TC1(C8orf4) Regulates Hematopoietic Stem/Progenitor Cells and Hematopoiesis

<div><p>Hematopoiesis is a complex process requiring multiple regulators for hematopoietic stem/progenitor cells (HSPC) and differentiation to multi-lineage blood cells. TC1(C8orf4) is implicated in cancers, hematological malignancies and inflammatory activation. Here, we report that Tc1 regulates hematopoiesis in mice. Myeloid and lymphoid cells are increased markedly in peripheral blood of <i>Tc1</i>–deleted mice compared to wild type controls. Red blood cells are small-sized but increased in number. The bone marrow of <i>Tc1</i>^−/− mice is normocellular histologically. However, Lin⁻Sca-1⁺c-Kit⁺ (LSK) cells are expanded in <i>Tc1</i>^−/− mice compared to wild type controls. The expanded population mostly consists of CD150⁻CD48⁺ cells, suggesting the expansion of lineage-restricted hematopoietic progenitor cells. Colony forming units (CFU) are increased in <i>Tc1</i>^−/− mice bone marrow cells compared to controls. In wild type mice bone marrow, Tc1 is expressed in a limited population of HSPC but not in differentiated cells. Major myeloid transcriptional regulators such as Pu.1 and Cebpα are not up-regulated in <i>Tc1</i>^−/− mice bone marrow. Our findings indicate that TC1 is a novel hematopoietic regulator. The mechanisms of TC1-dependent HSPC regulation and lineage determination are unknown.</p></div

Quantitative PCR primers.

<p>Quantitative PCR primers.</p

Expansion of LSK cells in <i>Tc1^−/−</i> mouse bone marrow.

<p>Representative flow cytometric analysis for Lin⁻ bone marrow cells for Sca-1⁺c-Kit⁺ cells from <i>Tc1^−/−</i> mice and matched control mice. Profiles for CD150 and CD48 co-expression in the gated LSK cells (R1) are shown, respectively.</p

<i>Tc1</i>-targeting strategy.

<p>Restriction maps of wild type allele, targeting construct and targeted allele. The probe used for Southern blot analysis is indicated. The top line represents the structure and partial restriction map from wild-type allele of <i>Tc1</i>. The middle and low lines depict the targeting construct and predicted structure of targeted allele, respectively.</p

Enhanced hematopoiesis in <i>Tc1</i>^−/− mice.

<p>(A) Numbers of white blood cells (WBC), lymphocytes (Lym) and neutrophils (Neut). Data for A, B, C, and F represent mean ± s.d. of 14 male, 7 to 9 week-old <i>Tc1</i>^−/− mice and, 14 sex- and age-matched control mice over 5 independent experiments. (B) The percentage of lymphocytes and neutrophils per total WBC. (C) Numbers of monocytes (Mon) and eosinophils (Eos). (D) Representative flow cytometric analysis of peripheral blood mononuclear cells (PBMC) for Cd11b and F4/80 expression in wild type and <i>Tc1</i>^−/− mice. (E) Left: qPCR analysis of Trem-1 expression in PBMC. Data represent mean ± s.d. of 6 male, 8 week-old <i>Tc1</i>^−/− mice, and 6 sex-, and age-matched control mice over 3 independent experiments. Right: qPCR analysis of TREM-1 expression in <i>TC1</i>-transfected HL-60 cells and vector-transfected control. Data of expression fold difference represent mean ± s.d. of 3 independent experiments. (F) The RBC number, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), hematocrit (HCT), and red cell distribution width (RDW) in peripheral blood from control and <i>Tc1</i>-deleted mice. <i>*p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001.</p

Hematopoietic lineage- and pluripotent stem cell-regulators.

<p>(A) qPCR analysis for Pu.1 expression in total Lin⁻ cells (left) or sorted Lin⁻Sca-1⁺ cell fraction (right) of <i>Tc1^−/−</i> and wild type mice bone marrow. Data represent mean ± s.d. of 6 male, 8 week-old <i>Tc1</i>^−/− mice, and 6 sex- and age-matched control mice over 3 independent experiments for the total Lin⁻ cell assay, and 10 male, 8 to 9 week-old <i>Tc1</i>^−/− mice, and 10 sex- and age-matched control mice over 2 independent experiments for the sorted-Lin⁻Sca-1⁺ fraction assay, respectively. (B–E) qPCR analysis for Cebpα (B), Gata-1 (C), c-Myc and Ccnd1 (D), and Klf4 (E) expression in Lin⁻ cells. Data represent mean ± s.d. of 6 male, 9 week-old <i>Tc1</i>^−/− mice, and 6 sex- and age-matched control mice over 3 independent experiments. **<i>p</i><0.01; ***<i>p</i><0.001.</p

<i>In vitro</i> colony-forming unit (CFU) assay.

<p>Data represent mean ± s.d. of colony numbers per plated cells as indicated of 8 male, 8 week-old <i>Tc1</i>^−/− mice, and 8 sex- and age-matched control mice over 3 independent experiments. (A) CFU-granulocyte-macrophage (CFU-GM). (B) CFU-granulocyte-erythroid-monocyte-megakaryocyte (CFU-GEMM). (C) CFU-erythroid (CFU-E). (D) burst-forming unit-erythroid (BFU-E). ***<i>P</i><0.001. (E) Representative flow cytometric analysis of recovered cells from the CFU plates of <i>Tc1^−/−</i> mice and matched control mice.</p