Stages Of Hematopoietic Stem Cells Research Articles

Dendritic cells derived from pluripotent stem cells: Potential of large scale production

Human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, are promising sources for hematopoietic cells due to their unlimited growth capacity and the pluripotency. Dendritic cells (DCs), the unique immune cells in the hematopoietic system, can be loaded with tumor specific antigen and used as vaccine for cancer immunotherapy. While autologous DCs from peripheral blood are limited in cell number, hPSC-derived DCs provide a novel alternative cell source which has the potential for large scale production. This review summarizes recent advances in differentiating hPSCs to DCs through the intermediate stage of hematopoietic stem cells. Step-wise growth factor induction has been used to derive DCs from hPSCs either in suspension culture of embryoid bodies (EBs) or in co-culture with stromal cells. To fulfill the clinical potential of the DCs derived from hPSCs, the bioprocess needs to be scaled up to produce a large number of cells economically under tight quality control. This requires the development of novel bioreactor systems combining guided EB-based differentiation with engineered culture environment. Hence, recent progress in using bioreactors for hPSC lineage-specific differentiation is reviewed. In particular, the potential scale up strategies for the multistage DC differentiation and the effect of shear stress on hPSC differentiation in bioreactors are discussed in detail.

Utility of LRF/Pokemon and NOTCH1 Protein Expression in the Distinction Between Nodular Lymphocyte-Predominant Hodgkin Lymphoma and Classical Hodgkin Lymphoma

Classical Hodgkin lymphoma (CHL) and nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) are considered separate entities with different prognosis and treatment. However, morphologic features can be similar and immunohistochemical studies are essential in the distinction; thus, determination of additional biomarkers is of utmost importance. LRF/Pokemon is a proto-oncogene, an interacting partner co-expressed with BCL6 in germinal centers and highly expressed in diffuse large B-cell lymphoma and follicular lymphoma. Conversely, loss of the LRF gene in mouse hematopoietic stem cells results in complete block of early B cell development with concomitant Notch de-repression, indicating its critical role in B versus T cell fate decision at the hematopoietic stem cell stage. For the first time, we show that LRF/Pokemon is predominantly expressed in NLPHL cases as is BCL6 with low to absent NOTCH1 protein expression; while Hodgkin Reed-Sternberg (HRS) cells in CHL show low to absent BCL6 and LRF/Pokemon expression with higher NOTCH1 expression. We illustrate a potential functional interaction between LRF and BCL6 in NLPHL pathogenesis, and differential expression of LRF/Pokemon and NOTCH1 proteins in CHL thus showing differential expression, making for an additional diagnostic marker and therapeutic target.

Zebrafish Cbfb Is Required For The Mobilization, But Not The Emergence, Of Hematopoietic Stem Cells In Embryos

CBFβ and RUNX1 form a DNA-binding heterodimer that plays a crucial role during definitive hematopoiesis at the stage of hematopoietic stem cells (HSCs). Both of them are targets of recurrent chromosomal translocations in human leukemia. In mammals and zebrafish, RUNX1 is required for the emergence of definitive HSCs from the hemogenic endothelium. Mouse knockouts for either Runx1 or Cbfb show similar phenotypes with complete lack of definitive hematopoiesis. Therefore, the impairment of all definitive hematopoietic lineages in both Runx1-/- and Cbfb-/- embryos suggested that the CBF heterodimer is required for HSC formation. However the exact role of the CBF complex in the development of HSCs remains unclear. The cellular mechanisms and the genetic pathways driving the HSC generation are highly conserved across vertebrates. Thus, we used the zebrafish model to dissect the role of cbfb and the CBF complex in the emergence and the maintenance of HSCs. We generated two independent cbfb knockouts (cbfb-/-) by zinc-finger nuclease (ZFN) - mediated targeted mutagenesis. The analysis of cbfb-/- embryos revealed a previously unknown role of cbfb during definitive hematopoiesis. Similar to the published zebrafish runx1 mutant embryos (runx1W84X/W84X), cbfb-/- embryos underwent primitive hematopoiesis and developed erythromyeloid progenitors (EMPs), but they lacked definitive hematopoiesis as the expression of markers for differentiated blood lineages such as rag1, lplastin and αe1globin was completely abrogated. Moreover, circulating thrombocytes were almost undetectable in cbfb-/-/tg(cd41:GFP) embryos. Unlike the runx1 mutants in which HSCs are not formed, however, the emergence of runx1+/c-myb+ HSCs from the hemogenic endothelium along the ventral wall of the dorsal aorta was unaffected in the cbfb-/- mutants. Rather, the subsequent translocation of the HSCs from aorta-gonad-mesonephros (AGM) to the caudal hematopoietic tissue (CHT) was blocked, as evidenced by the accumulation of runx1+ HSCs in the AGM and the concomitant absence of such cells in the CHT. Live imaging analysis of cbfb-/-/tg(c-myb:eGFP) embryos confirmed that HSCs egressed from the dorsal aorta but did not enter circulation through the axial vein. Moreover, embryos treated with a specific inhibitor of RUNX1-CBFβ interaction, Ro5-3335, phenocopied the hematopoietic defects observed in the cbfb-/- mutants, confirming that the function of RUNX1 and CBFβ during HSC development could be uncoupled. The Notch-Runx1 pathway is critical for the initial specification of HSCs during definitive hematopoiesis. Therefore, in order to gain insight into the genetic mechanisms that regulate cbfb expression we investigated the Notch pathway. We found that transient Notch activation enhanced cbfb expression and expanded it ectopically. On the other hand, in the Notch signaling mutant mind bomb, cbfb expression in hematopoietic regions was abrogated. Thus, our results suggest that cbfb is also downstream of the Notch pathway during hematopoiesis. Overall our data indicate that CBFβ and functional CBFβ-RUNX1 heterodimers are not required for the emergence of HSCs, but are essential for the mobilization of HSCs during early definitive hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

