pubmed.ncbi.nlm.nih.gov

Early cell fate decisions of human embryonic stem cells and mouse epiblast stem cells are controlled by the same signalling pathways - PubMed

  • ️Thu Jan 01 2009

Early cell fate decisions of human embryonic stem cells and mouse epiblast stem cells are controlled by the same signalling pathways

Ludovic Vallier et al. PLoS One. 2009.

Abstract

Human embryonic stem cells have unique value for regenerative medicine, as they are capable of differentiating into a broad variety of cell types. Therefore, defining the signalling pathways that control early cell fate decisions of pluripotent stem cells represents a major task. Moreover, modelling the early steps of embryonic development in vitro may provide the best approach to produce cell types with native properties. Here, we analysed the function of key developmental growth factors such as Activin, FGF and BMP in the control of early cell fate decisions of human pluripotent stem cells. This analysis resulted in the development and validation of chemically defined culture conditions for achieving specification of human embryonic stem cells into neuroectoderm, mesendoderm and into extra-embryonic tissues. Importantly, these defined culture conditions are devoid of factors that could obscure analysis of developmental mechanisms or render the resulting tissues incompatible with future clinical applications. Importantly, the growth factor roles defined using these culture conditions similarly drove differentiation of mouse epiblast stem cells derived from post implantation embryos, thereby reinforcing the hypothesis that epiblast stem cells share a common embryonic identity with human pluripotent stem cells. Therefore the defined growth factor conditions described here represent an essential step toward the production of mature cell types from pluripotent stem cells in conditions fully compatible with clinical use ant also provide a general approach for modelling the early steps of mammalian embryonic development.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Generation of extra-embryonic tissues using BMP4.

(A) Immunostaining analyses for the expression of pluripotency markers (Oct-4, SSEA-3), primitive endoderm markers (SSEA-1, GATA4), and trophectoderm markers (CDX2, Eomes) by H9 cells grown for 7 days in CDM+BMP4 10 ng/ml. Scale Bar 50 µM. (B) Dynamic expression of pluripotency markers (Oct-4, Nanog), trophectoderm markers (CDX2, Hand1), primitive endoderm markers (Sox7, GATA6) and definitive endoderm markers (Sox17, GSC) during differentiation of hESCs. H9 cells were differentiated following the protocol described above. Following the first day after plating, RNAs were extracted every day and expression of the denoted genes was analysed using Q-PCR. Normalized expression is shown as the mean±SD from two informative experiments. (C) A microarray gene expression heat map to compare human embryonic stem cells (ESC) grown in CDM and extra-embryonic cells generated in CDM (CDM) supplemented with BMP4 (BMP). For each gene (row) the heat map colours sample gene expression in units of standard deviation from the mean across all samples (columns). Up-regulation is coloured in shades of red and down-regulation in shades of blue according to the scale shown at the bottom of the heat map. (D) Fraction of cells expressing the pluripotency markers Tra-1-60 and Oct-4 after growth in the presence of BMP4 for 7 days. H9 cells were grown in CDM+BMP4 10 ng/ml, and then the fraction of cells expressing Oct-4, and Tra-1-60 was determined using FACS. H9 cells grown in CDM+Activin 10 ng/ml+FGF2 12 ng/ml were used as positive control. (E) Fraction of cells expressing CDX2 after extra-embryonic differentiation. H9 cells were grown in CDM+BMP4 10 ng/ml for 7 days, and then the fraction of cells expressing CDX2 was determined using FACS. H9 cells grown in CDM+Activin 10 ng/ml+FGF2 12 ng/ml were used as negative control.

Figure 2
Figure 2. Generation of neuroectoderm precursors by inhibiting Activin signalling in the presence of FGF2.

