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Identifying differences in differentiation between adherent and suspension culture for iPSC derived beta cells

ABSTRACT: Stage 3 cells from a suspension environment and adherent culture were compared and evaluated through RNA sequencing. RNA sequencing data in heatmap and volcano plot evaluation of core endocrine genes showed that expression levels in adherent cells were lower than those expressed in 3D environment. Several genes that are essential for insulin transcription such as NEUROD1, MAFA, PPARGC1A and NKX2.2 along with INS were expressed with higher transcript levels in bioreactor samples. It was observed that genes usually expressed in earlier stages such as FOXA2 and HNF1b had higher expression levels in adherent culture indicating that 2D environment differentiation might be lagging.

ORGANISM(S): Homo sapiens

PROVIDER: GSE264176 | GEO | 2024/04/26

REPOSITORIES: GEO

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Project description:Heterozygous mutations in HNF1B cause the complex syndrome Renal Cysts and Diabetes (RCAD), characterized by developmental abnormalities of kidney, genital tracts and pancreas and a variety of renal, pancreas and liver dysfunctions. The pathogenesis underlying this syndrome remained unclear since mice with Hnf1b heterozygous null mutations have no phenotype, while constitutive/conditional Hnf1b-ablation led to more severe phenotypes. We generated a novel mouse model carrying an identified human mutation at the intron-2 splice donor-site. Reflecting the disease and unlike previous heterozygous, heterozygous for the splicing mutation exhibited decreased HNF1B protein levels and bilateral renal cysts from embryonic stage E15, originated from glomeruli, early proximal tubules (PT) and intermediate nephron segments, concurrently with a delayed PT differentiation, hydronephrosis and rare genital tract anomalies. To further elucidate the cellular and molecular components sensitive to HNF1B levels, we performed RNA-sequencing on WT and heterozygous mutant kidneys at E14.5 (considered morphologically as pre-disease kidneys) and at disease stages (E15.5, E17.5 and P1). Consistent with the histological and immunhistochemical findings, these analysis showed that most down-regulated genes in embryonic kidneys were primarily expressed in early PTs and Henle’s Loop and involved in ion/drug transport, organic acid and lipid metabolic processes, while previous targets identified upon Hnf1b-ablation, including the cystic disease genes, were weakly or no affected. Postnatal analyses revealed renal abnormalities, ranging from glomerular cysts to hydronephrosis and rarely multicystic dysplasia. Urinary proteomics uncovered a particular profile predictive of progressive decline in kidney function, injury and fibrosis, and displayed common features with a recently reported urine proteome in a RCAD pediatric cohort. Our results show that HNF1B reduced levels lead to developmental disease phenotypes associated with the deregulation of a subset of its targets and further suggests that this new model represents a unique clinical/pathological viable model of the RCAD disease.

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Identifying differences in differentiation between adherent and suspension culture for iPSC derived beta cells

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