A genetically inducible endothelial niche enables vascularization of human kidney organoids with multilineage maturation and emergence of renin expressing cells

Abstract

Vascularization plays a critical role in organ maturation and cell-type development. Drug discovery, organ mimicry, and ultimately transplantation hinge on achieving robust vascularization of in vitro engineered organs. Here, focusing on human kidney organoids, we overcame this hurdle by combining a human induced pluripotent stem cell (iPSC) line containing an inducible ETS translocation variant 2 (ETV2) (a transcription factor playing a role in endothelial cell development) that directs endothelial differentiation in vitro, with a non-transgenic iPSC line in suspension organoid culture. The resulting human kidney organoids show extensive endothelialization with a cellular identity most closely related to human kidney endothelia. Endothelialized kidney organoids also show increased maturation of nephron structures, an associated fenestrated endothelium with de novo formation of glomerular and venous subtypes, and the emergence of drug-responsive renin expressing cells. The creation of an engineered vascular niche capable of improving kidney organoid maturation and cell type complexity is a significant step forward in the path to clinical translation. Thus, incorporation of an engineered endothelial niche into a previously published kidney organoid protocol allowed the orthogonal differentiation of endothelial and parenchymal cell types, demonstrating the potential for applicability to other basic and translational organoid studies.

Keywords: endothelial; genetic engineering; organoids; podocytes; renin; scRNAseq.

Figure 1 |. Genetically inducible ETV2 human induced pluripotent stem cells (iETV2-hiPSCs) undergo synthetic endothelial differentiation.

(a) Schematic of iETV2-hiPSC genetic circuit and path to differentiation. (b) iETV2-hiPSCs differentiated with doxycycline (dox) induction and representative immunofluorescent images from day 0 to day 9 from 3 biological replicates; bar = 200 um. (c) Per-cell immunofluorescent intensity quantification of green fluorescent protein (GFP), melanoma cell adhesion molecule 1 (MCAM), and platelet and endothelial cell adhesion molecule 1 (PECAM1) channels. N = 3 biological replicates; ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. (d) Graphical schematic of single-cell RNA sequencing (scRNAseq) carried out on iETV2-hiPSCs after they have been exposed to doxycycline for 4 days. (e) Uniform manifold approximation and projection (UMAP) of differentiated iETV2-hiPSCs generated 3 distinct cellular populations. (f) Pseudotime trajectory analysis with Monocle3 (Trapnell Lab) demonstrated developmental lineage from early endothelial-like progenitor population (P1) → mid–endothelial-like progenitor cells (P2) → late endothelial-like progenitor cells (P3). (g) Differentially expressed genes for P1, P2, P3. DAPI, 4’ ,6-diamidino-2-phenylindole; ERG, erythroblast transformation-specific-related gene; TRE, tetracycline responsive element. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Figure 2 |. Vascularization of human kidney organoids.

(a) Method for vascularizing human kidney organoids by combining genetically inducible ETV2 human induced pluripotent stem cells (iETV2-hiPSCs) with wild-type iPSCs. (b) Representative immunofluorescent platelet and endothelial cell adhesion molecule 1 (PECAM1) images of endothelial cell network between the MANZ2-2 control and vascularized kidney organoids from 3 independent biological replicates. (c) AngioTool quantification and diameter measurement between control and vascularized kidney organoids; organoids from 3 biological replicates. ****P < 0.0001. (d) Representative immunofluorescence images of endothelial interaction with podocytes, proximal tubule, distal tubule, and stroma between control and vascularized (Vasc.) kidney organoid from 3 biological replicates. Organoid images, bar = 200 mm; inset bar = 50 mm. CDH1, cadherin-1; dox, doxycycline; HNF4A, hepatocyte nuclear factor 4 alpha; NPHS1, nephrin. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Figure 3 |. Single-nuclei RNA sequencing (snRNAseq) of control and vascularized human kidney organoids.

(a) Graphical schematic demonstrating MANZ2-2 control and vascularized human kidney organoids analyzed via snRNAseq. (b) Cells from control and vascularized kidney organoid aggregated and were well overlapped on uniform manifold approximation and projection (UMAP). (c) Cells on UMAP clustered by cell type as podocyte (POD), endothelial (ENDO), tubular (TUB), and interstitial (INT-1/2). (d) Differentially expressed genes per cluster were identified with dot plot. Cellular populations were quantified to analyze composition by control or vascularization origin and graphed as pie chart per cell type (red = vascularized; blue = control). (e) Feature dot plot demonstrated enhanced green fluorescent protein (EGFP) expression was localized to the endothelial population.

Figure 4 |. Increased maturation of podocytes with vascularization.

