The Growth and Differentiation of Human Induced Pluripotent Stem Cell-Derived Kidney Organoids Within Self-Assembling Peptide Hydrogels

Integrating fully synthetic hydrogels with the culture of human induced pluripotent stem cell (hiPSC)-derived kidney organoids offers the potential to bring these renal structures forward as next generation therapeutics for chronic kidney disease (CKD). Kidney disease remains a leading cause of death worldwide with mortality due to its chronic form on the rise. Irrespective of the underlying cause, the progressive decline in renal function inevitably leaves patients requiring dialysis or transplantation. However, supply does not meet demand with regards to kidney transplantations and new therapeutic approaches are needed. hiPSC-derived kidney organoids hold great promise for regenerative medicine applications. Much focus has been placed on the biochemical factors that induce hiPSCs to a renal cell fate, however the biophysical environment kidney organoids are exposed to is also proving important. Here, fully synthetic self-assembling peptide hydrogels (SAPHs) were used for the growth and differentiation of hiPSC-derived kidney organoids. SAPHs were shown to structurally resemble extracellular matrix architecture while rheological characterisation highlighted their tuneable mechanical properties. In response to differentiating kidney organoids and cell culture conditions, SAPHs were shown to display soft and stiff mechanical profiles with regards to the reported G’ stiffness of healthy human kidneys. In order to culture kidney organoids within hydrogels, a methodology was developed whereby nephron progenitor aggregates were cultured in suspension for 48 hours prior to encapsulation. Subsequently, kidney organoids differentiated within SAPHs were shown to contain key renal cell types organised into complex structures. Notably, SAPH-derived kidney organoids were shown to be structurally comparable to those formed on Transwell inserts at the air-liquid interface and within the animal-derived matrix Matrigel. In order to investigate the influence of growth environment on organoid differentiation further comparisons were made using single cell RNA-sequencing (scRNA-seq). 13,179 cells were analysed revealing kidney organoids comprised 14 distinct cellular populations or clusters. While Transwell-grown organoids were shown to contain the largest proportion of nephron cell types, the degree of podocyte maturity was highest in organoids grown within Alpha5, the stiffer of the SAPHs used. A key finding was the role of the 3D microenvironment in reducing the formation of off-target cell types which is a known limitation in the kidney organoid field. After demonstrating the role of the 3D bio-interface in refining renal cell type differentiation, the lack of appropriately patterned vasculature, another limitation in the kidney organoid field, was addressed. hiPSC-derived vascular organoids comprising endothelial networks and supporting cell types were generated through suspension culture, obviating the requirement for Matrigel. A vascular : kidney organoid co-culture model was developed and while endothelia was not increased in differentiated Alpha5-derived kidney organoids, optimisation of the methodology could pave the way for vascularised kidney organoids grown within a fully synthetic matrix. Importantly, the work presented demonstrates for the first time the growth of stem cell-derived kidney organoids within a fully synthetic environment. It highlights self-assembling peptide hydrogels of defined stiffness as a viable 3D biomimetic matrix for the directed differentiation of hiPSC-derived kidney organoids.