SIRT1 deficiency compromises mouse embryonic stem cell hematopoietic differentiation, and embryonic and adult hematopoiesis in the mouse

Abstract

SIRT1 is a founding member of a sirtuin family of 7 proteins and histone deacetylases. It is involved in cellular resistance to stress, metabolism, differentiation, aging, and tumor suppression. SIRT1(-/-) mice demonstrate embryonic and postnatal development defects. We examined hematopoietic and endothelial cell differentiation of SIRT1(-/-) mouse embryonic stem cells (ESCs) in vitro, and hematopoietic progenitors in SIRT1(+/+)(+/-), and (-/-) mice. SIRT1(-/-) ESCs formed fewer mature blast cell colonies. Replated SIRT1(-/-) blast colony-forming cells demonstrated defective hematopoietic potential. Endothelial cell production was unaltered, but there were defects in formation of a primitive vascular network from SIRT1(-/-)-derived embryoid bodies. Development of primitive and definitive progenitors derived from SIRT1(-/-) ESCs were also delayed and/or defective. Differentiation delay/defects were associated with delayed capacity to switch off Oct4, Nanog and Fgf5 expression, decreased β-H1 globin, β-major globin, and Scl gene expression, and reduced activation of Erk1/2. Ectopic expression of SIRT1 rescued SIRT1(-/-) ESC differentiation deficiencies. SIRT1(-/-) yolk sacs manifested fewer primitive erythroid precursors. SIRT1(-/-) and SIRT1(+/-) adult marrow had decreased numbers and cycling of hematopoietic progenitors, effects more apparent at 5%, than at 20%, oxygen tension, and these progenitors survived less well in vitro under conditions of delayed growth factor addition. This suggests a role for SIRT1 in ESC differentiation and mouse hematopoiesis.

Figure 1

Blast cell colony development from WT (SIRT1^+/+) and SIRT1-deficient (SIRT1^−/−) ESCs. (A) Kinetics of blast and transitional colony development in SIRT1^+/+ and SIRT1^−/− EBs. Data are shown as mean ± SD. N = 3; *P < .01. Days (D) of differentiation are indicated. Δ = no colony grown. (B) fluorescence-activated cell sorting analysis of Flk1 and c-Kit expression on SIRT1^+/+ and SIRT1^−/− EB-derived cells. Days of differentiation are indicated. Numbers in each quadrant represent the percent of total population in each fraction. (C) Secondary EB colonies generated by SIRT1^+/+ and SIRT1^−/− EB cells. Days of differentiation are indicated. Data are shown as mean ± SD. N = 3; *P < .01. (D) Immunostaining and Dil-Ac-LDL uptake of adhesive cells generated from a single blast colony from SIRT1^+/+ and SIRT1^−/− EB-derived cells. VE-cadherin expression is indicated by green fluorescence and LDL uptake by red fluorescence. (E) Evaluation of the hematopoietic and endothelial potentials of BL-CFCs. The number of colonies that yielded secondary CFCs or adherent endothelial cells is divided by the total number of replated colonies. Bars represent SEM number from at least 3 experiments; *P < .01.

Figure 2

Kinetic analysis of primitive and definitive hematopoietic colonies generated from WT (SIRT1^+/+) and knockout (KO; SIRT1^−/−) EB cells under normoxia (20% O₂) and 5%O₂. Primitive erythroid colonies generated from SIRT1^+/+ and SIRT1^−/− EB-derived cells under (A) normoxia and (B) lower (5%) O₂ tension. Definitive GEMM, multilineage progenitors of granulocytes, erythroid cells, macrophages, megakaryocytes; GM, multilineage progenitors of granulocytes and macrophages; and secondary EBs generated from SIRT1^+/+ and SIRT1^−/− EB-derived cells under normoxia condition and lower (5%) O₂ tension at day 6 (C) and day 8 (D). Data are shown as mean ± SD. N = 3; *P < .05. Relative mRNA expression of embryonic globin (E) and adult globin (F) genes from SIRT1^+/+ and SIRT1^−/− day 0, 2, 4, 6, 8, and 10 EBs by real-time RT-PCR. Error bars indicate SDs from the average of 3 independent experiments, each performed in triplicate; ***P < .001; **P < .01; *P < .05.

Figure 3

Effect of SIRT1 deletion on in vitro vascular sprout formation in differentiating EBs. (A) Representative micrographs of 3-dimensional in vitro angiogenesis assays with collagen gel-embedded spheroids generated from WT (SIRT1^+/+) or KO (SIRT1^−/−) day 6 EBs. (B) Analysis of the percentage of angiogenic EBs was performed after 8 days of secondary culture in collagen gel. (C) Representative micrographs of in vitro matrigel assays with SIRT1^+/+ or SIRT1^−/− day 6 EBs. (D) Percentage of each class of vascular sprouting (see “Vascular sprout formation assay”). Data are shown as mean ± SEM. N = 3; *P = .01-.04. Image acquisition details: Nikon Diaphot microscope, 10×/0.25 numeric aperature objective lens, Nikon F3 camera, NIS-Elements D2.30 software.

