.
Project description:Retinitis pigmentosa (RP) is an irreversible and inherited retinopathy. RPGR mutations are the most common causes of this disease. It remains challenging to decipher the mechanism of RPGR mutation because of the lack of appropriate study models. The substitution of patient-specific diseased retina without ethical restrictions is desired and iPSC-derived 3D retina is the best choice. In our experiment, we generated iPSCs from one RP patient with 2-bp frameshift mutation in the exon14 of RPGR gene, which were differentiated into retinal organoids. Also we generated iPSCs from a normal control and differentiated those control-iPSCs into healthy retinal organoids. Samples of patient- and control-retinal organoids at W0, W7, W13 (two replicates), W18 (two replicates) and W22 (two replicates for patient) were collected for RNA-seq. Corrected-iPSC were derived from CRISPR/Cas9-mediated gene correction. Then we collected the corrected-iPSC derived retinal organoids at W0, W7, W13 (two replicates), W18 (two replicates) and W22 (two replicates) for RNA-seq. Through the RNA-seq data, we demonstrate that patient-specific iPSC-dervied 3D retinae can recapitulate disease progress of Retinitis Pigmentosa through presenting defects in photoreceptors' gene profile. CRISPR/Cas9-mediated gene correction can rescue photoreceptor gene profile. Those transcriptome are consistent with the phenotype and function.
Project description:Purpose: A proof of concept study examining the disease modelling capabilities of patient iPSC derived kidney organoids. Methods: A proband was diagnosed by genome sequencing with compound heterozygous IFT140 mutations. A one-step reprogramming/gene-editing protocol of proband fibroblasts was used to derive both uncorrected patient and isogenic gene-corrected induced pluripotent stem cells (iPSC) which were differentiated to kidney organoids. Organoids were examined by immunofluorescence. Additionally, epithelial cells magnetically sorted from whole kidney organoids underwent transcriptional profiling and spheroid culture. Results: Differential expression analysis of organoid epithelial cell fractions demonstrated apicobasal polarity, cell-cell junction and dynein motor assembly downregulation in patient organoids. Defective ciliary morphology and spheroid culture were rescued in gene corrected organoids. Conclusions: This study validates patient iPSC-derived kidney organoids as a novel, faithful and patient-specific model to further the study of inherited renal disease in regenerated, human, in vitro tissue.
Project description:Alport syndrome (AS) is a hereditary glomerulonephritis caused by COL4A3, COL4A4 or COL4A5 gene mutations and characterized by abnormalities of glomerular basement membranes (GBMs). Due to a lack of curative treatments, the condition proceeds to end-stage renal disease even in adolescents. Hampering drug discovery is the absence of effective in vitro methods for testing the restoration of normal GBMs. Here, we aimed to develop kidney organoid models from AS patient iPSCs for this purpose. We established iPSC-derived collagen α5(IV)-expressing kidney organoids and confirmed that kidney organoids from COL4A5 mutation-corrected iPSCs restore collagen α5(IV) protein expression. Importantly, our model recapitulates the differences in collagen composition between iPSC-derived kidney organoids from mild and severe AS cases. Furthermore, we demonstrate that a chemical chaperone, 4-phenyl butyric acid, has the potential to correct GBM abnormalities in kidney organoids showing mild AS phenotypes. This iPSC-derived kidney organoid model will contribute to drug discovery for AS.