Ascorbate regulates haematopoietic stem cell function and leukaemogenesis.

Stem-cell fate can be influenced by metabolite levels in culture, but it is not known whether physiological variations in metabolite levels in normal tissues regulate stem-cell function in vivo. Here we describe a metabolomics method for the analysis of rare cell populations isolated directly from tissues and use it to compare mouse haematopoietic stem cells (HSCs) to restricted haematopoietic progenitors. Each haematopoietic cell type had a distinct metabolic signature. Human and mouse HSCs had unusually high levels of ascorbate, which decreased with differentiation. Systemic ascorbate depletion in mice increased HSC frequency and function, in part by reducing the function of Tet2, a dioxygenase tumour suppressor. Ascorbate depletion cooperated with Flt3 internal tandem duplication (Flt3ITD) leukaemic mutations to accelerate leukaemogenesis, through cell-autonomous and possibly non-cell-autonomous mechanisms, in a manner that was reversed by dietary ascorbate. Ascorbate acted cell-autonomously to negatively regulate HSC function and myelopoiesis through Tet2-dependent and Tet2-independent mechanisms. Ascorbate therefore accumulates within HSCs to promote Tet activity in vivo, limiting HSC frequency and suppressing leukaemogenesis.

P-Selectin Sustains Extramedullary Hematopoiesis in the Gata1 low Model of Myelofibrosis.

Splenomegaly is a major manifestation of primary myelofibrosis (PMF) contributing to clinical symptoms and hematologic abnormalities. The spleen from PMF patients contains increased numbers of hematopoietic stem cells (HSC) and megakaryocytes (MK). These MK express high levels of P-selectin (P-sel) that, by triggering neutrophil emperipolesis, may cause TGF-ß release and disease progression. This hypothesis was tested by deleting the P-sel gene in the myelofibrosis mouse model carrying the hypomorphic Gata1(low) mutation that induces megakaryocyte abnormalities that recapitulate those observed in PMF. P-sel(null) Gata1(low) mice survived splenectomy and lived 3 months longer than P-sel(WT) Gata1(low) littermates and expressed limited fibrosis and osteosclerosis in the marrow or splenomegaly. Furthermore, deletion of P-sel disrupted megakaryocyte/neutrophil interactions in spleen, reduced TGF-ß content, and corrected the HSC distribution that in Gata1(low) mice, as in PMF patients, is abnormally expanded in spleen. Conversely, pharmacological inhibition of TGF-ß reduced P-sel expression in MK and corrected HSC distribution. Spleens, but not marrow, of Gata1(low) mice contained numerous cKIT(pos) activated fibrocytes, probably of dendritic cell origin, whose membrane protrusions interacted with MK establishing niches hosting immature cKIT(pos) hematopoietic cells. These activated fibrocytes were not detected in spleens from P-sel(null) Gata1(low) or TGF-ß-inhibited Gata1(low) littermates and were observed in spleen, but not in marrow, from PMF patients. Therefore, in Gata1(low) mice, and possibly in PMF, abnormal P-sel expression in MK may mediate the pathological cell interactions that increase TGF-ß content in MK and favor establishment of a microenvironment that supports myelofibrosis-related HSC in spleen.

Transcription factor Oct1 is a somatic and cancer stem cell determinant.

Defining master transcription factors governing somatic and cancer stem cell identity is an important goal. Here we show that the Oct4 paralog Oct1, a transcription factor implicated in stress responses, metabolic control, and poised transcription states, regulates normal and pathologic stem cell function. Oct1(HI) cells in the colon and small intestine co-express known stem cell markers. In primary malignant tissue, high Oct1 protein but not mRNA levels strongly correlate with the frequency of CD24(LO)CD44(HI) cancer-initiating cells. Reducing Oct1 expression via RNAi reduces the proportion of ALDH(HI) and dye efflux(HI) cells, and increasing Oct1 increases the proportion of ALDH(HI) cells. Normal ALDH(HI) cells harbor elevated Oct1 protein but not mRNA levels. Functionally, we show that Oct1 promotes tumor engraftment frequency and promotes hematopoietic stem cell engraftment potential in competitive and serial transplants. In addition to previously described Oct1 transcriptional targets, we identify four Oct1 targets associated with the stem cell phenotype. Cumulatively, the data indicate that Oct1 regulates normal and cancer stem cell function.