The orphan nuclear receptor Ear-2 (Nr2f6) is a novel negative regulator of T cell development

We describe a novel role for the orphan nuclear receptor Ear-2 in regulating T cell development. Retrovirus-mediated overexpression of Ear-2 (EAR-2++) in a bone marrow (BM) transplantation assay resulted in limited T cell development and a greater than tenfold decrease in thymus size and cellularity relative to controls. Ear-2-transduced murine BM hematopoietic stem cells (HSCs) in OP9-DL1 cultures showed a proliferation deficit during days 1-5 after induction of differentiation, which corresponded to increased expression of the cell cycle regulators p21 (cdkn1a) and p27 (cdkn1b), as well as increased expression of Hes1, Notch3, Egr1, and Scl (Tal1) and decreased expression of Gli1, Gfi-1, HoxA9, PU.1, Nrarp, and Tcf1. In addition, there was a block in differentiation at the DN4 to double-positive (DP) transition accompanied by an increase in apoptosis, similar to the deficit seen in the RORγt null mouse. Gene expression profiling revealed that, like the RORγt-deficient mouse, EAR-2++ DP cells had decreased expression of BclXL and increased expression of the proapoptosis gene Bad. In addition, EAR-2++ DP cells had decreased expression of Bcl11b, PU.1, and HoxA9, and increased expression of Id2. Based on these findings, we conclude that EAR-2++ cells were able to migrate to, but not fully repopulate, the thymus because of a cell-intrinsic defect in the proliferation of DN1 cells followed by a block in differentiation from the DN4 to DP stage of T cell development. We conclude that Ear-2 is a novel negative regulator of T-cell development and that downregulation of Ear-2 is indispensable for the proliferation of DN1 cells and the survival of DN4-DP cells.

Immune Injury by Allogeneic CD4+ T Cells Leads to Host Hematopoietic Stem Cell Dormancy and Prevents Engraftment of Donor Cells.

Abstract 2366The bone marrow (BM) is a complex microsystem that supports lifelong blood production and dynamically responds to injury, blood cell loss and senescence. At steady-state most hematopoietic stem cells (HSC) are quiescent. However, in situations of increased demand, their activation is triggered by an array of signals, such as cytokines released during infections. The susceptibility of BM to injury by T cells (TC) is long recognized, e.g. in aplastic anemia, and BM failure syndromes, and the inhibitory effect of IFNg on the proliferative activity of (HSC) is established. How donor TC given as part of an allogeneic hematopoietic cell transplantation (HCT) affect the recipient BM microenvironment and influence HSC activity has not been elucidated. Yet, graft TC are believed to be essential to overcome host barriers, especially following lower intensity (non-myeloablative) conditioning that spares host HSC and residual host immunity. We examined in an MHC-matched, minor-antigen-mismatched mouse model of non-myeloablative HCT the impact of graft TC subsets on HSC and hematopoietic reconstitution. BALB.K (H2k) and BALB.B (H2b) mice were prepared with sublethal radiation at a dose that allows donor cells (AKR/J [H2k] and C57BL/6 [H2b]) to engraft but also permits survival of host HSC and immune populations. Grafts consisted of purified HSC (KTLS; cKit+Thy1.1loLin−Sca-1+) given alone or supplemented with donor TC or TC subsets. Rather than improve engraftment, donor TC, specifically CD4+ cells, resulted in detainment of host HSC in their niches and consequent failure of donor HSC to engraft. These events appeared to be initiated specifically by conventional alloreactive donor CD4+CD25− cells. These CD4+CD25− cells interacted with immune-competent host cells, stimulating CD11c+ dendritic cells to upregulate the activation markers CD40 and MHC II. Following their activation dendritic cells produced excessive amounts of IL-12. IL-12, in turn, activated donor CD4+ cells to produce IFNg. This Th1-response was prominent in BM but absent in the spleen. In this Th1-cytokine environment, high levels of quiescent host HSC were observed, arrested at the short-term HSC stage, while more mature, multipotent progenitors were absent. Moreover, HSC arrest correlated with marrow aplasia, analogous to that seen in BM failure syndromes. This hypocellularity was only observed in recipients of CD4+ containing grafts, while recipients given HSC alone or HSC in combination with CD8+ TC had prompt blood regeneration. When resting HSC were FACS-purified from the BM of recipients of HSC+CD4+ and infused into secondary recipients they promptly expanded and gave rise to all blood lineages. Although recipients of HSC+CD4+ cells ultimately recovered blood formation, B lymphopoiesis was markedly delayed. No clinical signs of graft-versus-host disease were noted in these mice. Lymphocytes that recovered in these recipients of HSC+CD4+ cells were of host type only. In contrast, recipients of pure HSC demonstrated prompt donor cell engraftment, and mixed chimerism in all lymphoid lineages, including T cells. Our model corroborates the importance of the cytokine microenvironment in hematopoietic reconstitution and marrow failure post-transplant. Moreover, our findings underscore the importance of IFNg in BM aplasia and support the idea that IFNg can, under physiologic conditions, exert regulatory effects on hematopoiesis by controlling the exit of ST-HSC out of the marrow niche. Our studies shed new light on understanding the physiology of engraftment and marrow failure after HCT using nonmyeloablative conditioning, and indicate that the composition of the graft is of critical importance due to its effects on the marrow microenvironment We postulate that in some naturally occurring settings of BM inflammation (e.g., infections) detainment of resting HSC in their niche may provide protection of hematopoiesis and that comparable events are relevant for engraftment post-HCT. Disclosures:No relevant conflicts of interest to declare.

