Single cell gene expression analysis of X-linked Alport syndrome kidney organoid models and isogenic controls

In study we investigated podocyte maturation, with a particular focus on type IV collagen maturation in the glomerular basement membrane (GBM) of kidney organoids over extended culture periods (comparing Control Organoid d22 R2 with Control Organoid d38). The secondary aim was to compare the gene expression profiles of podocytes in X-linked Alport syndrome (XLAS) organoid models with isogenic controls at an early culture time point (day 22). Two distinct batches of sample collection were conducted to capture early and later stages of GBM development. In the first batch, three libraries were generated by collecting 16 organoids from one isogenic control, one severe XLAS model, and one moderate XLAS model on day 22 of differentiation. In the second batch, two libraries were created to evaluate the impact of culture duration on GBM maturation by harvesting an additional isogenic control organoid at both day 22 and day 38 of culture. Organoids were dissociated using a two-step enzymatic and mechanical protocol to obtain single-cell suspensions, with cell viability ranging between 70% and 85%. This experimental design enabled a comparative analysis of GBM maturation dynamics between early and late time points and across different severities of XLAS. Findings from this study provided insights into the cellular mechanisms underlying GBM abnormalities in Alport syndrome and supported the potential for GBM maturation in kidney organoids over time. Overall design: Two separate batches of sample collection were performed for this project. In the first batch, 16 organoids were collected on day 22 of differentiation, including one isogenic control organoids, one severe XLAS organoid, and one moderate XLAS organoid, resulting in three libraries. This project aimed to compare XLAS severe and moderate organoid models at early time point (day 22 of differentiation). An additional isogenic control was harvested on day 22 (Control_d22_2) and kept in culture for 38 days (Control_d38) and harvested at this later time point (two library, 5 libraries in total). This batch was used to compare isogenic controls at day 22 and day 38 of culture to study the gene expression profile in podocytes to confirm the glomerular basement membrane maturation in prolonged culture. Libraries obtained from these two batches were sequenced together.