Zebrafish screen identifies novel compound with selective toxicity against leukemia.

To detect targeted antileukemia agents we have designed a novel, high-content in vivo screen using genetically engineered, T-cell reporting zebrafish. We exploited the developmental similarities between normal and malignant T lymphoblasts to screen a small molecule library for activity against immature T cells with a simple visual readout in zebrafish larvae. After screening 26 400 molecules, we identified Lenaldekar (LDK), a compound that eliminates immature T cells in developing zebrafish without affecting the cell cycle in other cell types. LDK is well tolerated in vertebrates and induces long-term remission in adult zebrafish with cMYC-induced T-cell acute lymphoblastic leukemia (T-ALL). LDK causes dephosphorylation of members of the PI3 kinase/AKT/mTOR pathway and delays sensitive cells in late mitosis. Among human cancers, LDK selectively affects survival of hematopoietic malignancy lines and primary leukemias, including therapy-refractory B-ALL and chronic myelogenous leukemia samples, and inhibits growth of human T-ALL xenografts. This work demonstrates the utility of our method using zebrafish for antineoplastic candidate drug identification and suggests a new approach for targeted leukemia therapy. Although our efforts focused on leukemia therapy, this screening approach has broad implications as it can be translated to other cancer types involving malignant degeneration of developmentally arrested cells.

Localized production of IL-10 suppresses early inflammatory cell infiltration and subsequent development of IFN-γ-mediated Lyme arthritis.

IL-10 is a nonredundant inflammatory modulator that suppresses arthritis development in Borrelia burgdorferi-infected mice. Infected C57BL/6 (B6) IL-10(-/-) mice were previously found to have a prolonged IFN-inducible response in joint tissue. Infection of B6 IL-10 reporter mice identified macrophages and CD4(+) T cells as the primary sources of IL-10 in the infected joint tissue, suggesting that early local production of IL-10 dampened the proarthritic IFN response. Treatment of B6 IL-10(-/-) mice with anti-IFN-γ reduced the increase in arthritis severity and suppressed IFN-inducible transcripts to wild-type levels, thereby linking dysregulation of IFN-γ to disease in the B6 IL-10(-/-) mouse. Arthritis in B6 IL-10(-/-) mice was associated with elevated numbers of NK cell, NKT cell, α/ß T cell, and macrophage infiltration of the infected joint. FACS lineage sorting revealed NK cells and CD4(+) T cells as sources of IFN-γ in the joint tissue of B6 IL-10(-/-) mice. These findings suggest the presence of a positive-feedback loop in the joint tissue of infected B6 IL-10(-/-) mice, in which production of inflammatory chemokines, infiltration of IFN-γ-producing cells, and additional production of inflammatory cytokines result in arthritis. This mechanism of arthritis is in contrast to that seen in C3H/He mice, in which arthritis development is linked to transient production of type I IFN and develops independently of IFN-γ. Due to the sustained IFN response driven by NK cells and T cells, we propose the B6 IL-10(-/-) mouse as a potential model to study the persistent arthritis observed in some human Lyme disease patients.

Overexpression of Snai3 suppresses lymphoid- and enhances myeloid-cell differentiation.

The altered expression of transcription factors in hematopoietic stem cells and their subsequent lineages can alter the development of lymphoid and myeloid lineages. The role of the transcriptional repressor Snai3 protein in the derivation of cells of the hemato-poietic system was investigated. Snai3 is expressed in terminal T-cell and myeloid lineages, therefore, we chose to determine if expressing Snai3 in the early stages of hematopoietic development would influence cell-lineage determination. Expression of Snai3 by retroviral transduction of hematopoietic stem cells using bone marrow chimera studies demonstrated a block in lymphoid-cell development and enhanced expansion of myeloid-lineage cells. Analysis of Snai3-expressing hematopoietic precursor cells showed normal numbers of immature cells, but a block in the development of cells committed to lymphoid lineages. These data indicate that the overexpression of Snai3 does alter bone marrow cell development and that the identification of genes whose expression is altered by the presence of Snai3 would aid in our understanding of these developmental pathways.

Targeted deletion of the mouse Mitoferrin1 gene: from anemia to protoporphyria.

