Differentiation of embryonic stem cells into fibroblast-like cells in three-dimensional type i collagen gel cultures

Shinsaku Togo, Tadashi Sato, Hisatoshi Sugiura, Xingqi Wang, Hesham E Basma, Amy Nelson, Xiang-de Liu, Tom W. Bargar, John G Sharp, Stephen I. Rennard

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Fibroblasts are heterogeneous mesenchymal cells that play important roles in the production and maintenance of extracellular matrix. Although their heterogeneity is recognized, progenitor progeny relationships among fibroblasts and the factors that control fibroblast differentiation are poorly defined. The current study was designed to develop a reliable method that would permit in vitro differentiation of fibroblast-like cells from human and murine embryonic stem cells (ESCs). Undifferentiated ESCs were differentiated into embryoid bodies (EBs) with differentiation media. EBs were then cast into type I collagen gels and cultured for 21 d with basal media. The spindle-shaped cells that subsequently grew from the EBs were released from the gels and subsequently cultured as monolayers in basal media supplemented with serum. Differentiated cells showed a characteristic spindle-shaped morphology and had ultrastructural features consistent with fibroblasts. Immunocytochemistry showed positive staining for vimentin and alpha-smooth muscle actin but was negative for stage-specific embryonic antigens and cytokeratins. Assays of fibroblast function, including proliferation, chemotaxis, and contraction of collagen gels demonstrated that the differentiated cells, derived from both human and murine ESCs, responded to transforming growth factor-β1 and prostaglandin E 2 as would be expected of fibroblasts, functions not expected of endothelial or epithelial cells. The current study demonstrates that cells with the morphologic and functional features of fibroblasts can be reliably derived from human and murine ESCs. This methodology provides a means to investigate and define the mechanisms that regulate fibroblast differentiation.

Original languageEnglish (US)
Pages (from-to)114-124
Number of pages11
JournalIn Vitro Cellular and Developmental Biology - Animal
Volume47
Issue number2
DOIs
StatePublished - Feb 1 2011

Fingerprint

Fibroblasts
Embryonic Stem Cells
Stem cells
Cell culture
Collagen
Gels
Embryoid Bodies
Stage-Specific Embryonic Antigens
Endothelial cells
Transforming Growth Factors
Vimentin
Chemotaxis
Keratins
Collagen Type I
Prostaglandins E
Extracellular Matrix
Smooth Muscle
Muscle
Actins
Monolayers

Keywords

  • Embryonic stem cell differentiation
  • Fibroblasts
  • Three-dimensional collagen gel cultures

ASJC Scopus subject areas

  • Developmental Biology
  • Cell Biology

Cite this

Differentiation of embryonic stem cells into fibroblast-like cells in three-dimensional type i collagen gel cultures. / Togo, Shinsaku; Sato, Tadashi; Sugiura, Hisatoshi; Wang, Xingqi; Basma, Hesham E; Nelson, Amy; Liu, Xiang-de; Bargar, Tom W.; Sharp, John G; Rennard, Stephen I.

In: In Vitro Cellular and Developmental Biology - Animal, Vol. 47, No. 2, 01.02.2011, p. 114-124.

Research output: Contribution to journalArticle

Togo, Shinsaku ; Sato, Tadashi ; Sugiura, Hisatoshi ; Wang, Xingqi ; Basma, Hesham E ; Nelson, Amy ; Liu, Xiang-de ; Bargar, Tom W. ; Sharp, John G ; Rennard, Stephen I. / Differentiation of embryonic stem cells into fibroblast-like cells in three-dimensional type i collagen gel cultures. In: In Vitro Cellular and Developmental Biology - Animal. 2011 ; Vol. 47, No. 2. pp. 114-124.
@article{8284a168b35d42f0967aebcf368056eb,
title = "Differentiation of embryonic stem cells into fibroblast-like cells in three-dimensional type i collagen gel cultures",
abstract = "Fibroblasts are heterogeneous mesenchymal cells that play important roles in the production and maintenance of extracellular matrix. Although their heterogeneity is recognized, progenitor progeny relationships among fibroblasts and the factors that control fibroblast differentiation are poorly defined. The current study was designed to develop a reliable method that would permit in vitro differentiation of fibroblast-like cells from human and murine embryonic stem cells (ESCs). Undifferentiated ESCs were differentiated into embryoid bodies (EBs) with differentiation media. EBs were then cast into type I collagen gels and cultured for 21 d with basal media. The spindle-shaped cells that subsequently grew from the EBs were released from the gels and subsequently cultured as monolayers in basal media supplemented with serum. Differentiated cells showed a characteristic spindle-shaped morphology and had ultrastructural features consistent with fibroblasts. Immunocytochemistry showed positive staining for vimentin and alpha-smooth muscle actin but was negative for stage-specific embryonic antigens and cytokeratins. Assays of fibroblast function, including proliferation, chemotaxis, and contraction of collagen gels demonstrated that the differentiated cells, derived from both human and murine ESCs, responded to transforming growth factor-β1 and prostaglandin E 2 as would be expected of fibroblasts, functions not expected of endothelial or epithelial cells. The current study demonstrates that cells with the morphologic and functional features of fibroblasts can be reliably derived from human and murine ESCs. This methodology provides a means to investigate and define the mechanisms that regulate fibroblast differentiation.",
keywords = "Embryonic stem cell differentiation, Fibroblasts, Three-dimensional collagen gel cultures",
author = "Shinsaku Togo and Tadashi Sato and Hisatoshi Sugiura and Xingqi Wang and Basma, {Hesham E} and Amy Nelson and Xiang-de Liu and Bargar, {Tom W.} and Sharp, {John G} and Rennard, {Stephen I.}",
year = "2011",
month = "2",
day = "1",
doi = "10.1007/s11626-010-9367-2",
language = "English (US)",
volume = "47",
pages = "114--124",
journal = "In Vitro Cellular and Developmental Biology - Plant",
issn = "1054-5476",
publisher = "Springer New York",
number = "2",

