Umbilical Cord Blood-Derived Stem Cells Cultured Under Low Oxygen Tension Enhance the Migration and Homing Efficiency through the Increased Expression of CXCR4.

Abstract 3715

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. Recently, we reported that the increase of the cell surface expression of CXCR4 on CD34 cells by short time cultured enhanced the homing activity and engraftment (Ohno et al, Stem Cells Dev. 2009). It has been reported that CXCR4 expression has been enhanced by hypoxia through the enforcement of hypoxia-inducible factor-1α (HIF-1α). In the present study, we examined the effect of short-term culture of CB-derived CD34⁺ cells under low oxygen tension on CXCR4 expression and on migration, homing, and engraftment capability using xenotransplantation into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. CB-derived CD34⁺-enriched samples were divided into four aliquots and cultured. The first aliquot was incubated for 6hs in RPMI-1640 medium alone, the second was incubated for 6hrs in RPMI-1640 medium under low oxygen tension (5% CO₂, O₂ < 1%), whereas the third was incubated for 6hs in RPMI-1640 medium containing the specific HIF-1 antagonist, Rapamycin under low oxygen tension. As a control, the fourth sample received neither treatments nor incubations. As shown in Table, surface CXCR4 expression on CD34⁺ cells significantly increased after incubating the cells for 6hrs compared with control. The incubation under low oxygen tension led to further enhancement of CXCR4 expression. This enhancement was completely inhibited by the addition of Rapamycin. No difference in CXCR4 mRNA expression was noted after incubating CD34⁺ cells for 6hrs, however, CXCR4 expression as well as HIF-1 mRNA expressions in cells cultured under low oxygen tension was significantly increased by using quantitative real-time PCR. The incubation of the increase in the cell surface CXCR4 expression appears to be caused by translocation of CXCR4 from the interior of the cell to the cell surface and the increase in HIF-1 expression. CD34⁺ cells cultured under low oxygen tension for 6hrs showed significantly increased transmigrational activity toward SDF-1.Finally, we assessed the homing activity of cells cultured under low oxygen using the transplantation into NOD/SCID mice. Homing activity was examined by the detection of human CD45 and CD34 positive cells 16hrs after transplantation. The CD34⁺ cells ia cultured under low oxygen tension demonstrated significantly increased homing activity in the bone marrow of NOD/SCID mice compared with that of control cells, cells cultured for 6hs, and cells cultured under low oxygen tension in the presence of Rapamycin (P < 0.01). These observations suggest that the culture of CD34⁺ cells derived from CB under low oxygen tension led to the increase in the expression of CXCR4 in cells, resulting in the acceleration of homing capability in the bone marrow. The culture of hematopoietic cells under low oxygen tension may be useful technique for applying to hematopoietic stem cell transplantation.