Mitoferrin1 is 1 of 2 homologous mitochondrial iron transporters and is required for mitochondrial iron delivery in developing erythroid cells. We show that total deletion of Mfrn1 in embryos leads to embryonic lethality. Selective deletion of Mfrn1 in adult hematopoietic tissues leads to severe anemia because of a deficit in erythroblast formation. Deletion of Mfrn1 in hepatocytes has no phenotype or biochemical effect under normal conditions. In the presence of increased porphyrin synthesis, however, deletion of Mfrn1 in hepatocytes results in a decreased ability to convert protoporphyrin IX into heme, leading to protoporphyria, cholestasis, and bridging cirrhosis. Our results show that the activity of mitoferrin1 is required to manage an increase in heme synthesis. The data also show that alterations in heme synthesis within hepatocytes can lead to protoporphyria and hepatotoxicity.

Enrichment of functionally distinct mouse hematopoietic progenitor cell populations using CD62L.

The details of the bifurcation of the lymphoid and myeloid lineages following commitment by multipotent progenitor cells (MPP) remain a topic of controversy. We report that the surface glycoprotein CD62L can be characterized as a novel marker of this and other stages of early hematopoietic differentiation. Cell isolation and transplant studies demonstrated CD62L(neg/low) long-term hematopoietic stem cells and CD62L(high) MPP within the traditionally defined c-kit(pos)Lin(neg/low)Sca-1(pos) stem/progenitor cell population. Within the MPP population, previously defined as c-kit(pos)Lin(neg/low)Sca-1(pos)-Thy-1.1(neg)Flt3(pos), Sca-1 and CD62L resolved four populations and segregated Sca-1(high)CD62L(neg/low) MPP from Sca-1(high)CD62L(high) leukocyte-biased progenitors. Using a novel transplantation method that allows tracking of erythroid and platelet engraftment as an alternative to the classical method of in vitro colony formation, we characterized Sca-1(high)CD62L(neg/low) cells as MPP, based on transient engraftment of these lineages. These data establish CD62L as a useful tool in the study of early hematopoiesis and emphasize the power of trilineage-engraftment studies in establishing the lineage potential of MPP subsets.

Erythropoietin receptor signaling regulates both erythropoiesis and megakaryopoiesis in vivo.

Transgenic expression of a gain-of-function truncated mouse erythropoietin receptor gene (EpoR) leads to expansion of the HSC pool in response to human erythropoietin (Epo). We have re-examined this observation using a knock-in mouse model, wherein the mouse EpoR gene was replaced in its proper genetic locus by a single copy of either a wild-type human or a polycythemia-inducing truncated human EPOR gene. Bone marrow cells obtained from knock-in mice were transplanted together with competitor bone marrow cells in a model that allows tracking of erythroid, platelet, and leukocyte contributions by each genotype. Secondary transplants were also performed. Stem/progenitor cells were identified phenotypically and isolated for colony-forming assays to evaluate cytokine responsiveness by cells with the wild-type human or truncated human EPOR gene. Augmented Epo signaling increased erythroid repopulation post-transplant as expected, but had no effect on short-term or long-term leukocyte repopulation. However, the wild-type human EPOR knock-in mouse showed decreases in both erythroid and platelet repopulation compared to marrow cells from the mutant human EPOR knock-in mouse or normal B6 animals. These results provide evidence supporting a role for Epo signaling in megakaryopoiesis in vivo and suggest a role for Epo signaling early in hematopoietic development.

Transcription factor Oct1 protects against hematopoietic stress and promotes acute myeloid leukemia.

A better understanding of the development and progression of acute myelogenous leukemia (AML) is necessary to improve patient outcome. Here we define roles for the transcription factor Oct1/Pou2f1 in AML and normal hematopoiesis. Inappropriate reactivation of the CDX2 gene is widely observed in leukemia patients and in leukemia mouse models. We show that Oct1 associates with the CDX2 promoter in both normal and AML primary patient samples, but recruits the histone demethylase Jmjd1a/Kdm3a to remove the repressive H3K9me2 mark only in malignant specimens. The CpG DNA immediately adjacent to the Oct1 binding site within the CDX2 promoter exhibits variable DNA methylation in healthy control blood and bone marrow samples, but complete demethylation in AML samples. In MLL-AF9-driven mouse models, partial loss of Oct1 protects from myeloid leukemia. Complete Oct1 loss completely suppresses leukemia but results in lethality from bone marrow failure. Loss of Oct1 in normal hematopoietic transplants results in superficially normal long-term reconstitution; however, animals become acutely sensitive to 5-fluorouracil, indicating that Oct1 is dispensable for normal hematopoiesis but protects blood progenitor cells against external chemotoxic stress. These findings elucidate a novel and important role for Oct1 in AML.

