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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

Directed differentiation into prostatic fate determination.Prostatic fate determination images illustrate morphologic changes over 96 hours of treatment with 500 ng/ml WNT10B and FGF10 compared to untreated hESC as control shown in inset. After 48 and 72 hours of growth factor culture, spheroid-like structures attached to the cell monolayer were observed. At 96 hours, prior to Matrigel culture, 3-D structures with a budding-like phenotype were observed. Phase contrast images were obtained using the EVOS microscope. Scale bars represent 200 μm.
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pone.0133238.g002: Directed differentiation into prostatic fate determination.Prostatic fate determination images illustrate morphologic changes over 96 hours of treatment with 500 ng/ml WNT10B and FGF10 compared to untreated hESC as control shown in inset. After 48 and 72 hours of growth factor culture, spheroid-like structures attached to the cell monolayer were observed. At 96 hours, prior to Matrigel culture, 3-D structures with a budding-like phenotype were observed. Phase contrast images were obtained using the EVOS microscope. Scale bars represent 200 μm.

Mentions: Studies using ESCs to derive a variety of human tissues have shown that the coordinated temporal activation and repression of specific WNT and fibroblast growth factor (FGF) signaling pathways are essential for tissue specification and patterning. Hence, following endoderm differentiation, cells were driven into prostatic fate determination by culture in the presence of the secreted canonical WNT10B protein [40, 41], the earliest known secreted protein expressed by prostate epithelium immediately prior to bud formation [42], and FGF10, a urogenital mesenchyme-secreted growth factor essential for prostate epithelial budding and morphogenesis [43]. Four days of culture with both human growth factors was identified as the critical window for prostatic determination. Shorter culture was insufficient whereas exposure for 5–6 days in these morphogens markedly reduced organoid efficiency (data not shown). Notably, substitution of human WNT3A protein (500 ng/ml) was insufficient to drive prostatic structures (S1 Fig) which suggests a specificity for WNT10B actions that cannot be substituted by β-catenin activation through another canonical WNT. Similarly, culture in WNT10B or FGF10 protein alone was insufficient for derivation of differentiated prostate structures. At 24 hours of WNT10B and FGF10 treatment, the cell monolayer maintained a cobblestone-like morphology (Fig 2). Multilayers with attached 3-D spheroid-like structures were first observed at 48 hours and these continued to develop with 72 to 96 hours of culture with several spheroid-like structures giving rise to budding tubular-like structures (Fig 2).


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)

Directed differentiation into prostatic fate determination.Prostatic fate determination images illustrate morphologic changes over 96 hours of treatment with 500 ng/ml WNT10B and FGF10 compared to untreated hESC as control shown in inset. After 48 and 72 hours of growth factor culture, spheroid-like structures attached to the cell monolayer were observed. At 96 hours, prior to Matrigel culture, 3-D structures with a budding-like phenotype were observed. Phase contrast images were obtained using the EVOS microscope. Scale bars represent 200 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133238.g002: Directed differentiation into prostatic fate determination.Prostatic fate determination images illustrate morphologic changes over 96 hours of treatment with 500 ng/ml WNT10B and FGF10 compared to untreated hESC as control shown in inset. After 48 and 72 hours of growth factor culture, spheroid-like structures attached to the cell monolayer were observed. At 96 hours, prior to Matrigel culture, 3-D structures with a budding-like phenotype were observed. Phase contrast images were obtained using the EVOS microscope. Scale bars represent 200 μm.
Mentions: Studies using ESCs to derive a variety of human tissues have shown that the coordinated temporal activation and repression of specific WNT and fibroblast growth factor (FGF) signaling pathways are essential for tissue specification and patterning. Hence, following endoderm differentiation, cells were driven into prostatic fate determination by culture in the presence of the secreted canonical WNT10B protein [40, 41], the earliest known secreted protein expressed by prostate epithelium immediately prior to bud formation [42], and FGF10, a urogenital mesenchyme-secreted growth factor essential for prostate epithelial budding and morphogenesis [43]. Four days of culture with both human growth factors was identified as the critical window for prostatic determination. Shorter culture was insufficient whereas exposure for 5–6 days in these morphogens markedly reduced organoid efficiency (data not shown). Notably, substitution of human WNT3A protein (500 ng/ml) was insufficient to drive prostatic structures (S1 Fig) which suggests a specificity for WNT10B actions that cannot be substituted by β-catenin activation through another canonical WNT. Similarly, culture in WNT10B or FGF10 protein alone was insufficient for derivation of differentiated prostate structures. At 24 hours of WNT10B and FGF10 treatment, the cell monolayer maintained a cobblestone-like morphology (Fig 2). Multilayers with attached 3-D spheroid-like structures were first observed at 48 hours and these continued to develop with 72 to 96 hours of culture with several spheroid-like structures giving rise to budding tubular-like structures (Fig 2).

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