Engulfment of hematopoietic stem cells caused by down-regulation of CD47 is critical in the pathogenesis of hemophagocytic lymphohistiocytosis

AbstractHemophagocytic lymphohistiocytosis (HLH) is characterized by deregulated engulfment of hematopoietic stem cells (HSCs) by BM macrophages, which are activated presumably by systemic inflammatory hypercytokinemia. In the present study, we show that the pathogenesis of HLH involves impairment of the antiphagocytic system operated by an interaction between surface CD47 and signal regulatory protein α (SIRPA). In HLH patients, changes in expression levels and HLH-specific polymorphism of SIRPA were not found. In contrast, the expression of surface CD47 was down-regulated specifically in HSCs in association with exacerbation of HLH, but not in healthy subjects. The number of BM HSCs in HLH patients was reduced to approximately 207 of that of healthy controls and macrophages from normal donors aggressively engulfed HSCs purified from HLH patients, but not those from healthy controls in vitro. Furthermore, in response to inflammatory cytokines, normal HSCs, but not progenitors or mature blood cells, down-regulated CD47 sufficiently to be engulfed by macrophages. The expression of prophagocytic calreticulin was kept suppressed at the HSC stage in both HLH patients and healthy controls, even in the presence of inflammatory cytokines. These data suggest that the CD47-SIRPA antiphagocytic system plays a key role in the maintenance of HSCs and that its disruption by HSC-specific CD47 down-regulation might be critical for HLH development.

JS5-5 - Hematopoietic Stem Cell is the Primary Oncogenic Target in Human Leukemias

ABSTRACT Malignant neoplasms might originate from a small abnormal cell population with deregulated self-renewal capacity, impaired differentiation activity and/or reinforced cell survival. This ‘cancer stem cell’ hypothesis is well supported in the field of hematologic disorders such as acute leukemias, based on identification of a small malignant stem cell fraction that can reconstitute human diseases in xenograft mouse models. In hematological malignancies, for perpetual propagation of leukemic mutations, these mutational events should occur in a cell population that can self-renew, otherwise the short life of a committed cell would not sustain these oncogenic events. We have successfully tracked the step-wise acquisition of mutations such as FLT3-ITD and AML1-ETO in hematopoietic stem cells (HSCs), which eventually developed de novo acute myelogeneous leukemia (AML). In contrast, in lymphoid malignancies, HSCs are not likely to play such a role in accumulation of mutations, because the clonality of lymphoid malignancies is usually found after the stage of rearrangement of T-cell receptor or immunoglobulin genes. However, we recently found that even in chronic lymphocytic leukemia (CLL), a mature B cell malignancy, the initial event for leukemic transformation could be traced up to HSCs. We found that mice transplanted with the CD34 + CD38- HSC fraction from CLL patients displayed expansion of monoclonal or oligoclonal CD5+ B cells. In contrast, normal HSCs always developed polyclonal CD5- B cells, suggesting that the primary deficit has occurred at the HSC stage in CLL patients. These data strongly support the hypothesis that HSCs are the cells that accumulate genetic abnormality to enable fully malignant transformation.

Conditional Knockout of Sox4 Markedly Affects Early B Lymphopoiesis in Adult Mice

