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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., We demonstrate for the first time that induced pluripotent stem cells (iPSCs) can generate human prostate tissue both in vivo and in vitro, overcoming some significant limitations with patient‐derived xenografts and primary culture to provide a complimentary approach. Furthermore, the in vitro model provides a new platform to create a living biobank of prostate cancer organoids derived from gene editing of iPSCs for preclinical drug testing.

Primary culture of human prostate organoids is slow, inefficient and laborious. To overcome this, we demonstrate a new high-throughput model where rapidly proliferating and easily handled induced pluripotent stem cells, for the first time, enable generation of human prostate tissue in vivo and in vitro. Using a co-culture technique with urogenital sinus mesenchyme, we recapitulated the in situ prostate histology, including the stromal compartment and the full spectrum of epithelial differentiation. This approach overcomes major limitations in primary cultures of human prostate stem, luminal and neuroendocrine cells, as well as the stromal microenvironment. These models provide new opportunities to study prostate development, homeostasis and disease.

One of the key issues hampering the development of effective treatments for prostate cancer is the lack of suitable, tractable, and patient-specific in vitro models that accurately recapitulate this disease. In this review, we address the challenges of using primary cultures and patient-derived xenografts to study prostate cancer. We describe emerging approaches using primary prostate epithelial cells and prostate organoids and their genetic manipulation for disease modelling. Furthermore, the use of human prostate-derived induced pluripotent stem cells (iPSCs) is highlighted as a promising complimentary approach. Finally, we discuss the manipulation of iPSCs to generate ‘avatars’ for drug disease testing. Specifically, we describe how a conceptual advance through the creation of living biobanks of “genetically engineered cancers” that contain patient-specific driver mutations hold promise for personalised medicine.

Stem cell characteristics have been associated with treatment resistance and poor prognosis across many cancer types. The ability to induce and regulate the pathways that sustain these characteristic hallmarks of lethal cancers in a novel in vitro model would greatly enhance our understanding of cancer progression and treatment resistance. In this work, we present such a model, based simply on applying standard pluripotency/embryonic stem cell media alone. Core pluripotency stem cell master regulators (OCT4, SOX2 and NANOG) along with epithelial-mesenchymal transition (EMT) markers (Snail, Slug, vimentin and N-cadherin) were induced in human prostate, breast, lung, bladder, colorectal, and renal cancer cells. RNA sequencing revealed pathways activated by pluripotency inducing culture that were shared across all cancers examined. These pathways highlight a potential core mechanism of treatment resistance. With a focus on prostate cancer, the culture-based induction of core pluripotent stem cell regulators was shown to promote survival in castrate conditions-mimicking first line treatment resistance with hormonal therapies. This acquired phenotype was shown to be mediated through the upregulation of iodothyronine deiodinase DIO2, a critical modulator of the thyroid hormone signalling pathway. Subsequent inhibition of DIO2 was shown to supress expression of prostate specific antigen, the cardinal clinical biomarker of prostate cancer progression and highlighted a novel target for clinical translation in this otherwise fatal disease. This study identifies a new and widely accessible simple preclinical model to recreate and explore underpinning pathways of lethal disease and treatment resistance.

Cancer stem cells are defined as a self-renewing and self-protecting subpopulation of cancer cells able to differentiate into morphologically and functionally diverse cancer cells with a limited lifespan. To purify cancer stem cells, two basic approaches can be applied, the marker-based approach employing various more of less-specific cell surface marker molecules and a marker-free approach largely based on various self-protection mechanisms. Within the context of urothelial carcinoma, both methods could find use. The cell surface markers have been mainly derived from the urothelial basal cell, a probable cell of origin of muscle-invasive urothelial carcinoma, with CD14, CD44, CD90, and 67LR representing successful examples of this strategy. The marker-free approaches involve side population sorting, for which a detailed protocol is provided, as well as the Aldefluor assay, which rely on a specific overexpression of efflux pumps or the detoxification enzyme aldehyde dehydrogenase, respectively, in stem cells. These assays have been applied to both non-muscle-invasive and muscle-invasive bladder cancer samples and cell lines. Urothelial carcinoma stem cells feature a pronounced heterogeneity as to their molecular stemness mechanisms. Several aspects of urothelial cancer stem cell biology could enter translational development rather soon, e.g., a specific CD44+-derived gene expression signature able to identify non-muscle-invasive bladder cancer patients with a high risk of progression, or deciphering a mechanism responsible for repopulating activity of urothelial carcinoma stem cells within the context of therapeutic resistance.

