LKS Cells Research Articles

3149 – COMPARISON OF PRE-ENRICHMENT STRATEGIES FOR SORTING OF MOUSE LIN-CKIT+SCA1+ CELLS

The short life span of blood cells requires hematopoiesis to be a continuous ongoing process. In adult mammals, blood production takes place in the bone marrow. This tissue is home to hematopoietic stem cells (HSCs) that possess the ability to both self-renew and differentiate into mature blood cells. HSCs are widely studied in cancer research, as they are the source of various hematological malignancies. Such cancers develop in response to a progressive accumulation of mutations that confer a selective growth advantage to HSCs. In the case of leukemia, the mutations will lead to a differentiation blockade, causing dysfunctional blood cells to accumulate. Mouse models are the system of choice to study blood cancers given the complexity of the hematopoietic system. Thus, in order to study HSCs, we need to be able to isolate them from mice. Several HSC subpopulations have been defined, however, the most widely studied are the Lin-cKit+Sca1+ (LKS) cells. The best way to isolate LKS cells is by FACS sorting. Yet, the frequency of LKS cells in the bone marrow being very low (< 0.1%), a pre-enrichment step is necessary to decrease the sorting time. With the aim of identifying the most suitable pre-enrichment strategy, we tested several magnetic kits and compared them based on enrichment efficiency and final LKS yield. Four approaches were tested: lineage depletion (Lin-), cKit-enrichment (cKit+), Sca1-enrichment (Sca1+) and a combination of lineage depletion followed by Sca1-enrichment (Lin- Sca1+). Our results show that the best strategy are lineage depletion and Sca1-enrichment. Combining these two methods highly increased the LKS enrichment, however, the final LKS yield was low due to increased cell loss from performing a second enrichment step.

Overexpression of Calreticulin Impairs Self-Renewal Capacity of Hematopoietic Stem Cells

Mutations in JAK2, c-MPL and CALR have all been identified as drivers of MPN pathogenesis. JAK2 and c-MPL are critical for hematopoietic stem cell self-renewal and differentiation, however CALR's physiologic role in hematopoiesis is unknown. To investigate CALR's role in normal and malignant hematopoiesis lethally irradiated C57B/6 mice were transplanted with bone marrow cells infected with CALRDEL, CALRWT or empty MSCV-IRES-hCD4 vector retrovirus. As expected, mice transplanted with CALRDEL developed thrombocytosis, mice transplanted with CALRWT and empty vector had normal peripheral blood counts. At 6 months post-transplant mice were sacrificed. The percentage of hCD4+ whole bone marrow cells (marks cells expressing gene of interest) was 76.6% for empty vector, 73.1% for CALRWT, and 8.6% for CALRDEL. hCD4+ hematopoietic stem/progenitor cells (LKS), common myeloid progenitors (CMP), granulocyte monocyte progenitors (GMP), and megakaryocyte erythroid progenitors (MEP) from empty vector, CALRWT and CALRDEL mice were sorted into methylcellulose supplemented with cytokines (mIL-3, hEPO, and mSCF). MEP from CALRWT mice formed fewer colonies compared to empty vector and CALRDEL (p>0.004). GMP, CMP, and LKS from empty vector, CALRWT and CALRDEL formed equivalent numbers of colonies. To assess self-renewal function colonies were amalgamated and serially replated into methylcellulose at a concentration of 10,000 cells/ml. Upon serial replating CALRWT LKS cells formed fewer colonies compared to CALRDEL and empty vector (p>0.001). To more specifically assess the effect of CALR on HSC self renewal potential whole bone marrow from mice transplanted with CALRDEL, CALRWT or empty MSCV-IRES-GFP (MIG) vector was serially transplanted into lethally irradiated C57B/6 secondary recipient mice and the percentage of GFP-positive cells in the peripheral blood was measured by flow cytometry over time. Mice transplanted with CALRWT bone marrow cells quickly lost GFP positive cells in the peripheral blood over time whereas MIG empty vector and CALRDEL mice retained GFP positive cells for at least 8 months post-transplant.Because mutant CALRDEL interacts with and activates the thrombopoietin receptor (c-MPL) to drive MPN and because the defect in hematopoietic stem cell self-renewal that we observed in mice overexpressing CALRWT is reminiscent of the phenotype of mice lacking c-MPL we hypothesize that CALRWT interacts with c-MPL and negatively affects its function. Using co- immunoprecipitation of Ba/F3 cells doubly transduced with GFP tagged c-MPL and hCD4 tagged empty vector, CALRWT or CALRDEL, we found that CALRWT and CALRDEL interacts with c- MPL.In summary, these data demonstrate that overexpression of CALRWT negatively affects the self-renewal capacity of hematopoietic stem cells, evidenced by impaired ability to reconstitute secondary hosts and reduced serial re-plating ability in methylcellulose. This may be due to CALRWT binding to c-MPL and negatively regulating its function. DisclosuresNo relevant conflicts of interest to declare.

Acute Alcohol Intoxication Impairs Sonic Hedgehog-Gli1 Signaling and Activation of Primitive Hematopoietic Precursor Cells in the Early Stage of Host Response to Bacteremia.