Abstract 913The development of mature B cells from self-renewing pluripotent hematopoietic stem cells (HSCs) involves a complex process that is regulated in a hierarchical order by various proteins, particularly transcription factors. Sox4 is an SRY-related HMG-box-containing transcription factor and is known to be implicated in B cell development. However, Sox4's role in B cell development has not been systematically investigated. In this project, we used conditional knockout mouse strains to study the effect of Sox4 deletion on B lymphopoiesis in adult mice.To determine when in the B cell developmental process Sox4 is required, we crossed Sox4-floxed mice with 5 Cre-recombinase-expressing mouse strains that were expected to result in deletion of the floxed Sox4 gene at different developmental stages, as follows: Vav-Cre (HSC stage in early embryos), Mx1-Cre (HSC stage in adults, induced by pIpC injection), MB1-Cre (early progenitor B cell stage), CD19-Cre (pro-B cell stage), and CD21-Cre (mature B cell stage). We demonstrated that deletion of Sox4 caused arrest of B lymphopoiesis at the transition from pre-pro-B cell stage (fraction A) to pro-B cell stage (fraction B); fraction B and later-stage B cells were nearly absent. There was a significant reduction in the number of hematopoietic stem (LSK) cells upon Sox4 deletion, but the number of common lymphocyte progenitors (CLPs) was not significantly changed and that of fraction A cells was only slightly reduced, suggesting that reduction in HSCs was not directly responsible for the B cell deficiency. Deletion of Sox4 in the early embryonic stage (Vav-Cre) and in adults (Mx1-Cre) resulted in similar phenotypes with regard to B lymphopoiesis, except that the number of peritoneal B1 cells was reduced more significantly in Vav-Cre than in Mx1-Cre mice. MB1-Cre yielded results similar to those of Vav-Cre, but B lymphopoiesis arrest was not as dramatic. Sox4f/f CD19-Cre and Sox4f/f CD21-Cre mice produced no significant difference in B cell phenotype compared with Sox4f/+ CD19-Cre and Sox4f/+ CD21-Cre mice, respectively. These data suggest that Sox4 is required for early B cell development at the transition from pre-pro-B cells to pro-B cells and is not required for mature B cell development.To further understand the mechanism by which Sox4 deletion induced pro-B cell deficiency, we performed semi-quantitative RT-PCR on residual pro-B cells and showed that Sox4 deletion did not change the mRNA expression of the well-known genes involved in early B cell development (EBF1, E2A, Rag1, CXCR4, IL7R, EZH2, PAX5, FoxP1, STAT5), indicating that Sox4 might be functioning in early B lymphopoiesis through a novel mechanism. We next studied pro-B cell viability in Sox4f/+ and Sox4f/f Vav-Cre mice. We found that, when Sox4 was depleted, bone marrow pro-B cells, but not pre-pro-B cells, underwent remarkable apoptosis, as shown by increased annexin V staining. In accordance, the level of the antiapoptotic protein Bcl2 in Sox4-deleted pro-B cells was much lower than that in control pro-B cells. Introduction of Bcl2 transgenic gene into the Sox4 conditional knockout mice decreased pro-B cell apoptosis and partially rescued the Sox4-deficient B cell development. We also observed striking molecular communication between Sox4 and the c-Kit-mediated cell survival signaling pathway; after intraperitoneal injection of the c-Kit inhibitor imatinib, the apoptotic rate was much higher in Sox4f/+Vav-Cre mice than in Sox4+/+ mice, suggesting that reduction of Sox4 level sensitized the cells to the inhibitor.Our data suggest that Sox4 deletion markedly decreases pro-B cell viability by modulating Bcl2 and that the c-Kit signaling pathway and Sox4 function is interlinked. We are currently investigating Sox4 transcription program and the mechanism of Sox4-induced Bcl2 protein reduction in progenitor B cells. Disclosures:No relevant conflicts of interest to declare.

Hemophagocytic Lymphohistiocytosis Is Caused by Engulfment of Hematopoietic Stem Cells by Macrophages Through Stem Cell-Specific Suppression of CD47 Under Influence of Cytokine Deregulation

Abstract 522Hemophagocytic lymphohistiocytosis (HLH) is a syndrome characterized by hemophagocytosis in the bone marrow with systemic inflammatory reactions. We have reported that the inflammatory cytokinemia including interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) might play a key role in activating macrophages to induce HLH (Akashi et al. Br J Haematol, 1994). Recent studies have shown that the engulfment of blood cells by macrophages is regulated in part by the interaction of CD47 and its ligand, signal regulatory protein alpha (SIRPA). Blocking the CD47 and SIRPA interaction by using anti-CD47 monoclonal antibodies can induce engulfment of blood cells, suggesting that the SIRPA signaling provide “don’t eat me signals” to prevent macrophages from digesting self blood cells. These data led us to hypothesize that HLH is caused by the disruption of self-recognition by macrophages through impairment of the CD47-SIRPA system. To test this hypothesis, we evaluated the expression level of CD47 in bone marrow cells in 24 patients with HLH. Interestingly, the expression of CD47 was significantly downregulated (by ∼2-fold reduction in average at the protein level) in the CD34+CD38− hematopoietic stem cell (HSC) fraction in HLH patients, whereas that of the CD34+CD38+ progenitor and other mature blood cell fractions was unchanged. We then purified the CD34+CD38− HSCs and CD34+CD38+ progenitor cells from HLH patients and normal controls, co-cultured them with human macrophages in the presence of IFN-gamma and lipopolysaccharide, and evaluated the percentage of phagocyting macrophages. The numbers of phagocyting macrophages were significantly higher in cultures of CD34+CD38− HSCs from HLH patients, as compared to those in cultures of normal HSCs and of progenitor populations, suggesting that the expression level of CD47 inversely correlated with the efficiency of hemophagocytosis. Furthermore, normal HSCs but not CD34+CD38+ progenitors or other mature blood cells down-regulated CD47 in vitro in response to IFN-gamma, TNF-alpha, and IL-6, suggesting that these cytokines plays a critical role in down-regulation of CD47 especially in HSCs. In contrast, the expression level of SIRPA did not differ in myeloid cells between HLH patients and normal controls, and mutations of SIRPA were not found in HLH patients. Thus, in HLH, inflammatory cytokines down-regulate CD47 selectively in HSCs, resulting in the engulfment of HSCs by macrophages. Our data strongly suggest that the cytokine deregulation can lead to HLH in human, through disruption of self-recognition guided by the CD47-SIRPA system at the HSC stage. Disclosures:No relevant conflicts of interest to declare.

