Differentiation of human embryonic stem cells into functional ovarian Granulosa-like cells

Chen Wei Lan, Mei Jou Chen, Pey Shynan Jan, Hsin Fu Chen, Hong Nerng Ho

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Context: Granulosa cells are important for the development and maturation of oocytes. The dysfunction of granulosa cells may contribute to abnormal folliculogenesis and steroidogenesis. Objective: Our objective was to establish an effective culture system to differentiate human embryonic stem cells (hESCs) into granulosa cells. Design: For differentiation of hESCs to granulosa cells, we used multistep approaches comprising in vitro treatments with cocktails of growth factors. Expression of mesendoderm/intermediate plate mesoderm markers, and granulosa cell markers were analyzed by real time-PCR, Western blotting, immunofluorescence, and flow cytometry. The production of estradiol and anti-Mullerian hormone (AMH) was analyzed by ELISA. Results: Gene expression analyses showed the progress of hESCs to primitive streak-mesendoderm, intermediate plate mesoderm, and finally to functional granulosa-like cells that expressed the granulosa cell-specific forkhead transcription factor FOXL2, estrogen synthetase cytochrome P450 19A1 (CYP19A1),AMH, the type 2AMHreceptor (AMHR2), and the FSH receptor (FSHR). However, they did not express the LH receptor (LHR). Western blot showed that AMHR2 and CYP19A1 levels in differentiated hESCs were higher than in undifferentiated cells. Flow cytometry showed that the percentage of AMHR2-, FSHR-, and CYP19A1-positive cells increased to 36%, 12%, and 34%, respectively, after differentiation for 12 days. These granulosa-like cells were also capable of producing AMH and aromatizing testosterone to estradiol, suggesting that they were biologically functional. Conclusions: We successfully established an effective protocol to generate functional ovarian granulosa-like cells from hESCs. The derivation of these cells opens new avenues for the further study and potential application of these cells in human folliculogenesis and steroidogenesis.

Original languageEnglish
Pages (from-to)3713-3723
Number of pages11
JournalJournal of Clinical Endocrinology and Metabolism
Volume98
Issue number9
DOIs
Publication statusPublished - Sep 1 2013

Fingerprint

FSH Receptors
Forkhead Transcription Factors
Transforming Growth Factor beta Receptors
Aromatase
Ovarian Follicle
Peptide Receptors
Granulosa Cells
Embryonic Stem Cells
Stem cells
Cell Differentiation
Anti-Mullerian Hormone
Cell Culture Techniques
Cell Line
Cytochrome P-450 Enzyme System
Flow cytometry
Estradiol
LH Receptors
Mesoderm
Cell culture
Gene expression

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Biochemistry
  • Endocrinology
  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

Differentiation of human embryonic stem cells into functional ovarian Granulosa-like cells. / Lan, Chen Wei; Chen, Mei Jou; Jan, Pey Shynan; Chen, Hsin Fu; Ho, Hong Nerng.

In: Journal of Clinical Endocrinology and Metabolism, Vol. 98, No. 9, 01.09.2013, p. 3713-3723.

Research output: Contribution to journalArticle

Lan, Chen Wei ; Chen, Mei Jou ; Jan, Pey Shynan ; Chen, Hsin Fu ; Ho, Hong Nerng. / Differentiation of human embryonic stem cells into functional ovarian Granulosa-like cells. In: Journal of Clinical Endocrinology and Metabolism. 2013 ; Vol. 98, No. 9. pp. 3713-3723.
@article{65757a0005a746fc93442fcf7ddb47cf,
title = "Differentiation of human embryonic stem cells into functional ovarian Granulosa-like cells",
abstract = "Context: Granulosa cells are important for the development and maturation of oocytes. The dysfunction of granulosa cells may contribute to abnormal folliculogenesis and steroidogenesis. Objective: Our objective was to establish an effective culture system to differentiate human embryonic stem cells (hESCs) into granulosa cells. Design: For differentiation of hESCs to granulosa cells, we used multistep approaches comprising in vitro treatments with cocktails of growth factors. Expression of mesendoderm/intermediate plate mesoderm markers, and granulosa cell markers were analyzed by real time-PCR, Western blotting, immunofluorescence, and flow cytometry. The production of estradiol and anti-Mullerian hormone (AMH) was analyzed by ELISA. Results: Gene expression analyses showed the progress of hESCs to primitive streak-mesendoderm, intermediate plate mesoderm, and finally to functional granulosa-like cells that expressed the granulosa cell-specific forkhead transcription factor FOXL2, estrogen synthetase cytochrome P450 19A1 (CYP19A1),AMH, the type 2AMHreceptor (AMHR2), and the FSH receptor (FSHR). However, they did not express the LH receptor (LHR). Western blot showed that AMHR2 and CYP19A1 levels in differentiated hESCs were higher than in undifferentiated cells. Flow cytometry showed that the percentage of AMHR2-, FSHR-, and CYP19A1-positive cells increased to 36{\%}, 12{\%}, and 34{\%}, respectively, after differentiation for 12 days. These granulosa-like cells were also capable of producing AMH and aromatizing testosterone to estradiol, suggesting that they were biologically functional. Conclusions: We successfully established an effective protocol to generate functional ovarian granulosa-like cells from hESCs. The derivation of these cells opens new avenues for the further study and potential application of these cells in human folliculogenesis and steroidogenesis.",
author = "Lan, {Chen Wei} and Chen, {Mei Jou} and Jan, {Pey Shynan} and Chen, {Hsin Fu} and Ho, {Hong Nerng}",
year = "2013",
month = "9",
day = "1",
doi = "10.1210/jc.2012-4302",
language = "English",
volume = "98",
pages = "3713--3723",
journal = "Journal of Clinical Endocrinology and Metabolism",
issn = "0021-972X",
publisher = "The Endocrine Society",
number = "9",