Activation of hematopoietic stem cells [HSCs, lineage(lin)- stem cell growth factor receptor (c-kit)+ stem cell antigen-1(Sca-1)+ , or LKS cells in mice] is critical for initiating the granulopoietic response. This study determined the effect of alcohol exposure on sonic hedgehog (SHH) signaling in the regulation of HSC activation during bacteremia. Acute alcohol intoxication was induced in mice by intraperitoneal (i.p.) injection of 20% alcohol (5g alcohol/kg body weight). Control mice received i.p. saline. Thirty minutes later, mice were intravenously (i.v.) injected with Escherichia coli (E.coli, 1 to 5×107 CFUs/mouse) or saline. SHH expression by lineage-negative bone marrow cells (BMCs) was significantly increased 24hours after E.coli infection. Extracellular signal-regulated kinase 1/2 (ERK1/2)-specificity protein 1 (Sp1) signaling promotes SHH expression. ERK1/2 was markedly activated in BMCs 8hours following E.coli infection. Alcohol suppressed both the activation of ERK1/2 and up-regulation of SHH expression following E.coli infection. E.coli infection up-regulated GLI family zinc finger 1 (Gli1) gene expression by BMCs and increased Gli1 protein content in LKS cells. The extent of Gli1 expression was correlated with the activity of proliferation in LKS cells. Alcohol inhibited up-regulation of Gli1 expression and activation of LKS cells in response to E.coli infection. Alcohol also interrupted the granulopoietic response to bacteremia. These data show that alcohol disrupts SHH-Gli1 signaling and HSC activation in the early stage of the granulopoietic response, which may serve as an important mechanism underlying the impairment of immune defense against bacterial infection in host excessively consuming alcohol.

3043 - Elevation of IL4 Signaling Contributes to Thrombocytopenia in AML

Thrombocytopenia is a major and fatal complication in the patients with acute leukemia. However, the mechanisms underlying defective thrombopoiesis in leukemia remains unclear. In this study, we employed the non-irradiated MLL-AF9 induced acute myeloid leukemia (AML) murine model (Cheng et al, 2015) to define how megakaryopoiesis is suppressed during AML development. We measured the frequency of hematopoietic stem and progenitor (HSPC) subsets in the residual hematopoietic cells along the path of megakaryopoiesis and found that the frequency of megakaryocytic/erythroid progenitors (preMegEs) declined dramatically (14 ± 4% of healthy state), whereas the frequency of Lin-Sca-1 + c-kit+CD150+ hematopoietic stem cells (HSCs) gradually increased (3.1 ± 0.4 folds of healthy control), suggesting a blockade of megakaryopoiesis from CD150+ HSCs. Consistently, we observed reduced megakaryocyte (MK) colony forming ability (72.3 ± 3.4 vs 89.0 ± 1.0%, p = 0.097) in vitro and defective platelet reconstitution potential after transplantation of CD150+ LKS cells (4.3 ± 0.8 vs 13.8 ± 2.3%, p = 0.002) from AML mice. To search for a novel soluble factor involved in the suppressive megakaryopoiesis, we detected elevated Interleukin 4 (IL4) protein level in bone marrow plasma and upregulated IL4 mRNA expression in bone marrow endothelial cells in AML mice. Notably, platelet counts325 ± 58 vs 991 ± 87 X1012/L, p < 0.001), the frequency of MKs0.008 ± 0.001 vs 0.02 ± 0.002%, p < 0.001and preMegEs0.089 ± 0.003 vs 0.19 ± 0.026%, p = 0.0043in the mice receiving IL4 dropped dramatically, which indicated that IL4 had negative effects on megakaryocytic differentiation of HSPCs and MK maturation, and IL4 treatment could phenocopy thrombocytopenia in AML to some extent. Moreover, administration of IL4 neutralizing antibody partially recovered the yield of MK colonies in the presence of leukemia bone marrow plasma32.5 ± 1.555 vs 46.5 ± 2.598, p = 0.0036.In summary, our current study demonstrates that defective megakaryopoiesis in AML was at least partially contributed by the suppression of HSC differentiation toward megakaryocytic lineage and also reveals a previously unrecognized link between IL4 signaling and megakaryopoiesis.

Embigin Regulates HSPC Homing and Quiescence and Acts As a Cell Surface Marker for a Niche Factor-Enriched Subset of Osteolineage Cells