There is considerable interest in the use of multi-potent stem cells in kidney tissue regeneration. We studied if spermatogonial stem cells have the ability to undergo kidney differentiation. Spermatogonial stem cell differentiation was induced using in vitro and ex vivo co-culture techniques. Conditioned media from human kidney fibroblasts induced the expression of epithelial and endothelial lineages in spermatogonial stem cells, consistent with nephrogenesis. Furthermore, we showed that these cells up-regulated renal tubular-specific markers alkaline phosphatase, mineralocorticoid receptor, renal epithelial sodium channel and sodium-glucose transporter-2 (p

Mechanistic studies of tumorigenesis point towards stem cells (SCs) being the cell of origin in prostate cancer initiation, suggesting stem pathways have a crucial role in the early stages of cancer development (Gu et al, 2007; Goldstein et al, 2010; Lawson et al, 2010). Furthermore, known stem pathways have been seen to be aberrantly expressed as the prostate cancer progresses (Linn et al, 2010; Jia et al, 2011; Kregel et al, 2013). Characterisation of the genes at play within prostate SCs, and characterising these same markers in the malignant setting may identify unique therapeutic opportunities for the treatment of prostate cancer. We have recently demonstrated the presence of the androgen receptor (AR) within normal human prostate SCs (Williamson et al, 2012), so identification of specific novel AR pathway processes within these cells will be of particular importance. Such mechanisms may explain the maintenance of androgen ‘addiction' in the malignant setting during cancer progression despite androgen deprivation, as it is established that the AR pathway has diverse roles depending on the degree of prostate differentiation (Gregory et al, 1998; Buchanan et al, 2001; Mousses et al, 2001; Chen et al, 2004; Taplin and Balk, 2004; Attard et al, 2009; Bonkhoff and Berges, 2010; Lee et al, 2012). Human prostate SCs can be enriched by their expressions of α2β1-integrins and CD133 (Richardson et al, 2004). In this work, we describe gene expression signatures associated with SCs (α2β1HI CD133+VE), transiently amplifying (TAP) cells (α2β1HI CD133−VE) and terminally differentiated (TD) cells (α2β1LOW CD133−VE) following the intuitive pathway analysis of transcriptome characterisation of human prostates. Within normal prostate SCs, we discovered a deficiency in class 1 UDP (uridine 5′-diphospho)-glucuronosyltransferase; UGT) enzymes (UGTA1), which are regulators of androgen catabolism. Our functional in vitro models of prostate cancer demonstrated that UGT1A knock down leads to upregulation of prostate-specific antigen (PSA) along with increased cell survival. Furthermore, reduced UGT1A expression was shown to be a feature of clinical prostate cancer that was associated with a poorer prognosis.

