Generation of kidney organoids from human pluripotent stem cells

Abstract

The human kidney develops from four progenitor populations-nephron progenitors, ureteric epithelial progenitors, renal interstitial progenitors and endothelial progenitors-resulting in the formation of maximally 2 million nephrons. Until recently, the reported methods differentiated human pluripotent stem cells (hPSCs) into either nephron progenitor or ureteric epithelial progenitor cells, consequently forming only nephrons or collecting ducts, respectively. Here we detail a protocol that simultaneously induces all four progenitors to generate kidney organoids within which segmented nephrons are connected to collecting ducts and surrounded by renal interstitial cells and an endothelial network. As evidence of functional maturity, proximal tubules within organoids display megalin-mediated and cubilin-mediated endocytosis, and they respond to a nephrotoxicant to undergo apoptosis. This protocol consists of 7 d of monolayer culture for intermediate mesoderm induction, followed by 18 d of 3D culture to facilitate self-organizing renogenic events leading to organoid formation. Personnel experienced in culturing hPSCs are required to conduct this protocol.

Figure 1. Schematic diagram of the timeline for generating kidney organoids from hPSCs

The protocol is based upon a step-wise differentiation protocol with sequential changing of culture media. hPSCs are cultured in MEF-CM (MEF-conditioned hESC medium). Differentiation begins in APEL/CHIR99021 (APEL medium supplemented with 8 μM CHIR99021) followed by APEL/FGF9/Heparin (APEL medium supplemented with 200 ng ml⁻¹ FGF9 and 1 μg ml⁻¹ heparin) and all growth factors are withdrawn in the final step. A pulse of CHIR99021 (5 μM) stimulates nephrogenesis in the organoids.

Figure 2. Bright field images of hPSCs differentiation to kidney organoids

Bright field images of the cells during differentiation. a, hPSC colonies on MEF feeder layer at day −3, before transfer onto Matrigel. b, hPSCs on Matrigel at day 0 before adding CHIR99021 to the medium. c, hPSCs differentiated into the posterior primitive streak cells with CHIR99021 induction showed a triangular shaped cell morphology. d, Monolayer culture at day 4 of differentiation e, Cells at day 7 of differentiation. Ideally, cultures nearly reach confluence at this time and form distinct areas of thick and thin layers across the surface. f, At day 7 of differentiation, cells were trypsinized and centrifuged to form an aggregate. This panel represents an aggregate at 30 min after transfer to transwell filter. g,h, A whole kidney organoid at day 10 of differentiation (3 days in organoid culture). A black square indicates the field of the right panel (h), which shows the formation of small renal vesicles. i,j, A whole kidney organoid at day 25 of differentiation (18 days in organoid culture). A black square indicates the field of the right panel (j). If the differentiation has been successful, glomerular structures (gl) can be recognized under microscope. Scale bars, 200 μm (a-e), 1 mm (f,g,i), 100 μm (h,j).

Figure 3. Immunological characterization of structures within kidney organoids

a, Staining for anterior intermediate mesoderm cells (GATA3) and posterior intermediate mesoderm cells (HOXD11) on day 7 of the differentiation (Step 29). b-l, Whole mount immunostaining of kidney organoids (Step 57). b, Co-staining for markers of collecting ducts (PAX2, GATA3 and ECAD). c, Staining for loop of Henle (UMOD and ECAD). d, Staining for proximal tubule markers (LTL and CUBN) shows evidence of an apical brush border (CUBN). e, Staining for cortical interstitium (MEIS1 and FOXD1, a white arrowhead) and medullary interstitium (MEIS1 only). FOXD1 also marks podocytes (WT1). f, Evidence for the formation of segmenting nephrons showing staining for early podocytes (WT1 and NPHS1), distal tubules (ECAD) and intervening unstained segments at day 18 of differentiation. An involute cluster of podocyes can be seen at the end of a forming nephron (center). g, At day 25 of differentiation, maturing glomeruli develop glomerular basement membrane (LAM, blank arrowheads) in the middle of podocytes (NPHS1). h, Staining for early mesangial cells (PDGFRA) invaginating into podocytes (NPHS1). i, Staining for the endothelial network (CD31 and SOX17). j, Staining of a representative whole mount kidney organoid illustrating the endothelial network (CD31) and glomerular podocytes (NPHS1). Cell nuclei stained by DAPI are shown in the inset panel. k, An example of a small whole kidney organoid (< 3 mm). l, An example of a larger kidney organoid (> 4 mm) Scale bars, 50 μm (a-i), 1 mm (j-l).