Figure 4

Gene expression analysis of WT (SIRT1^+/+) and KO (SIRT1^−/−) EBs by real-time PCR analysis. (A) Quantitative RT-PCR (qRT-PCR) analysis of stem cell marker Oct4 and Nanog mRNA levels in day 0-10 SIRT1^+/+ and SIRT1^−/− EBs. (B) qRT-PCR analysis of mRNA levels of epiblast marker Fgf5, mesoderm markers T, Wnt3, and transcription factor Tal1/Scl in day 0-10 SIRT1^+/+ and SIRT1^−/− EBs. (C) qRT-PCR analysis of mRNA levels of trophectoderm marker Cdx2, neuroectoderm marker Sox1, and cardiac lineage marker Nkx2-5 in day 0-10 SIRT1^+/+ and SIRT1^−/− EBs. (D) qRT-PCR analysis of mRNA levels of endoderm/mesoderm markers GATA-4, GATA6, and Sox17 in day 0-10 SIRT1^+/+ and SIRT1^−/− EBs. Day (D) 0 represents undifferentiated ESCs. Graphs in panels A through D were plotted in logarithmic scale. Data are relative to SIRT1^+/+ D0 control and mRNA levels of each gene were compared between SIRT1^+/+ and SIRT1^−/− EBs at each time point. Results were the average of 3 independent experiments, each performed in triplicate. *P < .05.

Figure 5

Western Blot analysis of Erk signaling pathways and qRT-PCR analysis of gene expression in day 0-10 SIRT1^+/+ and SIRT1^−/− EBs. (A) Representative blot and (B) quantification showing phosphorylation of Erk and Erk1/2 protein expression at different time points during EB development. Data are shown as mean ± SEM. N = 3; *P < .05. (C) qRT-PCR analysis of mRNA levels of genes (BMPr2, Smad8, Smad3, Smad7) involved in BMP pathways in day 0-10 SIRT1^+/+ and SIRT1^−/− EBs. D0 represents undifferentiated ESCs. Graphs in panel C were plotted in logarithmic scale. Data are relative to SIRT1^+/+ D0 control and mRNA levels of each gene were compared between SIRT1^+/+ and SIRT1^−/− EBs at each time point. Results were of 3 independent experiments performed in triplicate. *P < .05.

Figure 6

Ectopic expression of exogenous SIRT1 in SIRT1^−/− ESCs. (A) Diagram of pLenti6-SIRT1-Venus lentiviral vector. (B) Transfected SIRT1^−/− ESCs were sorted, and expression of SIRT1 protein was determined by Western blotting. (C) Number of primitive erythroid colonies generated by day 4.5 and day 5 SIRT1^+/+, SIRT1^−/− ESCs. (D-E) qRT-PCR analysis of mRNA levels of embryonic globin (Hbb-bh1) and adult globin (Hbb-b1) in day (D) 0-8 SIRT1^+/+, SIRT1^−/− plus null and SIRT1^−/− +SIRT1 venus EBs. Data represent fold changes in EBs, relative to SIRT1^+/+ D0 control and comparison between SIRT1^+/+, SIRT1^−/− plus null, and SIRT1^−/− plus SIRT1 EBs at each time point. Results are average of 3 independent experiments. Each data point denotes 6 biological replicates; *P < .05; **P < .01; ***P < .001.

Figure 7

Hematopoietic progenitor cell colony forming assay from yolk sac and bone marrow of SIRT1^+/+, SIRT1^+/−, and SIRT1^−/− mice. (A) Primitive erythroid colony formation from yolk sac (YS) cells generated from E8 to E8.25 SIRT1^+/+, SIRT^+/−, and SIRT1^−/− embryos (SIRT1^+/+, n = 11; heterozygous, n = 10; SIRT1^−/−, n = 4) were grown in methylcellulose-based medium. Data are shown as mean ± SD; *P < .01. (B) PCR shows the genotyping of SIRT1 mutant embryos. The primers are: forward-5′-TCCTTGCCACAGTCACTCAC-3′, reverse for wild-type 5′-CATCTAAACTTTGTTGGCTGC-3′, reverse for deletion 5′-ACAGTCCCATTCCCATACC-3′. (C) Hematopoietic progenitor cell colony formation from bone marrow of 5-week-old SIRT1 heterozygote (SIRT1^+/−) or wild-type controls (SIRT1^+/+) under normoxia (20% O₂) and 5% O₂ conditions. Data are shown as mean ± SD; *P < .01; ND = SIRT1^−/− cells were not available for use and were not done. (D) Hematopoietic progenitor cell colony formation from bone marrow of 12-month-old SIRT1 heterozygote (SIRT1^+/−) or homozygote (SIRT1^−/−) null mice or from wild-type controls (SIRT1^+/+) under normoxia (20% O₂) or 5% O₂ conditions. (E) Cycling status of mouse bone marrow progenitor cells generated from 12-month-old SIRT1^+/+, SIRT1^+/−, and SIRT1^−/− mice under normoxia (20% O₂) and 5% O₂ conditions. This shows percent progenitors in DNA synthesis (S-phase) as determined by high specific activity tritiated thymidine kill technique. P values were calculated with Student t test. ND = not done. Data are the average of 3-5 mice/group. (F) Survival of hematopoietic progenitor cells in vitro after 1 day delayed addition of cytokines as denoted by decreased colony formation. Results are expressed as mean ± ISEM for 4-5 mice per group.