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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

Model of human embryonic stem cell directed differentiation into prostate tissue in vitro.(A) Definitive endoderm and mesoderm differentiation was driven by culturing hESC with activin A (100 ng/ml) for 3 days. In the next 4 consecutive days, differentiated cells were cultured with WNT10B (500 ng/ml) and FGF10 (500 ng/ml) to direct them into prostatic fate and organoid formation. Organoids were transferred and grown in Matrigel to allow their 3-D growth in prostatic media containing T (1.7 μM) and ATRA (10 nM), which permits differentiation and expansion of prostatic organoids. (B) Definitive endoderm differentiation images show morphological changes at 24 and 72 hours following activin A treatment, compared to untreated hESC (control). Phase-contrast images were obtained using the EVOS microscope. Scale bars represent 200 μm. After 3 days of treatment with Activin A, Day 3 DE cultures were immunostained for (C) FOXA2 (red), an endodermal specific marker and (D) Brachyury (green), a mesendodermal marker with nuclear staining (DAPI, blue). The merged image (E) show definitive endoderm staining (red-yellow; arrowheads) in the majority of cells while a subpopulation was Brachyury+ but FOXA2- (arrows) implicating a mesodermal component in a minority of cells. Scale bars represent 50 μm.
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pone.0133238.g001: Model of human embryonic stem cell directed differentiation into prostate tissue in vitro.(A) Definitive endoderm and mesoderm differentiation was driven by culturing hESC with activin A (100 ng/ml) for 3 days. In the next 4 consecutive days, differentiated cells were cultured with WNT10B (500 ng/ml) and FGF10 (500 ng/ml) to direct them into prostatic fate and organoid formation. Organoids were transferred and grown in Matrigel to allow their 3-D growth in prostatic media containing T (1.7 μM) and ATRA (10 nM), which permits differentiation and expansion of prostatic organoids. (B) Definitive endoderm differentiation images show morphological changes at 24 and 72 hours following activin A treatment, compared to untreated hESC (control). Phase-contrast images were obtained using the EVOS microscope. Scale bars represent 200 μm. After 3 days of treatment with Activin A, Day 3 DE cultures were immunostained for (C) FOXA2 (red), an endodermal specific marker and (D) Brachyury (green), a mesendodermal marker with nuclear staining (DAPI, blue). The merged image (E) show definitive endoderm staining (red-yellow; arrowheads) in the majority of cells while a subpopulation was Brachyury+ but FOXA2- (arrows) implicating a mesodermal component in a minority of cells. Scale bars represent 50 μm.

Mentions: In vivo, the prostate gland originates from the endodermal urogenital sinus (UGS). Prostate determination occurs prior to morphologic evidence of a developing structure and involves sequential expression of molecular signals that commit a specific field of UGS cells to a prostatic fate [2]. Phenotypic prostate development commences as UGS epithelial cells form buds that penetrate into the surrounding mesenchyme that further instructs branching and differentiation. The presence of steroids, in particular testosterone and its metabolite DHT, is essential for prostate specification, development and functional maturation. In the present study, careful consideration was given to each of these features in an attempt to grow prostate organoids from hESC in vitro. Fig 1A provides a schematic summary that identifies the four key stages of prostate organoid culture in the current study; hESC colony culture, induction of definitive endoderm (DE), prostatic fate determination and prostate organoid growth and differentiation.


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)

Model of human embryonic stem cell directed differentiation into prostate tissue in vitro.(A) Definitive endoderm and mesoderm differentiation was driven by culturing hESC with activin A (100 ng/ml) for 3 days. In the next 4 consecutive days, differentiated cells were cultured with WNT10B (500 ng/ml) and FGF10 (500 ng/ml) to direct them into prostatic fate and organoid formation. Organoids were transferred and grown in Matrigel to allow their 3-D growth in prostatic media containing T (1.7 μM) and ATRA (10 nM), which permits differentiation and expansion of prostatic organoids. (B) Definitive endoderm differentiation images show morphological changes at 24 and 72 hours following activin A treatment, compared to untreated hESC (control). Phase-contrast images were obtained using the EVOS microscope. Scale bars represent 200 μm. After 3 days of treatment with Activin A, Day 3 DE cultures were immunostained for (C) FOXA2 (red), an endodermal specific marker and (D) Brachyury (green), a mesendodermal marker with nuclear staining (DAPI, blue). The merged image (E) show definitive endoderm staining (red-yellow; arrowheads) in the majority of cells while a subpopulation was Brachyury+ but FOXA2- (arrows) implicating a mesodermal component in a minority of cells. Scale bars represent 50 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4519179&req=5

pone.0133238.g001: Model of human embryonic stem cell directed differentiation into prostate tissue in vitro.(A) Definitive endoderm and mesoderm differentiation was driven by culturing hESC with activin A (100 ng/ml) for 3 days. In the next 4 consecutive days, differentiated cells were cultured with WNT10B (500 ng/ml) and FGF10 (500 ng/ml) to direct them into prostatic fate and organoid formation. Organoids were transferred and grown in Matrigel to allow their 3-D growth in prostatic media containing T (1.7 μM) and ATRA (10 nM), which permits differentiation and expansion of prostatic organoids. (B) Definitive endoderm differentiation images show morphological changes at 24 and 72 hours following activin A treatment, compared to untreated hESC (control). Phase-contrast images were obtained using the EVOS microscope. Scale bars represent 200 μm. After 3 days of treatment with Activin A, Day 3 DE cultures were immunostained for (C) FOXA2 (red), an endodermal specific marker and (D) Brachyury (green), a mesendodermal marker with nuclear staining (DAPI, blue). The merged image (E) show definitive endoderm staining (red-yellow; arrowheads) in the majority of cells while a subpopulation was Brachyury+ but FOXA2- (arrows) implicating a mesodermal component in a minority of cells. Scale bars represent 50 μm.
Mentions: In vivo, the prostate gland originates from the endodermal urogenital sinus (UGS). Prostate determination occurs prior to morphologic evidence of a developing structure and involves sequential expression of molecular signals that commit a specific field of UGS cells to a prostatic fate [2]. Phenotypic prostate development commences as UGS epithelial cells form buds that penetrate into the surrounding mesenchyme that further instructs branching and differentiation. The presence of steroids, in particular testosterone and its metabolite DHT, is essential for prostate specification, development and functional maturation. In the present study, careful consideration was given to each of these features in an attempt to grow prostate organoids from hESC in vitro. Fig 1A provides a schematic summary that identifies the four key stages of prostate organoid culture in the current study; hESC colony culture, induction of definitive endoderm (DE), prostatic fate determination and prostate organoid growth and differentiation.

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