No abstract

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.

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.

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.