Hematopoietic stem cells (HSCs) are thought to divide infrequently based on their resistance to cytotoxic injury targeted at rapidly cycling cells1, 2 and have been presumed to retain labels such as the nucleotide analogue 5-bromodeoxyuridine (BrdU). However, recently it has been demonstrated that BrdU-retention is neither sensitive nor specific for HSCs3. Here we show that transient, transgenic expression of a Histone2B (H2B)-Green Fluorescent Protein (GFP) fusion protein in mice allows superior labeling of HSCs and permits improved analysis of their turnover in combination with other markers. Mathematical modeling of H2B-GFP dilution in HSCs, identified with a highly stringent marker combination (L−K+S+CD48−CD150+)4, revealed unexpected heterogeneity in their proliferation rates and suggests that ~ 20% of HSCs turn over at an extremely low rate (≤ 0.8–1.8% per day). Prospective isolation and transplantation of L−K+S+CD48−CD150+ HSCs with different H2B-GFP levels revealed that higher H2B-GFP label retention correlates with superior long-term repopulation potential.

The reprogramming of somatic cells into induced pluripotent stem (iPS) cells upon overexpression of the transcription factors Oct4, Sox2, Klf4 and cMyc is extremely inefficient. It has been assumed that the somatic differentiation state provides a barrier for efficient reprogramming; however, direct evidence for this notion is lacking. Here, we have tested the susceptibilities of hematopoietic cells at different stages of differentiation to be reprogrammed into iPS cells. Surprisingly, hematopoietic stem and progenitor cells give rise to iPS cells up to 300 times more efficiently compared with terminally differentiated B and T cells, yielding reprogramming efficiencies of up to 28%. Our data provide evidence that the differentiation stage of the starting cell has a critical influence on the efficiency of reprogramming into iPS cells. Moreover, we identify adult hematopoietic progenitors as an attractive cell type for applications of iPS technology in research and therapy.

The pathophysiology of microthrombocytopenia in the Wiskott-Aldrich syndrome (WAS) and its milder form, X-linked thrombocytopenia (XLT), is unclear. Although quantitative defects are correctable by splenectomy, residual platelet abnormalities are suggestive of intrinsic disturbances of production. In contrast to human patients, murine models of WASp deficiency exhibit only mild thrombocytopenia, and platelets are of normal size. Here, we have identified a critical role for WASp during murine platelet biogenesis.

The physiologic role of CXCR4 on hematopoietic stem/progenitor cells (HSPCs) is not fully understood. Here, we show that radioprotection of lethally irradiated mice by embryonic day 14.5 (E14.5) CXCR4 ؊/؊ fetal liver (FL) cells was markedly impaired when compared with CXCR4 ؉/؉ counterparts, but this defect was rescued when hosts were engrafted with high cell numbers. This quantitative defect contrasted with a similar content in hematopoietic colony-forming cells (CFCs), splenic colony-forming units (CFUs-S), and Lin ؊ Sca-1 ؉ c-kit ؉ cells in E14.5 CXCR4 ؊/؊ and CXCR4 ؉/؉ livers. In addition, the homing of HSPCs in the bone marrow was not altered as detected with a CFSE-staining assay. In contrast, a 30-fold increase in CFCs was seen in the circulation of mice stably reconstituted with CXCR4 ؊/؊ FL cells and this increment was already observed before hematopoiesis had reached a steady-state level. Together, the data strongly suggest that impaired retention may, at least in shortterm hematopoietic reconstitution, lead to a diminution in the number of available progenitors required for radioprotection. IntroductionThe CXC chemokine ligand 12 (CXCL12)/stromal cell-derived factor-1 (SDF-1)/pre-B cell growth-stimulating factor (PBSF) 1,2 mediates the homing of hematopoietic stem/progenitor cells (HSPCs) to the bone marrow (BM) by binding to its receptor CXCR4. 3 SDF-1/CXCR4 interactions play pleiotropic roles during development, as revealed by the phenotype of mice with a homozygous targeted gene disruption that die from defects in multiples organs. [4][5][6][7] In fetal hematopoiesis, CXCR4 Ϫ/Ϫ or SDF-1 Ϫ/Ϫ embryos display more profound defects in the marrow than in the fetal liver (FL), suggesting a major role for the CXCR4/SDF-1 axis in marrow colonization. [4][5][6][7] In adult hematopoiesis, CXCR4 receptors are involved in numerous biologic processes, including cell migration, 8 proliferation, and survival. [9][10][11][12][13][14] Several data suggest that SDF-1 and CXCR4 play a critical role in the homing of HSPCs with SDF-1 acting as a major chemoattractant for severe combined immunodeficiency (SCID)-repopulating cells and leukemic cells. 8,15,16 Blocking CXCR4 function on CD34 ϩ cells with anti-CXCR4 antibodies prevented hematopoietic reconstitution in nonobese diabetic (NOD)/SCID mice, 17 whereas CXCR4 overexpression increased their abilities to engraft NOD/SCID mice. 9 In mice, it has been reported that CXCR4 Ϫ/Ϫ FL cells can successfully reconstitute hematopoiesis of lethally irradiated mice, 18-20 whereas secondary transplantation experiments showed defective reconstitution. 20 A critical role for CXCR4 in mobilization, the egress of HSPCs from BM to the peripheral blood, was recently suggested. For instance, a rapid mobilization of HSPCs was obtained by treatment with the CXCR4 antagonist AMD3100, 21,22 as well as by induction of SDF-1 plasma elevation. 23,24 In addition, G-CSF-induced HSPC mobilization coincides in vivo with a decrease in BM SDF-1 concentration related to SDF-1 cleavage by marrow seri...

