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Figure 3. Setd2Δ/Δ mice had depletion of phenotypic and functional hematopoietic stem cells (HSCs). (A) Flow cytometry analysis of Setd2f/f and Setd2f/f/Vav1-Cre mice bone marrow (BM) cells. (B) Absolute number of HSC populations. (Setd2f/f =4, Setd2f/f/Vav1-Cre=5; mean±Standard Error of Mean (SEM)] (C). Experimental strategy: Setd2f/f or Setd2f/f/Mx1-Cre (pIpC injected) CD45.2 BM cells (1.5×106 cells each) was injected into irradiated (7.5+4.25Gy) B6-CD45.1 recipients, with B6-CD45.1 com- petitor BM (1.5×106 cells each). Peripheral blood (PB) was analyzed 2-16 weeks after competitive transplantation. Representative data were from 2 independent exper- iments. (N=8 each genotype; mean±SEM). (D) CD45.1/CD45.2 chimerism in BM LSKs, Gr1+CD11b+ myeloid cells, B220+ B cells, CD3+ T cells 16 weeks after transplan- tation. Representative data were from 2 independent experiments. (N=8 each genotype; mean±SEM). (E) Experimental strategy: Setd2f/f or Setd2f/f/Vav1-Cre CD45.2 BM cells (1.5×106 cells each) was injected into irradiated (7.5+4.25Gy) B6-CD45.1 recipients, with B6-CD45.1 competitor BM (1.5×106 cells each). PB was analyzed 4-16 weeks after competitive transplantation. Representative data were from 2 independent experiments. (N=8 each genotype; mean±SEM). (F and G) Experimental strategy: Setd2f/f or Setd2f/f/Mx1-Cre CD45.2 BM was injected into irradiated (7.5+4.25Gy) B6-CD45.1 recipients (2×106 cells per genotype), with B6-CD45.1 helper BM (1×105 cells). pIpC was injected two weeks after BMT. Peripheral blood were analyzed 0-10 weeks after pIpC injection. Representative data were from 2 independent experi- ments. (N=8 each genotype; mean±SEM). (H) Experimental strategy: Setd2f/f or Setd2f/f/Vav1-Cre CD45.2 BM was injected into irradiated (7.5+4.25Gy) B6-CD45.1 recip- ients (2×106 cells per genotype), with B6-CD45.1 helper BM (1×105 cells), survival conditions were monitored. (N=6 each genotype; mean±SEM).
Setd2 regulates hematopoietic stem cells
with increased frequencies of 3-, 2-, and 1-lineage clones (Figure 4E). These results indicated that Setd2Δ/Δ SLAM- HSCs lost normal clonogenicity and multi-lineage differ- entiation potential, which indicated that Setd2Δ/Δ SLAM- HSCs were in a more differentiated state.
AB
Setd2Δ/Δ HSPCs show a loss of stem cell identity and an increase in differentiation toward progenitors
There was a 4-fold reduction of SLAM-HSC numbers in Setd2Δ/Δ mice but a complete loss of HSC functions in the BMT assay. Thus, we consider it unlikely that the reduc-
haematologica | 2018; 103(7)
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