Splitomicin, a SIRT1 Inhibitor, Enhances Hematopoietic Differentiation of Mouse Embryonic Stem Cells

Abstract

Background and objectives: Embryonic stem (ES) cells have pluripotent ability to differentiate into multiple tissue lineages. SIRT1 is a class III histone deacetylase which modulates chromatin remodeling, gene silencing, cell survival, metabolism, and development. In this study, we examined the effects of SIRT1 inhibitors on the hematopoietic differentiation of mouse ES cells.

Methods and results: Treatment with the SIRT1 inhibitors, nicotinamide and splitomicin, during the hematopoietic differentiation of ES cells enhanced the production of hematopoietic progenitors and slightly up-regulated erythroid and myeloid specific gene expression. Furthermore, treatment with splitomicin increased the percentage of erythroid and myeloid lineage cells.

Conclusions: Application of the SIRT1 inhibitor splitomicin during ES cell differentiation to hematopoietic cells enhanced the yield of specific hematopoietic lineage cells from ES cells. This result suggests that SIRT1 is involved in the regulation of hematopoietic differentiation of specific lineages and that the modulation of the SIRT1 activity can be a strategy to enhance the efficiency of hematopoietic differentiation.

Keywords: Hematopoietic differentiation; Mouse embryonic stem cells; Sirt1; Sirt1 inhibitor; Splitomicin.

Fig. 1

Schematic representation of the culture system used for hematopoietic cell differentiation from mouse ES cells. For hematopoietic EB formation, ES cells were differentiated with the methylcellulose medium with SCF for 10 days. For secondary differentiation, EBs were harvested and disrupted into single cells and replated with cytokines (SCF, IL-3, IL-6, and EPO) in the presence or absence of SIRT1 inhibitors. Counting of the colony numbers, RT-PCR and FACS analyses were performed at the indicated time points.

Fig. 2

SIRT1 inhibitors enhance hematopoietic colony forming ability during hematopoietic differentiation of mES cells. (A) Colony-forming potential of mES-derived cells in the presence of nicotinamide 0.5 mM (NAM) and splitomicin 60 μM (SP). ES cells were differentiated by EB formation for 10 days. EBs were disrupted into single cells and re-plated in 1% methylcellulose medium containing a cocktail of hematopoietic cytokines. Phase-contrast images were taken from representative BFU-E, CFU-E, CFU-GM and CFU-GEMM colonies after 7 days. Scale bar. 100 μm. (B) After the colony-forming assays, the numbers of BFU, CFU-E, CFU-GM, CFU-GEMM, and 2^nd EB were scored by microscopy and compared. Each bar is expressed as the mean±standard error of the mean. *p<0.05, **p<0.01, ***p<0.001 (vs. control) of n=9 independent experiments. (C) The results are shown in percentage terms. Percentages represent the proportion of each colony type.

Fig. 3

Effects of SIRT1 inhibitors on the expression of mesodermal and hematopoietic marker genes during the hematopoietic differentiation of mES cells. (A) RNA samples were extracted on days 7, 14, and 21 during second hematopoietic differentiation, and gene expression was analyzed by RT-PCR. GAPDH is shown as a control. ES: mES cells, EB 10D: embryoid bodies obtained on day 10 of primary differentiation. Mesodermal marker, brachyury; hematopoietic stem cell and progenitor cell markers, Scl, Flk1, and HoxB4; hematopoietic lineage cell markers, GATA1 and GATA2; erythroid specific markers, βH-1 and β-major; early myeloid and lymphoid progenitor marker, Pu.1. The density of each band was measured using the ImageJ program and normalized with the density of GAPDH used as a loading control for each sample. The relative band density values were compared with the controls on day 7 (shown as fold expression). (B) RNA samples were extracted on days 7 and 14 during second hematopoietic differentiation, and gene expression was analyzed by RT-PCR. IL-7R, Rag2, and GATA3 were used as lymphoid lineage markers.

Fig. 4

Effects of SIRT1 inhibitors on the expression of hematopoietic surface antigens during hematopoietic differentiation. Expression of surface antigens was analyzed by flow cytometry. (A) Expression of CD41 and CD117 (hematopoietic progenitors), CD45 (hematopoietic), TER-119 (erythroid), CD11b (monocyte/macrophage) and Gr-1 (granulocyte) markers was monitored on day 7 of secondary hematopoietic differentiation. (B) Expression of TER-119, CD11b and Gr-1 markers was monitored on day 14 of secondary hematopoietic differentiation. EB 10D, embryoid bodies obtained on day 10 of primary differentiation. Each bar is expressed as the mean±standard error of the mean of three experiments. *p<0.05, **p<0.01 (vs. control).