BACKGROUND: Primary culture and animal and cell-line models of prostate and bladder development have limitations in describing human biology, and novel strategies that describe the full spectrum of differentiation from foetal through to ageing tissue are required. Recent advances in biology demonstrate that direct reprogramming of somatic cells into pluripotent embryonic stem cell (ESC)-like cells is possible. These cells, termed induced pluripotent stem cells (iPSCs), could theoretically generate adult prostate and bladder tissue, providing an alternative strategy to study differentiation.\ud \ud OBJECTIVE: To generate human iPSCs derived from normal, ageing, human prostate (Pro-iPSC), and urinary tract (UT-iPSC) tissue and to assess their capacity for lineage-directed differentiation.\ud \ud DESIGN, SETTING, AND PARTICIPANTS: Prostate and urinary tract stroma were transduced with POU class 5 homeobox 1 (POU5F1; formerly OCT4), SRY (sex determining region Y)-box 2 (SOX2), Kruppel-like factor 4 (gut) (KLF4), and v-myc myelocytomatosis viral oncogene homolog (avian) (MYC, formerly C-MYC) genes to generate iPSCs.\ud \ud OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The potential for differentiation into prostate and bladder lineages was compared with classical skin-derived iPSCs. The student t test was used.\ud \ud RESULTS AND LIMITATIONS: Successful reprogramming of prostate tissue into Pro-iPSCs and bladder and ureter into UT-iPSCs was demonstrated by characteristic ESC morphology, marker expression, and functional pluripotency in generating all three germ-layer lineages. In contrast to conventional skin-derived iPSCs, Pro-iPSCs showed a vastly increased ability to generate prostate epithelial-specific differentiation, as characterised by androgen receptor and prostate-specific antigen induction. Similarly, UT-iPSCs were shown to be more efficient than skin-derived iPSCs in undergoing bladder differentiation as demonstrated by expression of urothelial-specific markers: uroplakins, claudins, and cytokeratin; and stromal smooth muscle markers: α-smooth-muscle actin, calponin, and desmin. These disparities are likely to represent epigenetic differences between individual iPSC lines and highlight the importance of organ-specific iPSCs for tissue-specific studies.\ud \ud CONCLUSIONS: IPSCs provide an exciting new model to characterise mechanisms regulating prostate and bladder differentiation and to develop novel approaches to disease modelling. Regeneration of bladder cells also provides an exceptional opportunity for translational tissue engineering.

[1] Takahashi K, Yamanaka A. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663–76. [2] Moad M, Pal D, Hepburn AC, et al. A novel model of urinary tract differentiation, tissue regeneration, and disease: reprogramming human prostate and bladder cells into induced pluripotent stem cells. Eur Urol 2013;64:753–61. [3] Collins AT, Habib FK, Maitland NJ, Neal DE. Identification and isolation of human prostate epithelial stem cells based on alpha(2) beta(1)-integrin expression. J Cell Sci 2001;114:3865–72. [4] Tian H, Bharadwaj S, Liu Y, Ma PX, Atala A, Zhang Y. Differentiation of human bone marrow mesenchymal stem cells into bladder cells: potential forurological tissueengineering. TissueEngPartA2010;16: 1769–79. [5] Ghosh Z, Wilson KD, Wu Y, Hu S, Quertermous T, Wu JC. Persistent donor cell gene expression among human induced pluripotent stem cells contributes to differences with human embryonic stem cells. PLoS One 2010;5:e8975. http://www.plosone.org/article/info:doi/ 10.1371/journal.pone.0008975

Side population (SP) and ABC transporter expression enrich for stem cells in numerous tissues. We explored if this phenotype characterised human bladder cancer stem cells (CSCs) and attempted to identify regulatory mechanisms. Focusing on non-muscle invasive bladder cancer (NMIBC), multiple human cell lines were used to characterise SP and ABC transporter expression. In vitro and in vivo phenotypic and functional assessments of CSC behaviour were undertaken. Expression of putative CSC marker ABCG2 was assessed in clinical NMIBC samples (n = 148), and a role for MAPK signalling, a central mechanism of bladder tumourigenesis, was investigated. Results showed that the ABCG2 transporter was predominantly expressed and was up-regulated in the SP fraction by 3-fold (ABCG2(hi)) relative to the non-SP (NSP) fraction (ABCG2(low)). ABCG2(hi) SP cells displayed enrichment of stem cell markers (Nanog, Notch1 and SOX2) and a three-fold increase in colony forming efficiency (CFE) in comparison to ABCG2(low) NSP cells. In vivo, ABCG2(hi) SP cells enriched for tumour growth compared with ABCG2(low) NSP cells, consistent with CSCs. pERK was constitutively active in ABCG2(hi) SP cells and MEK inhibition also inhibited the ABCG2(hi) SP phenotype and significantly suppressed CFE. Furthermore, on examining clinical NMIBC samples, ABCG2 expression correlated with increased recurrence and decreased progression free survival. Additionally, pERK expression also correlated with decreased progression free survival, whilst a positive correlation was further demonstrated between ABCG2 and pERK expression. In conclusion, we confirm ABCG2(hi) SP enriches for CSCs in human NMIBC and MAPK/ERK pathway is a suitable therapeutic target.