ALBERT

Description: Key Points Scl operates both downstream of Kit to control the survival of Kit+ multipotent and erythroid progenitors and upstream of Kit to determine Kit expression levels. Scl and Kit establish a positive feedback loop in hematopoietic progenitors.

Description: The majority of long-term reconstituting hematopoietic stem cells (LT-HSCs) in the adult is in G0, whereas a large proportion of progenitors are more cycling. We show here that the SCL/TAL1 transcription factor is highly expressed in LT-HSCs compared with short-term reconstituting HSCs and progenitors and that SCL negatively regulates the G0-G1 transit of LT-HSCs. Furthermore, when SCL protein levels are decreased by gene targeting or by RNA interference, the reconstitution potential of HSCs is impaired in several transplantation assays. First, the mean stem cell activity of HSCs transplanted at approximately 1 competitive repopulating unit was 2-fold decreased when Scl gene dosage was decreased. Second, Scl+/− HSCs were at a marked competitive disadvantage with Scl+/+ cells when transplanted at 4 competitive repopulating units equivalent. Third, reconstitution of the stem cell pool by adult HSCs expressing Scl-directed shRNAs was decreased compared with controls. At the molecular level, we found that SCL occupies the Cdkn1a and Id1 loci in primary hematopoietic cells and that the expression levels of these 2 regulators of HSC cell cycle and long-term functions are sensitive to Scl gene dosage. Together, our observations suggest that SCL impedes G0-G1 transition in HSCs and regulates their long-term competence.

Description: Abstract 505 Few markers have thus far been identified on native mesenchymal stem cells (MSCs), both in the mouse and human systems. Most markers cited in the literature are indeed based on expression analyses on heterogeneous cultured cell populations, which may not have self-renewal properties if rigorously tested by transplantation assays. Previous studies using Nestin (Nes)-Gfp transgenic mice showed that Nes-GFP+ cells are self-renewing MSCs, a major constituent of the hematopoietic stem cell (HSC) niche in the bone marrow (BM) (Nature 2010; 466:829). However, the cytoplasmic location of Nestin precludes prospective live cell isolation outside of the transgenic mice. Hence, finding a combination of surface markers labeling Nestin+ cells in situ would be valuable to isolate bona fide MSCs and characterize niche cells. Screening analyses toward this end revealed that PDGFRα and CD51 expression among CD45− Ter119− CD31− BM stromal cells comprised a large fraction (∼60%) of Nes-GFP+ cells. Upon gating first on PDGFRα+ and CD51+, double-positive cells were also highly enriched in Nes-GFP+ cells (∼75%), and represented a rare fraction (∼2%) of the stromal population. Endogenous Nestin expression was also enriched in PDGFRα+ CD51+ cells, compared to single-positive or double-negative stromal cells (control subsets). Cell sorting of BM PDGFRα+ CD51+ and control subsets revealed that PDGFRα+ CD51+ significantly enriched (〉 10-fold, p 7-fold, p

Description: Hematopoietic stem cells (HSC) reside in specific bone marrow niches comprised of perisinusoidal Nestin-GFP+ (Nes-GFP+) and leptin receptor (LepR)+ stromal cells which highly overlap with each other, as well as osteolineage and endothelial cells. These different cellular constituents regulate HSC maintenance and retention in the bone marrow (BM). Recently, our laboratory further identified rare periarteriolar Nes-GFPbright cells which have been identified to be pericytes and functionally crucial for HSC quiescence and maintenance, from the more abundant reticular Nes-GFPdim population (Kunisaki et al, unpublished data). The function of Nes-GFP+ niche cells is tightly regulated by the sympathetic nervous system (SNS) via β2- and β3-adrenergic receptors. In acute myeloid leukemia (AML), BM infiltration by leukemic blasts is known to lead to hematopoietic failure. Although cytopenias are thought to result from the BM occupation by AML, the mechanisms remain unclear. In this study, we investigated the impact of AML on BM niche constituents, and evaluated the influence of SNS signals in AML progression using a syngeneic murine MLL-AF9 transplantation model. We observed a ∼4.4-fold expansion of Nes-GFP+ stromal cells (p