The initial steps in the pathogenesis of acute leukemia remain incompletely understood. The TEL-AML1 gene fusion, the hallmark translocation in Childhood Acute Lymphoblastic Leukemia and the first hit, occurs years before the clinical disease, most often in utero. We have generated mice in which TEL-AML1 expression is driven from the endogenous promoter and can be targeted to specific populations. TEL-AML1 renders mice prone to malignancy after chemical mutagenesis when expressed in hematopoietic stem cells (HSCs), but not in early lymphoid progenitors. We reveal that TEL-AML1 markedly increases the number of HSCs and predominantly maintains them in the quiescent (G(0)) stage of the cell cycle. TEL-AML1(+) HSCs retain self-renewal properties and contribute to hematopoiesis, but fail to out-compete normal HSCs. Our work shows that stem cells are susceptible to subversion by weak oncogenes that can subtly alter their molecular program to provide a latent reservoir for the accumulation of further mutations.

Regulators of G-protein signaling (RGS) constitute a family of proteins involved in the negative regulation of signaling through heterotrimeric G protein-coupled receptors (GPCRs). Several RGS proteins have been implicated in the down-regulation of chemokine signaling in hematopoietic cells. The chemokine stromal-cell-derived factor 1 (SDF-1) activates migration of hematopoietic progenitors cells but fails to activate mature megakaryocytes despite high levels of CXC chemokine receptor 4 (CXCR4) receptor expression in these cells. This prompted us to analyze RGS expression and function during megakaryocyte differentiation. We found that RGS16 and RGS18 mRNA expression was up-regulated during this process. Overexpressing RGS16 mRNA in the megakaryocytic MO7e cell line inhibited SDF-1-induced migration, mitogenactivated protein kinase (MAPK) and protein kinase B (AKT) activation, whereas RGS18 overexpression had no effect on CXCR4 signaling. Knocking down RGS16 mRNA via lentiviral-mediated RNA interference increased CXCR4 signaling in MO7e cells and in primary megakaryocytes. Thus, our data reveal that RGS16 is a negative regulator of CXCR4 signaling in megakaryocytes. We postulate that RGS16 regulation is a mechanism that controls megakaryocyte maturation by regulating signals from the microenvironment. ( IntroductionChemokines and their receptor(s) are broadly expressed in different tissues and regulate cell migration as well as several other important biologic processes. 1 The chemokine stromal-cell-derived factor 1 (SDF-1) is a stromal-cell-derived factor that interacts with a specific receptor CXC chemokine receptor 4 (CXCR4) and plays a role in B lymphopoiesis and bone marrow myelopoiesis. Studies using mutant mice with targeted gene disruption have revealed that SDF-1 and CXCR4 are essential for B-cell differentiation, for colonization of bone marrow by hematopoietic stem cells (HSCs) and myeloid lineage during ontogeny as well as for blood-vessel formation in gastrointestinal tract, cardiac ventricular septum formation, and cerebellar differentiation. 2,3 SDF-1CXCR4 signaling appears to be essential for the homing of hematopoietic stem/progenitor cells because treatment of immature human hematopoietic progenitor cells with anti-CXCR4 antibodies prevents their short-term engraftment into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. [4][5][6] In addition, transplantation of CXCR4 Ϫ/Ϫ fetal liver cells results in low numbers of B-lymphoid and myeloid lineage precursors in bone marrow but increased numbers in the peripheral blood compared with control animals. 7 Proteolytic cleavage of the N-terminus of CXCR4 and of SDF-1 is one mechanism that has been identified for the regulation of CXCR4/SDF1 signaling in circulating and mobilized blood cells. 8 The process of megakaryopoiesis occurs within a complex bone marrow microenvironment where chemokines, cytokines, and adhesive interactions play a major role. At the end of their maturation, polyploid megakaryocytes (MKs) migrate through bone m...