}

TY - JOUR

T1 - Differentiation of human embryonic stem cells into functional ovarian Granulosa-like cells

AU - Lan, Chen Wei

AU - Chen, Mei Jou

AU - Jan, Pey Shynan

AU - Chen, Hsin Fu

AU - Ho, Hong Nerng

PY - 2013/9/1

Y1 - 2013/9/1

N2 - Context: Granulosa cells are important for the development and maturation of oocytes. The dysfunction of granulosa cells may contribute to abnormal folliculogenesis and steroidogenesis. Objective: Our objective was to establish an effective culture system to differentiate human embryonic stem cells (hESCs) into granulosa cells. Design: For differentiation of hESCs to granulosa cells, we used multistep approaches comprising in vitro treatments with cocktails of growth factors. Expression of mesendoderm/intermediate plate mesoderm markers, and granulosa cell markers were analyzed by real time-PCR, Western blotting, immunofluorescence, and flow cytometry. The production of estradiol and anti-Mullerian hormone (AMH) was analyzed by ELISA. Results: Gene expression analyses showed the progress of hESCs to primitive streak-mesendoderm, intermediate plate mesoderm, and finally to functional granulosa-like cells that expressed the granulosa cell-specific forkhead transcription factor FOXL2, estrogen synthetase cytochrome P450 19A1 (CYP19A1),AMH, the type 2AMHreceptor (AMHR2), and the FSH receptor (FSHR). However, they did not express the LH receptor (LHR). Western blot showed that AMHR2 and CYP19A1 levels in differentiated hESCs were higher than in undifferentiated cells. Flow cytometry showed that the percentage of AMHR2-, FSHR-, and CYP19A1-positive cells increased to 36%, 12%, and 34%, respectively, after differentiation for 12 days. These granulosa-like cells were also capable of producing AMH and aromatizing testosterone to estradiol, suggesting that they were biologically functional. Conclusions: We successfully established an effective protocol to generate functional ovarian granulosa-like cells from hESCs. The derivation of these cells opens new avenues for the further study and potential application of these cells in human folliculogenesis and steroidogenesis.

AB - Context: Granulosa cells are important for the development and maturation of oocytes. The dysfunction of granulosa cells may contribute to abnormal folliculogenesis and steroidogenesis. Objective: Our objective was to establish an effective culture system to differentiate human embryonic stem cells (hESCs) into granulosa cells. Design: For differentiation of hESCs to granulosa cells, we used multistep approaches comprising in vitro treatments with cocktails of growth factors. Expression of mesendoderm/intermediate plate mesoderm markers, and granulosa cell markers were analyzed by real time-PCR, Western blotting, immunofluorescence, and flow cytometry. The production of estradiol and anti-Mullerian hormone (AMH) was analyzed by ELISA. Results: Gene expression analyses showed the progress of hESCs to primitive streak-mesendoderm, intermediate plate mesoderm, and finally to functional granulosa-like cells that expressed the granulosa cell-specific forkhead transcription factor FOXL2, estrogen synthetase cytochrome P450 19A1 (CYP19A1),AMH, the type 2AMHreceptor (AMHR2), and the FSH receptor (FSHR). However, they did not express the LH receptor (LHR). Western blot showed that AMHR2 and CYP19A1 levels in differentiated hESCs were higher than in undifferentiated cells. Flow cytometry showed that the percentage of AMHR2-, FSHR-, and CYP19A1-positive cells increased to 36%, 12%, and 34%, respectively, after differentiation for 12 days. These granulosa-like cells were also capable of producing AMH and aromatizing testosterone to estradiol, suggesting that they were biologically functional. Conclusions: We successfully established an effective protocol to generate functional ovarian granulosa-like cells from hESCs. The derivation of these cells opens new avenues for the further study and potential application of these cells in human folliculogenesis and steroidogenesis.

UR - http://www.scopus.com/inward/record.url?scp=84883675070&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84883675070&partnerID=8YFLogxK

U2 - 10.1210/jc.2012-4302

DO - 10.1210/jc.2012-4302

M3 - Article

VL - 98

SP - 3713

EP - 3723

JO - Journal of Clinical Endocrinology and Metabolism

JF - Journal of Clinical Endocrinology and Metabolism

SN - 0021-972X

IS - 9

ER -