Description: Vitamin K is an essential nutrient involved in the regulation of blood clotting and tissue mineralization. Vitamin K oxidoreductase (VKORC1) converts vitamin K epoxide into reduced vitamin K, which acts as the co-factor for the γ-carboxylation of several proteins, including coagulation factors produced by the liver. VKORC1 is also the pharmacological target of warfarin, a widely used anticoagulant. Vertebrates possess a VKORC1 paralog, VKORC1-like 1 (VKORC1L1), but until very recently, the importance of VKORC1L1 for protein γ-carboxylation and hemostasis in vivo was not clear. Here, we first review the current knowledge on the structure, function and expression pattern of VKORC1L1, including recent data establishing that, in the absence of VKORC1, VKORC1L1 can support vitamin K-dependent carboxylation in the liver during the pre- and perinatal periods in vivo. We then provide original data showing that the partial redundancy between VKORC1 and VKORC1L1 also exists in bone around birth. Recent studies indicate that, in vitro and in cell culture models, VKORC1L1 is less sensitive to warfarin than VKORC1. Genetic evidence is presented here, which supports the notion that VKORC1L1 is not the warfarin-resistant vitamin K quinone reductase present in the liver. In summary, although the exact physiological function of VKORC1L1 remains elusive, the latest findings clearly established that this enzyme is a vitamin K oxidoreductase, which can support γ-carboxylation in vivo.

Description: Abstract 2520 Poster Board II-497 The life-long production of blood cells depends on the regenerative capacity of a rare bone marrow population, the hematopoietic stem cells (HSCs). In the adult, the majority of HSCs are quiescent while a large proportion of progenitors are more cycling. The state of quiescence in HSCs is reversible and these cells can be triggered into cycle by chemotoxic injuries, exposure to cytokines in vitro, as well as transplantation in vivo. SCL/TAL1 is a bHLH transcription factor that has a critical role in generating HSCs during development. However, the role of SCL in adult HSCs is still a matter of debate. In the present study, we took several approaches to address this question. Scl expression was monitored by quantitative PCR analysis in a population that contains adult long-term reconstituting HSCs (LT-HSCs) at a frequency of 20–50%: Kit+Sca+Lin-CD150+CD48-. RT-PCR results were confirmed by β-galactosidase staining of these cells in Scl-LacZ mice. We show that Scl is highly expressed in LT-HSC and that its expression correlates with quiescence, i.e. Scl levels decrease when LT-HSCs exit the G0 state. In order to assess stem cell function, we performed several transplantation assays with adult bone marrow cells in which SCL protein levels were decreased at least two-fold by gene targeting or by RNA interference. 1) The mean stem cell activity of HSCs transplanted at ∼1 CRU was two-fold decreased in Scl heterozygous (Scl+/−) mice. 2) In competitive transplantation, the contribution of Scl+/− cells to primitive populations as well mature cells in the bone marrow was significantly decreased 8 months after transplantation. 3) In secondary transplantation assays, Scl+/− HSCs were severely impaired in their ability to reconstitute secondary recipient in stem cells and progenitor populations and in almost all mature lineages. 4) Reconstitution of the stem cell pool by adult HSCs expressing Scl-directed shRNAs was significantly decreased compared to controls. We therefore conclude that SCL levels regulate HSC long term competence. Since Scl levels decrease when LT-HSCs exit the G0 state, we addressed the question whether the cell cycle state of LT-HSCs is sensitive to Scl gene dosage. We stained bone marrow cell populations with Hoechst and Pyronin Y. At steady state, percentage LT-HSCs in G1 fraction appears to be significantly increased in mice lacking one allele of Scl. Furthermore, a three-fold increase in G1 fraction was also observed when cells were infected with Scl-directed shRNA, suggesting that a decrease in Scl levels facilitates G0-G1 transition. At the molecular level, we show by chromatin immunoprecipitation that SCL occupies the Cdkn1a and Id1 loci. Furthermore, in purified Kit+Sca+Lin-CD150+CD48- cells, the expression levels of these two regulators of HSC cell cycle and long-term functions are sensitive to Scl gene dosage. Together, our observations suggest that SCL impedes G0-G1 transition in HSCs and regulates their long-term competence. Disclosures: No relevant conflicts of interest to declare.