DNA Methylation Profile of Runx1 Regulatory Regions Is Correlated with Transition From Primitive to Definitive Hematopoietic Potential In Vitro and In Vivo

Abstract 389 Introduction:The Runt-related transcription factor Runx1 (AML1) is a central regulator of mammalian hematopoiesis and is required for the generation of hematopoietic stem cells (HSC) from hemogenic endothelium in the embryo. It has been shown that Runx1 is alternatively expressed from two promoters in a temporal fashion, and that their differential activities are influenced by a conserved intronic enhancer (+23) element. Intriguingly, promoter usage follows a pattern whereby the proximal (P2) initiates early in primitive hematopoiesis, while the distal (P1) becomes active later at the time of HSC emergence and is the predominant isoform expressed in fetal liver and adult HSC. While some transcription factor binding sites and cis-regulatory elements have been identified, an explanation for the alternative promoter usage remains elusive. We hypothesized that this regulation may be at the level of chromatin accessibility, and therefore investigated the DNA methylation status of Runx1 cis-elements. Methods/Results:We analyzed bisulfite-treated genomic DNA from E14.5 fibroblast (MEF), E8.5 yolk sac CD41+ (YS), E14.5 fetal liver Lin-Sca-1+CD48-CD150+ (FL), and adult marrow Lin-cKit+Sca-1+ (KLS); representing non-hematopoietic, primitive hematopoietic, and two stages of definitive HSC respectively. In addition, we also examined methylation in hematopoietic populations derived in vitro from murine embryonic stem cells (mESC). Initial exploratory analysis focused on classically defined CpG islands upstream of each promoter, however no significant differential methylation was observed within these regions. Subsequent analysis focused on CpGs near the transcription start site (TSS) and within the +23 enhancer. The P2 promoter was uninformative as it was unmethylated in all populations analyzed, whereas methylation within the +23 enhancer differentiated between hematopoietic and non-hematopoietic cell populations. At the P1 promoter, methylation status was remarkably correlated with primitive vs. definitive status. P1 was highly methylated in MEFs (77%), mESC embryoid body (EB) derived cKit+CD41+ (66%), and E8.5 YS CD41+ (58%); but significantly less methylated in vivo in FL HSC (8.1%) and adult KLS cells (18%). We are currently using this correlation of demethylation and definitive HSC potential to identify conditions that may drive definitive HSC generation from mESC-derived blood progenitors. Since overexpression of HoxB4 coupled with OP9 co-culture is the only confirmed method capable of producing definitive HSC from mESC, and HoxB4 has been shown to bind within the P1 promoter region of Runx1, we cultured HoxB4 or control EB-derived hematopoietic progenitors on OP9 stroma. We observed progressive demethylation in the HoxB4 arm: after 6 days of co-culture 47% vs. 71% in controls, and after 11 days 27% in the HoxB4 arm while the control population failed to proliferate past day 6. Isoform specific RT-PCR confirmed that HoxB4 overexpression resulted in Runx1 expression from the P1 promoter whereas the control vector did not. Within P1, we identify a single CpG that is most highly correlated with definitive HSC potential in vivo, and most significantly demethylated upon HoxB4 overexpression in vitro. Conclusions:These data indicate that differential methylation occurs at Runx1 regulatory regions during hematopoietic development in vitro and in vivo. The +23 enhancer is demethylated in cells with hematopoietic potential, whereas demethylation of the Runx1 P1 promoter is highly correlated with definitive HSPC populations and is promoted in vitro by HoxB4. These data are the first to identify a role for DNA methylation in the regulation of alternative promoter usage at the Runx1 locus, and may serve as a novel biomarker of HSC potential during embryonic development. Disclosures:No relevant conflicts of interest to declare.

Surprise! HSC Are Aberrant in Chronic Lymphocytic Leukemia

In this issue of Cancer Cell, Kikushige etal. report the surprising finding that, in human chronic lymphocytic leukemia (CLL), hematopoietic stem cells (HSC) aberrantly generate clonal B cells with CLL-like phenotypes, which implicate HSC in the pathogenesis of this mature lymphoid malignancy and has major implications for CLL therapies.

