Kidney organoids: accurate models or fortunate accidents

Abstract

There are now many reports of human kidney organoids generated via the directed differentiation of human pluripotent stem cells (PSCs) based on an existing understanding of mammalian kidney organogenesis. Such kidney organoids potentially represent tractable tools for the study of normal human development and disease with improvements in scale, structure, and functional maturation potentially providing future options for renal regeneration. The utility of such organotypic models, however, will ultimately be determined by their developmental accuracy. While initially inferred from mouse models, recent transcriptional analyses of human fetal kidney have provided greater insight into nephrogenesis. In this review, we discuss how well human kidney organoids model the human fetal kidney and how the remaining differences challenge their utility.

Keywords: collecting duct; kidney development; kidney organoid; metanephros; nephron progenitor; pluripotent stem cell; podocyte; single-cell transcriptional profiling.

Figure 1.

An overview of mammalian kidney development. (A) Positioning of the metanephric mesenchyme and mesonephric tubules within the developing mouse embryo at ∼10 d of embryonic development. The mesonephros and metanephric mesenchyme lie adjacent to the nephric duct, from which the ureteric bud (UB) arises. Adapted from http://www.gudmap.org/tutorials/urogenital-dev/devmus.html. (B) Comparison of gene expression and nephron induction in the mesonephric and metanephric mesenchyme. Solid arrows indicate known regulatory relationships. (MT) Mesonephric tubules; (RV) renal vesicle; (UB) ureteric bud. Adapted from Georgas et al. (2011). (C) Expression of key nephron markers in E10.5 mesonephric tubules (left panel) and E13.5 metanephric nephrons (right panel). Adapted from Georgas et al. (2011). (D) 3D rendering of the nephrogenic cord (NC), adjacent to the nephric duct (ND), and urogenital sinus (UGS). Reprinted from Wainwright et al. (2015) with permission from Elsevier. (E) Overview of reciprocal signaling and inductive relationships in the nephrogenic niche, nephron patterning, and maturation. (NZS) Nephrogenic zone stroma; (NP) nephron progenitor; (UT) ureteric tip; (PA) pretubular aggregate; (MET) mesenchyme to epithelial transition; (RV) renal vesicle; (CSB and SSB) comma- and S-shaped body; (cns) connecting segment. (F) Comparison of timeline and gross morphology of kidney development in humans and mice. Adapted from Little (2015). (G) Illustration of nephron arcading in the human kidney (https://abdominalkey.com/development-of-the-kidney). (H) Illustration of live imaging setup for developing mouse kidney explant cultures from Lawlor et al. (2019). (I) Tracking nephron progenitor cell movement (red dots and lines) over time. Ureteric tip is shown in green. (I–K) Cells labeled (red) with an inducible Cre driven by the Wnt4 promoter in the early committing nephron can integrate back into the progenitor population by cell migration. Green in I and J is GFP expression in the tip, green in K is NCAM staining, and white staining in K shows expression of nephron progenitor marker SIX2. I is from Combes et al. (2019a); J and K are from Lawlor et al. (2019).

Figure 2.

A comparison between the seminal protocols in the field for the directed differentiation of human pluripotent stem cells to kidney tissue, highlighting the congruence of timing between these approaches. Immunofluorescence images illustrate the presence of segmented nephrons in all instances. While the observation of a surrounding vasculature was highlighted in Takasato et al. (2015), the protocols of Taguchi et al. (2014) (R Nichinakamura, pers. comm.), Morizane et al. (2015) (as illustrated here), and Freedman et al. (2015) (as illustrated here) also show evidence of spontaneous endothelial cell formation. Factors and media used to direct differentiation: (A) Activin A (following number indicates micromolar concentration); (APEL) APEL differentiation medium (Stem Cell Technologies); (C) CHIR99201 (Wnt pathway agonist; following number indicates micromolar concentration); (B4) BMP4; (B7) BMP7; (B27) B-27 neural supplement medium; (F2) FGF2; (F9) FGF9; (no GFs) no growth factors added to basal medium; (mTESR-E6) E6 minimal differentiation medium (Stem Cell Technologies); (noggin) BMP antagonist; (R) retinoic acid, (RPMI) Roswell Park Memorial Institute 1640; (Y) Y27632 (Rho kinase inhibitor). Stages of differentiation: (AIM) Anterior intermediate mesoderm; (EB) embryoid body; (IM) intermediate mesoderm; (MM) metanephric mesenchyme; (PIM) posterior intermediate mesoderm; (PS) primitive streak; (PPS) posterior primitive streak; (PTA) pretubular aggregate; (RV) renal vesicle. Original figures are shown for the first two protocols (Takasato et al. 2014; Taguchi and Nishinakamura 2015). Reprinted with permission from Taguchi and Nishinakamura (2015) with permission from Elsevier. Unpublished images are presented for the final three protocols. Unpublished images were contributed by Lorna Hale (Takasato protocol), Tomoyo Miyoshi, Ryuji Morizane, Navin Gupta, Kimberley Homan and Jennifer Lewis (Morizane protocol), and Nelly Cruz (Freedman protocol). Scale bars, 50 µM.

