Relatively slower speed of engraftment in bone marrow of recipients makes cord blood (CB) transplantation (CBT) more difficult in a clinical setting. Selectins and their ligands are known to play a role in the homing of hematopoietic stem/progenitor cell (HSPC) to the bone marrow. Fucosyltransferase VI (FTVI) mediated ex vivo fucosylation improves human CB engraftment in NOD-SCID IL-2Rγnull (NSG) mice. FTVI is expressed in many cell types, while fucosyltransferase VII (FTVII) is primarily expressed in hematopoietic cells. FTVII deficient leukocytes do not bind the P- and E-selectins and are not recruited to sites of inflammation. We hypothesize that FTVII is more efficient than FTVI in surface fucosylation of CB-HSPC and thus enhances homing and engraftment of CB-HSPC (CD34+) in NSG mice.
Purified CB CD34+ cells were obtained from fresh CB in healthy full-term newborns by immunomagnetic microbeads sorting. CD34+ cells were treated with FTVI or FTVII respectively, the expression level of sLex (HECA-452) and binding to P- or E-selectin were detected by flow cytometry. Interactions of FTVI-, FTVII- or combined FTVI/FTVII-fucosylated CB CD34+cells with P- or E-selectin were measured under physiological flow conditions using a parallel-plate flow chamber assay. CBT was performed in NSG mice of 5 groups (negative control, sham-control, FTVI-treated, FTVII-treated, and FTVI/FTVII-treated). From 2 weeks post transplantation, mice were bled every 2 weeks for anti-human CD45 screening by flow cytometry. We used this method to evaluate the engraftment progress of CB-HSPC with different treatments. Ten weeks after transplantation, all mice were sacrificed and peripheral blood and bone marrow cells were isolated. Hematopoiesis reconstitution of CB cells was analyzed by flow cytometry after staining the cells with antibodies to various human hematopoietic subset markers, which included CD45 for total leukocytes, CD19 for B-cells, CD33 for myeloid cells, CD41 for megakaryocytes, CD3 for T-cells, and CD34 for HSPCs.
Under in vitro conditions, surface fucosylation with FTVI or FTVII results in an enhanced expression of HECA-452 epitopes on CD34+ cells from fresh CB. Increased P- and E-selectin binding to fresh CB CD34+ cells after either FTVI or FTVII treatment compared to sham-treated. However, FTVII-treated fresh CB CD34+ cells had a much higher binding to P-selectin than that of FTVI-treated fresh CB CD34+ cells. FTVI/FTVII-fucosylated CD34+ cells isolated from fresh CB accumulated more on P- or E-selectin coated plates than that of single enzyme fucosylated CD34+ cells under flow conditions. Although it is not statistically significant, more FTVII-treated fresh CB CD34+ cells rolled on both P- and E-selectin surface than FTVI-treated fresh CB CD34+ cells did. FTVII- or FTVI/FTVII-treated CD34+cells from fresh CB engrafted significantly better in NSG mice than the FTVI-treated cells at 4 and 6 weeks. Ten weeks after transplantation, the bone marrows of mice that received FTVII-treated cells had significantly improved engraftment of human hematopoietic progenitors of some lineages (CD3, CD33, and CD41) compared to that of mice that received FTVI-treated cells.
FTVII-treated CB-HSPCs have greater engraftment progress during early time after transplantation compared to FTVI. FTVII mediated fucosylation did not affect self-renewal of CB-HSPC. Hematopoiesis reconstitution of T-lymphocytes, myeloid cells, and megakaryocytes of FTVII is better than FTVI. Finally, FTVI and FTVII may have a cooperative effect on CB-HSPC homing to bone marrow.
Miyaji:Kyowa Hakko Kirin Co., Ltd.: Research Funding. Sato:Kyowa Hakko Kirin Co., Ltd.: Research Funding. Miller:Kyowa Hakko Kirin Co., Ltd.: Research Funding. Xia:Kyowa Hakko Kirin Co., Ltd.: Research Funding.
Asterisk with author names denotes non-ASH members.