Background. Discovery of niche-derived HSPC regulators is critical for further development of novel therapeutic approaches to promote HSPC regeneration. We have previously reported a proximity-based approach to the study of the bone marrow niche, which is based on the transcriptional comparison of osteolineage cells (OLCs) located close (proximal OLCs) or further away from transplanted HSPC (Blood 2014;124: 773), which lead to identification of IL18 as a quiescence regulator of early progenitors. We now report the results of functional validation for another molecule - Embigin - identified through this strategy as a hematopoietic regulator.Results. Embigin is a cell adhesion molecule with poorly characterized function. Our analysis showed that Embigin expression in proximal OLCs was significantly higher. Since the genetic tools for this molecule do not exist, we used a neutralizing antibody against Embigin to investigate the effect of Embigin blockade on primitive hematopoietic cells. We found that injection of anti-Embigin resulted in mobilization of myeloid progenitors and colony-forming cells (CFC) into the blood. In contrast, Embigin blockade was associated with a homing defect when LKS cells [known to express Embigin] were either pre-incubated with anti-Embigin antibody or injected into anti-Embigin pretreated animals, overall suggesting that Embigin regulates retention and localization of primitive hematopoietic cells in the bone marrow. Moreover, animals treated with anti-Embigin antibody had a higher frequency and proliferative activity of primitive hematopoietic cells, as demonstrated by cell cycle and BrdU incorporation studies and an increased CFC, consistent with Embigin-mediated localization also affecting HSPC cell cycling. Bone marrow from anti-Embigin treated mice reconstituted poorly when competitively transplanted with untreated animal marrow into irradiated recipients, likely due to the impaired homing and increased cell cycling. Finally, pre-treating irradiated recipients with anti-Embigin resulted in increased proliferation of transplanted WT LKS cells. Collectively, these data are consistent with Embigin serving as a regulator of HSPC localization and quiescence.Given a functional role of Embigin in the bone marrow niche and an overlapping pattern of expression with VCAM1, a known niche-derived HSPC regulator, in proximal OLCs we explored the use of Embigin in conjunction with VCAM1 as cell surface markers for prospective isolation of niche factor-enriched OLC subset by flow cytometry. Using this strategy, we were able to purify a rare OLC subset of CD45- Ter119-V CAM-1+E mbigin+ cells (termed VE cells), which were enriched for niche factor expression as compared to their non-VE counterparts. VE cells were transcriptionally distinct from other, previously defined niche subsets such as nestin-GFPdim mesenchymal stem cells, nestin-GFPbright pericytes and N-cadherin-positive osteoblastic cells. In particular, VE cells expressed higher levels of most niche factors than nestin-GFPbright cells - a cell population recently characterized as regulating HSPC quiescence. Interestingly, the expression profile of VE cells from animals transplanted with LT-HSCs and those which were irradiated but injected with saline alone demonstrated upregulation of cell adhesion molecules in the LT-HSC-injected group, suggesting that VE cells are involved in bidirectional communication within the niche.To investigate a functional role of VE cells, we performed co-culture experiments in which we compared the effect of VE and non-VE cells on HSPC proliferation and engraftment. We found that in the presence of VE cells, HSPCs proliferated at slower rate and generated a lower hematopoietic colony number, consistent with quiescence-inducing effect. Upon transplantation, VE-cultured HSPCs generated a higher level of chimerism compared to those cultured on non-VE layer, indicating a superior ability of VE cells to support engraftment and reconstitution properties of HSPC during the in vitro culture.Conclusion. Our work defines Embgin as a previously unrecognized hematopoietic regulator and a cell surface marker for a niche factor-enriched subset of the osteolineage cells which regulates HSPC quiescence. Pharmacological blockade of Embigin signaling may serve as a potential therapeutic tool to enhance hematopoietic regeneration. DisclosuresNo relevant conflicts of interest to declare.

Depletion of Ptch2 Activates Canonical and Non-Canonical HH Signaling within the Niche Leading to Myeloproliferation, Stem Cell Exhaustion and Accelerates JAK2V617F Driven Disease

MPNs often transform into secondary AML associated with poor prognosis and high mortality due to limited therapeutic options. Here we show that aberrant Hedgehog ligand secretion in MPN and CML not only induces constitutive Smoothened-dependent canonical HH signaling causing GLI1 transcription, but also causes PTCH1-dependent non-canonical HH signaling leading to constitutive ERK activation.Our investigations show, that depletion of the Ptch2 receptor in vitro and in vivo recapitulates overactivation of both pathways, while depletion of Ptch1 only reflects constitutive Gli1 activation pinpointing the important role of Ptch1 for Erk activation (Figure 1). Ptch2-/- mice develop a pronounced hematopoietic phenotype with leukocytosis driven by an increase in neutrophils, anemia, thrombocytopenia, loss of T cells combined with a strong increase of cKit+ progenitor and stem cells in the peripheral blood and increased extramedullary hematopoiesis causing splenomegaly reflecting a MPN phenotype. HSCs (Lin- cKit+ Sca1+) residing within the BM (bone marrow) showed enhanced cycling properties causing exhaustion and loss of LKS cells over time (Figure 2), but improved stress hematopoiesis after 5-FU treatment. Niche change experiments show that cytopenias and loss of LKS cells are caused by overactivated HH signaling within the niche cells, causing depletion of osteoblasts and alterations of essential niche factors like Cxcl12, Angiopoietin, Stem cell factor, Thrombopoietin or Jagged1 (Figure 2). In contrast, the hematopoietic Ptch2-/- is responsible for leukocytosis and even promotes HSC expansion and replating capacity in vitro.Interestingly, depletion of Ptch2 in the niche or within hematopoietic cells dramatically altered JAK2V617F-driven pathogenesis causing transformation of a non-lethal chronic myeloproliferative disease into an aggressive AML-like disease with up to 30% blasts in the peripheral blood (Figure 3). In contrast, the BCR-ABL-driven leukemia was exclusively accelerated by the cell intrinsic Ptch2-/-, but not by cell extrinsic HH activation.In conclusion, combined constitutive canonical and non-canonical HH activation induced by depletion of Ptch2 causes a MPN phenotype driven by cell intrinsic, but mainly cell extrinsic mechanisms and accelerates myeloproliferative diseases caused by JAK2V617F and BCR-ABL into acute leukemias. Therefore HH ligand-blocking antibodies or a combination of ERK and SMO inhibitors might be a potential treatment option to prevent transformation of MPNs. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Twist-1 Increases Hematopoietic Stem Cell Self-Renewal and Causes Myeloid Skewing

