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In addition to its well-known effect on migration and homing of hematopoietic stem/progenitor cells (HSPCs), CXCL12 chemokine also exhibits a cell cycle and survival-promoting factor for human CD34(+) HSPCs. CXCR4 was suggested to be responsible for CXCL12-induced biological effects until the recent discovery of its second receptor, CXCR7. Until now, the participation of CXCR7 in CXCL12-induced HSPC cycling and survival is unknown. We show here that CXCL12 was capable of binding CXCR7 despite its scarce expression at CD34(+) cell surface. Blocking CXCR7 inhibited CXCL12-induced Akt activation as well as the percentage of CD34(+) cells in cycle, colony formation, and survival, demonstrating its participation in CXCL12-induced functional effects in HSPCs. At steady state, CXCR7 and β-arrestin2 co-localized near the plasma membrane of CD34(+) cells. After CXCL12 treatment, β-arrestin2 translocated to the nucleus, and this required both CXCR7 and CXCR4. Silencing β-arrestin expression decreased CXCL12-induced Akt activation in CD34(+) cells. Our results demonstrate for the first time the role of CXCR7, complementary to that played by CXCR4, in the control of HSPC cycling, survival, and colony formation induced by CXCL12. We also provide evidence for the involvement of β-arrestins as signaling hubs downstream of both CXCL12 receptors in primary human HSPCs.

Cell cycle regulation plays a fundamental role in stem cell biology. A balance between quiescence and proliferation of hematopoietic stem cells in interaction with the microenvironment is critical for sustaining long-term hematopoiesis and for protection against stress. We analyzed the molecular mechanisms by which stromal cell-derived factor-1 (SDF-1) exhibited a cell cycle-promoting effect and interacted with transforming growth factor-β (TGF-β), which has negative effects on cell cycle orchestration of human hematopoietic CD34+ progenitor cells. We demonstrated that a low concentration of SDF-1 modulated the expression of key cell cycle regulators such as cyclins, cyclin-dependent kinase inhibitors, and TGF-β target genes, confirming its cell cycle-promoting effect. We showed that a cross-talk between SDF-1- and TGF-β-related signaling pathways involving phosphatidylinositol 3-kinase (PI3K)/Akt phosphorylation participated in the control of CD34+ cell cycling. We demonstrated a pivotal role of two downstream effectors of the PI3K/Akt pathway, FoxO3a and mammalian target of rapamycin, as connectors in the SDF-1-/TGF-β-induced control of the cycling/quiescence switch and proposed a model integrating a dialogue between the two molecules in cell cycle progression. Our data shed new light on the signaling pathways involved in SDF-1 cell cycle-promoting activity and suggest that the balance between SDF-1- and TGF-β-activated pathways is critical for the regulation of hematopoietic progenitor cell cycle status. Disclosure of potential conflicts of interest is found at the end of this article.

