Tet2 Uniquely Regulates Self-Renewal and Long Term Repopulating Capacity of Fetal Liver and Bone Marrow Hematopoietic Stem Cells

Abstract 4739

Ten-eleven-translocation 2 (TET2) gene is one of the frequent targets of mutation in various hematologic malignancies. These observations suggest critical roles of TET2 dysfunction in their molecular pathogenesis. To investigate physiological roles of TET2 in hematopoiesis, we previously analyzed fetal liver (FL) hematopoiesis of Tet2 gene-trap (Tet2^gt) mice and showed that Tet2^gt/gt FL cells displayed enhanced self-renewal and long term repopulating (LTR) capacity with expansion of Lineage(−)Sca-1(+)c-Kit(+) (LSK) and common myeloid progenitor (CMP) fractions. However, there remain several questions unanswered. First, self-renewal capacity was examined only by using bulk FL cells and therefore effects of Tet2 loss on purified cell populations such as hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) remain elusive. Second, because other groups have reported myeloid transformation in Tet2 conditional knockout mice, it is possible that Tet2 loss confers self-renewal capacity to non-self-renewing myeloid progenitors such as CMPs. Third, effects of Tet2 haploinsufficiency on adult hematopoiesis was not examined using purified HSCs or HPCs.

To address these issues, we analyzed E14.5 FL and adult bone marrow (BM) cells from Tet2^gt mice. We first performed serial replating assay of FL-LSK cells in methylcellulose containing interleukin (IL)-3, IL-6, stem cell factor (SCF) and erythropoietin (Epo). In this assay, Tet2^gt/gt FL-LSK cells showed significantly higher replating capacity as compared to that of WT cells. Interestingly, Tet2^gt/gt FL-LSK cells formed various types of colonies including granulocyte-macrophage (GM) and erythrocyte-megakaryocyte (EM) colonies, whereas WT FL-LSK cells generated only GM colonies at the second time of replating, showing that multipotent differentiation capacity was maintained in Tet2^gt/gt cells even in the presence of lineage-acting cytokines.

Next we examined the self-renewal capacity of highly purified FL-HSCs (CD34⁺LSK or CD150⁺LSK cells) by competitive repopulation assay. As expected, the recipients of Tet2^gt/gt CD34⁺LSK cells showed significantly higher donor chimerism in peripheral blood as compared to those receiving WT cells. Furthermore, CD150⁺LSK cells from Tet2^+/gt and Tet2^gt/gt FLs demonstrated higher peripheral blood repopulation in the secondary and tertiary recipient mice as compared to that of WT recipients in serial transplantation assay. These results indicate that the enhanced self-renewal and LTR capacity of Tet2-mutant FL cells was uniquely associated with highly purified HSCs. This conclusion also applied to the BM LSK cells from adult mice, since Tet2^+/gt BM LSK cells also showed significantly higher peripheral blood contribution compared to the WT cells in serial transplantation assays. This result demonstrates that Tet2 haploinsufficiency is sufficient to confer the enhanced self-renewal and LTR capacity to HSCs in adult hematopoiesis.

Lastly, we examined self-renewal capacity of FL CMPs by serial replating assay. Interestingly, Tet2^gt/gt FL CMP cells displayed increased replating capacity as compared to WT cells. However, in vivo repopulation assay using Tet2^+/+, Tet2^+/gt, and Tet2^gt/gt FL CMP cells showed no significant difference in peripheral blood chimerism among these recipients.

Taken together, enhanced self-renewal and LTR capacity by Tet2 ablation is uniquely associated with HSCs in FL and adult BM, but not with myeloid progenitors, indicating that Tet2 regulates self-renewal program intrinsic to HSCs. In addition, Tet2 haploinsufficiency is sufficient to enhance self-renewal and LTR capacity of HSCs, which explains pathological relation between high incidence of heterozygous TET2 mutations and hematologic malignancies.