Ex vivo fucosylation improves human cord blood engraftment in NOD-SCID IL-2Rγ(null) mice

Abstract

Delayed engraftment remains a major hurdle after cord blood (CB) transplantation. It may be due, at least in part, to low fucosylation of cell surface molecules important for homing to the bone marrow microenvironment. Because fucosylation of specific cell surface ligands is required before effective interaction with selectins expressed by the bone marrow microvasculature can occur, a simple 30-minute ex vivo incubation of CB hematopoietic progenitor cells with fucosyltransferase-VI and its substrate (GDP-fucose) was performed to increase levels of fucosylation. The physiologic impact of CB hematopoietic progenitor cell hypofucosylation was investigated in vivo in NOD-SCID interleukin (IL)-2Rγ(null) (NSG) mice. By isolating fucosylated and nonfucosylated CD34(+) cells from CB, we showed that only fucosylated CD34(+) cells are responsible for engraftment in NSG mice. In addition, because the proportion of CD34(+) cells that are fucosylated in CB is significantly less than in bone marrow and peripheral blood, we hypothesize that these combined observations might explain, at least in part, the delayed engraftment observed after CB transplantation. Because engraftment appears to be correlated with the fucosylation of CD34(+) cells, we hypothesized that increasing the proportion of CD34(+) cells that are fucosylated would improve CB engraftment. Ex vivo treatment with fucosyltransferase-VI significantly increases the levels of CD34(+) fucosylation and, as hypothesized, this was associated with improved engraftment. Ex vivo fucosylation did not alter the biodistribution of engrafting cells or pattern of long-term, multilineage, multi-tissue engraftment. We propose that ex vivo fucosylation will similarly improve the rate and magnitude of engraftment for CB transplant recipients in a clinical setting.

Figure 1

A. Endogenous fucosylation of BM and PB CD34⁺ cells is significantly greater (*P≤0.05) than fresh or frozen CB CD34⁺ cells. B. Fucosylated (HECA⁺) CD34⁺ cells are responsible for engraftment. C. FT-VI activity is limiting at higher cell and lower enzyme concentrations. D. Representative CLA antibody staining of untreated and FT-VI-treated CB CD34⁺ cells.

Figure 2

A. K562 were engineered to express PSGL-1 (K562/PSGL-1). B. K562 and K562/PSGL-1 have low endogenous levels of fucosylation and become heavily fucosylated following treatment with FT-VI. C. Only FT-VI-treated (fucosylated) K562/PSGL-1 cells showed improved homing to femoral marrow.

Figure 3

A. No evidence of an FT-VI-associated improvement in the homing of CB CD34⁺ cells to BM of NSG mice detected. (Solid bar - Untreated. Open bar - FT-VI treated). B. Representative flow cytometric analyses of BM from mice receiving: no cells (Grp1) or CFSE-stained, untreated (Grp2) or CFSE-stained, FT-V-treated CB CD34⁺ cells (Grp5).

Figure 4

A-C. Evidence that FT-VI treatment (fucosylation) improves the homing of CB CD34⁺ cells to BM of NSG mice. Significantly greater numbers of human CFU-GM, BFU-E and CFU-GEMM were found in BM of NSG mice receiving CB CD34⁺ cells treated with FT-VI when assessed 7, 14 and 21 days after transplant.

Figure 5

A. Fucosylated CD34⁺ cells engraft more rapidly and with greater magnitude. B-D. Increased CD19⁺ and CD33⁺ cell engraftment is observed in PB and spleen in recipients of fucosylated CB. No differences observed in BM. E. FT-VI treatment (fucosylation) improved the rate and magnitude of engraftment without altering biodistribution.

Figure 6

A-B. Colony-forming unit (CFU) and viability assays provide evidence of manganese (Mn²⁺) toxicity when used in the reaction buffer at >1mM. C. Use of Mn²⁺ at <1mM does not compromise the activity of FT-VI. The enzyme is active in the absence of Mn²⁺.