Propagation of human prostate tissue from induced pluripotent stem cells

Abstract

Primary culture of human prostate organoids and patient-derived xenografts is inefficient and has limited access to clinical tissues. This hampers their use for translational study to identify new treatments. To overcome this, we established a complementary approach where rapidly proliferating and easily handled induced pluripotent stem cells enabled the generation of human prostate tissue in vivo and in vitro. By using a coculture technique with inductive urogenital sinus mesenchyme, we comprehensively recapitulated in situ 3D prostate histology, and overcame limitations in the primary culture of human prostate stem, luminal and neuroendocrine cells, as well as the stromal microenvironment. This model now unlocks new opportunities to undertake translational studies of benign and malignant prostate disease.

Keywords: androgen receptor; differentiation; induced pluripotent stem cells; organoids; prostate; prostate cancer; stem cells.

Figure 1

In vivo generation of human prostate tissue. A, Schema overview (n = 12 mice, 4 mice × 3 induced pluripotent stem cell [iPSC] clones). All mice xenografts generated human prostate tissue. Urogenital sinus mesenchyme (UGM) cells injected alone did not develop into glands (data not shown). Scale bars 1 mm (left panel), 50 μm (middle panel), and 15 μm (right panel). B, A reference example of in situ human prostate histology, which is indistinguishable from the xenograft glands shown in A. C, Epithelial cells were confirmed of human origin and not mouse contamination by immunolocalization of antihuman mitochondria (anti‐hu‐mitochondria). D‐E, Luminal and basal epithelium was confirmed by specific differentiation markers CK8/18 and p63, respectively. F‐H, The presence of fully differentiated human prostate was confirmed by expression of androgen receptor, NKX3.1 and prostate‐specific antigen. I, The full breadth of prostate epithelial differentiation was confirmed by the presence of sporadic neuroendocrine cells expressing chromogranin A (ChrA). Scale bar = 50 μm. Nuclei counterstained with DAPI

Figure 2

In vitro generation of human prostate organoids. A, Schema outlining the differentiation process (n = 3 induced pluripotent stem cell [iPSC] clones). Briefly, human prostate‐derived iPSCs were differentiated to definitive endoderm (DE) cells using activin A and fetal bovine serum (FBS) over 3 days. Resultant cells were subsequently cocultured with rat urogenital sinus mesenchyme (UGM). At 5 weeks, early prostate organoids were observed having a predominantly basal phenotype while occasionally contained small lumens and expressed luminal markers. Multilayered organoids with large lumens demonstrating classical prostate‐like histology by forming an outer basal and inner luminal layer were observed by 12 weeks. B, Histology of organoids resembled prostate glands. C, Epithelium was identified as human by antihuman mitochondria staining. D, CK8/18 on the cell surface confirmed luminal cells. E, Nuclear p63 confirmed basal cells. F‐H, Androgen receptor (AR), prostate‐specific antigen, and NKX3.1 expression by luminal cells confirmed terminal differentiation. I, Neuroendocrine cells were identified by ChrA expression (0.64 ± 0.21% respectively, n = 7600 cells, n = 12 organoids). J, A subpopulation of basal cells expressed the somatic stem cell marker DLK1 (3.0 ± 1.3%, n = 650 cells, n = 3 experiments). Scale bar = 50 μm. K, Reproducible expression of differentiation markers in mature prostate organoids is shown (n = 183 organoids [164 ± 33 cells/organoid], n = 3 separate experiments; see description of calculations in Materials and Methods). L, Following passage, early clone formation from 1 to 2 cells was observed on day 3, while by day 13 clear canalization was noted of tubular structures associated with dense bud tips reminiscent of tubular branching patterns seen in organogenesis. Scale bar = 25 μm

Figure 3

In vitro induced pluripotent stem cell (iPSC)‐derived prostate organoids shared gene expression profiles of mature human prostate cells. A, Gene Set Enrichment Analysis (GSEA) demonstrating enrichment of basal and luminal gene expression in mature organoids (Wk12) in comparison to definitive endoderm (DE) cells (nominal P‐value< .001, n = 3 repeats). B, The same as in A, but Wk12 vs Wk5 organoids (nominal P‐value <.001). C, Heatmap demonstrating neuroendocrine marker expression. Data is Log2 transformed. D, GSEA Hallmark analysis identified eight statistically significant enriched pathways in mature prostate organoids

Figure 4

Prostate induced pluripotent stem cells (iPSCs) generated a self‐maintaining stromal compartment in mature prostate organoids. A, Human antimitochondria and α‐smooth muscle actin (SMA) colocalization was seen in stromal cells within areas of the kidney containing the xenograft. Scale bars = 5 mm (left panel) and 50 μm (middle and right panels) (n = 12, 4 mice × 3 iPSC clones). B, Stromal cells surrounded mature prostate organoids. Scale bar = 25 μm. C‐D, Overlapping expressions of vimentin, antihuman mitochondria, α‐SMA and p63 demonstrated capacity of iPSCs to generate the stromal compartment. Scale bar = 25 μm (n = 3 iPSC clones, n = 3 repeats)