Abstract 3448 Introduction SDF-1/CXCL12 chemokine exhibits a well-known effect on retention, migration and homing of hematopoietic stem/progenitor cells (HSC/HP). We have previously demonstrated that it is also a key regulator of hematopoiesis homeostasis, acting, at low concentrations, as a survival and cell cycle promoting factor for human CD34+ HP. It has long been considered that CXCR4 was responsible for SDF-1 induced biological effects until the recent discovery of its second receptor, CXCR7. In the present study, we explored the respective role of CXCR4 and CXCR7 in the cell cycling and survival promoting effect of SDF-1/CXCL12 on human CD34+HP. Material and Methods We used CD34+ HP purified from the peripheral blood (PB) of healthy un-mobilized donors since they are mainly in G0. This allows to study the role of CXCR4 and CXCR7 receptors in 0.5ng/ml SDF-1/CXCL12 induced G0-G1 transition in synchronized quiescent cells. Gene expression was detected by RT-QPCR. Protein expression was detected and quantified using confocal microscopy, flow cytometry, immunoblotting and immunoprecipitation. Cell cycling experiments were performed using a Ki67 antibody and CXCR7 binding assay was performed using SDF-1/CXCL12AF647. Neutralization experiments were performed using a specific CXCR4 antibody or CXCR7 chemical inhibitors, a kind gift from ChemoCentryx, Inc (CCX771 and CCX733) and their respective controls (IgG and CCX704). Results Flow cytometry and confocal analysis showed that CXCR7 and CXCR4 are differentially distributed in PB CD34+ cells. In contrast to CXCR4 that is present at both the plasma membrane and intracellular level, CXCR7 expression is mainly restricted to the intracellular compartment. Confocal analysis suggested the presence of CXCR4/CXCR7 heterodimers on these cells the presence of which were confirmed by immunoprecipitation in a HP cell line. Despite its very low expression at the surface of CD34+ cells, we found that CXCR7 is capable of binding to exogenous SDF-1/CXCL12. Indeed, pretreatment with CXCR7 antibody or a chemical inhibitor reduces the mean fluorescence of bound fluorescent SDF-1/CXCL12AF647, a fully functional and specific chemokine with similar effects compared to unlabeled SDF-1/CXCL12. Neutralizing either CXCR4 or CXCR7 in PB CD34+ cells strongly reduced Akt activation induced by SDF-1/CXCL12 (0.5 ng/ml) as well as the percentage of cells in cycle (G1 and S + G2/M), colony formation and cell survival. This demonstrates that both receptors cooperate in SDF-1/CXCL12 induced functional effects. We further analyzed the respective role of CXCR4 and CXCR7 in SDF-1/CXCL12 signalization. In contrast to CXCR4, CXCR7 is reported not to activate G protein signaling pathways in response to SDF-1/CXCL12. However, it can transduce cell signaling through the β-arrestin pathway. In the present study, we showed that CXCR7 and β-arrestin 2 colocalize near the plasma membrane in freshly purified PB CD34+ cells, suggesting that CXCR7 is constitutively activated. After SDF-1/CXCL12 treatment, the majority of β-arrestin 2 was translocated to the nucleus and only a partial colocalization persisted in the cytoplasm. Using neutralizing antibodies and specific inhibitors, we showed that β-arrestin 2 nuclear translocation was dependent on both CXCR7 and CXCR4 receptors. Reducing β-arrestin 2 expression using siRNA decreased SDF-1/CXCL12 induced Akt activation in PB CD34+cells indicating the involvement of β-arrestin 2 in this process. Conclusion Altogether, our results demonstrate for the first time the role of CXCR7 together with CXCR4 in SDF-1/CXCL12-induced CD34+ cell cycling/proliferation. They also suggest the involvement of β-arrestin 2 as signalling hubs, downstream of both receptors. Disclosures: No relevant conflicts of interest to declare.

Control of cell cycling is a key process in stem cell fate. A balance between cell quiescence and proliferation within the stem cell niche in interaction with microenvironment is critical for sustaining long-term hematopoiesis and for protection against stress. Among growth factors regulating haematopoietic stem/progenitor cell (HSC/HP) cell cycling, TGF-β is known to be a major negative regulator by blocking cell cycle progression during the G1 phase. Besides its effect on HSC/PH and lymphocyte trafficking, the SDF-1/CXCL12 chemokine is another key regulator which helps maintaining hematopoiesis homeostasis. We have previous demonstrated that SDF-1 acts as a survival and cell cycle promoting factor for CD34+ HSC/HP by triggering the G0-G1 transition and then regulating early cell cycle phases suggesting that it may act as an antagonist of TGF-β on cell cycle progression (Lataillade et al., Blood 2000 and 2002). The general aim of this study was to investigate the mechanisms of this antagonist effect on cell cycle. This work was performed on CD34+ cells purified from the peripheral blood of healthy un-mobilized donors. Since they are mainly in G0, they allow dissecting the early cell cycle phases including the G0-G1 transition. We demonstrated that SDF-1 and TGF-β exert an opposite effect on the expression of cell cycle key regulators such as cyclins and CDKI. We showed that cross-talk between SDF-1 and TGF-β signaling pathways involving PI3K/Akt phosphorylation participates in the control of CD34+ cell cycling. We demonstrated a pivotal role of FoxO3 and mTOR in the TGF-β/SDF-1 control of the quiescence/cycling of hematopoietic progenitors. A model integrating a pivotal role for the activation of two Akt substrates, FoxO3 and mTORC1, in the control of CD34+ cells quiescence/cycling by TGF-β and SDF-1 is proposed. Altogether, our results shed new light on the intracellular signaling mechanisms of SDF-1 and of its implication, together with TGF-β, in cell cycle promotion in primary CD34+ cells. Regarding their opposite effect on cell cycle and survival, we suggest that SDF-1 and TGF-β can be considered as natural mutual competitors, both acting as organizers of the stem cell niche where a balance between HSC hibernation and activation is an essential part of the stemness control.