Twist-1 protein belongs to the large family of basic Helix-Loop-Helix transcription factors with diverse physiological functions in mesoderm-derived tissues. Growing evidences now link Twist-1 to the acquisition of stem-cell-like properties. However, there is little information available regarding its expression pattern and functional role in the hematopoietic system. We analyzed Twist-1 expression patterns in different hematopoietic cell populations from normal mouse bone marrow, and found that Twist-1 was most highly expressed in long-term hematopoietic stem cells (LT-HSCs) but showed a low abundance in more differentiated descendants. To investigate Twist-1 gene function, retroviral-mediated overexpression or removal experiments were performed. Competitive repopulation studies demonstrated that enforced expression of Twist-1 in HSC-enriched Lin−c-Kit+Sca-1+ (LKS) cells resulted in an increase in the size of the G0 population, and in their reconstitution ability after the first and a second transplantation. Conversely, removal of Twist-1 in LKS cells impaired their ability to repopulate. In addition, increased Twist-1 expression caused a shift toward production of myeloid cells. Twist-1 transduction in LKS cells activated myeloid lineage-determining factors PU.1 and GATA-1 and down-regulated lymphoid factor GATA-3 in vitro, suggesting that Twist-1-mediated myeloid skewing occurs in hematopoietic stem and progenitor cells (HSPCs). These findings indicate that Twist-1 is not only involved in the maintenance of HSC dormancy and self-renewal capacity but also implicated in the myeloid lineage fate choice of HSPCs. Exploration of the underlying mechanisms revealed that Twist-1 overexpression lead to altered expression of Runx1/c-Mpl/Tie2 regulatory pathway, and quiescence-associated N-cadherin and Hes1 in LKS cells, which may contribute to the phenotypes observed in Twist-1-overexpressing mice. These studies shed additional light on the mechanisms involved in the maintenance of normal HSC and myeloid lineage differentiation, and may also provide clues to the mechanisms controlling pathogenesis and preservation of leukemic stem cells. DisclosuresNo relevant conflicts of interest to declare.

Proximity-Based Single Cell Analysis of the Bone Marrow Niche Identifies Interleukin-18 As a Quiescence Regulator of Early Hematopoietic Progenitors

