Induction of kidney organoids from human pluripotent stem cells (hPSCs): hPSCs were dissociated into single cells with Accutase (STEMCELL Technologies), and plated onto Matrigel (BD Biosciences)-coated plates at a density of ∼15,000 to ∼24,000 cells/cm2. The plated cells were cultured in TeSR supplemented with 10 μM Y-27632 (Sigma) for 24 hours, followed by another 48-72 hours culture in TeSR to reach a confluence of 50%–60%. To induce primitive streak, cells were cultured in basal differentiation medium (Advanced RPMI 1640, 1x L-GlutaMAX, 1 x NEAA) (Life Technologies) supplemented with 10 μM CHIR99021 (Sigma) for 4 days with daily media change. Next, to induce nephrogenic intermediate mesoderm, cells were cultured in basal differentiation medium, without any growth factor for 3 days with daily media change. Subsequently, to induce nephron progenitors, cells were cultured in basal differentiation medium supplemented with 50 ng/mL FGF9 (Peprotech) and 3 μM CHIR99021 with daily media change for 2 days. From day 10 of differentiation onward, cells were cultured in basal differentiation medium supplemented with 50 ng/mL FGF9 until day 20. On day 14 of differentiation, the organoid can be cultured in differentiation medium supplemented with 1 μM CHIR99021 for 1 to 10 days (patterning CHIR) to modulate the proportion of proximal-versus-distal segments. From day 20 of differentiation onward, basal differentiation medium was used for organoid culture.
Generation and patterning of three-dimensional (3D) kidney organoids.To generate 3D kidney organoids, day 10-12 differentiating cells were dissociated into single cells by- Accutase. 2.5-5 × 104 cells (per well) were aggregated in basal differentiation medium supplemented with 50 ng/mL FGF9 and 10 μM Y-27632 using round bottom ultra-low attachment 96-well plates (Corning). 24 hours later, Y-27632 was removed. To generate kidney organoids with a high glomerulus-to-tubule ratio, no patterning CHIR was added. To generate kidney organoids with a high tubule-to-glomerulus ratio, 10 days patterning CHIR was applied from day 14 to day 24 of differentiation. 3D kidney organoids were transferred onto the upper chamber of Transwell (Corning) for liquid-air interface culture on day 16-18 of differentiation (5-7 days after aggregation), with daily media change at the bottom chamber.
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