Intermediate-Term Hematopoietic Stem Cells with Extended but Time-Limited Reconstitution Potential

Sustained blood cell production depends on divisions by hematopoietic stem cells (HSCs) that yield both differentiating progeny as well as new HSCs via self-renewal. Differentiating progeny remain capable of self-renewal, but only HSCs sustain self-renewal through successive divisions securely enough to maintain clones that persist life-long. Until recently, the first identified next stage consisted of "short-term" reconstituting cells able to sustain clones of differentiating cells for only 4-6 weeks. Here we expand evidence for a numerically dominant "intermediate-term" multipotent HSC stage in mice whose clones persist for 6-8 months before becoming extinct and that are separable from both short-term as well as permanently reconstituting "long-term" HSCs. The findings suggest that the first step in stem cell differentiation consists not in loss of initial capacity for serial self-renewal divisions, but rather in loss of mechanisms that stabilize self-renewing behavior throughout successive future stem cell divisions.

Conditional Knockout of Sox4 Markedly Affects B Lymphopoiesis in Adult Mice.

Abstract 2524Poster Board II-501B cells constitute an integral part of the immune system. The development of mature B cells from hematopoietic stem cells is a complex process that is regulated in a hierarchical order by various proteins, particularly transcription factors. Sox4 is a SRY-related HMG box containing transcription factor and is known to be involved in B cell development. However, the role of Sox4 in various stages of B cell development has not been systematically investigated. In this study we used a conditional knockout mouse strain and studied the effect of Sox4 deletion in B lymphopoiesis in adult mice. We crossed the Sox4-floxed mice with different Cre mouse strains that were expected to delete the floxed Sox4 gene at different B cell developmental stages. These Cre strains included Vav-iCre (expressed in hematopoietic stem cell stage, starting from early embryos), MX1-Cre (expression in hematopoietic stem cells, induced by pIpC injection in adults), MB1-Cre (expressed in B cells, starting from early progenitor cells in embryos), and CD21-Cre (expressed in mature B cells). We demonstrated that deletion of Sox4 caused an arrest of B lymphopoiesis at the transition from pre-pro-B cell (fraction A) stage to pro-B cell stage (fraction B): fraction A cells are slightly reduced in number whereas fraction B and later stage cells are nearly absent. The pre-pro-B cells from the Sox4 knockout mice retain a population of AA4.1+ cells, which are considered to be developed into B cells. Deletion of Sox4 in early embryonic stage (Vav-iCre) or in adults (Mx1-Cre) results in a similar phenotype on B lymphopoiesis, except that peritoneal B1 cells appear to be affected with Vav-iCre, but not with Mx1-Cre. MB1-Cre gave rise to similar results as did Vav-iCre, but the arrest was not as dramatic as with Vav-iCre. CD21-Cre produced no significant difference in B cell phenotype. These data suggested that Sox4 is required for early B cell development at the transition from pre-pro-B cells to pro-B cells and is not required for mature B cells. We are currently investigating the transcription program of this transcription factor in B cell development. Disclosures:No relevant conflicts of interest to declare.

Severe block in hematopoiesis in the absence of NKAP (138.2)

Abstract During hematopoiesis, proper differentiation of the long-term, multi-potent, self-renewing hematopoietic stem cell (HSC) results in the cellular components of blood. The lineage fate decisions that occur during hematopoiesis result from the coordinated regulation of transcriptional activators, repressors, and chromatin modifiers. We have identified NKAP as a novel regulator of hematopoiesis, that functions at least in part as a transcriptional repressor. In this study we employ a combination of conditional and inducible mouse models of NKAP deletion to explore the role of NKAP in hematopoietic development starting at the HSC stage. Our models allow for both the pan-deletion of NKAP starting in the HSC stage by using a vav-cre transgenic, as well as temporally regulated deletion of NKAP in the hematopietic compartment through the use of the interferon-inducible mx1-cre model system. These complimentary approaches allow us to conclude that NKAP functions in a cell intrinsic manner, as a member of a unique group of transcriptional regulators, and is necessary in the earliest stage of definitive hematopoiesis.

Stepwise Development of Hematopoietic Stem Cells from Embryonic Stem Cells

The cellular ontogeny of hematopoietic stem cells (HSCs) remains poorly understood because their isolation from and their identification in early developing small embryos are difficult. We attempted to dissect early developmental stages of HSCs using an in vitro mouse embryonic stem cell (ESC) differentiation system combined with inducible HOXB4 expression. Here we report the identification of pre-HSCs and an embryonic type of HSCs (embryonic HSCs) as intermediate cells between ESCs and HSCs. Both pre-HSCs and embryonic HSCs were isolated by their c-Kit+CD41+CD45− phenotype. Pre-HSCs did not engraft in irradiated adult mice. After co-culture with OP9 stromal cells and conditional expression of HOXB4, pre-HSCs gave rise to embryonic HSCs capable of engraftment and long-term reconstitution in irradiated adult mice. Blast colony assays revealed that most hemangioblast activity was detected apart from the pre-HSC population, implying the early divergence of pre-HSCs from hemangioblasts. Gene expression profiling suggests that a particular set of transcripts closely associated with adult HSCs is involved in the transition of pre-HSC to embryonic HSCs. We propose an HSC developmental model in which pre-HSCs and embryonic HSCs sequentially give rise to adult types of HSCs in a stepwise manner.

