Identification of mesenchymal genes involved in prostate organogenesis by LongSAGE analysis

Growth and development of the prostate are regulated by androgens and mesenchymalepithelial interactions. Prostate growth starts in the embryo, continues throughout neonatal life, and is completed at puberty. The male and female embryonic urogenital sinus (UGS) both contain a prostate precursor, but due to the action of testosterone only the male will develop a prostate. Testosterone acts in concert with paracrine signalling from inductive mesenchyme to regulate epithelial growth and branching of the prostate. The ventral mesenchymal pad (VMP) is an area of inductive mesenchyme that is rich in regulatory growth factors of which few have been identified at present. Recently, the role of mesenchyme/stroma in cancer has been established and developmental factors may be involved in mediating the effects of cancer stroma. The aim of this thesis was to identify mesenchymal factors and characterise their expression and function in the developing prostate, and to investigate their expression in prostate cancer stroma.The LongSAGE technique was used to generate a comprehensive transcriptional profile of the neonatal rat prostate rudiment. LongSAGE is an open-ended and unbiased geneprofiling method. Two libraries were made from tissues in the female UGS; one library was prepared from the whole female prostate rudiment and the other library was prepared from the inductive mesenchyme (VMP) isolated from within the rudiment. Several essential factors known to be involved in prostate development were identified in the libraries, including low abundance cDNAs such as AR and FgflO. This suggested that the approach had sufficient sensitivity to identify key mesenchymal factors. The two libraries were compared, and the comparison was statistically analysed, highlighting genes that were VMP-enriched (P<0.05). Candidate mesenchymal transcripts were selected from the VSU and VMP Libraries by two different approaches, either because of their status as VMP -enriched (P<0.05) transcripts, or by an 'intuitive' approach, because the transcripts were associated with genes that are known members of developmental pathways and/or have been associated with prostate cancer. The expression and abundance of candidate transcripts were quantified by qRT-PCR in the male and female neonatal UGS. Subsequently, candidate transcripts were verified as VMP-enriched and were quantified during prostate development by Northern blot analysis. The protein distribution of selected candidates were localised within the neonatal rat UGS by immunocytochemistry, and the effect of testosterone treatment on the protein distribution was studied. To test the function of one of the candidates on prostate growth and development, recombinant protein was added to prostate rudiments grown in vitro. Finally, candidate transcripts were investigated in human prostate cancer associated fibroblast cells (CAFs) and normal prostate fibroblast cells (NPFs) by RT-PCR and Northern blot analysis.SAGE analysis, qRT-PCR and Northen blot analysis identified six candidate transcripts as VMP-enriched; Dlkl, Notch2, Ptn Nell2, MMP2 and MMP14. The transcript expression of each candidate was most abundant in the developing rat prostate during the perinatal period. Proteins for Dlkl, Ptn and Notch2 were localised to mesenchymal cells of the neonatal VMP and ventral prostate (VP). Ptn expression was also associated with the basement membrane and cell-surface of the epithelial duct cells of the VP. Treatment of VP organs with recombinant DLK1 in vitro increased the organ size and epithelial branching. Also, PTN, NOTCH2, MMP2 and MMP14 transcript expression was observed in CAFs and NPFs. PTN and NOTCH2 showed a decrease in CAFs compared to NPFs suggesting a tumour-suppressive role.In summary, a comprehensive gene profiling technique was used to identify mesenchyme specific/enriched transcripts in the developing prostate. The expression, distribution and function of candidate transcripts and proteins were investigated in the developing prostate and in prostate cancer stroma

Establishment of Bovine 3D Enteroid-derived 2D monolayers

Three-dimensional (3D) intestinal enteroids are powerful in vitro models for studying intestinal biology. However, due to their closed structure direct access to the apical surface is impeded, limiting high-throughput applications of exogenous compounds and pathogens. In this study, we describe a method for generating confluent 2D enteroids from single-cell suspensions of enzymatically-dissociated ileum-derived bovine 3D enteroids. Confluent monolayers were first achieved using IntestiCult media but to establish a defined, cost-effective culture media, we also developed a bovine enteroid monolayer (BEM) medium. The monolayers cultured in BEM media proliferated extensively and formed confluent cell layers on both Matrigel-coated plastic plates and transwell inserts by day 3 of culture. The 2D enteroids maintained the epithelial cell lineages found in 3D enteroids and ileum tissue. In addition, the monolayers formed a functional epithelial barrier based on the presence of the adherens and tight junction proteins, E-cadherin and ZO-1, and electrical resistance across the monolayer was measured from day 3 and maintained for up to 7 days in culture. The method described here will provide a useful model to study bovine epithelial cell biology with ease of access to the apical surface of epithelial cells and has potential to investigate host–pathogen interactions and screen bioactive compounds

Transcriptional profiling of inductive mesenchyme to identify molecules involved in prostate development and disease