Hematopoietic stem cells (HSC) are characterized by their potential to reconstitute in vivo haematopoiesis in NOD/SCID mice and to give rise to differentiated cells of all haematopoietic lineages. They are usually defined by a CD34+CD38−CD90+ antigenic profile; however taking into account the versatility of antigen membrane expression (Lataillade et al., J. Leukoc. Biol. 2005), more reliable methods based on their selective functional/metabolic activities such as ABCG2 activity and Aldehyde Deshydrogenase expression have been developed. Actually, HSC can be purified on basis of Hoechst 33342 die exclusion property due to the membrane ABCG2/BRCP1 transporter expression (SP cells). SP cells could be isolated from a wide variety of human and mammalian tissues and in many cases have been shown to contain multipotent stem cells; SP cells are heterogeneous since the lowest Hoechst fluorescent profile exhibited the highest primitive hierarchical level. Hematopoietic progenitors also expressed ALDH enzymatic activity and the use of BODIPY-AminoAcetAldehyde (BAAA), a fluorescent substrate of the ALDH enzyme, allows analyzing its expression by using Fluorescence Activated Cell Sorting (FACS). It has been reported that there is a close association between high ALDH expression (ALDHbr) and high HSC activity. Based on the hypothesis that the more primitive HSC should express both ALDH high expression level and SP profile, we developed a new multiparamatric flow cytometric assay combining an ALDH/SP co-labelling to identify different haematopoietic progenitor subpopulations. Our strategy allows identifying six lineage negative (Lin−) haematopoietic sub-populations from human bone marrow cells according to the co-expression of ALDH levels and SP phenotype: SP+ALDHBr, SP−ALDHBr, SP+ALDHMid, SP−ALDHMid, SP+ALDHLo and SP-ALDHLo. We showed that these different sub-populations exhibited specific antigenic patterns and demonstrated variable differentiation properties in long term cultures and clonogenic assays. Whatever the ALDH expression level, SP+ and SP−cells expressed the CD34 antigen, however, the SP phenotype identify a higher proportion of CD34+CD38− cells. Furthermore, in contrast to the other subpopulations, Lin-SP+ALDHBr cells were highly enriched with CD34+CD38−CD90+ progenitor cells and demonstrated a higher amplification and high clonogenic efficiency in liquid LTC-IC cultures than SP-ALDHBr cells. This subpopulation allowed obtaining all myeloid haematopoietic lineage differentiated cells after 30 days culture in liquid culture, including osteoclastic cells. Interestingly, in contrast to ALDHBr cells, ALDHLo sub-populations were highly enriched with megakaryocytic CD34+CD41+ progenitors that gave rise to CD41+CD42a+ cells. SP+ALDHMid and SP-ALDHMid have intermediate amplification, clonogenic and differentiation abilities as compared with the other subpopulations. In conclusion, our results show that the combined SP/ALDH technology allows discriminating a hierarchy within the human hematopoietic stem/progenitor compartment, the most primitive one being SP+ALDHBr. The stemness of these subpopulations is currently explored by testing their capability to reconstitute long term haematopoiesis in NOD/SCID mice.