Slug Is Required for a Functional Hematopoietic Stem Cell (HSC) Niche

Abstract 2579

Slug belongs to the highly conserved Slug/Snail family of zinc-finger transcription factors found in diverse species ranging from C. elegans to humans. Our previous study has already demonstrated that Slug serves intrinsic role in the regulation of HSC fates (Blood, 2010, 115(9):1709-1717). There is bone defect in Slug-/- mice, while Slug is expressed in osteoblast cells which are niche cells for HSCs, suggesting that Slug might be also extrinsically required for HSC regulation. To address this issue, the following experiments and assays were performed. Firstly, the cell proliferation of wild-type (WT) hematopoietic stem/progenitor cells (HS/PCs) (loaded with carboxyfluorescein succinimidyl ester (CFSE)) placed on bone marrow (BM) stromal layers (prepared from either Slug-/- or WT mice) was analyzed. Co-culture proliferation assay illustrated a clear increasing in the ability of Slug-/-BM stroma to support normal proliferation of WT HS/PCs, suggesting a significant and qualitative change in Slug-/- BM stromal cells so that they were able to support normal proliferation by Slug-competent HS/PCs. Secondly, BM microenvironment altered by Slug deficiency was defined by RT-PCR. Slug-/- BM had altered cell cycle profiles associated with increased stromal Notch1, N-cadherin, Vcam and Angiopoietin-1 expression. These findings indicate that Slug may participate in signaling in BM niche cells and thus is capable of influencing their function. Thirdly, the transplantation analysis was performed to address whether Slug impact was stem cell stroma dependent. WT hematopoietic cells in Slug knockout environment (8 weeks after transplantation) displayed a dramatic increase in LSK and LSK-Flk-2 ratio and in total number in BM, but not in spleen, indicating a stroma-determined effect by Slug on HSCs. It also showed a mobilization phenotype similar to that originally observed in Slug+/+ environment. Moreover, the annexin V apoptosis assay and cell-cycle analysis by the Edu incorporation assay suggested that Slug deficiency microenvironment promoted HSC expansion largely by increasing their proliferation, but not cell survival. Fourthly, each cell lineage in BM and spleen after WT BM transplanted into Slug+/+ and Slug-/- recipients was fully analyzed. The total number and ratio of T cells (CD3e, CD4+CD8-, CD4-CD8+ and CD4+CD8+) was markedly decreased in BM, but not in spleen, while the other lineages (granulocytes CD11b+Gr-1+, macrophages CD11b+, immature B lymphoid IgM-B220+, mature B lymphoid IgM+B220+, mature Erythroid CD71-Ter119+, and immature erythroid CD71+Ter119+) had an equivalent number and ratio in BM and spleen. Overall, Slug-/- BM microenvironment results in T cell loss. Finally, the recent study shows BM adipocytes as negative regulators of haematopoietic microenvironment, while the in vivo and in vitro evidences indicate that Slug is a key regulator of the adipocyte differentiation. Thus, in this study, we try to address whether Slug as a key factor adjusts adipocytes in BM niche. The fat of the whole body and the femurs and tibias were measured by DXA (Dual-energy X-ray Absorptiometry). Our data showed Slug-/- mice at 8 wk of age had lowest total fat, as well as ROI (region of interest) fat compared to Slug+/+ mice. In vitro adipocyte differentiation assay indicated there was less adipocyte formation from Slug-/- MSCs from BM. Furthermore, a dramatic loss of adipocytes in the femur in Slug-/- recipient was observed in 8 weeks after transplanting WT hematopoietic cells into Slug+/+ and Slug-/- recipients. Combining the above-mentioned data from WT hemtopoisis in Slug-/- microenvironment showed a dramatic increase in LSK and LSK-Flk-2 ratio and total numbers, lack of BM adipocytes after irradiation in fatless mice (Slug-/- recipient), suggesting a role of Slug-/- BM microenvironment in enhancing haematopoietic progenitor expansion via inhibition of BM adipocytes and post-transplant recovery. Taken together, our previously and present findings demonstrate that Slug serves intrinsic and extrinsic roles in the regulation of HSC fates.