}

TY - JOUR

T1 - Differentiation of embryonic stem cells into fibroblast-like cells in three-dimensional type i collagen gel cultures

AU - Togo, Shinsaku

AU - Sato, Tadashi

AU - Sugiura, Hisatoshi

AU - Wang, Xingqi

AU - Basma, Hesham E

AU - Nelson, Amy

AU - Liu, Xiang-de

AU - Bargar, Tom W.

AU - Sharp, John G

AU - Rennard, Stephen I.

PY - 2011/2/1

Y1 - 2011/2/1

N2 - Fibroblasts are heterogeneous mesenchymal cells that play important roles in the production and maintenance of extracellular matrix. Although their heterogeneity is recognized, progenitor progeny relationships among fibroblasts and the factors that control fibroblast differentiation are poorly defined. The current study was designed to develop a reliable method that would permit in vitro differentiation of fibroblast-like cells from human and murine embryonic stem cells (ESCs). Undifferentiated ESCs were differentiated into embryoid bodies (EBs) with differentiation media. EBs were then cast into type I collagen gels and cultured for 21 d with basal media. The spindle-shaped cells that subsequently grew from the EBs were released from the gels and subsequently cultured as monolayers in basal media supplemented with serum. Differentiated cells showed a characteristic spindle-shaped morphology and had ultrastructural features consistent with fibroblasts. Immunocytochemistry showed positive staining for vimentin and alpha-smooth muscle actin but was negative for stage-specific embryonic antigens and cytokeratins. Assays of fibroblast function, including proliferation, chemotaxis, and contraction of collagen gels demonstrated that the differentiated cells, derived from both human and murine ESCs, responded to transforming growth factor-β1 and prostaglandin E 2 as would be expected of fibroblasts, functions not expected of endothelial or epithelial cells. The current study demonstrates that cells with the morphologic and functional features of fibroblasts can be reliably derived from human and murine ESCs. This methodology provides a means to investigate and define the mechanisms that regulate fibroblast differentiation.

AB - Fibroblasts are heterogeneous mesenchymal cells that play important roles in the production and maintenance of extracellular matrix. Although their heterogeneity is recognized, progenitor progeny relationships among fibroblasts and the factors that control fibroblast differentiation are poorly defined. The current study was designed to develop a reliable method that would permit in vitro differentiation of fibroblast-like cells from human and murine embryonic stem cells (ESCs). Undifferentiated ESCs were differentiated into embryoid bodies (EBs) with differentiation media. EBs were then cast into type I collagen gels and cultured for 21 d with basal media. The spindle-shaped cells that subsequently grew from the EBs were released from the gels and subsequently cultured as monolayers in basal media supplemented with serum. Differentiated cells showed a characteristic spindle-shaped morphology and had ultrastructural features consistent with fibroblasts. Immunocytochemistry showed positive staining for vimentin and alpha-smooth muscle actin but was negative for stage-specific embryonic antigens and cytokeratins. Assays of fibroblast function, including proliferation, chemotaxis, and contraction of collagen gels demonstrated that the differentiated cells, derived from both human and murine ESCs, responded to transforming growth factor-β1 and prostaglandin E 2 as would be expected of fibroblasts, functions not expected of endothelial or epithelial cells. The current study demonstrates that cells with the morphologic and functional features of fibroblasts can be reliably derived from human and murine ESCs. This methodology provides a means to investigate and define the mechanisms that regulate fibroblast differentiation.

KW - Embryonic stem cell differentiation

KW - Fibroblasts

KW - Three-dimensional collagen gel cultures

UR - http://www.scopus.com/inward/record.url?scp=79952242730&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=79952242730&partnerID=8YFLogxK

U2 - 10.1007/s11626-010-9367-2

DO - 10.1007/s11626-010-9367-2

M3 - Article

C2 - 21107747

AN - SCOPUS:79952242730

VL - 47

SP - 114

EP - 124

JO - In Vitro Cellular and Developmental Biology - Plant

JF - In Vitro Cellular and Developmental Biology - Plant

SN - 1054-5476

IS - 2

ER -