Transcriptomic profiling of prolonged-culture iPSC-derived kidney organoids and X-linked Alport syndrome organoid models carrying COL4A5 deep-intronic variants

Kidney organoids serve as an invaluable platform for modeling hereditary renal diseases and developing therapeutic interventions. While various kidney organoid differentiation protocols have been developed, the protocol introduced by Morizane et al. was among the first to generate kidney organoids from induced pluripotent stem cells (iPSCs). By using a modified version of this protocol, we successfully generated kidney organoids in 21 days. Most studies on kidney organoids focus on early stages, typically between days 21 and 29, leaving the gene expression dynamics during prolonged culture less explored. In this study, we cultured healthy iPSC-derived kidney organoids for 22, 32, and 42 days and performed bulk RNA sequencing to investigate gene expression regulation during organoid culture. We also developed X-linked Alport syndrome (XLAS) organoid models carrying deep-intronic variants (severe and moderate models) to develop Antisense oligonucleotide (ASO) therapeutic approaches. Kidney organoids contain over 15 distinct cell types, making them a complex model for studying cell-type-specific maturation. By comparing organoids at early (day 22), mid (day 32), and late (day 42) time points, we observed significant changes in gene expression, particularly in genes related to the extracellular matrix, alongside the downregulation of podocyte-specific genes. Notably, we confirmed the upregulation of podocyte-specific collagen IV genes (COL4A3 and COL4A4), which are critical for forming the glomerular basement membrane (GBM). Overall, these findings underscore the importance of maintaining organoids in culture for at least 15 days beyond the last day of differentiation (day 21) to ensure the development of a more mature GBM, essential for BM disease modeling such as Alport syndrome.