Human umbilical cord blood (CB) is rich source of hematopoietic stem cells (HSCs) and provides an attractive alternative to bone marrow or mobilized peripheral blood transplantation. However, major disadvantage of CB transplantation is the relatively low number of HSCs in each CB unit, which severely limits its usefulness in clinical transplantation. A part of CD34+ cells expresses cell surface CXCR4, which has been found to be critical for bone marrow engraftment by human hematopoietic stem cells. In the present study, we examined the effect of short-term culture on CXCR4 expression and engraftment in CB-derived CD34+ cells transplanted into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. CB-derived CD34+-enriched samples were divided into three aliquots and cultured. The first aliquot was incubated for 2 h in RPMI-1640 medium alone, whereas the second was incubated for 2h in RPMI-1640 medium containing the specific CXCR4 antagonist, AMD3100. As a control, the third sample was neither treated nor incubated. Surface CXCR4 expression on CD34+ cells significantly increased after incubating the cells in medium alone for 2 h (7.7 to 19.8%); this increase was blocked by the addition of AMD3100. No difference in CXCR4 mRNA expression was noted after incubating CD34+ cells for 2 h. The short-term incubation seems to influence the translocation of CXCR4 from the interior of the cell to the cell surface. CD34+ cells cultured for 2 h showed significantly increased transmigrational activity toward SDF-1 and homing activity in NOD/SCID mice. However, those were lower in non-cultured cells and cells treated with AMD3100. Next, we assessed the engraftment of cells cultured into NOD/SCID mice. Engraftment analysis was performed 8 weeks after transplantation. The presence of human cells was detected by the cell surface expression of CD45, CD34, CD33, and CD19. The CD34+ cells cultured demonstrated significantly increased engraftment of human cells in the bone marrow of NOD/SCID mice compared with that of CD34+ cells without culture or with culture in the presence of AMD3100 (p < 0.01). The recipients exhibited CD19+ B-lymphoid cells, CD33+ myeloid cells, and CD34+ immature cells, but neither CD3+ T cells nor CD45+ cells in bone marrow. Finally, we performed a secondary transplant of engrafted cells from previously transplanted mice to confirm long-term engraftment and self-renewal of cultured CB cells. The successful engraftment was observed in mice injected with primary cultured cells, demonstrating that these cultured cells are self-renewing and capable of long-term engraftment. These observations suggest that the increase of the cell surface expression of CXCR4 on CD34 cells resulted in the retention of human umbilical cord blood cells within the bone marrow through the engraftment of homing activity.

Disclosures: No relevant conflicts of interest to declare.

Author notes

Corresponding author