Tuneable gelatin methacryloyl (GelMA) hydrogels for the directed specification of renal cell types for hiPSC-derived kidney organoid maturation

Abstract

Diabetic Kidney Disease (DKD) represents a significant global health burden and is recognised as the leading cause of end-stage renal disease. Kidney organoids derived from human induced Pluripotent Stem Cells (hiPSCs) have the potential to transform how we model renal disease and may provide personalised replacement tissues for patients with renal failure. However, kidney organoids remain poorly reproducible, and are structurally and functionally immature. Three-dimensional cultures that more appropriately mimic the complexity of the in vivo microenvironment are required to improve organoid maturation and structural authenticity. Here, we describe the application of semi-synthetic Gelatin Methacryloyl (GelMA) hydrogels as extracellular support matrices for the differentiation of hiPSC-derived kidney organoids. Hydrogels of defined mechanical strengths were generated by varying the concentration of GelMA solution in the presence of low concentration photo-initiator. After confirming a high level of mechanical stability of the hydrogels over extended culture periods, their effect on kidney organoid maturation was investigated. Organoids differentiated within GelMA hydrogels generated typical renal cell types including podocytes, tubular epithelia, renal interstitial cells, and some nascent vascularisation. Interestingly, kidney organoids derived within hydrogels that closely approximate the stiffness of the adult human kidney (∼5000-10,000 Pa) demonstrated improved podocyte maturation and were shown to upregulate renal vesicle-associated genes at an earlier stage following encapsulation when compared to organoids derived within softer hydrogels (∼400 Pa). A model of TGFβ-induced injury was also developed to investigate the influence of the mechanical environment in propagating early, fibrotic-like features of DKD within organoids. Growth within the softer matrix was shown to reduce pSMAD3 expression following TGFβ1 treatment, and accordingly ameliorate the expression of the myofibroblast marker α-Smooth Muscle Actin (α-SMA). This work demonstrates the suitability of GelMA hydrogels as mechanically-stable, highly-tuneable, batch-to-batch reproducible three-dimensional supports for hiPSC-derived kidney organoid growth and differentiation, and further substantiates the role of the biophysical environment in guiding processes of cell fate determination and organoid maturation.

Keywords: Gelatin methacryloyl; Hydrogels; Mechanoregulation; Stiffness; hiPSC-derived kidney organoids.