Figure 3.

Cellular complexity of kidney organoids generated via directed differentiation of human pluripotent stem cells using the Takasato protocol. (A–C) Human kidney organoids generated using Takasato et al. (2015) at day 25 of culture. (A,B) Immunofluorescence staining of whole kidney organoid (A; diameter = 3 mm) and higher magnification (B) shows evidence for glomeruli (NPHS1; white), proximal tubules (LTL; blue), distal tubules (CDH1; green), and a connecting epithelial network positive for GATA3 (red) and CDH1 (green). (C) Evidence of an extensive endothelial network (CD31; green) between organoid glomeruli (NPHS1; magenta). (D) Diagram of kidney differentiation from the intermediate mesoderm (IM) highlighting the ureteric epithelium (UE) as the origin of the collecting duct and the metanephric mesenchyme (MM) as the origin of the nephron. Evidence exists for stromal and vascular progenitors; however, their origins are less clear. (DT) Distal tubule; (LoH) loop of Henle; (PT) proximal tubule; (POD) podocyte; (VASC) vasculature; (STROM) stroma. (E) Immunofluorescence images of the cellular elements present within a kidney organoid generated using Takasato et al. (2015). (F) Immunofluorescence analysis of isolated organoid glomeruli showing polarized colocalization of the slit diaphragm proteins NEPHRIN (green) and NEPH1 (red) proteins compared with the nonpolarized protein PODXL (magenta). Image from Hale et al. (2018). (G) Bright-field image through intact organoid glomeruli with capillary loop structure evident at the left. Image from Hale et al. (2018). (H) Heat map showing relative expression of key podocyte genes in the immortalized podocyte cell line compared with organoid derived podocytes (OrgPod) after 2D culture and 3D isolated organoid glomeruli (OrgGlom). Adapted from Hale et al. (2018). (I–N) Reporter lines illustrating the presence of individual cell types within kidney organoids. In each example, the promoter into which the fluorescent reporter has been placed is indicated in italics while regular text is used for antibody staining. These reporters demonstrate the presence of endothelial cells (I, Sox17mCherry) (Ng et al. 2016), proximal tubules (J, HNF4AYFP; K, Lrp2mTAGBFP), connecting segment/ureteric epithelium (L, GATA3mCherrry), podocytes (M, MAFBmTAGBFP), and a double reporter with both GATA3mCherry and MAFBmTAGBFP (N). Images from Vanslambrouck et al. (2019). (O) Targeting constructs for the generation of a human iPSC SIX2 lineage tracing reporter line. Tamoxifen (4-OHT) will result in the production of Cre recombinase within any cell expressing SIX2. As a result, loxP-Cre mediated excision of the EGFP cassette present within the GAPDH safe harbor locus will cause a switch in cell color from green to red (mCherry). Howden et al. (2019). (P) High-resolution immunofluorescence of nephrons within kidney organoids subjected to early (day 7 + 10) or date (day 7 + 18) 4-OHT. mCherry indicates cells that have initiated SIX2 expression. mCherry⁺ cells within nephrons after early induction shows that a SIX2-expressing progenitor contributes to nephron formation early in organoid patterning. The absence of mCherry within nephrons after late induction shows that this is no longer the case at later time points. Glomeruli (NPHS1; white), proximal tubules (LTL; blue), nephron epithelium (EpCAM; green). Adapted from Howden et al. (2019). Scale bars, 50 µM.