(A) Immunostaining analyses for the expression of a pluripotency marker (Oct-4), neuroectoderm markers (Sox2, Nestin, N-CAM) or mesendoderm markers (Brachyury, Sox17, FoxA2, αFP) by H9 cells grown for 7 days in CDM+Activin 100 ng/ml or in CDM+SB431542 10 µM+FGF2 12 ng/ml. Scale Bar 50 µM. Note homogenous expression of both Oct-4 and Sox2 in Activin (Yellow staining), but only Sox2 in FGF+SB (Green Staining). (B) Dynamic expression of a pluripotency marker (Oct-4) and neuroectoderm markers (Sox2, Sox1, Gbx2) during differentiation of hESCs. H9 cells were differentiated following the protocol described above. Following the first day after plating, RNAs were extracted every 2 days and expression of the denoted genes was analysed using Q-PCR. Normalized expression is shown as the mean±SD from two informative experiments. (C) Fraction of cells expressing the pluripotency markers Oct-4, Tra-1-60 and Sox2 and the neuronal markers Sox2 and N-CAM after growth in the presence of SB431542 for 7 days. H9 cells were grown in CDM+SB431542 10 µM+FGF2 12 ng/ml (SB+FGF), and then the fraction of cells expressing Oct-4, Sox2 and N-CAM was determined using FACS. H9 cells grown in CDM+Activin 10 ng/ml+FGF2 12 ng/ml (Activin+FGF) were used as control. (D) Expression of neuronal markers by fully differentiated neuroectoderm progenitors. H9 cells were induced to differentiate into neuroectoderm in CDM+SB431542 10 µM+FGF2 12 ng/ml. The resulting neuroectoderm progenitors were then grown as embryoid bodies (EBs) in non-adherent conditions for 14 days to allow further differentiation. The resulting EBs were then plated back on plastic and grown for 14 additional days before assessment. Nuclei are shown by Hoechst staining. Scale Bar 100 µM.

Figure 3
Figure 3. Generation of mesendoderm using a combination of Activin, FGF2 and BMP4.

(A) Colony morphologies formed in response to the three-step protocol to differentiate hESCs into mesendoderm progeny. H9 cells were grown for 2 days in CDM/PVA+Activin 10 ng/ml+FGF2 12 ng/ml, then for 72 hours in CDM/PVA+SU5402 10 µM+Activin 5 ng/ml. The next 4 days, cells were grown in CDM/PVA+Activin 30 ng/ml+FGF2 20 ng/ml+BMP4 10 ng/ml. Images of the same colonies were captured every day for 9 days. Scale Bar 200 µM. (B) Effect of different combinations and doses of Activin, FGF2 and BMP4 on the differentiation of hESCs grown in CDM/PVA. Following the third day in CDM/PVA+SU5402 10 µM, H9 cells were induced to differentiated in CDM/PVA supplemented with different combination of growth factors. RNAs were extracted after 3 days and expression of the denoted genes was analysed using Q-PCR. Normalized expression is shown as the mean±SD from two informative experiments. hESCs grown in CDM+Activin+FGF or differentiated in CDM+SB431542+FGF2 were used as negative controls. (C) Expression of specific markers for mesendoderm in hESCs differentiated in CDM/PVA supplemented with Activin, FGF2 and BMP4 in the three step protocol. Nuclei are shown by Hoechst staining. Scale Bar 100 µM. (D) Microarray gene expression heat map to compare human embryonic stem cells (ESC) grown in CDM supplemented with Activin and FGF and mesendoderm cells generated in CDM/PVA supplemented with Activin, FGF and BMP4 (LE). Up-regulation is coloured in shades of red and down-regulation in shades of blue according to the log z scale shown at the bottom of the heat map. Gene names marked with an asterisk denote genes that pass a significant differential regulation threshold. (See Material and Methods). (E) Fraction of cells expressing the definitive endoderm marker CXCR4 and the mesendoderm/mesoderm marker PDGFαR after induction of differentiation in CDM PVA in the presence of increasing doses of Activin. H9 cells were differentiated following the three step protocol described above in the presence of Activin (30 or 100 ng/ml). Fraction of cells expressing CXCR4 or PDGFαR was determined using FACS 8 days after plating. (F) RT-PCR analyses for the expression of liver markers (Albumin, HNF4, αFP), gut marker (CDX2) and cardiac markers (ANF, α Actinin, α-1 Channel) in endoderm progenitors grown in media containing serum. Endoderm progenitors generated using the three step protocol were differentiated in media containing three different FBS lots. RNAs were extracted after 5 and 10 days of differentiation and the expression of genes expressed was analysed using RT-PCR.

