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R5108 --> 665.13 --> Human induced pluripotent stem cell (hiPSC)‐derived kidney organoids have prospective applications ranging from basic disease modelling to personalised medicine. However, there remains a necessity to refine the biophysical and biochemical parameters that govern kidney organoid formation. Differentiation within fully‐controllable and physiologically relevant 3D growth environments will be critical to improving organoid reproducibility and maturation. Here, we matured hiPSC‐derived kidney organoids within fully synthetic self‐assembling peptide hydrogels (SAPHs) of variable stiffness (storage modulus, G′). The resulting organoids contained complex structures comparable to those differentiated within the animal‐derived matrix, Matrigel. Single‐cell RNA sequencing (scRNA‐seq) was then used to compare organoids matured within SAPHs to those grown within Matrigel or at the air‐liquid interface. A total of 13,179 cells were analysed, revealing 14 distinct clusters. Organoid compositional analysis revealed a larger proportion of nephron cell types within Transwell‐derived organoids, while SAPH derived organoids were enriched for stromal‐associated cell populations. Notably, differentiation within a higher G' SAPH generated podocytes with more mature gene expression profiles. Additionally, maturation within a 3D microenvironment significantly reduced the derivation of offtarget cell types, which are a known limitation of current kidney organoid protocols. This work demonstrates the utility of synthetic peptide‐based hydrogels with a defined stiffness, as a minimally complex microenvironment for the selected differentiation of kidney organoids. [ABSTRACT FROM AUTHOR]

R3064 --> 665.4 --> The generation of human induced pluripotent stem cell (hiPSC)‐derived kidney organoids have facilitated novel insights into renal developmental processes and have the potential to provide personalised treatment strategies for patients with end‐stage renal disease. Traditional protocols for the directed differentiation of hiPSC‐derived kidney organoids have predominantly focused on biochemical cues to specify hiPSCs towards a mesonephric lineage, but have not considered the influence of the surrounding biophysical properties of the extracellular environment on organoid formation. We hypothesised that Gelatin methacryloyl (GelMA), a derivative of collagen with mechanically amendable hydrogel stiffness profiles, could be used to investigate the influence of extracellular matrix stiffness on kidney organoid specification. hiPSC‐derived kidney organoids were differentiated within photo‐crosslinked GelMA hydrogels of defined mechanical strengths. Enrichment of renal cell types in response to the various mechanical microenvironments was subsequently investigated using immunocytochemistry, qRT‐PCR, transmission electron microscopy (TEM) and histological staining methods. Rheological analysis of formulated hydrogels confirmed the generation of matrices with distinct stiffness (Young's Modulus, G') profiles. Hydrogels comparable to the stiffness of the gastrulation‐stage embryo (G' = 400 Pa) and adult human kidney tissue (G' = 5‐8 kPa) were generated. Importantly, the mechanical properties of the hydrogels showed remarkable stability even with prolonged time in culture. PCNA proliferation and cleaved caspase‐3 apoptotic staining of organoids embedded within scaffolds demonstrated high cell proliferation and viability in the hydrogel conditions by day 24 of differentiation. The formation of glomerular, proximal tubular and distal tubular structures, supported by basement membrane and interstitial cells were confirmed in conditions using immunofluorescent imaging. Interestingly, qRT‐PCR analysis revealed significant upregulation of nephron‐associated genes (including PAX8, NPHS2, NPHS1, SLC3A1 and AQP1) in organoids differentiated within extracellular environments that approximated adult human kidney tissue, when compared to organoids differentiated within the much softer microenvironments. Our results illustrate the influence of the extracellular environment on appropriate cell fate determination and propose GelMA hydrogels as faithful extracellular supports for the specification of hiPSC‐derived kidney organoids. [ABSTRACT FROM AUTHOR]

TGFβ1 plays a regulatory role in the determination of renal cell fate and the progression of renal fibrosis. Here we show an association between SMAD3 and the histone methyltransferase, EZH2, during cell differentiation; ChIP-seq revealed that SMAD3 and EZH2 co-occupy the genome in iPSCs and in iPSC-derived nephron progenitors. Through integration of single cell gene expression and epigenome profiling, we identified de novo ACTA2+ve/POSTN+ve myofibroblasts in kidney organoids treated with TGFβ1, characterised by increased SMAD3-dependent cis chromatin accessibility and gene expression associated with fibroblast activation. We have identified fibrosis-associated regulons characterised by enrichment of SMAD3, AP1, the ETS family of transcription factors, and NUAK1, CREB3L1, and RARG, corresponding to enriched motifs at accessible loci identified by scATACseq. Treatment with the EZH2 specific inhibitor GSK343, blocked SMAD3-dependent cis co-accessibility and inhibited myofibroblast activation. This mechanism, through which TGFβ signals directly to chromatin, represents a critical determinant of fibrotic, differentiated states. Multimodal single-cell analysis on iPSC-derived kidney organoids shows that inhibiting EZH2 attenuates TGFβ1-induced fibrotic gene expression and changes in chromatin accessibility, highlighting a potential therapeutic strategy for renal fibrosis. [ABSTRACT FROM AUTHOR]

R2854 --> 515.5 --> Differential methylation patterns and changes in post‐translational histone modifications are observed in patients with or without progressive diabetic kidney disease (DKD). TGFβ1 plays a central regulatory role in the determination of renal cell fate and the progression of DKD. Using ChIP‐seq, we identified an association between SMAD3 and the histone methyltransferase, EZH2, in iPSCs and in iPSC derived nephron progenitors. Using the 10X Genomics platform, we performed single cell RNA‐seq and ‐ATAC‐seq on human iPSC‐derived kidney organoids treated with the EZH2 specific inhibitor, GSK343, prior to treatment with TGFβ1. Integration of single cell RNAseq and ‐ATACseq identified de novo ACTA2+ve/POSTN+ve myofibroblasts in TGFβ1‐treated kidney organoids, characterised by increased SMAD3‐dependent cis chromatin accessibility and the expression of genes associated with fibroblast activation, which are similarly overexpressed in DKD patients. This fibrosis‐associated regulon is characterised by enrichment of SMAD3 and AP‐1, corresponding to FOS/JUN motifs at accessible loci identified by scATACseq. Treatment with GSK343 blocked SMAD3‐dependent cis co‐accessibility and inhibited myofibroblast activation. We propose that that the enzymatic function of the polycomb repressive complex is necessary for TGFβ1 induced increase in chromatin accessibility and its subsequent gene regulatory functions. Manipulation of the association between SMAD3 and EZH2 may be a useful therapeutic strategy for the resolution of renal fibrosis. [ABSTRACT FROM AUTHOR]

Cell differentiation is directed by extracellular cues and intrinsic epigenetic modifications, which control chromatin organization and transcriptional activation. Central to this process is PRC2, which modulates the di- and trimethylation of lysine 27 on histone 3; however, little is known concerning the direction of PRC2 to specific loci. Here, we have investigated the physical interactome of EZH2, the enzymatic core of PRC2, during retinoic acid-mediated differentiation of neuroepithelial, pluripotent NT2 cells and the dedifferentiation of neuroretinal epithelial ARPE19 cells in response to TGF-β. We identified Smad3 as an EZH2 interactor in both contexts. Co-occupation of the CDH1 promoter by Smad3 and EZH2 and the cooperative, functional nature of the interaction were established. We propose that the interaction between Smad3 and EZH2 targets the core polycomb assembly to defined regions of the genome to regulate transcriptional repression and forms a molecular switch that controls promoter access through epigenetic mechanisms leading to gene silencing. [ABSTRACT FROM AUTHOR]