Heparan sulfate is required for embryonic stem cells to exit from self-renewal

Abstract

Pluripotent embryonic stem cells (ESCs) must select between alternative fates of self-renewal and lineage commitment at each division during continuous proliferation. Heparan sulfate (HS) is a highly sulfated polysaccharide and is present abundantly on the ESC surface. In this study, we investigated the role of HS in ESC self-renewal by examining Ext1(-/-) ESCs that are deficient in HS. We found that Ext1(-/-) ESCs retained their self-renewal potential but failed to transit from self-renewal to differentiation upon removal of leukemia inhibitory factor. Furthermore, we found that the aberrant cell fate commitment is caused by defects in fibroblast growth factor signaling, which directly retained high expression of the pluripotency gene Nanog in Ext1(-/-) ESCs. Therefore, our studies identified and defined HS as a novel factor that controls ESC fate commitment and also delineates that HS facilitates fibroblast growth factor signaling, which, in turn, inhibits Nanog expression and commits ESCs to lineage differentiation.

FIGURE 1.

Ext1^−/− ESCs are deficient in HS. A, PCR analysis of genomic DNA isolated from Ext1^+/+ and Ext1^−/− ESC clones. Amplification of the Ext1^flox allele yields a 460-bp product. Amplification of the Ext1⁻ allele yields a 500-bp product. B, RT-PCR analysis of Ext1 and Ext2 transcripts from Ext1^+/+ and Ext1^−/− ESCs (ES) and EBs. C, detection of HS by flow cytometry using an anti-HS antibody (H10E4). Background controls are represented by shaded peaks.

FIGURE 2.

Ext1^−/− ESCs can be maintained in the self-renewing state. A, Ext1^+/+ and Ext1^−/− ESCs were examined for their colony morphology after 20 days (5 passages) in feeder-free conditions in the presence of LIF. Bar, 300 μm. B, Ext1^+/+ and Ext1^−/− ESCs were stained for AP activity after 20 days of culture in the presence of LIF. Bar, 200 μm. C, ESCs were cultured for 15 days and then plated out at clonal density. The percentage of AP-positive colonies was quantitated. Error bars indicate the S.E. generated from triplicates of the same experiment, which is representative of at least three independent experiments. D, semiquantitative PCR of pluripotency genes. RNA was extracted from ESCs cultured for 20 days in feeder-free conditions. E, immunofluorescence of OCT-4 expression in colonies formed after 20 days in culture in feeder-free conditions in the presence of LIF. Bar, 150 μm. DAPI, 4′,6-diamidino-2-phenylindole.

FIGURE 3.

HS is required for ESC differentiation commitment. A, Ext1^+/+ and Ext1^−/− ES cells were plated at clonal density and cultured at various LIF concentrations for 5 days. The percentage of undifferentiated colonies was examined by AP assays. Error bars indicate S.E. generated from triplicates of the same experiment, which is representative of at least three independent experiments. B, phase-contrast microscopy of day 10 EBs. Bar, 200 μm. C, RNA expression levels of pluripotency and differentiation markers during in vitro differentiation of ESCs. Semiquantitative RT-PCR analysis was performed on RNA extracted from either undifferentiated ESCs or EBs throughout a differentiation period of 10 days (days 2–10). β-Actin transcripts were used as an internal control. D, confocal microscopy images of day 8 EBs immunostained for OCT-4. Bar, 100 μm. U, units.

FIGURE 4.

FGFR inhibition blocks cell fate commitment and recapitulates the Ext1^−/− phenotype. A, ESCs were treated with 100 ng/ml PD in serum-containing or serum-free culture conditions. Western blot shows phosphorylation of ERK1/2 (pErk); total ERK1/2 levels were used as internal controls. B, ESCs were differentiated for 2 days in adherent culture in the absence of LIF with and without PD173074. Phase-contrast micrographs show colony morphology. Bar, 150 μm. C, ESCs were differentiated for 5 days in absence of LIF with PD173074 or solvent control (DMSO). Immunofluorescence staining shows OCT-4 levels. Bar, 150 μm. D, real-time RT-PCR for Nanog, Wnt-3, Brachyury, Nestin, and Gata-6 was performed at day 5 of differentiation. Transcript levels are relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Error bars indicate S.E. generated from triplicates of the same experiment, which is representative of at least three independent experiments. DAPI, 4′,6-diamidino-2-phenylindole.

FIGURE 5.

FGF signaling is impaired in Ext1^−/− ESCs. A, ESCs were incubated with biotinylated FGF-2. Cell surface binding was analyzed by flow cytometry. Negative control was incubated with secondary antibody only. B, Western blot showing phosphorylation of ERK1/2 (pErk) after FGF-2 stimulation of serum-starved ESCs. Total ERK1/2 levels were used as internal controls. C, Western blot showing steady-state phospho-ERK1/2 levels. Lysate from ESCs cultured in presence of LIF (time point 0) and from ESCs in absence of LIF (time points 2–6 h) were analyzed. Total ERK1/2 levels were used as internal controls.

FIGURE 6.

Heparin/HS rescues Ext1^−/− cell fate commitment. A, Western blot showing steady-state phospho-ERK1/2 levels upon the addition of heparin in serum-containing medium. Total ERK1/2 levels were used as internal controls. B, as in A with the addition of HS isolated from Ext1^+/+ ESCs instead of heparin. C, ESCs were differentiated for 5 days. Ext1^−/− ESCs were treated with (+) or without (−) heparin (HEP), HS, and PD. Shown are fluorescence microscopy pictures of OCT-4 staining. Bar, 200 μm. D, ESCs were plated at clonal density and cultured for 5 days in the absence of LIF supplemented with heparin or HS and/or PD. Subsequently, colonies were analyzed for AP activity. The proportion of AP-positive colonies was quantified. Error bars indicate the S.E. generated from triplicates of the same experiment, which is representative of at least three independent experiments. In parallel, colony morphology was examined by phase-contrast microscopy. Bar, 200 μm. E, real-time RT-PCR for Nanog, Wnt-3, Brachyury, Nestin, and Gata-6 was performed at day 5 of differentiation. Transcript levels are relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Error bars indicate S.E. generated from triplicates of the same experiment, which is representative of at least three independent experiments. F, ESCs were transferred into serum-free medium supplemented with 10 ng/ml FGF-4 with or without PD. Ext1^−/− ESCs were treated with 1 μg/ml heparin in the absence or presence of PD. After 24 h, RNA was collected, and Nanog mRNA levels were examined by real-time RT-PCR.