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background The mesenchymal compartment plays a key role in organogenesis, and cells within the mesenchyme/stroma are a source of potent molecules that control epithelia during development and tumorigenesis. We used serial analysis of gene expression (SAGE) to profile a key subset of prostatic mesenchyme that regulates prostate development and is enriched for growth-regulatory molecules. Results SAGE libraries were constructed from prostatic inductive mesenchyme and from the complete prostatic rudiment (including inductive mesenchyme, epithelium, and smooth muscle). By comparing these two SAGE libraries, we generated a list of 219 transcripts that were enriched or specific to inductive mesenchyme and that may act as mesenchymal regulators of organogenesis and tumorigenesis. We identified Scube1 as enriched in inductive mesenchyme from the list of 219 transcripts; also, quantitative RT-PCR and whole-mount in situ hybridization revealed Scube1 to exhibit a highly restricted expression pattern. The expression of Scube1 in a subset of mesenchymal cells suggests a role in prostatic induction and branching morphogenesis. Additionally, Scube1 transcripts were expressed in prostate cancer stromal cells, and were less abundant in cancer associated fibroblasts relative to matched normal prostate fibroblasts. Conclusion The use of a precisely defined subset of cells and a back-comparison approach allowed us to identify rare mRNAs that could be overlooked using other approaches. We propose that Scube1 encodes a novel stromal molecule that is involved in prostate development and tumorigenesis.Published versio

Reduction of pro-tumorigenic activity of human prostate cancer-associated fibroblasts using Dlk1 or SCUBE1

SUMMARY Human prostatic cancer-associated fibroblasts (CAFs) can elicit malignant changes in initiated but non-tumorigenic human prostate epithelium, demonstrating that they possess pro-tumorigenic properties. We set out to reduce the pro-tumorigenic activity of patient CAFs using the Dlk1 and SCUBE1 molecules that we had previously identified in prostate development. Our hypothesis was that mesenchymally expressed molecules might reduce CAF pro-tumorigenic activity, either directly or indirectly. We isolated primary prostatic CAFs and characterised their expression of CAF markers, expression of Notch2, Dlk1 and SCUBE1 transcripts, and confirmed their ability to stimulate BPH1 epithelial cell proliferation. Next, we expressed Dlk1 or SCUBE1 in CAFs and determined their effects upon tumorigenesis in vivo following recombination with BPH1 epithelia and xenografting in SCID mice. Tumour size was reduced by about 75% and BPH1 proliferation was reduced by about 50% after expression of Dlk1 or SCUBE1 in CAFs, and there was also a reduction in invasion of BPH1 epithelia into the host kidney. Inhibition of Notch signalling, using inhibitor XIX, led to a reduction in BPH1 cell proliferation in CAF-BPH1 co-cultures, whereas inhibition of Dlk1 in NIH3T3-conditioned media led to an increase in BPH1 growth. Our results suggest that pro-tumorigenic CAF activity can be reduced by the expression of developmental pathways

Identification of genes expressed in a mesenchymal subset regulating prostate organogenesis using tissue and single cell transcriptomics

Abstract Prostate organogenesis involves epithelial growth controlled by inductive signalling from specialised mesenchymal subsets. To identify pathways active in mesenchyme we used tissue and single cell transcriptomics to define mesenchymal subsets and subset-specific transcript expression. We documented transcript expression using Tag-seq and RNA-seq in female rat Ventral Mesenchymal Pad (VMP) as well as adjacent urethra comprised of smooth muscle and peri-urethral mesenchyme. Transcripts enriched in female VMP were identified with Tag-seq of microdissected tissue, RNA-seq of cell populations, and single cells. We identified 400 transcripts as enriched in the VMP using bio-informatic comparisons of Tag-seq and RNA-seq data, and 44 were confirmed by single cell RNA-seq. Cell subset analysis showed that VMP and adjacent mesenchyme were composed of distinct cell types and that each tissue contained two subgroups. Markers for these subgroups were highly subset specific. Thirteen transcripts were validated by qPCR to confirm cell specific expression in microdissected tissues, as well as expression in neonatal prostate. Immunohistochemical staining demonstrated that Ebf3 and Meis2 showed a restricted expression pattern in female VMP and prostate mesenchyme. We conclude that prostate inductive mesenchyme shows limited cellular heterogeneity and that transcriptomic analysis identified new mesenchymal subset transcripts associated with prostate organogenesis

A role for mospd1 in mesenchymal stem cell proliferation and differentiation

Mesenchymal stem cells (MSCs) isolated from many tissues including bone marrow and fat can be expanded in vitro and can differentiate into a range of different cell types such as bone, cartilage, and adipocytes. MSCs can also exhibit immunoregulatory properties when transplanted but, although a number of clinical trials using MSCs are in progress, the molecular mechanisms that control their production, proliferation, and differentiation are poorly understood. We identify MOSPD1 as a new player in this process. We generated MOSPD1‐null embryonic stem cells (ESCs) and demonstrate that they are deficient in their ability to differentiate into a number of cell lineages including osteoblasts, adipocytes, and hematopoietic progenitors. The self‐renewal capacity of MOSPD1‐null ESCs was normal and they exhibited no obvious defects in early germ layer specification nor in epithelial to mesenchymal transition (EMT), indicating that MOSPD1 functions after these key steps in the differentiation process. Mesenchymal stem cell (MSC)‐like cells expressing CD73, CD90, and CD105 were generated from MOSPD1‐null ESCs but their growth rate was significantly impaired implying that MOSPD1 plays a role in MSC proliferation. Phenotypic deficiencies exhibited by MOSPD1‐null ESCs were rescued by exogenous expression of MOSPD1, but not MOSPD3 indicating distinct functional properties of these closely related genes. Our in vitro studies were supported by RNA‐sequencing data that confirmed expression of Mospd1 mRNA in cultured, proliferating perivascular pre‐MSCs isolated from human tissue. This study adds to the growing body of knowledge about the function of this largely uncharacterized protein family and introduces a new player in the control of MSC proliferation and differentiation. Stem Cells 2015;33:3077–308