Deletion of Notch1 Converts Pro-T Cells to Dendritic Cells and Promotes Thymic B Cells by Cell-Extrinsic and Cell-Intrinsic Mechanisms

Notch1 signaling is required for T cell development and has been implicated in fate decisions in the thymus. We showed that Notch1 deletion in progenitor T cells (pro-T cells) revealed their latent developmental potential toward becoming conventional and plasmacytoid dendritic cells. In addition, Notch1 deletion in pro-T cells resulted in large numbers of thymic B cells, previously explained by T-to-B cell fate conversion. Single-cell genotyping showed, however, that the majority of these thymic B cells arose from Notch1-sufficient cells by a cell-extrinsic pathway. Fate switching nevertheless exists for a subset of thymic B cells originating from Notch1-deleted pro-T cells. Chimeric mice lacking the Notch ligand delta-like 4 (Dll4) in thymus epithelium revealed an essential role for Dll4 in T cell development. Thus, Notch1-Dll4 signaling fortifies T cell commitment by suppressing non-T cell lineage potential in pro-T cells, and normal Notch1-driven T cell development repels excessive B cells in the thymus.

Notch1 Expression Is Regulated at Post-Transcriptional Level by 3′UTR Region in Hematopoietic Stem Cell Development

In hematopoietic stem cell development, the expression of critical genes is precisely regulated in a stage specific manner, which supports normal hematopoietic development through adequately regulating timing of cell division, self-renewal, and lineage commitment. Regulation of gene expression is known to take place at least at transcriptional level. In addition to the transcriptional regulation, there are growing evidences that post-transcriptional control of critical genes may play an important role, suggesting an interesting possibility that post-transcriptional control may also play a role in hematopoiesis. Here, we provide the evidence that the expression of Notch1, a key factor in lymphoid development, is controlled at post-transcriptional level in hematopoietic stem cell (HSC). By quantitative PCR, Notch1 mRNA is substantially expressed at HSCs as well as common lymphoid progenitors (CLPs) or double negative (DN) thymocytes. However, Notch1 protein is detected at very low level in HSCs compared to CLPs or DN thymocytes, suggesting that Notch1 expression is regulated at post-transcriptional level in HSC. To investigate the effect of 3′UTR (untranslated region) on post-transcriptional regulation, we prepared a retrovirus sensor vector, in which 3′UTR of target gene is placed between the GFP coding region and the retrovirus 3′LTR, and found that induction of the sensor vector with the 3′UTR sequence of Notch1 showed marked suppression of the GFP intensity at the HSC stage. This effect was not observed when we introduced the vector into DN thymocytes. Suppression of Notch1 by its 3′UTR was further confirmed by using a retrovirus vector which has two distinct markers of YFP and GFP-3′UTR fusion genes under bi-directional EF1 promoter. Deletion mutant analysis showed that the responsible region required for this post-transcriptional suppression is confined to 120-bp sequence within Notch1 3′UTR so far. These data suggest that the expression of Notch1 should be regulated at post-transcriptional level by its 3′UTR at the HSC stage and our data provide the first evidence that the stage-specific translational regulation can play an important role in organization of hematopoietic development.

Delineation of the Common Developmental Pathway for Granulocyte/Monocyte and Lymphoid Lineages by Using an Expression Reporter for PU.1.

Understanding how multipotent cells commit to each of their terminal fate potentials is an important aspect of stem cell biology. Hematopoietic stem cells (HSCs) of Lin −Sca-1+c-Kit+ (LSK) phenotype have been purified, which were further divided into CD34-long-term and CD34+ short-term (ST)-HSCs. The existence of phenotypically isolatable common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs) downstream of ST-HSCs suggests that the first commitment step after the HSC stage is the bifurcation of lymphoid vs. myeloid pathway. Recent studies, however, suggest that the loss of MegE potential could be an early event in HSC stage. For example, LSK cells activating RAG-1 or Flt-3 expression retained granulocyte/monocyte (GM) but not megakaryocyte/erythrocyte (MegE) potential together with lymphoid potential, suggesting the existence of common progenitor for GM and lymphoid lineages. Here we report that a fraction of ST-LSK cells expressing high levels of PU.1, a transcription factor necessary for GM and lymphoid development, represents GM/lymphoid bipotent progenitors. In mice harboring knock-in GFP reporter for PU.1, LSK cells were divided into GFP high and GFP low subpopulations. Although PU.1low LSK cells were multipotent, PU.1high LSK cells differentiated only into GM and lymphoid cells in vitro and in vivo. We also found that single PU.1high LSK cells differentiated into GM, T and B cells in vivo. These data formally prove the existence of the third major early progenitor population activating PU.1 at a high level, the granulocyte/monocyte/lymphoid progenitor (GMLP). The existence of prospectively isolatable GMLPs strongly suggests that HSCs sequentially lose MegE then GM potential during their lymphoid commitment.

HFlt3 Expression Marks Long-Term Hematopoietic Stem Cells, but Not the Pathway Common to Granulocyte/Monocyte and Lymphoid Lineages in Human Hematopoiesis.