Figure 4
Figure 4. Contrary to mESCs, EpiSCs are responsive to culture conditions controlling differentiation of hESCs into extra-embryonic tissues, neuroectoderm and mesendoderm.

(A) Expression of pluripotency markers (Oct-4, Nanog) and neuroectoderm (Sox2, Sox1, Six3, Tuj1 and Gbx2), extra-embryonic markers (Cdx2, Hand1, Sox7, GATA6) and mesendoderm markers (Brachyury, Mixl1, Eomes, Sox17) during differentiation of mouse EpiSCs using the culture conditions developed with hESCs. EpiSCs were differentiated following the conditions described above for hESCs (SB+FGF for neuroectoderm, BMP4 for extra-embryonic tissues, three step protocol for mesendoderm). Following the ninth day after plating, RNAs were extracted and expression of the denoted genes was analysed using Q-PCR. Normalized expression is shown as the mean±SD from two informative experiments. (B) Expression of pluripotency markers (Oct-4, Nanog, SSEA-1), extra-embryonic markers (CDX2, Sox7, GATA4), neuroectoderm markers (Sox1, Nestin, βIII tubulin) and mesendoderm markers (Brachyury and Sox17) in EpiSCs differentiated using the conditions developed for hESCs. Expression of the genes denoted was analysed by immuno-fluoresence analyses. Nuclei are shown by Hoechst staining. Scale Bar 100 µM. (C) Expression of pluripotency markers (Oct-4), extra-embryonic markers (CDX2, GATA6), neuroectoderm markers (Sox2, Six3) and mesendoderm markers (Brachyury and Sox17) in mESCs differentiated using the method developed with hESCs. mESCs were differentiated in CDM as described for hESCs and then the expression of the genes denoted was analysed by Real-Time PCR. Normalized expression is shown as the mean±SD from three experiments.

Figure 5
Figure 5. Cell signalling pathways controlling cell fate specification of pluripotent cells in vitro.

A) Pluripotency of hESCs and mEpiSCs relies on Activin signalling and to a lesser extent on FGF signalling to maintain their pluripotent status. BMP signalling inhibition may be required to avoid extra-embryonic differentiation, depending on the level of endogenous BMP signalling activity of each cell type. B) Inhibition of Activin/Nodal signalling induces differentiation toward neuroectoderm in the presence of FGF2. C) BMP4 induces differentiation toward extra-embryonic tissues which is blocked by Activin and FGF2. D) BMP alternatively induces mesendoderm in cooperation with Activin (high dose) and FGF2. This model summarises results from hESCs and mEpiSCs and distinguish them from mESCs which remain pluripotent in LIF+BMP.

Similar articles

Cited by

References

    1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145–1147. - PubMed
    1. D'Amour KA, Agulnick AD, Eliazer S, Kelly OG, Kroon E, et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol. 2005;23:1534–1541. - PubMed
    1. D'Amour KA, Bang AG, Eliazer S, Kelly OG, Agulnick AD, et al. Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol. 2006;24:1392–1401. - PubMed
    1. Brons IG, Smithers LE, Trotter MW, Rugg-Gunn P, Sun B, et al. Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature. 2007;448:191–195. - PubMed
    1. Tesar PJ, Chenoweth JG, Brook FA, Davies TJ, Evans EP, et al. New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature. 2007;448:196–199. - PubMed

Publication types

MeSH terms

Substances