Background. Niche-derived signals are essential for maintenance and expansion of stem/progenitor cells in the bone marrow. Recently published studies used elegant genetic tools to dissect predominant cellular sources of known HSPC regulators, such as CXCL12 and kit-ligand (Ding et al, Nature 2012, Greenbaum et al, Nature 2013). However, strategies enabling identification of novel niche-derived factors are lacking. Here, we describe an approach which utilizes spatial proximity between osteolineage cells (OLC) and HSPC in the post-transplant bone marrow niche as a guide to niche factor discovery and reveals the role of Interleukin-18 (IL18) as a quiescence regulator of early hematopoietic progenitors.Results. We established a neonatal bone marrow (BM) transplantation model, in which adult BM-derived LKS CD34-Flk2- HSPCs fluorescently labeled with DiI were transplanted into irradiated newborn col2.3GFP recipients (in this mouse strain, the majority of OLCs are labeled with GFP). Forty-eight hours later, we obtained sections of femoral trabecular bone from transplanted mice and located rare OLCs harboring DiI-positive HSPCs in their immediate proximity. We harvested individual OLCs located within two cell diameters (proximal OLCs) and greater than five cell diameters (distal OLCs) from DiI-labeled HSPCs and performed comparative transcriptome analysis by single cell RNA-Seq. Remarkably, we found that proximal OLCs were distinct from their distal counterparts and showed a higher expression levels of known niche-associated molecules, as well as secreted factors not previously linked to regulatory niche function, including a pro-inflammatory cytokine IL18.Quantification of primitive subsets in the BM of IL18 knock-out (IL18KO) mice showed no abnormalities. However, cell cycle studies revealed that while long-term HSCs (which did not express the IL18 receptor) were unaffected by IL18 deletion, early progenitors - short-term HSC, multi-potent progenitor and common lymphoid progenitor – were more actively cycling. Accordingly, following sublethal exposure to 5-fluorouracil (5FU), IL18KO animals displayed a 2-fold increase in frequency of lin-kit+Sca1+ (LKS) cells, lin-kit+ myeloid progenitors and CLPs, as compared wild-type (WT) controls (Figure 1). On the other hand, exogenous administration of recombinant IL18 to 5FU-treated WT animals was associated with 25% mortality, while no deaths were observed in the vehicle-treated group. Taken together, these data illustrate that IL18 constrains the ability of the progenitor pool to respond to a genotoxic stress.Next, we tested if IL18 acts in a non cell-autonomous fashion. We transplanted progenitor-enriched bone marrow fraction (LKS cells) into IL18KO or WT hosts and observed an approximately 2-fold increase in both myeloid and lymphoid cells in peripheral blood of IL18KO animals. This finding was recapitulated in a reciprocal experiment, when LKS cells from IL18 receptor knock-out animals were transplanted into WT hosts, supporting the possibility that the effect of IL18 on progenitor proliferation is direct.Finally, we investigated if the loss of IL18-mediated quiescence can be therapeutically exploited in the transplant setting. To this end, we assessed survival of lethally irradiated WT and IL18KO mice following transplantation with a limiting BM dose, i.e. predicted to confer survival to 50% or less of WT animals. Notably, we observed reduced 30-day post-transplant mortality (33% versus 72%, p value = 0.05) in IL18KO animals, which likely resulted from accelerated progenitor expansion in the absence of IL18 in the host microenvironment (Figure 2).Conclusions.Our study demonstrates the capability of proximity-based single cell analysis of the post-transplant bone marrow microenvironment to identify a novel niche factor – IL 18. We show that IL18 specifically regulates quiescence of early hematopoietic progenitors, particularly under the conditions of stress hematopoiesis, such as exposure to 5FU or post-transplant hematopoietic expansion. The results presented here provide an insight into poorly understood molecular mechanisms of progenitor regulation by the niche. Moreover, improved post-transplant survival of animals in the absence of IL18 suggests that pharmacological inhibition of IL18 has the potential of improving clinical outcomes in transplant recipients. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Microanatomy of the Leukemic Stem Cell Niche in Murine Chronic Myelogenous Leukemia

Objectives and background: Constituents of the bone marrow microenvironment (BMM) influence the proliferation, differentiation and location of hematopoietic stem and progenitor cells (HSPC). Dependent on their maturation stage, different subsets of HSPC are localized at distinct sites in the BMM. This location depends on HSPC-intrinsic, as well as HSPC-extrinsic factors. The BMM protects leukemic stem cells (LSC) from treatment with tyrosine kinase inhibitors or chemotherapy. We, therefore, investigated the microanantomy of the LSC niche hypothesizing that it may differ from the normal HSPC niche.Methods: We used a combination of confocal and 2-photon intravital microscopy (IVM) of the murine calvarium and well-described retroviral models of BCR-ABL1+chronic myelogenous leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL).Results: We show here that BCR-ABL1+Lin–c-Kit+Sca-1+ (LKS) CD150+CD48– (LKS SLAM) cells, which harbor the LSC fraction in the CML model, homed to locations further away from the endosteum than their normal counterparts. Prior in-vitro treatment of BCR-ABL1+ LKS with imatinib mesylate, considered standard of care in CML, reversed this phenotype and the cells were found closer to the endosteum.Native BCR-ABL1, as well as the imatinib-resistant BCR-ABL1 point mutants BCR-ABL1Y253F, BCR-ABL1E255K, BCR-ABL1T315I and BCR-ABL1M351T had similar intrinsic catalytic activity, but the BCR-ABL1Y253F, BCR-ABL1E255K, and BCR-ABL1T315I mutants increased the IL-3-independent proliferative capacity of 32D cells relative to native BCR-ABL1. BCR-ABL1Y253F and BCR-ABL1M351T caused increased transformation of primary BM B-lymphoid progenitors in vitro and led to accelerated induction of B-ALL in mice. In the CML model, BCR-ABL1Y253F and BCR-ABL1T315Iinduced myeloproliferative neoplasia with shortened survival and features of accelerated phase disease compared to native BCR-ABL1, whereas BCR-ABL1T315I LKS cells homed closer to osteoblastic cells than LKS cells expressing native BCR-ABL1.Sequential in vivo tracking of leukemic progenitor growth by IVM showed a similar nadir in the number of cells per leukemic cell 'nest’ 11 days after irradiation and IV transplantation in recipients of DsRed+BCR-ABL1+ or empty vector control-transduced bone marrow. However, between days 18-25 after transplantation there was a significant increase in the number of cells per leukemic cell 'nest’ compared to the empty vector control group. Sequential immunohistochemistry and TUNEL assays of leukemic bone sections in imatinib- or vehicle-treated recipient mice with CML showed that initial BCR-ABL1+ growth tends to occur at locations further away from the endosteum, whereas erythroid islands were found closer to the endosteum and trabeculae. Apoptosis in response to imatinib appeared most prominent in the metaphysis. Lastly, we could demonstrate by IVM in the CML model that treatment of mice with a combination of imatinib plus granulocyte colony-stimulating factor led to 'emptying’ of the LSC niche and superior eradication of BCR-ABL1+ leukemic cells compared to treatment with imatinib alone.Conclusions: In summary, these data suggest that the microanatomy of the LSC niche in CML differs from the normal hematopoietic niche. BCR-ABL1 mutation status may affect the positioning of CML LSC in the microenvironment, and location in the niche may be altered pharmacologically, suggesting that niche location may influence clinical outcome. DisclosuresKrause:Glycomimetics. Inc.: Research Funding.

