Interaction with Human Stromal Cells Enhances CXCR4 Expression and Engraftment of Cord Blood Lin-CD34-Cells Via Wnt Signaling.

Ex vivo manipulation of hematopoietic stem/progenitor cells has been performed in preclinical and clinical settings for expansion of hematopoietic stem cells (HSCs) and enhancement of HSC engraftment. Because the absolute HSC dose in a cord blood (CB) unit is a limiting factor for its use as a graft for adult transplant recipients. Transplantation of hematopoietic stem cells (HSCs) is usually accomplished through intravenous injection, a complex process that requires recognition of bone marrow vasculature and migration to a supportive microenvironment. Hence, some populations of HSCs, including cord blood (CB) Lin-CD34-stem cells, do not engraft well in bone marrow (BM) of NOD/SCID mice. In this study, we examined the effect of human stromal interactions on the properties of CB Lin-CD34-cells. CD34 and CXCR4 expression on fresh CB Lin-CD34-cells and CB Lin-CD34-cells co-cultured with human stromal cells were analyzed. Homing activity and engraftment of these cells were assessed using NOD/SCID mice. Co-culture with human stromal cells induced expression of CD34 and CXCR4 on CB Lin-CD34-cells. Furthermore, these cells acquired homing activity and engrafted in the BM of primary and secondary recipients of NOD/SCID mice after intravenous injection. In an attempt to identify the stromal CXCR4-inducing factor, we conducted comparative experiments using stroma-free, contact culture or non-contact culture. As a result, CXCR4 expression on CB Lin-CD34-cells was induced even in the non-contact culture condition but not stroma-free condition, suggesting that this CXCR4-inducing factor is soluble. Because the function of hematopoietic stem/progenitor cells was modulated by several hematopoietic growth factors, angiogenic factors and morphogens, we next screened for soluble CXCR4 inducing factors using blocking antibodies for hedgehog protein, VEGF receptor (anti-KDR) and angiopoietin-1 receptor (anti-Tie2), BMP inhibitor (noggin), pan Wnt inhibitor (sFRP-1), Wnt/β-catenin signaling inhibitor (DKK1) and Wnt/RhoA signaling inhibitor. We found that DKK1 significantly reduced the expression of CXCR4. This indicated that CXCR4 could be regulated by the Wnt signaling pathway. Subsequently, we analyzed the expression of Wnt family members in human stromal cells by RT-PCR. High expression of Wnt1, Wnt2B, Wnt5A and Wnt11 were observed in human stromal cells. Thus, these Wnts may contribute to the induction of CXCR4 expression on CB Lin-CD34-CXCR4-cells. These findings may be useful for understanding the role of stromal cells in homing and engraftment of HSCs and may provide a new strategy to utilize CB and human stromal cells for HSC transplantation.