Directed Differentiation of Human Embryonic Stem Cells into Prostate Organoids <i>In Vitro</i> and its Perturbation by Low-Dose Bisphenol A Exposure

<div><p>Studies using rodent and adult human prostate stem-progenitor cell models suggest that developmental exposure to the endocrine disruptor Bisphenol-A (BPA) can predispose to prostate carcinogenesis with aging. Unknown at present is whether the embryonic human prostate is equally susceptible to BPA during its natural developmental window. To address this unmet need, we herein report the construction of a pioneer <i>in vitro</i> human prostate developmental model to study the effects of BPA. The directed differentiation of human embryonic stem cells (hESC) into prostatic organoids in a spatial system was accomplished with precise temporal control of growth factors and steroids. Activin-induced definitive endoderm was driven to prostate specification by combined exposure to WNT10B and FGF10. Matrigel culture for 20–30 days in medium containing R-Spondin-1, Noggin, EGF, retinoic acid and testosterone was sufficient for mature prostate organoid development. Immunofluorescence and gene expression analysis confirmed that organoids exhibited cytodifferentiation and functional properties of the human prostate. Exposure to 1 nM or 10 nM BPA throughout differentiation culture disturbed early morphogenesis in a dose-dependent manner with 1 nM BPA increasing and 10 nM BPA reducing the number of branched structures formed. While differentiation of branched structures to mature organoids seemed largely unaffected by BPA exposure, the stem-like cell population increased, appearing as focal stem cell nests that have not properly entered lineage commitment rather than the rare isolated stem cells found in normally differentiated structures. These findings provide the first direct evidence that low-dose BPA exposure targets hESC and perturbs morphogenesis as the embryonic cells differentiate towards human prostate organoids, suggesting that the developing human prostate may be susceptible to disruption by <i>in utero</i> BPA exposures.</p></div

Characterization of prostatic phenotype.

<p>(<b>A</b>) Twenty-eight day time course phase-contrast images of a representative Matrigel cultured organoid forming over time from DE-differentiated H9 cells. While images of M-d1, d-4 and d-8 show the entire organoid as it grew, images of M-d20, d-24 and d-28 represent focal areas of formation and elongation of a single duct with extended culture. The representative duct is composed of a putative layer of columnar epithelium (arrows) with central lumens (dotted green lines), surrounded by mesenchyme (arrowheads). All images were obtained at the same magnification, scale bars represent 50 μm. (<b>B</b>) M-d16 and d-22 phase-contrast images following a representative Matrigel cultured organoid differentiated from H1 hESC. The organoid exhibited growth, budding, elongation and increased complexity over 6 days. Scale bars represent 50 μm. Insets: Lower magnification photographs show the entire organoid composed of convoluted ductal structures and mesenchyme (arrowheads). Scale bars represent 200 μm.</p

Primary antibodies used for immunofluorescence.

<p>Primary antibodies used for immunofluorescence.</p

Characterization of functional differentiation of organoids.

<p>Immunostaining analysis by confocal microscopy of 28–30 day organoids for cytodifferentiation and functional differentiation markers. (<b>A and D</b>) Luminal cell cytodifferentiation markers AR (green) and (<b>B and E</b>) CK8/18 (red) and (<b>C and F</b>) merged images with DAPI (blue) reveals most luminal epithelial cells contain nuclear AR. (<b>G</b>) Merged NKX3.1 (green), a prostate specific epithelial cell marker, with CK8/18 (red), a luminal epithelial cell marker and DAPI (blue) shows nuclear NKX3.1 in all epithelial cells suggesting prostatic nature. Functional differentiation markers PSA (<b>H and I</b>) and androgen regulated gene TMPRSS2 (<b>J</b>) indicate the ability of cytodifferentiated luminal cells to produce secretory proteins specific to the prostate. Laminin (<b>K</b>), a basement membrane marker, delineates the normal acinar organization of the organoids. Staining for Vimentin (<b>L</b>) confirms the extra-acinar cells are derived from mesenchymally differentiated hESC cells, forming a stromal compartment. Normal IgG as negative controls for each probe is shown in insets. Lumens are indicated by white dotted lines (H-L) and epithelial ducts are outlined by green dotted lines (D-F). Scale bars represent 20 μm.</p

BPA effects during prostatic organoid maturation.

<p>Immunolocalization of the stem cell markers: (<b>A</b>) TROP2 and (<b>B</b>) CD49f show a dose-dependent increase in stem cell focal aggregates when treated with BPA compared to vehicle. (<b>C</b>) Organoids labeled with epithelial cytodifferentiation markers AR and CK8/18 show normal ductal morphology and epithelial differentiation after BPA treatment. Lumens are delineated by white and whole structures by green dotted lines. Scale bars represent 20 μm and all images are representative of n = 3.</p