Project description:The aim of the experiment is to elucidate changes in tumor heterogeneity upon chemotherapy treatment. The experiment includes non-treated samples, samples right after chemotherapy treatment (FOLFIRI) and organoids recovered from FOLFIRI. It was conducted in two different genetic backgrounds, AKP (Apc KO, KrasG12D and P53 KO) and APS (Apc KO, P53 and Smad4KO). In addition to that, the organoids carry an inducible Cre-ERT2 knock-in allele in the mex3a locus. Upon 4-OH-Tamoxifen induction, Mex3a cells will recombine the tracing allele stop-TdTomato, enabling the tracing of this subpopulation. To understand the fate of the traced cells, samples right after chemotherapy and in the recovery setting were sorted based on their tomato expression. This enables a single cell tracing experiment.
Project description:Mex3a is an RNA binding protein of unknown function. To elucidate the contribution of Mex3a to tumoral heterogeneity, Mex3a KO organoids engineered by CRISPR were sequenced in three different conditions. Live organoids (DAPI negative) were sorted in Control, after 2 days of FOLFIRI and after 5 days of treatment. Two WT organoids (parental and a derived clone) and two KO (KO1 and KO2, two independent clones) were used for this experiment.
Project description:To comprehensively capture changes in retinal transcriptome for the LCA7 organoids compared to control, we performed single cell RNA-sequencing (scRNAseq) using the 10X Genomics platform. Retinal organoids at D150 of differentiation were dissociated for scRNAseq analysis. scRNAseq data revealed significant dysregulation of specific photoreceptor genes between control and LCA7 organoids, as well as mutation-specific differences in various genes, including CRX, RCVRN, ARR3, and AIPL1.
Project description:Human induced pluripotent stem cell-derived kidney organoids have potential for disease modelling and regenerative medicine purposes. However, they lack a functional vasculature and remain immature in in vitro culture. Here, we transplanted kidney organoids at day 7+12 of differentiation in the coelomic cavity of chicken embryos and then compared them to their respective untransplanted controls at d7+13 and d7+20 using scRNAseq and imaging modalities. We demonstrate vascularization and enhanced maturation of transplanted kidney organoids.
Project description:Human embryonic stem cell (hESC) line Man-13 was edited by CRISPR resulting in a hESC line carrying a heterozygous frameshift mutation with a premature stop codon in exon-1 of the HNF1B gene ([p.Val61Argfs*18]), functionally equivalent to a heterozygous whole-gene deletion. Kidney organoids were then created by differentiation (as per Takasato et al, 2015; Bantounas et al, 2018; Bantounas et al 2021) of this line and an isogenic non-mutant control line. Single-cell RNAseq (scRNAseq) was then performed on day-25 of differentiation to identify transcriptomic differences, as well as differences in identity and numbers of the cell populations comprising the organoids, with the aim of mechanistically explaining developmental aberrations observed in patients with mutations in this gene.
Project description:As metabolic rewiring is crucial for cancer cell proliferation, metabolic phenotyping of patient-derived organoids is desirable to identify drug-induced changes and trace metabolic vulnerabilities of tumor subtypes. We established a novel protocol for metabolomic and lipidomic profiling of colorectal cancer organoids by LC-QTOF-MS facing the challenge of capturing metabolic information from minimal sample amount (< 500 cells/injection) in the presence of extracellular matrix (ECM). The best procedure of the tested protocols included ultrasonic metabolite extraction with acetonitrile/methanol/water (2:2:1, v/v/v) without ECM removal. To eliminate ECM-derived background signals, we implemented a data filtering procedure based on p-value and fold change cut-offs which retained features with signal intensities >120% compared to matrix-derived signals present in blank samples. As a proof-of-concept, the method was applied to examine the early metabolic response of colorectal cancer organoids to 5-fluorouracil treatment. Statistical analysis revealed dose-dependent changes in the metabolic profiles of treated organoids including elevated levels of 2'-deoxyuridine, 2'-O-methylcytidin, inosine and 1-methyladenosine and depletion of 2'-deoxyadenosine and specific phospholipids. In accordance with the mechanism of action of 5-fluorouracil, changed metabolites are mainly involved in purine and pyrimidine metabolism. The novel protocol provides a first basis for the assessment of metabolic drug response phenotypes in 3D organoid models.
Project description:We performed RNA-sequencing to investigate the gene expression profiles of colorectal cancer patient-derived organoids (PDO) and PDO-initiated spontaenous metastases mouse models