The in vitro differentiation of prostate epithelial cells and gonadal adipocytes from mouse embryonic stem cells

Abstract

Mouse embryonic stem (ES) cells are pluripotent, undifferentiated cells that can differentiate into any cell of the three germ layers: the ectoderm, the mesoderm and the endoderm. Mammalian development is challenging to study due to the in utero gestation and small size of early embryos. Mouse ES cells overcome this hurdle by recapitulating in vivo organogenesis in an in vitro setting. There are currently no in vitro models of prostate development from mouse ES cells. The prostate is an endoderm derived exocrine organ that functions to provide the seminal constituents for sperm. The initial aim of this study was to differentiate mouse ES cells into mature, prostatic epithelial cells in vitro in stepwise manner. The prostate is derived from the urogenital sinus which in turn is derived from the endoderm. The endoderm and urothelium have been generated from mouse ES cells in vitro with the addition of Activin-A and alltrans retinoic acid respectively. It was hypothesised that treatment of mouse ES cell-derived urothelial cells with dihydrotestosterone (DHT), TGFb1 and FGF10 would induce prostate epithelial cell differentiation. Gene expression of homeobox protein Nkx3.1 and prostate marker probasin (Pbsn) were identified in day 16 and day 22 differentiated cultures treated with the aforementioned growth factors. Additionally, day 22 cultures treated with DHT, TGFb1 and FGF10 developed cyst-like acinus structures with a lumen. In conjunction with prostate epithelial cell differentiation, adipocyte-like cells were unexpectedly generated. Brown and white adipocytes are thought to arise from the mesoderm germ layer. Mouse ES cell culture conditions used to generate prostate epithelial cells were primed for endoderm specification. The question was then asked if the adipocytes generated were indicative of a particular adipose depot and if the endoderm could be a source of adipocytes. In order to identify the adipocyte-like cells differentiated in vitro, a method was developed to characterise brown, subcutaneous and visceral white adipocytes by flow cytometry. Multi-parametric flow cytometric analyses of adipocytes stained with Nile Red, MitoTracker Deep Red, Nile Blue, fatty acid translocase CD36 and laminin receptor integrins a6b1 established adipocyte heterogeneity at the single cell level. Comparisons in dye uptake and surface protein expression between adipocytes revealed a code that was applied to the adipocyte-like cells differentiated under endoderm culture conditions. Adipocytes generated in mouse ES cell cultures treated with DHT, TGFb1 and FGF10 matched the profile of gonadal adipocytes. Here, I report the first protocol describing prostate epithelial cell differentiation from mouse ES cells. As mouse ES cells can be genetically modified, the development of prostate epithelial cells may serve as an alternative approach for the study of prostate disease in vitro. The generation of adipocytes from mouse ES cells under endoderm conditions prompted the development of a flow cytometric method to characterise adipocyte heterogeneity at the single cell level. The method can potentially be used for the diagnosis of a range of metabolic disorders stemming from obesity such as diabetes. Contrary to reports of a mesoderm origin of adipocytes, a possible endoderm origin of gonadal adipocytes is hypothesised, the adipose depot closest to the prostate. The development of the prostate in conjunction with gonadal adipocytes has yet to be reported.