Gradients of antigen expression and developmental potential in hematopoiesis.

Prospective isolation of hematopoietic stem and progenitor cell subsets depends upon the premise that expression of combinations of surface antigens reflects developmental potential. During the process of differentiation, however, the loss of antigens associated with stem cells and the concomitant gain of those associated with progenitor cells often occurs as a continuum rather than by discrete binary steps. Coupled with the fact that assay conditions can profoundly influence the developmental fates of prospectively isolated cells, gradients of antigen expression during differentiation have led to a variety of interpretations of lineage commitment in hematopoiesis.

Distinct roles of IL-7 and stem cell factor in the OP9-DL1 T-cell differentiation culture system.

OBJECTIVE: The OP9-DL1 culture system is an in vitro model for T-cell development in which activation of the Notch pathway by Delta-like 1 promotes differentiation of mature T cells from progenitors. The roles of specific cytokines in this culture system have not been well defined, and controversy regarding the role of IL-7 has recently emerged. We examined the roles played by IL-7, Flt3 ligand, and stem cell factor (SCF) in differentiation of adult bone marrow cells in the OP9-DL1 culture system. METHODS: Hematopoietic progenitor cells isolated from mouse bone marrow were cultured with OP9 or OP9-DL1 stromal cells and evaluated for T and B lymphocyte differentiation using immunofluorescent staining. RESULTS: IL-7 provided both survival/proliferation and differentiation signals in a dose-dependent manner. T-cell development from the CD4/CD8 double-negative (DN) stage to the CD4/CD8 double-positive (DP) stage required IL-7 provided by the stromal cells, while differentiation from the DP to the CD8 single-positive (SP) stage required addition of exogenous IL-7. SCF favored the proliferation of DN lymphoid progenitors and inhibited differentiation to the DP stage in a dose-dependent manner. Conversely, blocking the function of SCF expressed endogenously by OP9-DL1 cells inhibited proliferation of lymphoid progenitors and accelerated T-lineage differentiation. Flt3 ligand promoted proliferation without affecting differentiation. CONCLUSION: These results validate the OP9-DL1 model for the analysis of T-cell development from bone marrow-derived progenitor cells, and demonstrate specific roles of SCF, IL-7, and Flt3L in promoting efficient T-lineage differentiation.

Lymphoid potential of primitive bone marrow progenitors evaluated in vitro.

Bone marrow contains a heterogeneous mixture of mature and maturing precursors of blood cells, progenitor cells for myeloid and lymphoid lineages, and hematopoietic and mesenchymal stem cells. The differentiation potential of these different stem, progenitor, and precursor populations can be evaluated by using transplantation and cell culture assays. In this study, we used a stromal cell co-culture system to evaluate the B and T lineage potential of different subsets of mouse bone marrow. We enriched hematopoietic stem (Lin(-)Sca-1(+)c-kit(+)Thy1.1(low) [Thy1.1(low)]) cells and lymphoid progenitor (Lin(-)Sca-1(+)c-kit(+)Thy1.1(-) [Thy1.1(-)]) cells from mouse bone marrow and co-cultured these populations with OP9 or OP9-DL1 stromal cell lines. Development of the B and T lineages was evaluated over time. Both populations gave rise to B and T cells but with different kinetics. Thy1.1(-) lymphoid progenitors gave rise to B and T lineage cells earlier than did Thy1.1(low) stem cells; and at any given time, percentages of differentiating B and T cells were higher in Thy1.1(-) cultures than in Thy1.1(low) cultures. We also compared the lineage potential of Thy-1.1(-) lymphoid progenitors with that of the recently described common lymphoid progenitor 2 (isolated as Lin(-)Sca-1(+)c-kit(-)Thy1.1(-)B220(+) cells [B220(+)]). B220(+) cells produced B lineage progeny in OP9 cultures more rapidly than did Thy1.1(-) cells and produced higher percentages of differentiating T cells in OP9-DL1 cultures. These studies demonstrate the utility of the OP9 and OP9-DL1 co-culture systems for evaluation of lymphoid lineage potential and for determining the relative position of specific bone marrow populations within the hematopoietic hierarchy.

