Differentiation of human pluripotent stem cells into urothelial organoids via transient activation of WNT signaling

Abstract

The cloaca is a transient structure that forms in the terminal hindgut giving rise to the rectum dorsally and the urogenital sinus ventrally. Similarly, human hindgut cultures derived from human pluripotent stem cells generate human colonic organoids (HCOs) which also contain co-developing urothelial tissue. In this study, our goal was to identify pathways involved in cloacal patterning and apply this to human hindgut cultures. RNA sequencing (RNA-seq) data comparing dorsal versus ventral cloaca in e10.5 mice revealed that WNT signaling was elevated in the ventral versus dorsal cloaca. Inhibition of WNT signaling in hindgut cultures maintained their differentiation toward colonic organoids. WNT activation promoted differentiation toward human urothelial organoids (HUOs). HUOs contained developmental stage specific cell types present in mammalian urothelial tissue including co-developing mesenchyme. Therefore, HUOs offer a powerful in vitro model for dissecting the regulatory pathways that control the dynamic emergence of stage specific cell types within the human urothelium.

Keywords: developmental biology; molecular genetics; stem cells research.

Graphical abstract

Figure 1

Urothelial tissue is present in HCO differentiations (A and B) (A) Immunofluorescence staining and (B) quantification on day 35 H1 ESC derived HCOs for CDH1 (white), CDX2 (red), and GATA3 (green) and counterstained with DAPI (blue). A total of 29 organoids from 3 separate differentiations were analyzed. (C) Uniform manifold approximation and projection (UMAP) Seurat clustering of all urothelial cells from the d22 and d37 HCO cultures. (D) Relative proportions of predicted cell types as well as corresponding markers in each HCO sample. (E) Feature plots of select urothelial genes including uroplakin genes (UPK1A, 1B, 2, and 3A), keratin genes (KRT13, 20, 8, and 5) and transcription factors (GATA3, TBX3, TP63, and SOX2). See also Figure S1 and Table S1. Scale bars: (A) 50 μm.

Figure 2

WNT signaling regulates lineage fate of hindgut cultures (A) Schematic of WNT manipulation during patterning of human hindgut cultures. (B) Principal-component analysis comparing day 7 midgut/hindgut cultures, day 10 BMP2 + ICRT3, and day 10 BMP2 + CHIR cultures. Samples were from 3 to 4 separate differentiations. (C) Gene ontology analysis of genes upregulated in day 10 BMP2 + CHIR cultures compared to day 10 BMP2 + ICRT3 cultures. (D–F) Table of most upregulated transcription factors in day 10 BMP2 + CHIR cultures compared to day 10 BMP2 + ICRT3 cultures. Transcription factors in (D) that overlap with those upregulated in ventral cloaca are shown in bold. Immunofluorescence staining (E) and quantification (F) of d7 and 10 cultures stained for CDH1 (white), CDX2 (red), TP63 (green) and counterstained with DAPI (blue). N = 12 total organoids per group, 4 organoids for each of 3 separate differentiations. Organoids were derived from the IPSC72.3 cell line. See also Figure S3, Video S1 and Table S3. Scale bars: (E) 50 μm. Data in (F) are presented as mean ± SD. ∗∗∗∗ denotes p < 0.0001 based on ANOVA analysis.

Figure 3

WNT patterned hindgut cultures maintain urothelial identity (A) Schematic of long-term growth of BMP2 + CHIR cultures and BMP2 + ICRT3 cultures to 35 days. (B) Wholemount immunofluorescence staining of 35-day old organoids for CDH1 (green), CDH17 (yellow), GATA3 (red) and counterstained with DAPI (blue). (C) Sections from wholemounts in (B). Yellow arrows in (B and C) point to simple columnar epithelium while red arrows point to stratified transitional epithelium. (D) Quantification of organoids stained for CDX2 and GATA3. N = 41 for BMP2+ICRT3, N = 38 for BMP2, N = 41 for BMP2 + CHIR. (E) Principal-component analysis comparing day 35 BMP2 + ICRT3, and day 35 BMP2 + CHIR cultures. Samples from 4 separate differentiations are shown. (F) Principal-component analysis comparing day 35 cultures (N = 4 per condition), human fetal colon (N = 4), and human fetal bladder (N = 2). Organoids were derived from the IPSC72.3 cell line. See also Figures S4 and S5, Tables S4 and S5 and Video S2. Scale bars: (B and C) 100 μm.

Figure 4

Urothelial cell type emergence in HUOs resembles murine fetal development (A) Schematic depicting cell types and their associated markers during development of murine urothelium. (B–Q) Immunofluorescence staining and quantification of 10, 21, and 35-day-old BMP2 + CHIR (HUO) cultures stained for (B–E) CDH1 (white), FOXA2 (red), ISL1 (green), (F–I) CDH1 (white), P63 (red), UPK2 (green), (J–M) CDH1 (white), KRT5 (green), KRT20 (red), (N–Q) CDH1 (green), KI67 (red). (B–D, F–H, J–L, N and O) Samples were counterstained with DAPI (blue). (N′) and (O′) have DAPI excluded. Images are representative of 4 organoids per differentiation from 3 separate differentiations. Organoids were derived from the IPSC72.3 cell line. See also Figure S4 and Video S3. Scale bars: (B–D, F–H, J–L, N, and O) 50 μm. Data in (E, I, M, and Q) are presented as mean ± SD. # denotes p < 0.0001 based on ANOVA analysis.

Figure 5

HUOs generate stratified layers of basal, intermediate, and superficial cells (A) Immunofluorescence staining of 35-day old BMP2 + CHIR (HUO) cultures stained for (A) CDH1 (green) and UPK2 (red). (A′) and (A″) show higher magnification images of the boxed region in (A). (B) Staining of CDH1 (green), UPK3 (red) and GATA3 (magenta). (B′) shows GATA3 and (B″) shows CDH1/UPK3. (C) Staining for KRT5 (white), P63 (green) and ZO-1 (red). (C′) and (C″) show individual channels for KRT5 and ZO-1 respectively. (D) Staining for KRT5 (white), P63 (green) and KRT20 (red). (D′) and (D″) show higher magnification images of the boxed region in (D). (A–D) Samples were counterstained with DAPI (blue). Images are representative of 4 organoids per differentiation from 3 separate differentiations. Organoids were derived from the IPSC72.3 cell line. See also Figure S5 and Video S3. Scale bars: (A, B, B′, B″, and D) 50 μm and (A′, A″, C′, C″, D′, and D″) 20 μm.