Fig 2: Kidney organoids formed in all dynamic hydrogels, but a subset of renal cell types is absent in the 20 kPa hydrogel. Culture of the organoids for 4 days (from day 7+14 to 7+18) in hydrogels did not affect the presence of nephron segments in the 0.1 kPa (d) or 3 kPa (c) hydrogels when compared to organoids grown on an air–liquid interface until day 7+18 (a). Only the organoids encapsulated in the stiffest 20 kPa hydrogel (b) lacked loop of Henle segments (two left columns) and interstitial cells (two right columns). Staining for distal tubules (E-cadherin; ECAD); the loop of Henle (NKCC2; SLC12A1); proximal tubules (lotus tetragonolobus lectin; LTL); glomeruli (nephrin; NPHS1); and interstitial cells (homeobox protein Meis 1/2/3; MEIS1/2/3) as indicated. DAPI staining (blue) for nuclei. The white box denotes the area of interest enlarged in the respective right panel. Scale bars: 50 µm. Representative images of N = 3 organoid batches with n = 3 organoids per batch. See single channels in Figure S5, Supporting Information.

Fig 3: Differentiation protocol and subsequent characterisation of hiPSC-derived kidney organoids formed within SAPHs. A) hiPSCs were differentiated into kidney progenitors and day 7 aggregates were placed in suspension culture for 48 h. Compacted pellets were encapsulated within Alpha4 or Alpha5 until day 24. Resulting organoids comprised a dense centre with peripheral tubular structures. Day 0 scale bar = 250 µm, rest = 1 mm. B) Monolayer immunofluorescent characterisation from pluripotent hiPSCs to day 7 nephron progenitors. Scale bar = 100 µm. C) Viability of kidney organoids was compared on day 24 using live (calcein-AM) and dead (ethidium-homodimer-1) stains. Scale bar = 300 µm. Increased matrix stiffness (Matrigel < Alpha4 < Alpha5) had an inverse effect on cell viability. Data expressed as mean ± SD percentage of organoid area positive for live or dead stain with a one-way ANOVA and Tukey's multiple comparisons test used; ** for p < 0.01, **** for p < 0.0001. N = 18 for each condition from three independent experiments. D) H&E and immunofluorescent characterisation of Alpha4 and Alpha5-derived organoids demonstrated successful formation of complex structures and comprised typical cell types of the developing nephron (WT1+ve podocytes, LTL+ve proximal tubules, ECAD+ve distal tubules and ZO-1+ve epithelial tight junctions) and supporting cells (MEIS1/2/3+ve interstitium and LAM+ve basement membrane). H&E scale bar = 250 µm, immunofluorescence = 50 µm. E) Organoids showed functionality by the uptake of fluorescently labelled 10,000 MW dextran. Scale bar = 35 µm.

Fig 4: Genome-wide transcriptional analysis of the KM cell of WT and Tg(drl:hoxa9;hsp70:meis1) adult fish. (a) The volcano plot for differential gene expression between WT and Tg(drl:hoxa9;hsp70:meis1) from three biological replicates. (b) Numbers of up- and downregulated genes in Tg(drl:hoxa9;hsp70:meis1) relative to WT (greater than twofold change, adj p = 0.05). (c) GO enrichment analysis of differential expressed genes. (d) Heatmap display of the representative differentially expressed genes between WT and Tg(drl:hoxa9;hsp70:meis1). (e) qPCR assay on the mRNA levels of a number of genes in KW cells in Tg(drl:hoxa9;hsp70:meis1) compared to WT. Data are represented as mean ± s.d. *p < .05, **p < .01. (f–h) GSEA of the expressing profile of KM cells in WT and Tg(drl:hoxa9;hsp70:meis1) using cell cycle-associated upregulated signature, haematopoietic progenitor cell differentiation-associated downregulated signature and HSC differentiation-associated downregulated signature. KW, kidney marrow.

Fig 5: DHODH inhibitor leflunomide rescues defective haematopoiesis and improves the survival in Tg(drl:hoxa9;hsp70:meis1) fish. (a,b) Tg(drl:hoxa9;hsp70:meis1) embryos and control siblings were heat-shock induced at 24 hpf. Tg(drl:hoxa9;hsp70:meis1) embryos were treated with DMSO or Lef after heat shock. Embryos were assayed by WISH for the expression of cmyb, mpx and mfap4 at 3 dpf or 5 dpf. (c) Quantification of WISH results in (a,b) (n = 50–100 embryos per group). Data are represented as mean ± s.d. *p < .05, **p < .01, ***p < .001. (d) WT and Tg(drl:hoxa9;hsp70:meis1) were repeatedly heat-shock induced starting from 24 hpf, Tg(drl:hoxa9;hsp70:meis1) fish were treated with DMSO or Lef after the initial heat shock. Survival curves of WT and Tg(drl:hoxa9;hsp70:meis1) fish (n = 20–30 per group, Log-rank (Mantel-Cox) significance test). (e) WT and Tg(drl:hoxa9;hsp70:meis1) were repeatedly heat shock induced starting from 26 dpf, Tg(drl:hoxa9;hsp70:meis1) fish were treated with DMSO or Lef after the initial heat shock. Survival curves of WT and Tg(drl:hoxa9;hsp70:meis1) fish (n = 10 per group, Log-rank (Mantel-Cox) significance test). Lef, Leflunomide; hpf, hours post-fertilization; dpf, days post-fertilization.