Small Molecule Antagonist of Cell Surface Glycosaminoglycans Restricts Mouse Embryonic Stem Cells in a Pluripotent State

Abstract

Recently, the field of stem cell-based regeneration has turned its attention toward chemical approaches for controlling the pluripotency and differentiation of embryonic stem cells (ESCs) using drug-like small molecule modulators. Growth factor receptors or their associated downstream kinases that regulate intracellular signaling pathways during differentiation are typically the targets for these molecules. The glycocalyx, which plays an essential role in actuating responses to growth factors at the cellular boundary, offers an underexplored opportunity for intervention using small molecules to influence differentiation. Here, we show that surfen, an antagonist of cell-surface glycosaminoglycans required for growth factor association with cognate receptors, acts as a potent and general inhibitor of differentiation and promoter of pluripotency in mouse ESCs. This finding shows that drugging the stem cell Glycome with small molecules to silence differentiation cues can provide a powerful new alternative to existing techniques for controlling stem cell fate. Stem Cells 2018;36:45-54.

Keywords: Carbohydrate; Differentiation; Embryonic stem cell; Fibroblast growth factors; Heparan sulfate; Pluripotency.

Figure 1.

Targeting glycosaminoglycans to influence embryonic stem cell fate. Top: HS GAGs are required for FGF2-dependent induction of the MAPK pathway and neural differentiation in murine embryonic stem cells. Deactivation of HS GAGs disrupts MAPK signaling and inhibits differentiation. Bottom: Small molecule antagonists provide an attractive alternative to genetic and enzymatic methods for the attenuation of HS GAG activity. Abbreviation: ESC, embryonic stem cell; FGFR, FGF receptor; GAG, glycosaminoglycan; HS GAG, heparan sulfate glycosaminoglycan; MAPK, mitogen-activated protein kinase.

Figure 2.

Dual endpoint GFP-reporter assay for evaluating heparin sulfate glycosaminoglycan (HS GAG) antagonists as inhibitors of neural differentiation in murine embryonic stem cells (mESCs). (A): Oct4-GFP and Sox1-GFP mESC reporter lines were used to evaluate the ability of HS GAG antagonists to inhibit neural differentiation and promote pluripotency over 6 days in N2B27 culture. (B): Live cell fluorescence micrographs show loss of pluripotency (Oct4) and acquisition of neural phenotype (Sox1) after 6 days of differentiation. Scale bar: 100 μm. (C): In the presence of surfen (5 μM), Oct4 and Sox1 expression profiles indicate that mESCs continue to maintain high levels pluripotency even after 6 days in differentiation. (D): Surfen shows enhanced ability to promote pluripotency and inhibit differentiation compared to other known HS modulators, protamine (10 μM), and adhesamine (5 μM). Surfen activity is comparable to that of PD173074 (1 μM), an ATP-competitive FGF receptor inhibitor. (E): Surfen inhibits differentiation in both Oct4-GFP and Sox1-GFP cell lines with IC₅₀ ~2.0 μM. Each point represents technical duplicates (mean6SD), representative of two biological replicates. (F): Analysis of mRNA expression in Oct4-GFP mESCs on day 6 of treatment with surfen in N2B27 (5.0 μM) indicates cell arrest in the embryonic state. Relative mRNA expression is calculated from technical triplicates (mean6SD) normalized to untreated (N2B27) controls (defined as 1.0). This experiment is representative of two biological replicates. **, p <.0037; ***, p <.002; ****, p <.0001. Abbreviations: GFP, green fluorescent protein; LIF, leukemia inhibitory factor.

Figure 3.

Surfen is a reversible inhibitor of differentiation. (A): After 6 days in neural differentiation in the presence of surfen (5 μM) cells remain pluripotent, as evidenced by high Oct4-GFP and low Sox1-GFP expression levels via flow cytometry. Differentiation resumes after removal of surfen at day 6 producing high levels of Sox1-GFP expression with concurrent loss of Oct4-GFP by day 13. %GFP^+ve values are provided as technical duplicates (mean ± SD), representative of three biological replicates. *, p < .0335; **, p < .0048; ***, p .0001.(B): Immunostaining for neural markers, nestin, and β-III-tubulin in Oct4-GFP murine embryonic stem cells (mESCs) after 6 days of neural differentiation in N2B27 with or without surfen. (C): Nestin and b-III-tubulin expression in Oct4- GFP mESCs on day 13, 6 days after surfen removal. Scale bar: 100 μm. Abbreviation: GFP, green fluorescent protein.

Figure 4.

Surfen acts by inhibiting FGF2 signaling and its activity can be neutralized with soluble heparin. (A): Stimulation of Oct4- GFP murine embryonic stem cells (mESCs) with FGF2 in the presence of surfen leads to dose-dependent attenuation of Erk1/2 phosphorylation. (B): Erk1/2 phosphorylation in Oct4-GFP mESCs is recovered in the presence of heparin (5 μg/ml), a soluble competitor for cell surface heparan sulfate-bound surfen. (C): Added soluble heparin restores the ability of mESCs to undergo neural differentiation in the presence of surfen (5 μM). Dunnett’s multiple comparison test against surfen-treated control, ****, p < 0001. Shown are technical duplicates (mean ± SD), repeated with two biological replicates. Abbreviation: GFP, green fluorescent protein.

Figure 5.

Receptor tyrosine kinase (RTK) array analysis of embryonic Oct4-GFP murine embryonic stem cells in response to surfen treatment. Processed blots (A) and bar graphs (B) demonstrate that surfen inhibits phosphorylation of numerous RTKs. Bargraphs were generated using technical duplicates (means ± SD), and the experiment was performed once. Abbreviations: EGFR, epidermal growth factor receptor; GFP, green fluorescent protein; MSPR, macrophage-stimulating protein receptor.

Figure 6.

Surfen is a general inhibitor of neural and spontaneous differentiation in mESCs. Live cell fluorescence micrograph images (A) and flow cytometry analysis (B) show inhibition of neural differentiation and maintenance of pluripotency in EB culture 6 days after neural induction in N2B27 media. (C): Surfen maintains pluripotency in mESCs under spontaneous differentiation conditions 6 days after withdrawal of the leukemia inhibitory factor. Provided are technical duplicates (means ± SD), as a representative experiment of three biological replicates, ****, p < .0001. Scale bar: 100 μm. Abbreviations: GFP, green fluorescent protein; KSR, knockout serum replacement; mESCs, murine embryonic stem cells.