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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 development.Organoid quantitation (A-C) was performed 4 days following transfer to Matrigel in the absence or presence of 1 and 10 nM BPA. (A) 1 nM BPA increased budding (P < 0.001) whereas, 10 nM BPA reduced budding (P < 0.05) structure numbers compared to vehicle. (B) Nonbudding structure numbers were not affected by either dose of BPA. (C) While degenerating body numbers increased (P < 0.05) following 10 nM BPA exposure. (D) Differentiation gene expression of NKX3.1, CK18, AR and p63 was not altered by BPA treatment. In contrast, vimentin expression was significantly increased at 10 nM BPA (P < 0.01). (E) No difference in ERα, ERβ and GPER mRNA expression was noted. (F) Whereas, mRNA expression of 10 nM BPA treated organoids was significantly increased for the stem cell markers CD49f and NANOG (P < 0.05), with a similar trend noted for OCT4. TROP2 mRNA levels were not altered by BPA. From A-F bars represent means ± SEM (D-F n = 3), * P< 0.05, ** P < 0.01, *** P < 0.001. Scale bars represent 1 μm.
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pone.0133238.g005: BPA effects during prostatic organoid development.Organoid quantitation (A-C) was performed 4 days following transfer to Matrigel in the absence or presence of 1 and 10 nM BPA. (A) 1 nM BPA increased budding (P < 0.001) whereas, 10 nM BPA reduced budding (P < 0.05) structure numbers compared to vehicle. (B) Nonbudding structure numbers were not affected by either dose of BPA. (C) While degenerating body numbers increased (P < 0.05) following 10 nM BPA exposure. (D) Differentiation gene expression of NKX3.1, CK18, AR and p63 was not altered by BPA treatment. In contrast, vimentin expression was significantly increased at 10 nM BPA (P < 0.01). (E) No difference in ERα, ERβ and GPER mRNA expression was noted. (F) Whereas, mRNA expression of 10 nM BPA treated organoids was significantly increased for the stem cell markers CD49f and NANOG (P < 0.05), with a similar trend noted for OCT4. TROP2 mRNA levels were not altered by BPA. From A-F bars represent means ± SEM (D-F n = 3), * P< 0.05, ** P < 0.01, *** P < 0.001. Scale bars represent 1 μm.

Mentions: To evaluate and confirm that the differentiated organoids were prostatic in nature, mRNA for prostate epithelial genes and immunolocalization of several prostatic markers was performed using qRT-PCR and immunofluorescence confocal imaging on day 28–30 organoids. Protein marker examination (Fig 4A–4L) and gene expression analysis (Fig 5D–5F) documented epithelial and stromal cyto- and functional differentiation into prostatic-like structures resembling the adult human prostate gland. The luminal cells were marked by cytokeratin 8/18 (CK8/18; Fig 4B and 4E) and were largely androgen receptor (AR) positive (Fig 4A, 4C, 4D and 4F). Basal cell presence in all structures was indicated by p63 mRNA in all control cultures (Fig 5D). Their prostatic nature was confirmed by staining for multiple markers. NKX3.1 (Fig 4G), an epithelial transcription factor highly expressed in prostate epithelium and bulbourethral glands but not bladder, gut or seminal vesicles [48, 49], was found in all stained structures. TMPRSS2 (Fig 4J), an androgen-regulated proteases produced by human prostate epithelium was also found in all structures immunostained for this protein. Most importantly, prostate specific antigen (PSA; Fig 4H and 4I), another androgen-regulated protease produced exclusively by the prostate epithelium [50] was found in all immunostained structures, confirming the prostatic nature of the organoids. Laminin (Fig 4K), a basement membrane marker was used to delineate the normal acinar organization of the organoids which were surrounded by vimentin-positive stromal cells (Fig 4L). Some, but not all, of stromal-like cells were AR positive (Fig 4D, arrowheads) which is characteristic of prostate stroma [35]. Together, these data document the directed differentiation of hESC into functional prostatic organoids in vitro with operational capacity to respond to testosterone and produce secretory proteins.


