Fine tuning the extracellular environment accelerates the derivation of kidney organoids from human pluripotent stem cells

Abstract

The generation of organoids is one of the biggest scientific advances in regenerative medicine. Here, by lengthening the time that human pluripotent stem cells (hPSCs) were exposed to a three-dimensional microenvironment, and by applying defined renal inductive signals, we generated kidney organoids that transcriptomically matched second-trimester human fetal kidneys. We validated these results using ex vivo and in vitro assays that model renal development. Furthermore, we developed a transplantation method that utilizes the chick chorioallantoic membrane. This approach created a soft in vivo microenvironment that promoted the growth and differentiation of implanted kidney organoids, as well as providing a vascular component. The stiffness of the in ovo chorioallantoic membrane microenvironment was recapitulated in vitro by fabricating compliant hydrogels. These biomaterials promoted the efficient generation of renal vesicles and nephron structures, demonstrating that a soft environment accelerates the differentiation of hPSC-derived kidney organoids.

Fig. 1 |. Efficient generation of kidney organoids in 3D culture.

a, Schematic of the stepwise differentiation methodology for generating kidney organoids from hPSCs. b, Confocal microscopy images of glomerular structures in day 16 kidney organoids showing podocyte-like cells positive for PODXL, nephrin, NEPH1 and podocin, and the basement membrane protein laminin. Scale bars, 25 μm. c, Dendrogram representing the hierarchical clustering of day 0,5,8 and 16 kidney organoids with human fetal kidneys from 9, 13, 17 and 18 weeks of gestation (first trimester) and 22 weeks of gestation (second trimester). Data from Chuva de Sousa Lopes (SRP055513) (1) and McMahon (SRP111183) (2) are included in the analysis. d, qPCR analysis during kidney organoid differentiation and 13-, 16-, and 22-week human fetal kidneys (genes are indicated). Data are mean ± s.d. For SIX2, WT1, SALL1 and PAX2, day 0, day 5, n = 3; day 8, day 16, n = 2. For PODXL, SLC3A1, SYNPO and NPHS1, day 0, day 5, n = 1; day 8, day 16, n = 2. Each sample is a pool of six organoids. Three technical replicates are shown per sample. e,f, Semithin sections of day 16 kidney organoids showing glomerular (e) and tubular-like (f) structures. Scale bars, 100 μm. g–j, TEM of day 16 kidney organoids. g, Immature podocytes. Scale bar, 5 μm. h, A magnified view of the boxed region in g showing a detail of podocyte-related structures including the deposition of a basement membrane (bm), and primary (pp) and secondary cell processes (sp). Scale bar, 1 μm. i, Epithelial tubular-like cells with brush borders (bb), high mitochondrial (mit) content and tight junctions (tj). Scale bar, 2 μm. j, A magnified view of the boxed region in i showing a detail of brush borders. Scale bar, 1 μm.

Fig. 2 |. Kidney organoids model human kidney organogenesis in vitro.

a, Representation of the coculture of day 5 NPCs with mouse embryonic kidney cells. b–d, Bright-field images of reaggregates after 1 d (b), 4 d (c) and 6 d (d) in culture. Scale bars, 500 μm. e, Immunocytochemistry for PAX8, WT1 and HuNu of the reaggregate in d. Scale bar, 250 μm. f, Magnified views of d. Scale bars, 50 μm. g–i, Modulation of β-catenin signalling in kidney organoids with IWR1 and CHIR inhibitors. g, Immunocytochemistry for WT1 and LTL in day 16 kidney organoids with the indicated regimens. Scale bars, 50 μm. h, Corresponding quantification of the percentage of WT1⁺ cells and LTL⁺ structures. Data are mean ± s.d. n = 3 organoids per treatment. One-way analysis of variance with Tukeýs post hoc test. For % WT1, F (1.009, 2.017) = 213.6, P = 0.0045; vehicle versus CHIR, **P = 0.0082; CHIR versus IWR1, ****P = 0.000034; vehicle versus IWR1, n.s., not significant, P = 0.9995. For % LTL, F (1.002, 2.004) = 0.9976, P = 0.4232, not significant. i, Corresponding qPCR analysis (genes are indicated). Data are mean ± s.d. (three technical replicates). j–l, Energy metabolism profile of kidney organoids maintained in EGM or REGM: kinetic oxygen consumption rate (OCR) response (j), inner mitochondrial membrane proton leak and cellular ATP production (k) and basal respiration and spare respiratory capacity (l). Data are normalized to mitochondrial DNA copy number/sample. Data are mean ± s.d. n = 3 (EGM) and n = 2 (REGM) organoids. m, Immunocytochemistry for LTL and PODXL in day 16 kidney organoids under EGM or REGM regimen. Scale bars, 200 μm (EGM) and 400 μm (REGM). n, Corresponding quantification of the percentage of PODXL⁺ and LTL⁺ structures. Data are mean ± s.d. n = 2 organoids per condition.