Graph shows the mean ± SEM. n = 3. *P < 0.05. (G) Immunofluorescent staining of aortas from E10.5 embryos for KDM2B (red; nuclear) and c-Kit (green; cell surface). White arrow shows budding hemogenic endothelium and yellow arrow a circulating "stem-like" cell. Nuclei were stained with DAPI (blue). Scale bars: 10 μm.

IntroductionHematopoiesis is maintained by a pool of hematopoietic stem cells (HSCs) defined by their capacity to self-renew and, hence, to maintain the pool and to differentiate into all mature progenies. These properties underlie their ability to reconstitute the hematopoietic system. 1 HSCs are identified by molecular markers 2 and are divided into long-term HSCs (LT-HSCs), short-term HSCs (STHSCs), and multilineage progenitors (MPPs). 3,4 As they differentiate, HSCs give rise to committed progenitors, such as common lymphoid progenitors (CLPs) or common myeloid progenitors (CMPs), which generate mature cells within the different hematopoietic lineages. 5 The action of HSCs to self-renew or to differentiate is a tightly controlled process linked to the induction or repression of some crucial genes. 6 Gene-targeting experiments have provided support to this hypothesis by identifying transcription factors of various families or their coregulators that are implicated in HSC biology. 7,8 The basic helix-loop-helix (bHLH) family of transcription factors regulates a wide range of developmental events. 1,9 Some bHLHs, such as MyoD and NeuroD, display restricted tissue expression and control the differentiation of particular cellular lineages. 10 They form transactivating dimers by heteromerization with ubiquitous bHLH proteins, termed E proteins, such as E2A, E12, and E47 genes. 11,12 Conversely, the latter proteins are repressed by other HLH proteins, which often precludes differentiation. 10,13,14 The tal/scl gene (hereafter referred to as scl) encodes a bHLH protein. 15,16 It was identified through its implication in T-cell acute lymphocytic leukemia (T-ALL). Knock-out (KO) experiments 17,18 show that scl is autonomously required for primitive and definitive hematopoiesis. 19,20 Moreover, its enforced expression enhances megakaryocytopoiesis and erythropoiesis. 21,22 The Lyl-1 gene encodes a bHLH protein closely related to scl. 23 Its transcriptional activation upon translocation is also associated with T-ALL. 24 SCL and LYL-1 bHLH regions show 82% of amino acid identity, 24 suggesting that these 2 proteins share at least some target genes and biologic functions. 25,26 However, LYL-1 and SCL diverge largely outside the bHLH region and display a distinct expression pattern in hematopoietic cells. 23,27 From the Insitut National de la The biologic functions of SCL prompted us to investigate those of LYL-1 in hematopoiesis. In contrast to scl Ϫ/Ϫ mice, Lyl-1 Ϫ/Ϫ mice do not exhibit embryonic lethality but have a reduced number of B cells. Although the CLP compartment is normal, the immature B-cell compartments are reduced in adult mice. In addition, we show that Lyl-1 is highly expressed in stem/progenitor cells, correlating with an important role in the control of the size and function of this cell compartment. Similarly, the number of multipotent progenitor S 12 colony-forming units (CFU-S 12 s) is reduced in Lyl-1 Ϫ/Ϫ animals. Overall, these defects are distinct from those revealed upon scl KO, suggesting that...