Microenvironmental Cross-Talk of Wnt/Notch Signal in Hematopoietic Stem Cell

Microenvironment in the stem cell niche plays an important role to regulate self-renewal and differentiation of hematopoietic stem cells (HSCs). We previously showed opposing effects of b-catenin activation on HSC depending on target of activation in the bone marrow microenvironment. To further analyze the microenvironmental regulation of HSC by Wnt/b-catenin signal, we examined b-catenin activation mode in the trabecular bone marrows. In-situ immunohistochemistry of bone marrows revealed a compartmentalized enrichment of b-catenin in the spindle shaped, CD45(−) endosteal stroma of bone marrow (SNO cells) compared to CD45(+) hematopoietic cells. Receptors for canonical Wnt signals (Fz1, 2, 7, 8) or co-receptors (LRP5, 6) were also enriched in the CD45(−) mesenchymal stromal cells of bone marrows than hemapoietic cells. Moreover, accumulation of active form b-catenin was selectively observed in the “stimulated” bone marrows that had been irradiated or injected with Wnt 3a conditioned medium (Wnt 3a-CM), but not in the “steady state” bone marrows. To examine the effect of b-catenin activated stroma on HSCs, 5-FU bone marrow cells were co-cultured in-vitro for 5 days and transplanted into irradiated mice. A 3-fold higher expansion of primitive phenotype (Lin-Sca-1+c-kit+) cells were seen after culture without differences in cell cycle progression. Further, CRU analysis of the transplanted co-cultured cells displayed higher numbers of CRUs regenerated in the recipient bone marrows (65 CRU vs. 1155 CRUs for MIG vs. b-catenin MSC group, respectively). To directly test the effect of b-catenin activated stroma on HSC during normal reconstitution process, we compared HSC self-renewal in the marrows reconstituted with control or b-catenin activated MSCs; MIG or b-catenin transduced MSCs were directly injected into femur with bone marrow cells and each group mice marrows were then mixed (1:1) transplanted into secondary recipient mice for competitive CRU assay. A 3-fold higher CRU frequency was seen for the HSCs derived from marrows reconstituted with b-catenin/MSCs, indicating the physiological significance of b-catenin activation for in-vivo reconstitution. We next investigated the underlying mechanism for stromal b-catenin effects on HSCs. Expression analysis of b-catenin transduced or Wnt3a-stimulated MSCs revealed higher levels of notch ligands (jagged-1, dll-1), which was similarly observed in the trabecular endosteum of mice treated with Wnt3a-CM. A microarray-based expression analysis further supported up-regulation of notch ligands in b-catenin transduced MSCs, as evidenced by induction of dlk-1 and microfibrillar glycoprotein-2, a protein facilitating utilization of jagged-1. Importantly, induction of notch down-stream molecules (Hes-1 and Deltex-1) was demonstrated in the hematopoietic cells (Lin-Sca-1+) cells co-cultured on the b-catenin activated MSCs. Furthermore, enhancing effects of b-catenin/MSC for expanding undifferentiated cells were abrogated by treatment of gamma-secretase 2 inhibitors during the co-culture. These results show that b-catenin activated stromal cells activate notch signal in the contacting HSCs and that activated notch signal underlies the observed stimulatory effects of b-catenin activated stroma on HSCs. Taken together, Wnt/β-catenin activated stroma and the cross-talk with HSCs may function as a physiologically regulated microenvironmental cue for HSC self-renewal in the stem cell niche.