Fig. 3 |. In vivo vascularization of kidney organoids using chick CAM.

a, Methodology for the implantation of day 16 kidney organoids into chick CAM. b,c, Macroscopic views of implanted organoids maintained in ovo for 3 d (b) and 5 d (c). d, The implanted organoid in c after intravital injection of dextran–FITC through the chick vasculature. Scale bars, 1000 μm (c,d). e, Semithin sections of a kidney organoid (dashed line) implanted in the CAM mesenchyme (m) for 5 d. Magnified views of glomerular (G) and tubular (T) cells are shown. Scale bars, 200 μm, 100 μm (magnified views). f–k, TEM of implanted organoids. Magnified views of the boxed regions in f,h,j are shown in g,i,k, respectively). f, Differentiated podocytes (p) extending primary cell processes and apical microvilli (black triangles) are located on one side of the basement membrane and a vascular endothelial cell (end) is found on the opposite side. g, Slit diaphragm-like structures (red arrows) between secondary cell processes (sp). bm, basement membrane. h, Aligned podocytes showing primary cell processes and apical microvilli (black triangles). er, chicken erythrocytes. i, A detail of the basement membrane (bm), primary cell processes (pp) and a slit diaphragm-like structure (red arrow). j, Tubular-like cells. k, A detail of brush borders (bb). Scale bars, 2 μm (f), 500 nm (g), 10 μm (h), 2 μm (i), 5 μm (j), 1 μm (k). l–n, Confocal microscopy images of glomerular structures in implanted organoids. l, Immunohistochemistry for PODXL, nephrin, NEPH1, podocin and laminin. Scale bars, 25 μm. m, Immunohistochemistry for nephrin and CD34. Scale bars, 25 μm, 10 μm (magnified view). n, Immunohistochemistry for PODXL, CD31 and the human marker HuNu. Scale bars, 10 μm, 5 μm (magnified view). White arrows indicate endothelial-like cells in close contact with podocyte-like cells (m,n).

Fig. 4 |. Soft hydrogels accelerate the differentiation of kidney organoids.

a, Immunocytochemistry for PAX2 in RV-stage organoids generated using 1 kPa or 60 kPa hydrogels. Scale bars, 500 μm, 150 μm (magnified views). b, Quantification of a. The mean number of RVs and area percentage occupied by RVs were quantified. Data are mean ± s.d. n = 2 organoids per condition. c, Immunohistochemistry for LTL, WT1 and ECAD in day 16 kidney organoids from 1 kPa or 60 kPa. Scale bars, 500 μm and 50 μm (magnified views). d, Quantification of c. The percentages of WT1⁺ and LTL⁺ area were quantified. Data are mean ± s.d. n = 3 organoids per condition. For WT1⁺, t(4) = 5.8057, **P = 0.0044. For LTL⁺, t(4) = 4.6023, *P = 0.0100. Two-tailed Student’s t-test. e, qPCR analysis of day 16 kidney organoids from 1 kPa or 60 kPa (genes are indicated). Data are mean ± s.d. (technical replicates). f–i, TEM of day 16 kidney organoids from 1 kPa. f, Epithelial tubular-like cells with brush borders (bb). g, Podocyte-like cells with primary cell processes (pp). h,i, Magnified views of g. Secondary cell processes (black arrows) with slit diaphragm-like structures (red arrows). Red asterisks, podocyte membrane protrusions. bm, basement membrane. Scale bars, 2 μm (f), 5 μm (g), 500 nm (h), 200 nm (i). j, Day 16 kidney organoids from 1 kPa were implanted into the CAM. k–n, TEM of implanted kidney organoids from 1 kPa. k, Tubular-like cells with brush borders. l, Aligned podocyte-like cells extending primary cell processes near endothelial (en) cells and chicken erythrocytes (er). m,n, Magnified views of l. Secondary cell processes with slit diaphragm-like structures. Scale bars, 2 μm (k), 10 μm (l), 1 μm (m), 200 nm (n).