Twist-1, a novel regulator of hematopoietic stem cell self-renewal and myeloid lineage development.

Transcription factor Twist-1 plays essential roles in specification and differentiation of mesoderm-derived tissues. Growing evidences now link Twist-1 to the acquisition of stem-cell-like properties. However, the role of Twist-1 in hematopoietic stem cell (HSC) remains largely uncharacterized. We report that Twist-1 is more highly expressed in murine HSC and its expression declines with differentiation. To investigate Twist-1 gene function, retroviral-mediated overexpression or removal experiments are performed. Competitive repopulation studies demonstrate that enforced expression of Twist-1 in HSC-enriched Lin(-) c-Kit(+) Sca-1(+) (LKS) cells results in an increase in the size of the G(0) population, and in their reconstitution ability after the first and a second transplantation. Conversely, removal of Twist-1 in LKS cells impairs their ability to repopulate. In addition, increased Twist-1 expression causes a shift toward production of myeloid cells. Twist-1 transduction in LKS cells activates myeloid lineage-determining factors PU.1 and GATA-1 and downregulates lymphoid factor GATA-3 in vitro, suggesting that Twist-1-mediated myeloid skewing occurs in hematopoietic stem and progenitor cells (HSPCs). These findings indicate that Twist-1 is not only involved in the maintenance of HSC dormancy and self-renewal capacity but also implicated in the myeloid lineage fate choice of HSPCs. Exploration of the underlying mechanisms reveals that Runx1/c-Mpl/Tie2 regulatory pathway could possibly account for the observed effects caused by Twist-1 overexpression. Our study provides the first evidence supporting a role for Twist-1 in hematopoiesis.

Depletion of STAT5 blocks TEL-SYK-induced APMF-type leukemia with myelofibrosis and myelodysplasia in mice.

The spleen tyrosine kinase (SYK) was identified as an oncogenic driver in a broad spectrum of hematologic malignancies. The in vivo comparison of three SYK containing oncogenes, SYKwt, TEL–SYK and IL-2-inducible T-cell kinase (ITK)-SYK revealed a general myeloexpansion and the establishment of three different hematologic (pre)diseases. SYKwt enhanced the myeloid and T-cell compartment, without leukemia/lymphoma development. ITK–SYK caused lethal T-cell lymphomas and the cytoplasmic TEL–SYK fusion induced an acute panmyelosis with myelofibrosis-type acute myeloid leukemia (AML) with up to 50% immature megakaryoblasts infiltrating bone marrow, spleen and liver, additional MPN features (myelofibrosis and granulocyte expansion) and MDS stigmata with megakaryocytic and erythroid dysplasia. LKS cells were reduced and all subsets (LT/ST/MPP) showed reduced proliferation rates. SYK inhibitor treatment (R788) of diseased TEL–SYK mice reduced leukocytosis, spleen and liver infiltration, enhanced the hematocrit and prolonged survival time, but could not significantly reduce myelofibrosis. Stat5 was identified as a major downstream mediator of TEL–SYK in vitro as well as in vivo. Consequently, targeted deletion of Stat5 in vivo completely abrogated TEL–SYK-induced AML and myelofibrosis development, proving Stat5 as a major driver of SYK-induced transformation. Our experiments highlight the important role of SYK in AML and myelofibrosis and prove SYK and STAT5 inhibitors as potent treatment options for those diseases.

The Transcriptional corepressorTle4 Is a Novel Regulator Of Murine Hematopoietic Stem and Progenitor Cells and Their Niche