Flt3 is a tyrosine kinase receptor, whose signals support proliferation and differentiation of early hematopoietic progenitors in cooperation with other cytokine signals. The concept of myeloid vs. lymphoid commitment in murine hematopoiesis has been challenged based on the analysis of Flt3 expression (Adolfsson et al, Cell, 2006): Long-term murine hematopoietic stem cells (HSCs) with self-renewal activity do not express murine Flt3 (mFlt3), but mFlt3 is upregulated in a fraction of progenitors common to granulocyte/monocyte and lymphoid lineages, suggesting the existence of the 3rd pathway of murine hematolymphoid development. In order to test whether Flt3 expression can delineate such a developmental pathway also in human hematopoiesis, we have analyzed the expression of human Flt3 (hFlt3) in prospectively-purified human stem and progenitors (PNAS 99, 2002) by utilizing 7-color FACS and a highly efficient xenograft systems. Surprisingly, all single human hCD34+hCD38-Thy-1+Lin-long-term HSCs expressed Flt3 at a low level. Purified Flt3lo HSCs can reconstitute NOD-scid/IL2Rγnull newborns (Blood 106, 2005) for a long-term after adoptive transfer of only 100 cells, and generated all hematopoietic cells including GM, megakaryocyte/erythrocyte (MegE), T, B and NK cells, providing formal proof that hFlt3 is expressed in long-term multipotent human HSCs. In the myeloid pathway, hCD34+hCD38+hCD45RA-IL-3RαloLin-CMPs expressed low to negative levels of hFlt3, and hCD34+hCD38+CD45RA-IL-3Rα-Lin-MEPs did not express Flt3. However, in marked contrast to murine progenitors, hCD34+hCD38+hCD45RA+IL-3RαloLin-GMPs uniformly expressed hFlt3 at the highest level. We then separated CMPs into 2 fractions with negative and low level expression of Flt3. In vitro culture in the presence of cytokine cocktail revealed that hFlt3lo CMPs differentiated into hFlt3-CMPs, hFlt3-MEPs and hFlt3hi GMPs, while hFlt3-CMPs mainly generated hFlt3-MEPs in vitro, suggesting that hFlt3lo CMPs is located upstream of the hFlt3-CMP stage that has skewed differentiation potential into the MegE lineage. In vitro differentiation activity of these progenitors was not affected with or without hFlt3 ligand in the culture. hFlt3 ligand, however, appeared to be able to maintain survival of hFlt3-expressing stem and progenitor cells at least in vitro. Thus, in striking contrast to mice, hFlt3 expression is already initiated at the most primitive long-term HSC stage, and is progressively upregulated in accordance with GM lineage differentiation. These data collectively suggest that the role of Flt3 signaling in hematopoietic development might be quite different between human and mouse, and that hFlt3 signaling may especially play a critical role in maintenance of long-term HSCs and in development of GM cells in human hematopoiesis.

SNX5, a Novel Gene Linked to Fanconi Anemia Causes Hematopoietic Failure When Knocked down in Zebrafish.

Sorting nexin 5 (SNX 5) is a member of the sorting nexin family, a diverse group of cellular trafficking proteins unified by the presence of a phospholipid binding domain (PHOX) which is involved in protein-protein interactions. While the function of SNX5 is unknown, there is evidence from yeast two hybrid studies that SNX5 binds to FANCA, and SNX5 is one of the 20 genes located on a region on mouse chromosome 2 associated with accelerated stem cell aging. We have recently shown that SNX5 is significantly higher expressed in CD34+CD33-CD38-Rho+ (Rhohi) cells from umbilical cord blood (UCB) and bone marrow (BM), depleted of SCID repopulating cells (SRCs) than in CD34+CD33-CD38-cKIT+ Rho- cells (Rholo), containing all SRCs. A large scale high throughput functional analysis was carried out in zebrafish and genes differentially expressed between Rholo and Rhohi cells were knocked down using morpholino (MO) antisense oligonucleotides. Morpholinos are a useful tool which can specifically inhibit the translation of target mRNA. 16 out of 70 genes knocked down, including snx5, demonstrated decreased circulating blood in the zebrafish injected. Further analysis has shown that MO knock down of snx5 in zebrafish (n=152) results in hematopoietic failure with normal vasculature, with normal expression of scl and gata-1 by in situ hybridization, indicating a defect at or beyond the hematopoietic stem cell stage. Quantitative RT-PCR was used to further confirm the phenotype. Levels of hemoglobin, l-plastin and myeloperoxidase mRNA in snx5 morphants compared to uninjected controls, were significantly lower, consistent with a multi-lineage hematopoietic differentiation defect. The decrease in circulating blood could be partially rescued by overexpressing human SNX5 cDNA, confirming specificity of the morphant phenotype. To determine whether SNX5 might be linked to Fanconi anemia (FA), we measured levels of SNX5 mRNA and protein in EBV transformed cell lines from patients with Fanconi-complementation group A (FANCA), -complementation group C (FANCC) or unknown complementation group (FANC-NX). We have found no significant difference between SNX5 mRNA expression in 11 cell lines compared to Jurkat controls. However, levels of SNX5 protein appeared lower in FANCA cells suggesting a possible role of SNX5 in FANCA, which is being evaluated further. Ongoing studies evaluating the role of SNX5 in normal human hematopoiesis will also be presented.