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Current organoid models are limited by their inability to mimic mature organ architecture and associated tissue microenvironments1,2. Here we create multilayer bladder ‘assembloids’ by reconstituting tissue stem cells with stromal components to represent an organized architecture with an epithelium surrounding stroma and an outer muscle layer. These assembloids exhibit characteristics of mature adult bladders in cell composition and gene expression at the single-cell transcriptome level, and recapitulate in vivo tissue dynamics of regenerative responses to injury. We also develop malignant counterpart tumour assembloids to recapitulate the in vivo pathophysiological features of urothelial carcinoma. Using the genetically manipulated tumour-assembloid platform, we identify tumoural FOXA1, induced by stromal bone morphogenetic protein (BMP), as a master pioneer factor that drives enhancer reprogramming for the determination of tumour phenotype, suggesting the importance of the FOXA1–BMP–hedgehog signalling feedback axis between tumour and stroma in the control of tumour plasticity. Multilayer 3D reconstitution of bladder stem cells with stromal cells enables recapitulation of the architecture and molecular functions of bladder tissue.

Current organoid models are limited by their inability to mimic mature organ architecture and associated tissue microenvironments1,2. Here, we create multi-layered normal bladder assembloids and tumour assembloids by reconstituting tissue stem cell/tumour cell-derived organoids with multiple stromal components to represent an organized tissue architecture and functionality of adult bladder, and to mimic in vivo pathophysiological features of patient-derived urothelial carcinoma, respectively. Our assembloid model will facilitate development of an innovative model system for studying tissue regeneration and tumourigenesis at molecular and cellular levels, and also provide a unique experimental tool for establishing novel personalized therapeutic options, such as new drug screening strategies that are customized for different stages and types of cancer in individual patients.

Quantification of intratumoral heterogeneity is essential for designing effective therapeutic strategies in the age of personalized medicine. In this study, we used a piezoelectric inkjet printer to enable analysis of intratumoral heterogeneity in a bladder cancer for the first time. Patient-derived tumor organoids were dissociated into single cell suspension and used as a bioink. The individual cells were precisely allocated into a microwell plate by drop-on-demand inkjet printing without any additive or treatment, followed by culturing into organoids for further analysis. The sizes and morphologies of the organoids were observed, so as the expression of proliferation and apoptotic markers. The tumor organoids also showed heterogeneous responses against chemotherapeutic agent. Further, we quantified mRNA expression levels of representative luminal and basal genes in both type of tumor organoids. These results verify the heterogeneous expression of various genes among individual organoids. This study demonstrates that the fully automated inkjet printing technique can be used as an effective tool to sort cells for evaluating intratumoral heterogeneity.

Plants show great potential for producing recombinant proteins in a cost-effective manner. Many strategies have therefore been employed to express high levels of recombinant proteins in plants. Although foreign domains are fused to target proteins for high expression or as an affinity tag for purification, the retention of foreign domains on a target protein may be undesirable, especially for biomedical purposes. Thus, their removal is often crucial at a certain time point after translation. Here, we developed a new strategy to produce target proteins without foreign domains. This involved in vivo removal of foreign domains fused to the N-terminus by the small ubiquitin-related modifier (SUMO) domain/SUMO-specific protease system. This strategy was tested successfully by generating a recombinant gene, BiP:p38:bdSUMO : His:hLIF, that produced human leukemia inhibitory factor (hLIF) fused to p38, a coat protein of the Turnip crinkle virus; the inclusion of p38 increased levels of protein expression. The recombinant protein was expressed at high levels in the leaf tissue of Nicotiana benthamiana. Coexpression of bdSENP1, a SUMO-specific protease, proteolytically released His:hLIF from the full-length recombinant protein in the endoplasmic reticulum of N. benthamiana leaf cells. His:hLIF was purified from leaf extracts via Ni2+-NTA affinity purification resulting in a yield of 32.49 mg/kg, and the N-terminal 5-residues were verified by amino acid sequencing. Plant-produced His:hLIF was able to maintain the pluripotency of mouse embryonic stem cells. This technique thus provides a novel method of removing foreign domains from a target protein in planta.

