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Stem Cell Reviews and Reports

, Volume 11, Issue 5, pp 761–773 | Cite as

Wnt/β-Catenin and MEK-ERK Signaling are Required for Fibroblast-Derived Extracellular Matrix-Mediated Endoderm Differentiation of Embryonic Stem Cells

  • Kevin Dzobo
  • Matjaz Vogelsang
  • M. Iqbal ParkerEmail author
Article

Abstract

Human embryonic stem cells (hESCs) have the potential to differentiate into all cells of the three germ layers, thus making them an attractive source of cells for use in regenerative medicine. The greatest challenge lies in regulating the differentiation of hESCs into specific cell lineages by both intrinsic and extrinsic factors. In this study we determined the effect of a fibroblast-derived extracellular matrix (fd-ECM) on hESCs differentiation. We demonstrate that growth of hESCs on fd-ECM results in hESCs losing their stemness and proliferation potential. As the stem cells differentiate they attain gene expression profiles similar to the primitive streak of the in vivo embryo. The activation of both the MEK-ERK and Wnt/β-catenin signaling pathways is required for the fd-ECM-mediated differentiation of hESCs towards the endoderm and involves integrins α1, α2, α3 and β1. This study illustrates the importance of the cellular microenvironment in directing stem cell fate and that the nature and composition of the extracellular matrix is a crucial determining factor.

Keywords

Human embryonic stem cell Extracellular matrix Cellular differentiation Endoderm Mesoderm 

Notes

Acknowledgments

This work was supported by the International Centre for Genetic Engineering and Biotechnology (ICGEB), the South African Medical Research Council and the University of Cape Town. The funders had no role in the conduct of the research or the preparation of the manuscript.

Conflicts of Interest

The authors declare no potential conflicts of interest.

Supplementary material

12015_2015_9598_Fig9_ESM.gif (308 kb)
Supplemental Fig S1

Densitometric quantification of western blot gels, showing stem cell markers, Oct4, Sox2 and Ssea4 downregulation in ESCs cultured on fd-ECM a After 4 days of incubation b After 8 days of incubation c After 16 days of incubation d After 24 days of incubation. Data are presented as mean ± standard deviation (GIF 308 kb)

12015_2015_9598_MOESM1_ESM.tif (327 kb)
High resolution image (TIFF 327 kb)
12015_2015_9598_Fig10_ESM.gif (304 kb)
Supplemental Fig S2

a Experimental design of induced hESCs differentiation through the hanging drop method. b A representative image of hESCs BG01V cultured on feeder layer of MEFs c A representative image of the hanging drop method (GIF 304 kb)

12015_2015_9598_MOESM2_ESM.tif (650 kb)
High Resolution Image (TIFF 650 kb)
12015_2015_9598_Fig11_ESM.gif (359 kb)
Supplemental Fig S3

Effect of different ECM coatings on hESCs differentiation a hESCs BG01V cultured on feeder layer of MEFs then differentiated by the hanging drop (HD) method and suspension culture. Images are for embryoid bodies day 2,4 and 8 b hESCs BG01V cultured on fd-ECM and treated as in (A). Images are for embryoid bodies day 2,4 and 8 c hESCs BG01V cultured on Matrigel and treated as in (A). Images are for embryoid bodies day 2,4 and 8 d hESCs BG01V cultured on collagen and treated as in (a). Images are for embryoid bodies day 2,4 and 8. Scale bar: 25 μm (GIF 359 kb)

12015_2015_9598_MOESM3_ESM.tif (736 kb)
High Resolution Image (TIFF 736 kb)
12015_2015_9598_Fig12_ESM.gif (221 kb)
Supplemental Fig S4

a Western blot analysis of embryoid bodies samples after incubation for the indicated days using Oct4, Sox-2 and Ssea-4 antibodies. The levels of β-tubulin in the same sample were used as a loading control. (b to c) RT PCR analysis of OCT4, NANOG, AXIN2 and LEF1 expression in embryoid bodies from different ECM coatings. GAPDH was used as the normalizer. d Detection of integrin-binding sites on the fd-ECM. Integrin receptors were blocked and attached cells were quantified through the use of crystal violet staining. Quantification was done relative to cells that were not incubated with blocking antibodies which was taken as 100 % attachment. Data are presented as mean ± standard deviation, * p < 0.05 (GIF 220 kb)

12015_2015_9598_MOESM4_ESM.tif (271 kb)
High Resolution Image (TIFF 271 kb)
12015_2015_9598_MOESM5_ESM.doc (76 kb)
Supplemental Table S1 Molecular weights of proteins analyzed by western blot (DOC 76 kb)
12015_2015_9598_MOESM6_ESM.doc (258 kb)
Supplemental Table S2 Oligonucleotide primer sequences used for real time quantitative PCR (DOC 258 kb)

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Kevin Dzobo
    • 1
    • 2
  • Matjaz Vogelsang
    • 1
  • M. Iqbal Parker
    • 1
    • 2
    Email author
  1. 1.Cape Town Component, Wernher and Beit Building (South), UCT CampusInternational Centre for Genetic Engineering and Biotechnology (ICGEB)Cape TownSouth Africa
  2. 2.Division of Medical Biochemistry, Faculty of Health SciencesUniversity of Cape TownCape TownSouth Africa

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