Description: SCL/TAL1 is a member of the basic helix-loop-helix family of transcription factors that also includes E2A and HEB. SCL is essential at the onset of hematopoiesis and activates transcription through heterodimerization with E2A. Both genes are expressed in long-term and short-term repopulating hematopoietic stem cells (HSCs) as assessed by expression profiling. To determine whether these genes regulate lineage commitment in HSCs and possibly other HSCs functions, we proceeded to serial transplantation of E2A-deficient or SCL-impaired HSCs. All mice were on a C57Bl/6 background and transplanted donor cells were discriminated from host cells by differential expression of CD45 isoforms. Since SCL function is dosage-sensitive, we used bone marrow cells from SCL LacZ/wt mice in which one SCL allele has been invalidated. When transplanted mice were analyzed at 4 months for reconstitution in all hematopoietic organs, SCLLacZ/wt HSCs (CD45.2) showed normal repopulating capacity in competition with wild type cells (CD45.1) as expected. Strikingly, analysis at 8 months revealed that SCLLacZ/wt HSCs were markedly impaired compared to normal HSCs. The contribution of SCLLacZ/wt cells to the Kit+Sca+Lin− (KSL) and all myeloid progenitor (CMP, MEP and GMP) populations was less than 5%. This long term defect was also observed in secondary transplantation assays. Despite this near absence of HSCs and CMPs, common lymphoid progenitors (CLPs) were almost equally composed of SCLLacZ/wt and wild type cells, and the generation of B22O positive cells was expanded when SCL gene dosage was reduced. Together, our results indicate that long-term stem cell activity requires full SCL gene dosage. Furthermore, SCL operates at the branchpoint of the myeloid and lymphoid lineages to favor the myeloid lineages. Since E2A is an obligate SCL partner, we next addressed E2A function in HSCs. As E2A−/− embryos die before birth, we therefore transplanted E14.5 fetal liver cells. E2A deficiency did not affect the number of HSCs, as assessed by limiting dilution analysis. Nonetheless, serial transplantation assays revealed that HSCs from wild type littermates could be serially transplanted beyond the fourth passage while there was an early exhaustion of E2A-deficient HSCs at the third transplantation. Together, our analyses are consistent with the view that E2A is the functional partner of SCL in HSCs and that SCL/E2A does not control the size of the HSC pool but is required for long-term HSC activity. Similarly to SCL impairment, all progenitors were decreased by E2A deficiency. There were, however, two major differences. First, CLPs were severely impaired indicating that E2A activity is required for specification of the lymphoid lineages. Second, CD11b positive cells were expanded by E2A-deficiency and this was gene dosage-dependent, consistent with the view that E2A inhibits the monocyte-granulocyte lineage. Taken together, our results indicate that SCL collaborates with E2A in HSCs to maintain their long-term activities. In contrast, at the myeloid-lymphoid branchpoint, the functions of SCL and E2A are opposite: SCL favors the myeloid lineage at the expense of the lymphoid lineage, while E2A specifies the lymphoid lineage and inhibits granulocyte-macrophage differentiation.

Description: The c-Kit tyrosine kinase receptor and the SCL/Tal1 (Stem Cell Leukemia) transcription factor are crucial to hematopoiesis and are co-expressed by hematopoietic progenitors. In the present study, we directly address the question whether SCL and c-Kit establish a code for the fate of multipotent progenitors in the adult. We therefore proceeded to loss and gain of function experiments, via retroviral mediated gene transfer in primary hematopoietic cells. We show that SCL is functionally required to sustain the survival of c-Kit+ cells in response to Steel Factor (Kit ligand), but not to GM-CSF and IL-3. Interfering with SCL function using a DNA-binding defective SCL mutant (ΔbSCL) caused a 100-fold loss of multipotent progenitors, due to massive apoptotic death in the c-Kit+ fraction, indicating that the DNA binding function of SCL is essential in cell survival. An antisense SCL also caused apoptosis in c-Kit+ cells, which was rescued by co-delivering SCL in the sense orientation. Furthermore, SCL sets threshold for the response of multipotent progenitors to Steel factor without affecting their response to IL-3 or GM-CSF, demonstrating the specificity of SCL for c-Kit-dependent pathway. Moreover, SCL levels in purified hematopoietic progenitors (HSC, CMP, MEP, CLP) were 3.5 to 15-fold decreased in W41 W41 mice that have a hypomorphic c-Kit allele when compared to age-matched wild type controls. Conversely, ectopic SCL expression is sufficient to bypass c-Kit signalling to suppress apoptosis and increase the proliferative potential of multipotent progenitors, as anemia and other hematopoietic defects caused by a hypomorphic c-Kit allele were rescued by introducing an SCL transgene into the W41 W41 background. We conclude that SCL operates within the c-Kit pathway to suppress apoptosis and determine the clonal output of adult multipotent progenitors. To define the pathway through which SCL suppresses apoptosis in hematopoietic progenitors, we performed microarrays analysis on Δb-SCL expressing cells and identified novel survival genes that were confirmed to be direct SCL targets by chromatin immunoprecipitation. Finally, the expression of these target genes was decreased in purified progenitors from W41 W41 mice as assessed by quantitative PCR. Together, this study reveals a new and essential genetic pathway involving SCL downstream of c-Kit signalling for the survival of hematopoietic progenitors.