Future challenges for hematopoietic stem cell research.

This perspective summarizes several important advances in hematopoietic stem cell (HSC) biology in the past few years and places these advances in the context of future directions in stem cell research. The potential utility of stem cells for gene therapy, tissue engineering, and the treatment of neurological and other forms of disease is simply too significant to ignore, and yet our knowledge and ability to deliver these forms of therapy in a safe and efficacious manner will require additional advances in the understanding of the basic biology of stem cells.

When is a stem cell really a stem cell?

While bone marrow transplantation has long been established as an effective approach to the clinical management of a variety of malignant and nonmalignant diseases, the future application of pluripotent stem cells in transplant settings promises to deliver this therapy to a much broader range of indications. In this review, I summarize the emerging field of embryonic stem cell biology in the context of potential clinical applications and regulatory issues.

Major age-related changes of mouse hematopoietic stem/progenitor cells.

To study age-related changes of mouse bone marrow (BM) cells and hematopoietic stem cells (HSCs), we isolated rhodamine-123(low) (Rh(low)) Thy1.1(low) Lin(-)Sca-1(+) (TLS) HSCs from the BM of old mice and compared their functional characteristics to cells of the same phenotype isolated from young mice. We observed impaired recovery of B lymphocytes and decreased self-renewal in recipients of old Rh(low) cells compared to young Rh(low) cells. Blockade of Rh efflux using verapamil improved lymphoid reconstitution by enriched HSCs, and isolation of aged HSCs based on efflux of a fluorescent multi-drug resistance (MDR) substrate (Bodipy-verapamil) resulted in enrichment of HSC activity equivalent to that obtained with Rh. These observations suggest a complex relationship between MDR activity and HSC function during aging. To address whether the difference between young and aged donors was intrinsic to the HSC compartment or was due to a shift in HSC phenotype, we co-transplanted normal BM derived from young or old donors and followed repopulation simultaneously in the same recipient animals. In a parallel experiment, we co-transplanted HSCs purified from old donors with BM derived from young donors. In both experiments, transplants were given to both young and old recipients. The results show a clear defect in B-cell engraftment from either BM or HSCs of old donors, irrespective of the age of the recipient. In contrast, myeloid engraftment was predominantly derived from BM or HSCs derived from aged donors, again irrespective of recipient age. These data suggest a stem cell basis for B-cell immuno-senescence and the increased incidence of myelocytic leukemia in elderly people.

Early stages of hematopoietic differentiation.

Mouse bone marrow contains hematopoietic stem cells as well as progenitor cells, which are partially differentiated offspring of stem cells. We have utilized several approaches to separate progenitors from stem cells in order to characterize essential differences between these two stages of development. As a first approach, we utilized the supravital fluorescent dye rhodamine-123 (Rh-123) to distinguish quiescent stem cells (Rh-123(low)) from metabolically active progenitor cells (Rh-123(hi)). Analysis of megakaryocyte potential in a tissue culture assay demonstrated that Rh-123(hi) progenitor cells were capable of robust megakaryocyte differentiation, while Rh-123(low) stem cells produced fewer colonies containing megakaryocytes. Transplantation of the two cell populations into irradiated recipients revealed the opposite outcome, suggesting that the tissue culture assay failed to predict behavior in a transplant setting. We also evaluated functional potential of lymphoid progenitors isolated by selecting for differential expression of Thy-1.1 and c-kit. The potential of defined cell populations to differentiate as T or B lymphocytes in vivo was dependent upon the time post transplant at which animals were evaluated. These studies underscore the need for caution in the interpretation of lineage potentials evaluated by both in vitro and in vivo assays.

Effects of caspase inhibitors on hematopoietic engraftment after short-term culture.

