SIRT1 Is Required for Mouse Embroyonic Stem Cell Commitment to Hematopoietic Cell Differentiation.

Abstract 811

SIRT1 is a conserved NAD-dependent deacetylase that catalyzes deacetylation of acetyl-lysine residues of proteins such as histone and p53. SIRT1 plays an important role in a variety of biological processes including stress resistance, metabolism, differentiation and aging (Rodgers et al, Nature, 2005; 434:113). A role for SIRT1 in mouse (m) embryonic stem cell (ESC) maintenance is only beginning to be elucidated (Han et al, Cell Stem Cell, 2008; 2:241). Here we focus on a role for SIRT1 in differentiation of mESCs into hematopoietic cells. We hypothesized that SIRT1 is involved in mESC commitment to hematopoietic cell differentiation. We first confirmed that SIRT1 is highly expressed in the R1 mESC line. Hemoglobinized embryoid bodies (EBs) formed from SIRT1−/− R1 ESC were greatly decreased in number upon removal of LIF compared with that of wildtype parental (+/+) R1 cells, as assessed by primary differentiation assay. Differences in hemoglobinized cells were confirmed by gene analysis of βH1 globin (embryonic hemoglobin), markers for primitive erythroid cells. Next, the ability of SIRT1−/− ESCs to form primitive and definitive hematopoietic cells was evaluated and we found that primitive erythroid progenitors formed from SIRT1−/− R1 cells were greatly decreased. Moreover, after differentiation of SIRT1 −/− mESC there were also significant decreases in definitive erythroid (BFU-E), granulocyte-macrophage (CFU-GM), and multipotential (CFU-GEMM) progenitors. We next explored hematopoietic differentiation in EBs from SIRT1−/− cells by flow cytometry analysis of expression of surface antigens. CD41 defines the onset of primitive and definitive hematopoiesis in the murine embryo (Ferkowicz et al, Development, 2003; 130(18): 4393-403). There were much fewer CD41⁺ cells in SIRT1 d7 EBs compared with those in WT d7 EBs. To further investigate a role for SIRT1 in hematopoietic differentiation, SIRT1−/− ESCs were tested in an alternative in vitro hematopoietic system involving use of the OP9 stromal cell line and after ectopically-inducing HOXB4 to expand hematopoietic cell differentiation. Unlike WT cells, cells from day 6 EBs of SIRT1−/− ESCs did not differentiate into hematopoietic clusters, instead forming mesoderm-like colonies. This suggested that the defect in differentiation of SIRT1−/− ESCs into hematopoietic cells is before the onset of primitive erythropoiesis. Vascular endothelial growth factor (VEGF)-responsive blast cell colonies are known to contain both endothelial and hematopoietic precursors. This blast-colony-forming cell (BL-CFC) represents a transient population that is present in EBs between day 2.5 and day 3.5 of differentiation and represents the in vitro equivalent of the hemangioblast and as such, the earliest commitment step in the differentiation of mesoderm to the hematopoietic and endothelial lineages. We therefore assessed the ability of WT and SIRT1−/− cells to give rise to BL-CFC. When compared, SIRT1−/− cells generated significantly reduced number of blast colonies, while more colonies of tightly associated cells were observed than with WT cells in the BL-CFC clonogenic assay. Differentiation of mESC towards mesoderm and hemangioblasts (Blast-colony-forming) within the EBs was assessed by measuring brachyury and flk-1 expression respectively. There was no difference in expression of brachyury. However, flk-1 expression was remarkably reduced in SIRT1−/− EBs compared to +/+ EBs. These results indicated that SIRT1−/− cells differentiate properly into mesoderm, while they have a defect in differentiation into blast colonies. Reintroduction of WT SIRT1 into SIRT1−/− cells rescued the hemoglobinized EB formation of SIRT1−/− cells, suggesting that the defect of hematopoietic commitment is due to deletion of SIRT1, and not to genetic drifting of SIRT1−/− cells. Taken together, these results demonstrate that SIRT1 plays a role in hemangioblast development and the earliest stages of hematopoietic cell commitment.