We previously identified Transducin-like/Enhancer of Split 4 (TLE4) as a potential tumor suppressor gene in acute myeloid leukemia whose loss appears to complement AML1-ETO induced leukemia. In this study we examined the role of Tle4 in normal hematopoiesis. Using Tle4 knockout mice we demonstrated that Tle4 is critical for maintaining a supportive hematopoietic stem cell niche, and also has intrinsic effects on HSC survival and B-cell differentiation.Tle4 knockout mice exhibit profound defects in bone mineralizationTle4 knockout mice become runted by 2 weeks of age and generally die by the time of weaning. These mice exhibit a profound defect in mineralization of the bone and bone marrow failure by 3.5 weeks. At birth KO mice showed a significant reduction in the degree of calcification of the skull and long bones, despite no significant differences in overall body size or weight at this early time point. This was confirmed by Von Kossa staining for calcified bone of one-day old tibiae from KO and WT mice. At 21-28 days after birth trabecular bone was almost absent in tibiae and femurs with thin cortices of the long bones. These data indicate Tle4is critical for proper bone mineralization. In further support of this novel role of Tle4 in skeletal development, a mutation in TLE4 has recently been identified in a father and daughter with congenital vertebral malformations including delayed anterior fontanelle closure, shortened vertebral pedicles and kyphoscoliosis.Tle4 deficient bone marrow stroma is defective in supporting hematopoietic stem and progenitor cellsTo determine if the hematopoietic abnormalities in Tle4 KO mice might in part be due to the observed defects in the skeletal or stromal compartment of the BM, we cultured WT LKS cells on WT or KO stromal cells obtained at 2 weeks, prior to the observation of bone marrow hypoplasia. In WT cocultures, 6-15% of recovered cells were positive for both c-kit and Sca-1. In stark contrast, less than 1% of cells were C-kit+Sca-1+, when WT LKS cells were plated in KO stromal cocultures. Long-term coculture experiments in methylcellulose revealed an even more pronounced effect, with cells recovered from WT cocultures generating on average 10-fold more colonies than cells recovered from KO cocultures. Some KO cocultures failed to exhibit any colony forming ability. Therefore, these data suggest that Tle4-/- stromal cells cannot maintain and support HSPC growth as efficiently as WT stromal cells. In addition, Western blots of whole bone lysates from two week old mice showed that the majority of Tle4 KO mice had a reduction in Scf protein levels.Tle4 deficient hematopoietic stem cells exhibit cell intrinsic defects in B-cell differentiation and survivalDue to the progressive leukopenia in primary Tle4 KO mice, and the extrinsic effects noted above, we sought to determine whether loss of Tle4 intrinsically affected self-renewal and repopulation efficiency of HSC in serial transplantation assays using BM and fetal liver cells.Whole BM from two-week-old KO and WT animals transplanted into lethally irradiated allogeneic CD45.1 mice showed similar levels in donor chimerism in blood, but significant reductions in the frequency of B cells in KO transplanted mice at 16 weeks. At 32 weeks after transplant, recipient mice of KO BM developed significant leucopenia, mostly due to a decreased frequency of B cells. To exclude any effects of the bone marrow stroma we also performed serial transplantations from fetal livers of embryonic day 13.5 WT and KO fetuses. In this transplant model, both peripheral leukopenia and specific B cell lymphopenia were observed in primary transplant recipients, recapitulating the phenotype observed in recipients of KO BM. Although in the primary transplants no difference in the frequency of HSC between recipients of KO or WT BM was observed, progressively with secondary and tertiary transplants we observed a striking hematopoietic stem cell exhaustion with reduction in LKS cells, CD34-LKS cells, and LKS CD34-CD48-CD150+ long term HSC.Taken together, these data indicates Tle4 is a critical regulator of hematopoiesis acting by maintaining supportive function of the bone marrow niche as well as intrinsically affecting B-cell differentiation and the survival of hematopoietic stem cells. Disclosures:No relevant conflicts of interest to declare.

Gene Expression Profile and Functionality of ESC-Derived Lin-ckit+Sca-1+ Cells Are Distinct from Lin-ckit+Sca-1+ Cells Isolated from Fetal Liver or Bone Marrow

In vitro bioreactor-based cultures are being extensively investigated for large-scale production of differentiated cells from embryonic stem cells (ESCs). However, it is unclear whether in vitro ESC-derived progenitors have similar gene expression profiles and functionalities as their in vivo counterparts. This is crucial in establishing the validity of ESC-derived cells as replacements for adult-isolated cells for clinical therapies. In this study, we compared the gene expression profiles of Lin-ckit+Sca-1+ (LKS) cells generated in vitro from mouse ESCs using either static or bioreactor-based cultures, with that of native LKS cells isolated from mouse fetal liver (FL) or bone marrow (BM). We found that in vitro-generated LKS cells were more similar to FL- than to BM LKS cells in gene expression. Further, when compared to cells derived from bioreactor cultures, static culture-derived LKS cells showed fewer differentially expressed genes relative to both in vivo LKS populations. Overall, the expression of hematopoietic genes was lower in ESC-derived LKS cells compared to cells from BM and FL, while the levels of non-hematopoietic genes were up-regulated. In order to determine if these molecular profiles correlated with functionality, we evaluated ESC-derived LKS cells for in vitro hematopoietic-differentiation and colony formation (CFU assay). Although static culture-generated cells failed to form any colonies, they did differentiate into CD11c+ and B220+ cells indicating some hematopoietic potential. In contrast, bioreactor-derived LKS cells, when differentiated under the same conditions failed to produce any B220+ or CD11c+ cells and did not form colonies, indicating that these cells are not hematopoietic progenitors. We conclude that in vitro culture conditions significantly affect the transcriptome and functionality of ESC-derived LKS cells and although in vitro differentiated LKS cells were lineage negative and expressed both ckit and Sca-1, these cells, especially those obtained from dynamic cultures, are significantly different from native cells of the same phenotype.