In bladder, loss of mammalian Sonic Hedgehog (Shh) accompanies progression to invasive urothelial carcinoma, but the molecular mechanisms underlying this cancer-initiating event are poorly defined. Here, we show that loss of Shh results from hypermethylation of the CpG shore of the Shh gene, and that inhibition of DNA methylation increases Shh expression to halt the initiation of murine urothelial carcinoma at the early stage of progression. In full-fledged tumors, pharmacologic augmentation of Hedgehog (Hh) pathway activity impedes tumor growth, and this cancer-restraining effect of Hh signaling is mediated by the stromal response to Shh signals, which stimulates subtype conversion of basal to luminal-like urothelial carcinoma. Our findings thus provide a basis to develop subtype-specific strategies for the management of human bladder cancer., eLife digest In order to grow, cancer cells shut down or over-activate genes that normally maintain a cell’s health. The Sonic Hedgehog gene – named after a Japanese cartoon character – is associated with the cancer of several tissues, including the bladder. In 2014, researchers found that losing the Sonic Hedgehog gene, Shh for short, is necessary for bladder cancers to become aggressive: Shh signals prompt healthy cells near the tumor to inhibit the cancer cell growth, whilst aggressive bladder cancer cells turn off the Shh gene. Kim et al. – including many of the researchers involved in the 2014 work – now investigate how cancer cells switch off the Shh gene and what effect it has on bladder cancer cells and their surrounding tissue when turned back on. DNA sequencing bladder cancer cells derived from human patients showed that there were no genetic deletions or mutations within the gene. However, the sequence and nearby regions of DNA did contain methylations – a chemical modification that generally switches genes off. When mice with early stages of bladder cancer were treated with a drug that inhibits methylation, the Shh gene turned back on, the bladder cancers stopped growing and the tumors stayed at an early stage of development. When the same drug was used on mice with aggressive bladder cancer, this caused non-cancer cells in the surrounding tissue to respond to Shh and send restraining signals back to the tumor. These signals eventually stopped cancer growth and converted the tumor into a less aggressive type of bladder cancer. Additionally, Kim et al. saw that blocking methylation had the same effect on human bladder cancer cells that had been transplanted into mice. These results therefore indicate that Shh could be a new target for cancer treatments. For instance, drugs that decrease methylation and turn on the gene could be a way of managing cancer in patients with aggressive bladder cancers, which often show low activity of the gene. However, future studies are needed to understand what exactly happens within cancer cells during tumor conversion and to determine if this kind of intervention could have unintended consequences.

Branching morphogenesis is thought to be governed by epithelial-stromal interactions, but the mechanisms underlying specification of branch location remain largely unknown. Prompted by the striking absence of Hedgehog (Hh) response at the sites of nascent buds in regenerating tubules of the adult prostate, we investigated the role of Hh signalling in adult prostate branching morphogenesis. We find that pathway activity is localized to stromal cells, and that its attenuation by genetic or pharmacologic manipulation leads to increased branching. Decreased pathway activity correlates with increased stromal production of hepatocyte growth factor (Hgf), and we show that Hgf induces epithelial tubule branching. Regulation of Hgf expression by Hh signalling is indirect, mediated by Hh-induced expression of the microRNAs miR-26a and miR-26b, which in turn downregulate expression of Hgf. Prostate tubule branching thus may be initiated from regions of low Hh pathway activity, with implications for the prostatic hyperplasia commonly observed in late adulthood.

Double duty for mammary stem cell niche The stem cell niche is a complex local signaling microenvironment that regulates stem cell activity for tissue and organ maintenance and regeneration. As well as responding locally, during puberty, the mammary gland stem cell niche also responds to systemic hormonal signals. Zhao et al. have found that Gli2, a transcriptional effector of Hedgehog signaling, coordinates the niche-signaling program and activates expression of receptors for the mammatrophic hormones estrogen and growth hormone throughout the mammary gland (see the Perspective by Robertson). Disease may result not only from stem cell defects, but also from dysregulation of the microenvironment. Science , this issue p. eaal3485 ; see also p. 250

Understanding how malignancies arise within normal tissues requires identification of the cancer cell of origin and knowledge of the cellular and tissue dynamics of tumour progression. Here we examine bladder cancer in a chemical carcinogenesis model that mimics muscle-invasive human bladder cancer. With no prior bias regarding genetic pathways or cell types, we prospectively mark or ablate cells to show that muscle-invasive bladder carcinomas arise exclusively from Sonic hedgehog (Shh)-expressing stem cells in basal urothelium. These carcinomas arise clonally from a single cell whose progeny aggressively colonize a major portion of the urothelium to generate a lesion with histological features identical to human carcinoma in situ. Shh-expressing basal cells within this precursor lesion become tumour-initiating cells, although Shh expression is lost in subsequent carcinomas. We thus find that invasive carcinoma is initiated from basal urothelial stem cells but that tumour cell phenotype can diverge significantly from that of the cancer cell of origin.

