Nephron segmentation and patterning in kidney organoids can be modulated by distinct FGF subfamily members

Human pluripotent stem cell-derived kidney organoids replicate embryonic nephrogenesis in a 3D-culture system. Recent advances suggest that refining the culture environment to replicate spatiotemporal cues present during embryonic organogenesis improves patterning. Here, this paradigm was applied to FGF signalling, a key regulator of embryonic nephron progenitor maintenance, nephrogenesis and ureteric branching. Both FGF8b and FGF10 signalling is sufficient to support nephrogenesis, with each having distinct effects on nephron patterning. FGF10 enhanced the initial WT1+ mesenchymal population, leading to proximally biased nephrons, while FGF8b biased toward early distal patterning, leading to the formation of cells with connecting segment identity. The addition of both FGF8b and FGF10 together had an additive effect, leading to a balance of proximal and distal patterning. This differential patterning was retained in tissue transplanted under the murine renal capsule, with FGF8b-treated organoids displaying increased distal/connecting segments. These findings highlight plasticity during organoid nephrogenesis that can be modulated by FGF signalling and identify an approach to refine nephrogenesis toward key cell types. Overall design: Single cell suspensions of each condition were fixed with formaldehyde (ProSciTech C004) and additive according to the Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit (10x Genomics). Fixation was performed at 4oC overnight, and after addition of the buffer, fixed cells were stored at -80oC until processing. The four barcode version of the Chromium Fixed RNA Kit, Human Transcriptome (10x Genomics) was used to barcode all 8 samples, giving 2 libraries. Post hybridisation washes were completed individually for each barcoded sample, according to the manufacturer's protocol. Cell counting was completed using InvitrogenTM ReadyCountTM Green/Red Cell Viability Stain (Thermo Fisher Scientific) and the Countess 3 Automated Cell Counter (Thermo Fisher Scientific). A target of 10,000 cells per sample was combined for a total of 40,000 cells per library.