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

Calderon-Gierszal EL, Prins GS - PLoS ONE (2015)

Bottom Line: To address this unmet need, we herein report the construction of a pioneer in vitro human prostate developmental model to study the effects of BPA.Immunofluorescence and gene expression analysis confirmed that organoids exhibited cytodifferentiation and functional properties of the human prostate.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.

View Article: PubMed Central - PubMed

Affiliation: Departments of Urology and Physiology & Biophysics, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, United States of America.

ABSTRACT
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 in vitro 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 in utero BPA exposures.

No MeSH data available.


Related in: MedlinePlus

BPA effects during prostatic organoid maturation.Immunolocalization of the stem cell markers: (A) TROP2 and (B) CD49f show a dose-dependent increase in stem cell focal aggregates when treated with BPA compared to vehicle. (C) 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.
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pone.0133238.g006: BPA effects during prostatic organoid maturation.Immunolocalization of the stem cell markers: (A) TROP2 and (B) CD49f show a dose-dependent increase in stem cell focal aggregates when treated with BPA compared to vehicle. (C) 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.

Mentions: To assess whether BPA affects cellular differentiation of the hESC into mature organoids, 3-D culture was continued to day 30 and cellular differentiation markers and steroid receptor expression were evaluated by qRT-PCR and immunofluorescence. Luminal epithelial cell differentiation markers NKX3.1 and CK18 as well as stromal and luminal AR levels were unaffected at the mRNA (Fig 5D) or protein levels (Fig 6C) by either 1 or 10 nM BPA as compared to vehicle controls. Interestingly, p63 mRNA was elevated by 10 nM BPA exposure, although this was not statistically significant (Fig 5D). While vimentin expression was not changed by 1 nM BPA, mRNA levels were significantly increased with 10 nM BPA exposure suggesting an increase in the stromal component by the higher BPA dose (Fig 5D). The expression of estrogen receptors (ERα, ERβ, GPER), confirmed mediators of BPA actions [30, 51], was not altered by developmental BPA exposure (Fig 5E).


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

Calderon-Gierszal EL, Prins GS - PLoS ONE (2015)

BPA effects during prostatic organoid maturation.Immunolocalization of the stem cell markers: (A) TROP2 and (B) CD49f show a dose-dependent increase in stem cell focal aggregates when treated with BPA compared to vehicle. (C) 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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4519179&req=5

pone.0133238.g006: BPA effects during prostatic organoid maturation.Immunolocalization of the stem cell markers: (A) TROP2 and (B) CD49f show a dose-dependent increase in stem cell focal aggregates when treated with BPA compared to vehicle. (C) 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.
Mentions: To assess whether BPA affects cellular differentiation of the hESC into mature organoids, 3-D culture was continued to day 30 and cellular differentiation markers and steroid receptor expression were evaluated by qRT-PCR and immunofluorescence. Luminal epithelial cell differentiation markers NKX3.1 and CK18 as well as stromal and luminal AR levels were unaffected at the mRNA (Fig 5D) or protein levels (Fig 6C) by either 1 or 10 nM BPA as compared to vehicle controls. Interestingly, p63 mRNA was elevated by 10 nM BPA exposure, although this was not statistically significant (Fig 5D). While vimentin expression was not changed by 1 nM BPA, mRNA levels were significantly increased with 10 nM BPA exposure suggesting an increase in the stromal component by the higher BPA dose (Fig 5D). The expression of estrogen receptors (ERα, ERβ, GPER), confirmed mediators of BPA actions [30, 51], was not altered by developmental BPA exposure (Fig 5E).

Bottom Line: To address this unmet need, we herein report the construction of a pioneer in vitro human prostate developmental model to study the effects of BPA.Immunofluorescence and gene expression analysis confirmed that organoids exhibited cytodifferentiation and functional properties of the human prostate.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.

View Article: PubMed Central - PubMed

Affiliation: Departments of Urology and Physiology & Biophysics, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, United States of America.

ABSTRACT
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 in vitro 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 in utero BPA exposures.

No MeSH data available.


Related in: MedlinePlus