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Glycocalyx remodeling with proteoglycan mimetics promotes neural specification in embryonic stem cells.

Huang ML, Smith RA, Trieger GW, Godula K - J. Am. Chem. Soc. (2014)

Bottom Line: Interactions of GFs with their receptors are often mediated by heparan sulfate proteoglycans (HSPGs).There, the neoPGs assumed the function of native HSPGs, rescued FGF2-mediated kinase activity, and promoted neural specification.This glycocalyx remodeling strategy is versatile and may be applicable to other types of differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California , San Diego, California 92093-0358, United States.

ABSTRACT
Growth factor (GF) signaling is a key determinant of stem cell fate. Interactions of GFs with their receptors are often mediated by heparan sulfate proteoglycans (HSPGs). Here, we report a cell surface engineering strategy that exploits the function of HSPGs to promote differentiation in embryonic stem cells (ESCs). We have generated synthetic neoproteoglycans (neoPGs) with affinity for the fibroblast growth factor 2 (FGF2) and introduced them into plasma membranes of ESCs deficient in HS biosynthesis. There, the neoPGs assumed the function of native HSPGs, rescued FGF2-mediated kinase activity, and promoted neural specification. This glycocalyx remodeling strategy is versatile and may be applicable to other types of differentiation.

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Glycocalyx remodeling rescued FGF2-mediated signalingin Ext1–/– mESCs. NeoPGs 11A, D, and N (1 μM) inserted into membranesof Ext1–/– mESCs (green)and promotedFGF2 binding according to the structure of their glycans (red) (A). FGFs binding neoPG 11D enhanced Erk1/2phosphorylation (B).
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fig3: Glycocalyx remodeling rescued FGF2-mediated signalingin Ext1–/– mESCs. NeoPGs 11A, D, and N (1 μM) inserted into membranesof Ext1–/– mESCs (green)and promotedFGF2 binding according to the structure of their glycans (red) (A). FGFs binding neoPG 11D enhanced Erk1/2phosphorylation (B).

Mentions: To evaluate whether the ability of neoPGs to bind FGF2 establishedin our microarray screen can be recapitulated on the surface of mESCs,we synthesized analogs of neoPGs 7A, D,and N functionalized with a phospholid tail for membraneinsertion and an Alexa Fluor 488 (AF488) tag for imaging (neoPGs 11, Scheme 1). Incubation of Ext1–/– mESCs in solutions of neoPGs 11 in base media at 37 °C for 1 h led to a successful introductionof the polymers to the cell surface (Figure 3A). The amount of neoPG delivered to the cell surface is proportionalto the polymer concentration in the media and the incubation time,offering control over the extent of cell-surface remodeling (for optimizationof these variables for neoPG 11N, see Figure S40). The degree of remodeling by the different neoPGswas assessed by fluorescence microscopy (Figure 3A). Interestingly, the nonsulfated neoPG 11A exhibitedhigher levels of membrane incorporation relative to 11D and 11N, presumably due to its lower overall negativecharge. To sustain differentiation, the neoPGs need to remain activeon the cell surface for a period of several hours.4b To establish the membrane residence time for our polymers,we cultured Ext1–/– mESCs remodeled withneoPG 11D and monitored its clearance from the cell surfaceusing an anti-AF488 antibody. Satisfyingly, >50% of the neoPG stillremained localized to the cell surface after 8 h (Figure S42).


Glycocalyx remodeling with proteoglycan mimetics promotes neural specification in embryonic stem cells.

Huang ML, Smith RA, Trieger GW, Godula K - J. Am. Chem. Soc. (2014)

Glycocalyx remodeling rescued FGF2-mediated signalingin Ext1–/– mESCs. NeoPGs 11A, D, and N (1 μM) inserted into membranesof Ext1–/– mESCs (green)and promotedFGF2 binding according to the structure of their glycans (red) (A). FGFs binding neoPG 11D enhanced Erk1/2phosphorylation (B).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4121001&req=5

fig3: Glycocalyx remodeling rescued FGF2-mediated signalingin Ext1–/– mESCs. NeoPGs 11A, D, and N (1 μM) inserted into membranesof Ext1–/– mESCs (green)and promotedFGF2 binding according to the structure of their glycans (red) (A). FGFs binding neoPG 11D enhanced Erk1/2phosphorylation (B).
Mentions: To evaluate whether the ability of neoPGs to bind FGF2 establishedin our microarray screen can be recapitulated on the surface of mESCs,we synthesized analogs of neoPGs 7A, D,and N functionalized with a phospholid tail for membraneinsertion and an Alexa Fluor 488 (AF488) tag for imaging (neoPGs 11, Scheme 1). Incubation of Ext1–/– mESCs in solutions of neoPGs 11 in base media at 37 °C for 1 h led to a successful introductionof the polymers to the cell surface (Figure 3A). The amount of neoPG delivered to the cell surface is proportionalto the polymer concentration in the media and the incubation time,offering control over the extent of cell-surface remodeling (for optimizationof these variables for neoPG 11N, see Figure S40). The degree of remodeling by the different neoPGswas assessed by fluorescence microscopy (Figure 3A). Interestingly, the nonsulfated neoPG 11A exhibitedhigher levels of membrane incorporation relative to 11D and 11N, presumably due to its lower overall negativecharge. To sustain differentiation, the neoPGs need to remain activeon the cell surface for a period of several hours.4b To establish the membrane residence time for our polymers,we cultured Ext1–/– mESCs remodeled withneoPG 11D and monitored its clearance from the cell surfaceusing an anti-AF488 antibody. Satisfyingly, >50% of the neoPG stillremained localized to the cell surface after 8 h (Figure S42).

Bottom Line: Interactions of GFs with their receptors are often mediated by heparan sulfate proteoglycans (HSPGs).There, the neoPGs assumed the function of native HSPGs, rescued FGF2-mediated kinase activity, and promoted neural specification.This glycocalyx remodeling strategy is versatile and may be applicable to other types of differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California , San Diego, California 92093-0358, United States.

ABSTRACT
Growth factor (GF) signaling is a key determinant of stem cell fate. Interactions of GFs with their receptors are often mediated by heparan sulfate proteoglycans (HSPGs). Here, we report a cell surface engineering strategy that exploits the function of HSPGs to promote differentiation in embryonic stem cells (ESCs). We have generated synthetic neoproteoglycans (neoPGs) with affinity for the fibroblast growth factor 2 (FGF2) and introduced them into plasma membranes of ESCs deficient in HS biosynthesis. There, the neoPGs assumed the function of native HSPGs, rescued FGF2-mediated kinase activity, and promoted neural specification. This glycocalyx remodeling strategy is versatile and may be applicable to other types of differentiation.

Show MeSH
Related in: MedlinePlus