The induction of apoptosis during cytokine-induced proliferation of hematopoietic stem and progenitor cells (HSPC) may result in the loss of hematopoietic function. We tested the ability of several caspase inhibitors to maintain transplantation potential of mouse HSPC during in vitro culture. HSPC were isolated from mouse bone marrow by cell sorting and cultured in the presence of steel factor (STL) with or without various caspase inhibitors. After incubation, cells were harvested and tested for in vitro colony-forming cell (CFC) potential and transplantation activity in both short- and long-term in vivo assays. HSPC required STL to retain CFC activity during a 24-h culture at 37 degrees C, and none of three caspase inhibitors could substitute for STL in this respect. In transplant assays, a twofold higher frequency of animals showed donor-derived blood cells 12 weeks after competitive transplantation of 50 HSPC cultured for 4 h in the presence of STL plus n-acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone (ac-YVAD) compared with 50 cells cultured in STL alone. To evaluate the effect of ac-YVAD on short-term engraftment, 500 cultured HSPC were transplanted into lethally irradiated mice. Animals transplanted with cells cultured in the presence of ac-YVAD showed a higher survival rate and a faster recovery of platelets and hematocrit compared with animals transplanted with cells cultured in STL alone. We conclude that both the short-term and the long-term engraftment potentials of HSPC cultured in the presence of STL + ac-YVAD were superior to that obtained from cells cultured in STL alone.

Developing and mature human granulocytes express ELP 6 in the cytoplasm.

BACKGROUND: c3orf75 is a conserved open reading frame within the human genome and has recently been identified as the Elongator subunit, ELP6 [1]. The Elongator enzyme complex has diverse roles, including translational control, neuronal development, cell migration and tumorigenicity [2]. OBJECTIVE: To identify genes expressed early in human eosinophil development. METHODS: Eosinophilopoiesis was investigated by gene profiling of IL-5 stimulated CD34+ cells; ELP6 mRNA is upregulated. A monoclonal antibody was raised to the recombinant protein predicted by the open reading frame. RESULTS: ELP6 transcripts are upregulated in a human tissue culture model of eosinophil development during gene profiling experiments. Transcripts are expressed in most tissue types, as shown by reverse-transcriptase PCR. Western blot experiments show that human ELP6 is a 30 kDa protein expressed in the bone marrow, as well as in many other tissues. Flow cytometry experiments of human bone marrow mononuclear cells show that ELP6 is expressed intracellularly, in developing and mature human neutrophils, eosinophils and monocytes. CONCLUSIONS: ELP6 is expressed intracellularly in developing and mature granulocytes and monocytes but not in lymphocytes and erythrocytes.

Nicotinic receptor alpha7 expression identifies a novel hematopoietic progenitor lineage.

How inflammatory responses are mechanistically modulated by nicotinic acetylcholine receptors (nAChR), especially by receptors composed of alpha7 (α7) subunits, is poorly defined. This includes a precise definition of cells that express α7 and how these impact on innate inflammatory responses. To this aim we used mice generated through homologous recombination that express an Ires-Cre-recombinase bi-cistronic extension of the endogenous α7 gene that when crossed with a reporter mouse expressing Rosa26-LoxP (yellow fluorescent protein (YFP)) marks in the offspring those cells of the α7 cell lineage (α7(lin+)). In the adult, on average 20-25 percent of the total CD45(+) myeloid and lymphoid cells of the bone marrow (BM), blood, spleen, lymph nodes, and Peyers patches are α7(lin+), although variability between litter mates in this value is observed. This hematopoietic α7(lin+) subpopulation is also found in Sca1(+)cKit(+) BM cells suggesting the α7 lineage is established early during hematopoiesis and the ratio remains stable in the individual thereafter as measured for at least 18 months. Both α7(lin+) and α7(lin-) BM cells can reconstitute the immune system of naïve irradiated recipient mice and the α7(lin+):α7(lin-) beginning ratio is stable in the recipient after reconstitution. Functionally the α7(lin+):α7(lin-) lineages differ in response to LPS challenge. Most notable is the response to LPS as demonstrated by an enhanced production of IL-12/23(p40) by the α7(lin+) cells. These studies demonstrate that α7(lin+) identifies a novel subpopulation of bone marrow cells that include hematopoietic progenitor cells that can re-populate an animal's inflammatory/immune system. These findings suggest that α7 exhibits a pleiotropic role in the hematopoietic system that includes both the direct modulation of pro-inflammatory cell composition and later in the adult the role of modulating pro-inflammatory responses that would impact upon an individual's lifelong response to inflammation and infection.