Figure 4.

Investigating the reproducibility, cellular complexity, transcriptional and developmental accuracy of hPSC-derived kidney organoids. (A) 3D correlation analysis of global transcriptional profile across kidney organoid differentiation from day 0 (undifferentiated iPSC) through day 4 (posterior primitive streak), day 7 (intermediate mesoderm) and days 10, 18, and 25 of organoid culture using two distinct iPSC lines. This analysis suggests that the differentiation protocol is robust with replicates correlating with stage of differentiation even when generated using different iPSC lines and experiments performed at different times. From Phipson et al. (2019). (B) tSNE plot showing overlay of >8000 single cells generated from four different day 7 + 18 kidney organoids showing strong reproducibility between organoids at the single-cell level. From Phipson et al. (2019). (C) tSNE plot overlaying single cells isolated from iPSC-derived kidney organoids (blue) with human fetal kidney (pink). Reprinted from Combes et al. (2019b). (D) Analysis of the cell types present in the human fetal kidney and organoid data sets, as analyzed in Combes et al. (2019b). (E) Relative abundance of key cellular elements in human fetal kidney versus kidney organoid. Note that while stroma is a prevalent component in kidney organoids, this is also the case in human fetal kidney. Kidney organoids showed a higher level of proliferating cells (cell cycle). Reprinted from Combes et al. (2019b). (F) Comparison of organoid cell clustering in “organoid only” to “combined” organoid and human fetal kidney clusters. Overlap in samples within clusters from the different analyses is shown using the Jaccard Index (JI) with a score of 1 (yellow) indicating identical clusters and 0 (blue) indicating no cells in common. Combined (C) cluster identities are listed at the left of the heat map indicating JI score, some markers conserved between organoid and fetal kidney cells within each cluster are listed at the right.

Figure 5.

Comparison between fetal kidney and kidney organoids. This diagram highlights the developmental features in common, restricted to normal fetal kidney development (fetal kidney features) or present only in kidney organoids (organoid features).

Figure 6.

The challenge of a higher order kidney from induced pluripotent stem cells. (A) Immunofluorescence of putative collecting duct within human kidney organoid. PAX2 (green), GATA3 (purple), CDH1 (yellow). (B) Evidence for an intervening nephron segment (CDH1⁺) between the proximal nephron (LTL; blue) and the putative collecting duct (CDH1⁺GATA3⁺). (C) Evidence for an intervening nephron segment (CDH1⁺) between the thick ascending limb/distal straight tubule (SLC12A1; white) and the putative collecting duct (GATA3, red). (D) Sectioned kidney organoids showing evidence for a contiguous GATA3⁺CDH1⁺ epithelium connecting all nephrons within the kidney organoid. Reprinted from Higgins et al. (2018). (E) Lineage tracing shows contribution of SIX2-expressing cells to nephron segments other than the distal GATA3-expressing region. From Howden et al. (2019). (F) Comparison of expression between E18.5 mouse kidney distal nephron and collecting duct shows coexpression of many genes assumed to be collecting duct specific, including Hoxb7, Gata3, Calb1, Krt8, Krt18, and Aqp2. Reprinted from Combes et al. (2019a). (G) Evidence in mouse for expression of Gata3 and Hoxb7 within distal nephron (DN), including distal tubule (DT) and connecting segment (CnS), as well as ureteric epithelium (UE)/ureteric tip (UT). Lineage tracing using the SIX2Cre-TdTomato shows a nephron progenitor (NP) origin for the DT and CnS but not the UT. Reprinted from Combes et al. (2019a) with permission from Elsevier. (H,I) Immunofluorescence of a murine higher order kidney organoid generated after combining mouse ESC-derived nephron progenitor and collecting duct progenitors with Foxd1-sorted mouse stroma. (H) Evidence for a branching collecting duct tree (CK8; red) and a peripheral nephron progenitor (Six2) population. (I) Evidence for the initiation of nephrons which connect to the collecting duct tree. Reprinted from Taguchi and Nishinakamura (2017). (J) A branching presumptive collecting duct structure generated from human IPSC differentiated using directed differentiation to ureteric epithelium. Reprinted from Taguchi and Nishinakamura (2017).