Early steps in neural induction

Neural induction is the instructive interaction whereby signals emitted from the organizer direct cells in the ectoderm to a neural fate and thereby to form the neural plate. Recent work in many labs has suggested that it involves a hierarchy of molecular events. Here, I investigate the earliest steps in the neural induction cascade and the signals that define them. Many genes expressed during the neural induction cascade have been shown to be regulated by FGF. However the signals that induce three of the genes, Bert, TrkC and Obelix, are unknown. I therefore tested candidate signalling molecules by misexpression analysis. While Obelix is also regulated by FGF, none of many factors tested, including FGF, retinoic acid, somatostatin, noggin, insulin, and increasing intracellular calcium were able to induce expression of TrkC or Bert. BMP also plays an important role in neural induction, I therefore studied how cells may integrate TGFβ signalling through Smad1 and Smad2. I used a BiFCo approach to investigate Smad protein binding interactions in culture and in vivo, however this did not turn out to be a useful method for this question. Finally I investigated the ground-state of the epiblast at the start of the neural induction cascade. Culture of early epiblast explants showed, unexpectedly, that cells initially enter a state similar to that of the neural plate border, confirmed by their subsequent differentiation into lens. This correlates with the finding that BMP signalling in vivo only affects cells of the neural plate border region and suggests why explants can be neuralized by BMP. Overall, the experiments reveal a hitherto unknown importance of a neural border cell-state, and suggest that lens is the ground state at the start of the neural induction cascade

Prospective identification of tumorigenic prostate cancer stem cells

Existing therapies for prostate cancer eradicates the bulk of cells within a tumor. However, most patients go on to develop androgen-independent disease that remains incurable by current treatment strategies. There is now increasing evidence in some malignancies that the tumor cells are organized as a hierarchy originating from rare stem cells that are responsible for maintaining the tumor. We report here the identification and characterization of a cancer stem cell population from human prostate tumors, which possess a significant capacity for self-renewal. These cells are also able to regenerate the phenotypically mixed populations of nonclonogenic cells, which express differentiated cell products, such as androgen receptor and prostatic acid phosphatase. The cancer stem cells have a CD44+/2ß1hi/CD133+ phenotype, and we have exploited these markers to isolate cells from a series of prostate tumors with differing Gleason grade and metastatic states. Approximately 0.1% of cells in any tumor expressed this phenotype, and there was no correlation between the number of CD44+/2ß1hi/CD133+ cells and tumor grade. The identification of a prostate cancer stem cell provides a powerful tool to investigate the tumorigenic process and to develop therapies targeted to the stem cell

Experimental prostate epithelial morphogenesis in response to stroma and three-dimensional matrigel culture

To reproduce the structural and functional differentiation of human prostatic acini in vivo, prostatic epithelial and stromal cells derived from human primary cultures were cocultured in Matrigel. In the absence of stroma and serum, epithelial spheroids composed of solid masses of stratified and cuboidal cells formed. Outer cells of the spheroid expressed cytokeratins 1, 5, 10, and 14, whereas the inner cells expressed cytokeratin 18. The addition of 2% serum induced formation of a lumen surrounded by a layer of one or two cuboidal and columnar epithelial cells. The further addition of stromal cultures, dihydrotestosterone, and estrogen induced polarization of the epithelium and increased spheroid-forming efficiency. Epithelium expressed either cytokeratin 18 alone or additionally cytokeratins 1, 5, 14, and 10. All spheroid epithelium expressed prostate-specific antigen and prostate-specific membrane antigen. Androgen receptor was only detected in the presence of stroma, serum, and hormones. Thus, development of a functional and morphologically correct prostate gland in vitro is dependent on extracellular matrix, steroid hormones, and factors from stromal cells and serum