Inflammation disrupts tissue architecture and function, thereby contributing to the pathogenesis of diverse diseases; the signals that promote or restrict tissue inflammation thus represent potential targets for therapeutic intervention. Here, we report that genetic or pharmacologic Hedgehog pathway inhibition intensifies colon inflammation (colitis) in mice. Conversely, genetic augmentation of Hedgehog response and systemic small-molecule Hedgehog pathway activation potently ameliorate colitis and restrain initiation and progression of colitis-induced adenocarcinoma. Within the colon, the Hedgehog protein signal does not act directly on the epithelium itself, but on underlying stromal cells to induce expression of IL-10, an immune-modulatory cytokine long known to suppress inflammatory intestinal damage. IL-10 function is required for the full protective effect of small-molecule Hedgehog pathway activation in colitis; this pharmacologic augmentation of Hedgehog pathway activity and stromal IL-10 expression are associated with increased presence of CD4+Foxp3+ regulatory T cells. We thus identify stromal cells as cellular coordinators of colon inflammation and suggest their pharmacologic manipulation as a potential means to treat colitis.

Mechanosensitive sensory hair cells are the linchpin of our senses of hearing and balance. The inability of the mammalian inner ear to regenerate lost hair cells is the major reason for the permanence of hearing loss and certain balance disorders. Here we present a stepwise guidance protocol starting with mouse embryonic stem (ES) and induced pluripotent stem (iPS) cells, which were directed toward becoming ectoderm capable of responding to otic-inducing growth factors. The resulting otic progenitor cells were subjected to varying differentiation conditions, one of which promoted the organization of the cells into epithelial clusters displaying hair cell-like cells with stereociliary bundles. Bundle-bearing cells in these clusters responded to mechanical stimulation with currents that were reminiscent of immature hair cell transduction currents.

SummaryHedgehog (Hh) pathway inhibitors are clinically effective in treatment of basal cell carcinoma and medulloblastoma, but fail therapeutically or accelerate progression in treatment of endodermally derived colon and pancreatic cancers. In bladder, another organ of endodermal origin, we find that despite its initial presence in the cancer cell of origin Sonic hedgehog (Shh) expression is invariably lost during progression to invasive urothelial carcinoma. Genetic blockade of stromal response to Shh furthermore dramatically accelerates progression and decreases survival time. This cancer-restraining effect of Hh pathway activity is associated with stromal expression of BMP signals, which stimulate urothelial differentiation. Progression is dramatically reduced by pharmacological activation of BMP pathway activity with low-dose FK506, suggesting an approach to management of human bladder cancer.

The tight junction is an intricate seal between adjoining epithelial cells that also separates the apical and basolateral membranes within these cells. A paper in this issue of Cell by Umeda et al. (2006) demonstrates that loss of the ZO scaffolding proteins prevents the formation of tight junctions but surprisingly does not perturb apico-basal polarity.

Directional migration is important in wound healing by epithelial cells. Recent studies have shown that polarity proteins such as mammalian Partitioning-defective 6 (Par6), atypical protein kinase C (aPKC) and mammalian Discs large 1 (Dlg1) are crucial not only for epithelial apico-basal polarity, but also for directional movement. Here, we show that the protein associated with Lin seven 1 (PALS1)-associated tight junction protein (PATJ), another evolutionarily conserved polarity protein, is also required for directional migration by using a wound-induced migration assay. In addition, we found that aPKC and Par3 localize to the leading edge during migration of epithelia and that PATJ regulates their localization. Furthermore, our results show that microtubule-organizing centre orientation is disrupted in PATJ RNA interference (RNAi) MDCKII (Madin–Darby canine kidney II) cells during migration. Together, our data indicate that PATJ controls directional migration by regulating the localization of aPKC and Par3 to the leading edge. The migration defect in PATJ RNAi cells seems to be due to the disorganization of the microtubule network induced by mislocalization of polarity proteins.

The tight junction is an intracellular junctional structure that mediates adhesion between epithelial cells and is required for epithelial cell function. Tight junctions control paracellular permeability across epithelial cell sheets and also serve as a barrier to intramembrane diffusion of components between a cell's apical and basolateral membrane domains. Recent genetic and biochemical studies in invertebrates and vertebrates indicate that tight junction proteins play an important role in the establishment and maintenance of apico-basal polarity. Proteins involved in epithelial cell polarization form evolutionarily conserved multiprotein complexes at the tight junction, and these protein complexes regulate the architecture of epithelia throughout the polarization process. Accumulating information regarding the regulation of these polarity proteins will lead to a better understanding of the molecular mechanisms whereby cell polarity is established.