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 development.Organoid quantitation (A-C) was performed 4 days following transfer to Matrigel in the absence or presence of 1 and 10 nM BPA. (A) 1 nM BPA increased budding (P < 0.001) whereas, 10 nM BPA reduced budding (P < 0.05) structure numbers compared to vehicle. (B) Nonbudding structure numbers were not affected by either dose of BPA. (C) While degenerating body numbers increased (P < 0.05) following 10 nM BPA exposure. (D) Differentiation gene expression of NKX3.1, CK18, AR and p63 was not altered by BPA treatment. In contrast, vimentin expression was significantly increased at 10 nM BPA (P < 0.01). (E) No difference in ERα, ERβ and GPER mRNA expression was noted. (F) Whereas, mRNA expression of 10 nM BPA treated organoids was significantly increased for the stem cell markers CD49f and NANOG (P < 0.05), with a similar trend noted for OCT4. TROP2 mRNA levels were not altered by BPA. From A-F bars represent means ± SEM (D-F n = 3), * P< 0.05, ** P < 0.01, *** P < 0.001. Scale bars represent 1 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4519179&req=5

pone.0133238.g005: BPA effects during prostatic organoid development.Organoid quantitation (A-C) was performed 4 days following transfer to Matrigel in the absence or presence of 1 and 10 nM BPA. (A) 1 nM BPA increased budding (P < 0.001) whereas, 10 nM BPA reduced budding (P < 0.05) structure numbers compared to vehicle. (B) Nonbudding structure numbers were not affected by either dose of BPA. (C) While degenerating body numbers increased (P < 0.05) following 10 nM BPA exposure. (D) Differentiation gene expression of NKX3.1, CK18, AR and p63 was not altered by BPA treatment. In contrast, vimentin expression was significantly increased at 10 nM BPA (P < 0.01). (E) No difference in ERα, ERβ and GPER mRNA expression was noted. (F) Whereas, mRNA expression of 10 nM BPA treated organoids was significantly increased for the stem cell markers CD49f and NANOG (P < 0.05), with a similar trend noted for OCT4. TROP2 mRNA levels were not altered by BPA. From A-F bars represent means ± SEM (D-F n = 3), * P< 0.05, ** P < 0.01, *** P < 0.001. Scale bars represent 1 μm.
Mentions: To evaluate and confirm that the differentiated organoids were prostatic in nature, mRNA for prostate epithelial genes and immunolocalization of several prostatic markers was performed using qRT-PCR and immunofluorescence confocal imaging on day 28–30 organoids. Protein marker examination (Fig 4A–4L) and gene expression analysis (Fig 5D–5F) documented epithelial and stromal cyto- and functional differentiation into prostatic-like structures resembling the adult human prostate gland. The luminal cells were marked by cytokeratin 8/18 (CK8/18; Fig 4B and 4E) and were largely androgen receptor (AR) positive (Fig 4A, 4C, 4D and 4F). Basal cell presence in all structures was indicated by p63 mRNA in all control cultures (Fig 5D). Their prostatic nature was confirmed by staining for multiple markers. NKX3.1 (Fig 4G), an epithelial transcription factor highly expressed in prostate epithelium and bulbourethral glands but not bladder, gut or seminal vesicles [48, 49], was found in all stained structures. TMPRSS2 (Fig 4J), an androgen-regulated proteases produced by human prostate epithelium was also found in all structures immunostained for this protein. Most importantly, prostate specific antigen (PSA; Fig 4H and 4I), another androgen-regulated protease produced exclusively by the prostate epithelium [50] was found in all immunostained structures, confirming the prostatic nature of the organoids. Laminin (Fig 4K), a basement membrane marker was used to delineate the normal acinar organization of the organoids which were surrounded by vimentin-positive stromal cells (Fig 4L). Some, but not all, of stromal-like cells were AR positive (Fig 4D, arrowheads) which is characteristic of prostate stroma [35]. Together, these data document the directed differentiation of hESC into functional prostatic organoids in vitro with operational capacity to respond to testosterone and produce secretory proteins.

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