(a) Podocytes (POD) exist in nephrin (NPHS1⁺) clusters in kidney organoids on the exterior surface by immunofluorescence. They become highly vascularized (vasc.) with the vascularization protocol and lack vascular integration with the control (cont) kidney organoid. Representative immunofluorescent images of 10 biological replicates. (b) Vascularized MANZ2-2 kidney organoids contain green fluorescent protein (GFP)⁺ endothelial cells encasing the podocyte clusters from the external surface, and invaginating networks through the middle of the cluster. (c) Podocytes from both control and vascularized kidney organoid were (d) subclustered on uniform manifold approximation and projection (UMAP) for further analysis. (e) Podocytes distinctly cluster into 2 populations predominated largely by control or vascularized podocytes. (f) Podocyte-specific markers are present in both clusters, by feature plots; however, slit diaphragm and basement membrane markers are upregulated in cluster 0, predominated by vascularized podocytes. (g) Gene-set enrichment analysis identified upregulated pathways of basement membrane, glomerular development (dev), and endothelial vasculature differentiation (diff.) and migration (mig.) in the vascularized predominating podocyte cluster, with dot plots showing representative markers from each gene enrichment set ontology identifier. COL4A, collagen type IV alpha 2 chain; ETV2, ETS variant transcription factor 2; LAMB1, laminin subunit beta 1; NID2, nidogen 2; PECAM1, platelet and endothelial cell adhesion molecule 1; SLIT3, slit guidance ligand 3; WT1, Wilms’ tumor protein 1. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Figure 5 |. Vascularization of kidney organoid enables emergence of a renin (REN) cell population.

(a) Interstitial (INT) cells from MANZ2-2 control and vascularized kidney organoid were further analyzed in separate uniform manifold approximation and projections (UMAPs). (b) Interstitial cells clustered into 8 distinct populations. FIB-X, fibroblast-X; MYO, myoblast; MUR, mural; PROL-FIB, proliferative fibroblast; PROL-MYO, proliferative myoblast; VSM-REN, vascular smooth muscle renin. (c) Violin plots of key genes per cluster. (d) REN specifically localized to a population of cells on a feature plot that (e) originated largely from the vascularized kidney organoid as demonstrated on dot plot. (f) Control kidney organoid contains no REN⁺ cells on immunofluorescence, whereas vascularized kidney organoid contains many, spread across podocyte (nephrin [NPHS1]⁺) clusters; bar = 200 mm. (g) Vascularized kidney organoid podocyte clusters contain REN⁺ cells within the cluster,(h) juxtaposing but not colabelling with NPHS1⁺ podocytes or green fluorescent protein (GFP)⁺ platelet and endothelial cell adhesion molecule 1 (PECAM1)⁺ endothelial cells; bar = 50 mm. (i) 10-uM forskolin (FSK)—a pro–renin stimulatory drug—supplemented in organoid media enables a 200-fold increase in renin expression in the vascularized kidney organoid by reverse transcription quantitative polymerase chain reaction (RT-qPCR). avg. exp, average expressed; ENDO, endothelial; FAP, fibroblast activation protein alpha; FC, fold change; FIB, fibroblast; MEIS1, Meis homeobox 1; MYO, myoblast; NEXN, nexilin F-actin binding protein; pct. exp., percentage expressed; PDGFRA, platelet-derived growth factor receptor alpha; TUB, tubular; VSM, vascular smooth muscle. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Figure 6 |. Genetically inducible ETV2 human induced pluripotent stem cells (iETV2-hiPSCs) undergo maturation and organ specification.

(a) Endothelial (ENDO) population of MANZ2-2 control and vascularized (vasc.) kidney organoids reclustered on uniform manifold approximation and projection (UMAP). (b) Endothelial cells subcluster into 3 distinct identities. (c) Endothelial cells are + platelet and endothelial cell adhesion molecule 1 (PECAM1)⁺ cadherin 5 (CDH5)⁺ on feature plot. (d) By dot plot, each distinct cluster represents glomerular (GLOM)-, arterial (ART)-, and venous (VEN)-like subidentity by canonical markers previously published. (e) SingleCellNet (Cahan Lab) classification of each subendothelial cell type using the Tabula Sapiens organ-specific endothelial dataset demonstrates kidney specification of endothelial cells. (f) On scanning electron microscopy, endothelial cell surface of vascularized kidney organoid demonstrates equally spaced fenestrations (fen); bar = 1 um. (g) Vascularized kidney organoid with iETV2-hiPSC express fenestration marker PLVAP on immunofluorescence; bar = 100 um. GFP, green fluorescent protein; INT, interstitial; PLVAP, plasmalemma vesicle-associated protein; POD, podocyte; rand, random; TUB, tubular. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.