Evidence for the Involvement of CXCR4 Signaling in In Vivo Self-Renewal of Transplanted Hematopoietic Stem Cells

Abstract 1888

Hematopoietic stem cells (HSCs) represent the unique cell population capable of self-renewal and multi-lineage differentiation, thereby lifelong sustainment of the hematopoiesis. HSC transplantation has proven beneficial for various diseases, it is therefore important to elucidate the molecular determinants for successful HSC engraftment. Signaling through the chemokine receptor CXCR4 has been implicated in HSC engraftment by the observation that transplantation of HSCs lacking this molecule results in poor hematopoietic reconstitution. Because this impairment, however, can be attributed to the defects in any of the post-transplantation processes that include bone marrow (BM)-homing, -repopulation, or –retention, it is still unclear whether CXCR4 plays an essential role in HSC self-renewal upon transplantation.

To elucidate the role of CXCR4 signaling in HSC self-renewal in conjunction with transplantation, we used a purified CD34^neg/low c-Kit⁺ Sca-1⁺ Lineage-marker^neg population as the defined stem cell source. As a loss-of-function study, CXCR4 was conditionally deleted in HSCs before transplantation. As a gain-of-function study, we generated the HSC populations overexpressing either wild-type (wt)- or C-terminal truncated (δC)-CXCR4 (OE-HSCs), the latter of which is known to exhibit enhancement in the SDF-1 signaling, by gene transfer and subsequent cell sorting. We compared these cells with control HSCs in in vitro assays with regard to the biological characteristics including chemotaxis, proliferation, colony formation, and cobblestone-area (CA) forming ability. To dissect in vivo post-transplantation processes, we investigated hematopoietic repopulation kinetics in the recipient BM at the homing/lodging phase (within 1 wk) and the early repopulation phase (2–3 wks) for the above test HSCs. The self-renewal potential of each HSC population was estimated by competitive repopulation assay.

In vitro studies: OE-HSCs with wt- or δC-CXCR4 exhibited enhanced chemotaxis and proliferation in response to SDF1, confirming the gain-of-function effects of these modifications. CA forming ability was greater in OE-HSCs with δC-CXCR4 than control counterparts and absent in CXCR4-KO HSCs, suggesting the critical role of CXCR4-signaling in HSC proliferation in the presence of stromal support.

In vivo studies: 1) the homing/lodging phase. Unexpectedly, we did not find significant alteration in the numbers of early progenies detectable in recipient BM 3 days after transplantation of HSCs receiving either loss- or gain-of-function modification to CXCR4, indicating that this signaling is indispensable in HSC homing. 2) the early repopulation phase. Impairment of hematopoietic repopulation in BM became evident for CXCR4-KO HSCs through 2–3 wks. On the other hand, OE-HSCs with CXCR4, more remarkably of ΔC-mutation, showed enhanced BM repopulation kinetics at ∼3 wks post transplantation, suggesting the importance of CXCR4 signaling in HSC amplification at this post-transplantation phase. 3) long-term hematopoiesis. CXCR4-KO-HSCs showed poor hematopoietic reconstitution potentials, consistent with previous observations. Interestingly, impaired peripheral repopulation was also observed with OE-HSCs with wt- or ΔC-CXCR4. Further characterization revealed that the recipients of CXCR4-overexpressing HSCs did retain their progenies, which showed multilineage differentiation, but exhibited impaired release of mature leukocytes from the BM to the peripheral blood. Most importantly, however, test-cell chimerism in the long-term HSC fraction was significantly higher in the mice receiving OE-HSCs with CXCR4, especially of ΔC-type, than those transplanted with control HSCs, indicating that the augmentation of CXCR4 signaling enhanced competitive repopulation ability of HSCs. These modified HSCs demonstrated repopulation abilities also in secondary recipients.

We demonstrated that CXCR4 signaling is indispensible for HSC homing and that continuous overexpression of CXCR4 cannot benefit the peripheral reconstitution in contrary to the expectation. More importantly, our studies showed that augmentation of CXCR4 signaling leads to HSC expansion in vivo upon transplantation. We thus conclude that CXCR4 signaling has a role in HSC self-renewal and that its regulation may find the approach that will improve HSC transplantation outcomes.