Activation State of the Vascular Niche Regulates Homeostasis of the Normal and Malignant Hematopoietic Stem Cells.

Abstract 3624

Poster Board III-560

Hematopoietic stem cells (HSCs) reside in supportive microenvironment in bone marrow called niche. Growing evidence indicates that interaction between niche component cells and HSCs might be critical to balance the quiescence, expansion, and differentiation of HSCs. The osteogenic niche has been shown to be primarily important for the maintenance and modulation of the size of the HSCs mass. It has also been shown that vascular niche, which is composed of sinusoidal endothelial cells, plays a critical role in maintenance of HSCs and leukemic cells. Understanding the mechanism by which vascular niche controls fate and number of HSCs may be useful not only to identify as yet undetermined factors that control self-renewal or differentiation of HSCs but also to elucidate physiologically relevant molecular targets to treat malignant counterpart of HSCs. We have established a co-culture system of endothelial cells and hematopoietic stem cells mimicking the vascular niche by using primary endothelial cells (PECs) transduced with adenovirus gene E4ORF1, and succeeded in over 500-fold expansion of repopulating long-term (LT)-HSCs. In this study, we evaluated as to how activation state of endothelial cells changes the endothelial cell potential in supporting LT-HSCs or leukemic cell expansion.

Akt or/and p42/44 MAPK pathway activated endothelial cells were generated by transducing PECs with lenti viruses expressing E4ORF1, constitutive active Akt, constitutive active c-Raf, constitutive active K-ras or polyoma virus middle T antigen. Microarray and qPCR analysis showed there are clear differences between Akt-activated and MAPK-activated PECs in expression profile of genes involved in self-renewal or differentiation of HSCs such as jagged-1, CXCR4 and IGFBP-2. After 20 days of co-culture with mouse hematopoietic lineage negative (Lin-) cells, Akt-activated PECs supported over 200-fold increase of Lin- cells, whereas MAPK-activated PECs only supported less than 5-fold increase and Akt plus MAPK-activated PECs supported around 40-fold increase. On the contrary, MAPK-activated PECs supported hematopoietic lineage positive cells generation or leukemic cells expansion to the same extent as Akt-activated PECs. Conditioned medium of MAPK-activated, but not Akt-activated, PECs inhibited Lin- cell expansion on PECs without affecting lineage positive cell generation. We also confirmed Lin- cells cultured on Akt-activated PECs maintained more LT-HSCs activity than that cultured on MAPK-activated or Akt plus MAPK-activated PECs by competitive transplantation assays.

These results indicate that activation of Akt signaling in vascular niche can support HSCs self-renewal, whereas activation of p42/44 MAPK signaling can induce HSCs differentiation. Stable endothelial cells and angiogenic endothelial cells might differently regulate HSCs fate after injury such as myelosuppression in bone marrow. We are now trying to define growth factors that regulate HSC self-renewal and differentiation by using this co-culture system.