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

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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.

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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.

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The authors declare no potential conflicts of interest.

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Authors and Affiliations

  1. Cape Town Component, Wernher and Beit Building (South), UCT Campus, International Centre for Genetic Engineering and Biotechnology (ICGEB), Anzio Road, Observatory, 7925, Cape Town, South Africa

    Kevin Dzobo, Matjaz Vogelsang & M. Iqbal Parker

  2. Division of Medical Biochemistry, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa

    Kevin Dzobo & M. Iqbal Parker

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  1. Kevin Dzobo
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  2. Matjaz Vogelsang
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  3. M. Iqbal Parker
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Correspondence to M. Iqbal Parker.

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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)

High resolution image (TIFF 327 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)

High Resolution Image (TIFF 650 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)

High Resolution Image (TIFF 736 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)

High Resolution Image (TIFF 271 kb)

Supplemental Table S1

Molecular weights of proteins analyzed by western blot (DOC 76 kb)

Supplemental Table S2

Oligonucleotide primer sequences used for real time quantitative PCR (DOC 258 kb)

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Dzobo, K., Vogelsang, M. & Parker, M.I. Wnt/β-Catenin and MEK-ERK Signaling are Required for Fibroblast-Derived Extracellular Matrix-Mediated Endoderm Differentiation of Embryonic Stem Cells. Stem Cell Rev and Rep 11, 761–773 (2015). https://doi.org/10.1007/s12015-015-9598-4

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