Recent studies have revealed an important role for tight junction protein complexes in epithelial cell polarity. One of these complexes contains the apical transmembrane protein, Crumbs, and two PSD95/discs large/zonula occludens domain proteins, protein associated with Lin seven 1 (PALS1)/Stardust and PALS1-associated tight junction protein (PATJ). Although Crumbs and PALS1/Stardust are known to be important for cell polarization, recent studies have suggested that Drosophila PATJ is not essential and its function is unclear. Here, we find that PATJ is targeted to the apical region and tight junctions once cell polarization is initiated. We show using RNAi techniques that reduction in PATJ expression leads to delayed tight junction formation as well as defects in cell polarization. These effects are reversed by reintroduction of PATJ into these RNAi cells. This study provides new functional information on PATJ as a polarity protein and increases our understanding of the Crumbs–PALS1–PATJ complex function in epithelial polarity.

Prior work in our laboratory established a connection between the PALS1/PATJ/CRB3 and Par6/Par3/aPKC protein complexes at the tight junction of mammalian epithelial cells. Utilizing a stable small interfering RNA expression system, we have markedly reduced expression of the tight junction-associated protein PALS1 in MDCKII cells. The loss of PALS1 resulted in a corresponding loss of expression of PATJ, a known binding partner of PALS1, but had no effect on the expression of CRB3. However, the absence of PALS1 and PATJ expression did result in the decreased association of CRB3 with members of the Par6/Par3/aPKC protein complex. The consequences of the loss of PALS1 and PATJ were exhibited by a delay in the polarization of MDCKII monolayers after calcium switch, a decrease in the transepithelial electrical resistance, and by the inability of these cells to form lumenal cysts when grown in a collagen gel matrix. These defects in polarity determination may be the result of the lack of recruitment of aPKC to the tight junction in PALS1-deficient cells, as observed by confocal microscopy, and subsequent alterations in downstream signaling events.

Epithelial integrity in metazoan organs is maintained through the regulated proliferation and differentiation of organ-specific stem and progenitor cells. Although the epithelia of organs such as the intestine regenerate constantly and thus remain continuously proliferative1, other organs, such as the mammalian urinary bladder, shift from near-quiescence to a highly proliferative state in response to epithelial injury2–4. The cellular and molecular mechanisms underlying this injury-induced mode of regenerative response are poorly defined. Here we show in mice that the proliferative response to bacterial infection or chemical injury within the bladder is regulated by signal feedback between basal cells of the urothelium and the stromal cells that underlie them. We demonstrate that these basal cells include stem cells capable of regenerating all cell types within the urothelium, and are marked by expression of the secreted protein signal Sonic hedgehog (Shh). On injury, Shh expression in these basal cells increases and elicits increased stromal expression of Wnt protein signals, which in turn stimulate the proliferation of both urothelial and stromal cells. The heightened activity of this signal feedback circuit and the associated increase in cell proliferation appear to be required for restoration of urothelial function and, in the case of bacterial injury, may help clear and prevent further spread of infection. Our findings provide a conceptual framework for injury-induced epithelial regeneration in endodermal organs, and may provide a basis for understanding the roles of signalling pathways in cancer growth and metastasis.

Latent transforming growth factor (TGF)-beta-binding protein (LTBP) is required for the assembly, secretion, matrix association, and activation of latent TGF-beta complex. To elucidate the cell specific expression of the genes of LTBP-1 and their splice variants and the factors that regulate the gene expression, we cultured primary human glomerular endothelial cells (HGEC) under different conditions. Basal expression of LTBP-1 mRNA was suppressed in HGEC compared to WI-38 human embryonic lung fibroblasts. High glucose, H(2)O(2), and TGF-beta1 upregulated and vascular endothelial growth factor (VEGF) further downregulated LTBP-1 mRNA in HGEC. RT-PCR with a primer set for LTBP-1S produced many clones but no clone was gained with a primer set for LTBP-1L. Of 12 clones selected randomly, Sca I mapping and DNA sequencing revealed that only one was LTBP-1S and all the others were LTBP-1Sdelta53. TGF-beta1, but not high glucose, H(2)O(2) or VEGF, tended to increase LTBP-1Sdelta53 mRNA. In conclusion, HGEC express LTBP-1 mRNA which is suppressed at basal state but upregulated by high glucose, H(2)O(2), and TGF-beta1 and downregulated by VEGF. Major splice variant of LTBP-1 in HGEC was LTBP-1S 53. Modification of LTBP-1S 53 gene in HGEC may abrogate fibrotic action of TGF-beta1 but this requires confirmation.