Figure 3. Membrane remodeling with engineered SDC1 ectodomains provides insight into regulation of mESC differentiation by proteoglycans.

(A) HS-deficient (Ext1^−/−) mESCs fail to differentiate due to their reduced ability to bind FGF2 (blue). The formation of ternary complexes among FGF2, FGFR, and HSPGs promotes differentiation. (B) A two-step complexation strategy is used to present engineered SDC1 ectodomain variants onto cell surfaces. Live mESCs are first incubated with cholPEGNTA (10 μM, 1 hr, 37°C), followed by Ni(OAc)₂ (100 μM, teal) and the SDC1 ectodomain equipped with a poly-His tag (red tail; 2 μM, 1 hr, 37°C). Excess material is washed off at each step. (C) A six-day protocol for mESC differentiation, illustrating the loss of pluripotency marker Nanog and gain of SOX1 expression, indicative of neuroectoderm differentiation. Ext1^−/− cells are incubated with engineered SDC1 constructs, with or without prior cell surface engineering, for 1 hr on D0 or a single treatment with soluble heparin (5 μg/mL) until D2. (D) GAG-conjugated SDC1 ectodomains (2 μM) can rescue differentiation of Ext1^−/− mESCs, as evidenced by expression of neural precursor markers Sox1 (green) and Tubb3 (red). Scale bar: 50 μm. Data representative of three biological replicates. (E) RT-qPCR analysis at D6 of differentiation shows decreased Nanog expression of treated (heparin or SDC1) compared to untreated cells. Cells remodeled with cholPEGNTA before addition of SDC1 proteins demonstrated increased SOX1 expression. (F) RT-qPCR analysis at D6 of differentiation demonstrates increased expression of neural differentiation marker SOX1 with addition of glycosylated SDC1. All proteins are displayed on cell surface (+cholPEGNTA, 10 μM). All experiments performed in technical triplicate in two biological replicates. One-way ANOVA with Tukey’s post-hoc, (*) p <0.0332, (**) p <0.0021, (***) p <0.0002, (****) p <0.0001. Bar graphs represent means and error bars represent SEM.