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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

Characterization of prostatic phenotype.(A) 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) 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.
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pone.0133238.g003: Characterization of prostatic phenotype.(A) 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) 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.

Mentions: Following 4 days of specification culture, visible structures were collected and transferred to 3-D Matrigel culture for maturation to prostatic organoids. For this step, prostate specific media was developed and optimized to support branching morphogenesis and functional differentiation and included combined stromal and epithelial culture medias utilized for prostate cell culture, R-Spondin1 to potentiate endogenous Wnt signaling [44], Noggin to limit BMP signaling and permit branching morphogenesis [7], EGF to drive cell proliferation and augment budding, all-trans retinoic acid (ATRA) to enhance cytodifferentiation and testosterone (T), essential for prostate growth and differentiation [2]. In this system, the structures showed a rapid and steady increase in size, many exhibiting budding and outgrowth by Matrigel-day 8 (M-d8) (Fig 3A). An increase in the development of convoluted epithelial-like ducts with expanding morphologic complexity continued until harvesting on M-d28 (Fig 3A and 3B). Mature organoids at day 28–30 exhibited a complex network of epithelial-like ducts, composed of a layer of epithelium with a central lumen, surrounded by a basement membrane and stromal-type cells. No differences were observed between the H1 (XY) and H9 (XX) hESC lines in terms of capacity to produce differentiated organoids as assessed by morphology, immunostaining and RT-PCR for differentiation markers. While H9 is genetically XX, it is well established that the female UGS is fully capable of generating a functional prostate gland when exposed to T or DHT [45–47].


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)

Characterization of prostatic phenotype.(A) 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) 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4519179&req=5

pone.0133238.g003: Characterization of prostatic phenotype.(A) 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) 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.
Mentions: Following 4 days of specification culture, visible structures were collected and transferred to 3-D Matrigel culture for maturation to prostatic organoids. For this step, prostate specific media was developed and optimized to support branching morphogenesis and functional differentiation and included combined stromal and epithelial culture medias utilized for prostate cell culture, R-Spondin1 to potentiate endogenous Wnt signaling [44], Noggin to limit BMP signaling and permit branching morphogenesis [7], EGF to drive cell proliferation and augment budding, all-trans retinoic acid (ATRA) to enhance cytodifferentiation and testosterone (T), essential for prostate growth and differentiation [2]. In this system, the structures showed a rapid and steady increase in size, many exhibiting budding and outgrowth by Matrigel-day 8 (M-d8) (Fig 3A). An increase in the development of convoluted epithelial-like ducts with expanding morphologic complexity continued until harvesting on M-d28 (Fig 3A and 3B). Mature organoids at day 28–30 exhibited a complex network of epithelial-like ducts, composed of a layer of epithelium with a central lumen, surrounded by a basement membrane and stromal-type cells. No differences were observed between the H1 (XY) and H9 (XX) hESC lines in terms of capacity to produce differentiated organoids as assessed by morphology, immunostaining and RT-PCR for differentiation markers. While H9 is genetically XX, it is well established that the female UGS is fully capable of generating a functional prostate gland when exposed to T or DHT [45–47].

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