Ftvii

Murine AdMSCs were derived from C57BL/6 mice, the recipient strain for MHC-mismatched HSCT, whereas hAdMSCs and hBMMSCs were isolated from healthy human donors. Fucose was stereoselectively installed onto sialyllactosaminyl glycans of CD44 using an α(1,3)-linkage-specific fucosyltransferase, fucosyltransferase VII (FTVII; obtained from R&D Systems), in presence of donor fucose substrate (GDP-fucose; Sigma Aldrich): MSCs were resuspended at 2 × 10⁷ cells/ml and incubated for 60 min at 37 °C in FTVII reaction buffer composed of Hank’s Balanced Salt Solution (HBSS) (without Ca²⁺ and Mg²⁺) (Lonza) containing 20 mM HEPES (Lonza), 0.1% human serum albumin (HSA) (Grifols), 30 μg/ml FTVII (R&D Systems), and 1 mM GDP-fucose (Fucosylation-modified, “FucmAdMSCs”). Controls consisted of MSCs treated with reaction buffer alone (i.e., Unmodified MSCs, “UmAdMSCs” or, “UhAdMSCs” and “UhBMMSCs”). Exofucosylation efficacy was measured by analysis of HECA452 antibody (10 μg/ml, BD Biosciences, Cat#555946) staining and murine E-selectin-human Fc chimera (mE-Ig; 5 μg/ml, R&D Systems, Cat#575-ES-100) binding by flow cytometry and western blot.

Human BMMSCs and AdMSCs were exofucosylated as previously reported (21 (link)). Briefly, cells were resuspended at 2x10⁷ cells/mL in the exofucosylation reaction buffer composed of Hanks´ Balanced Salt Solution (HBSS) (Thermo Fisher Scientific) containing 40 μg/mL fucosyltransferase VII (FTVII) (Bio-Techne, R&D Systems, Minneapolis, MN, United States), 10 mmol/L HEPES (Merck Millipore, Burlington, MA, United States), 0.1% human serum albumin (HSA; Albutein, Grifols, Barcelona, Spain) and 1 mmol/L guanosine 5’-diphosphate (GDP)-fucose (Sigma-Aldrich), and treated at 37°C for 1 hour with gentle shaking (Figure 1, Step IV). After, treated MSCs were collected by centrifugation and washed twice with DPBS. Cell viability after exofucosylation was assessed by trypan blue exclusion (normally more than 90% live cells), and exofucosylation efficacy was evaluated using the phycoerythrin-labeled anti-sLe^X monoclonal antibodyHECA-452 (BD Biosciences) by flow cytometry.

Murine pericytes were modified by enzymatic exofucosylation as previously reported (Garcia-Bernal et al., 2020 (link)). Briefly, cells were resuspended at 2 × 10⁷ cells/ml in fucosyltransferase VII (FTVII) reaction buffer composed of Hanks Balanced Salt Solution (HBSS, Gibco) containing 30 μg/ml fucosyltransferase VII (FTVII, R&D Systems), 20 mM HEPES (Thermo Fisher Scientific), 0.1% human serum albumin (Grifols) and 1 mM guanosine 5′-diphospho-β-L-fucose sodium salt (GDP-fucose, Sigma Aldrich), and incubated for 60 min at 37°C and 5% CO₂. Unmodified controls pericytes were treated only with GDP-fucose (w/o FTVII) in the same conditions as above. Cell viability after exofucosylation was assessed by trypan blue exclusion (usually 95% live cells). Efficacy of exofucosylation was evaluated by analysis of HECA452 antibody (BD Biosciences) staining and calcium-dependent mouse E-selectin/human IgG chimera (R&D Systems) binding by flow cytometry.