Single Cell Proteomics Reveals Novel Cytokine-Producing Function of Hematopoietic Stem and Progenitor Cells

AbstractAbstract 26During infection, hematopoietic stem and progenitor cells (HSPCs) are called upon to proliferate and differentiate to produce more innate and adaptive immune cells to combat infection. Traditionally, HSPCs are thought to respond to depletion of downstream hematopoietic cells during infection. More recent evidence suggests that HSPCs may respond directly to infection and pro-inflammatory cytokines. However, little is known about the direct immune response of HSPCs and the molecular signaling regulating this response upon sensing an infection. In this study, we have combined transgenic and genetic knockout mouse models with a novel single cell barcode proteomics microchip technology to tackle these questions. We show that although long-term hematopoietic stem cells (HSCs) (defined by Lineage-cKit+Sca1+CD150+CD48-) do not secrete cytokines upon toll-like receptor (TLR) stimulation, short-term HSCs and multipotent progenitor cells (MPPs) (defined by Lineage-cKit+Sca1+, referred to as LKS thereafter) can produce copious amounts of cytokines upon direct TLR-4 and TLR-2 stimulation, indicating that LKS cells can directly participate in an immune response by producing a myriad of cytokines, upon a bacterial infection. Within the population of LKS cells we detect multiple functional subsets of cells, specialized in producing myeloid-like, lymphoid-like or both types of cytokines. Moreover, we show that the cytokine production by LKS cells is regulated by the NF-κB activity, as p50-deficient LKS cells show reduced cytokine production while microRNA-146a (miR-146a)-deficient LKS cells show significantly increased cytokine production.As long-term HSCs differentiate, they start to gain effector immune function much earlier than we had originally anticipated. In light of this finding, we should start to view the stepwise differentiation scheme of HSCs, and perhaps all other stem cells, as a strategy to sequentially gain functional capacity, instead of simply losing stemness and self-renewal ability. The remarkable ability of LKS cells to produce copious amounts of cytokines in response to bacteria may provide some protective immunity during severe neutropenia and lymphopenia or in the early stage of HSC transplantation. This study further extends the functions of NF-κB to include the regulation of primitive hematopoietic stem and progenitor cells and provides direct evidence of the bacteria-responding ability of HSPCs through the TLR/NF-κB axis. The single cell barcode proteomics technology can be widely applied to study proteomics of other rare cells or heterogeneous cell population at a single cell level. Disclosures:No relevant conflicts of interest to declare.

In Vivo Modulation of Mitochondrial Activity Determines HSC Engraftment and Post-Transplant Survival in Mice

AbstractAbstract 213Cellular metabolism is emerging as a potential fate determinant in cancer and stem cell biology, constituting a crucial regulator of the hematopoietic stem cell (HSC) pool [1–4]. The extremely low oxygen tension in the HSC microenvironment of the adult bone marrow forces HSCs into a low metabolic profile that is thought to enable their maintenance by protecting them from reactive oxygen species (ROS). Although HSC quiescence has for long been associated with low mitochondrial activity, as testified by the low rhodamine stain that marks primitive HSCs, we hypothesized that mitochondrial activation could be an HSC fate determinant in its own right. We thus set to investigate the implications of pharmacologically modulating mitochondrial activity during bone marrow transplantation, and have found that forcing mitochondrial activation in the post-transplant period dramatically increases survival.Specifically, we examined the mitochondrial content and activation profile of each murine hematopoietic stem and progenitor compartment. Long-term-HSCs (LT-HSC, Lin-cKit+Sca1+ (LKS) CD150+CD34-), short-term-HSCs (ST-HSC, LKS+150+34+), multipotent progenitors (MPPs, LKS+150-) and committed progenitors (PROG, Lin-cKit+Sca1-) display distinct mitochondrial profiles, with both mitochondrial content and activity increasing with differentiation. Indeed, we found that overall function of the hematopoietic progenitor and stem cell compartment can be resolved by mitochondrial activity alone, as illustrated by the fact that low mitochondrial activity LKS cells (TMRM low) can provide efficient long-term engraftment, while high mitochondrial activity LKS cells (TMRM high) cannot engraft in lethally irradiated mice. Moreover, low mitochondrial activity can equally predict efficiency of engraftment within the LT-HSC and ST-HSC compartments, opening the field to a novel method of discriminating a population of transitioning ST-HSCs that retain long-term engraftment capacity. Based on previous experience that a high-fat bone marrow microenvironment depletes short-term hematopoietic progenitors while conserving their long-term counterparts [5], we set to measure HSC mitochondrial activation in high-fat diet fed mice, known to decrease metabolic rate on a per cell basis through excess insulin/IGF-1 production. Congruently, we found lower mitochondrial activation as assessed by flow cytometry and RT-PCR analysis as well as a depletion of the short-term progenitor compartment in high fat versus control chow diet fed mice. We then tested the effects of a mitochondrial activator known to counteract the negative effects of high fat diet. We first analyzed the in vitro effect on HSC cell cycle kinetics, where no significant change in proliferation or division time was found. However, HSCs responded to the mitochondrial activator by increasing asynchrony, a behavior that is thought to directly correlate with asymmetric division [6]. As opposed to high-fat diet fed mice, mice fed with the mitochondrial activator showed an increase in ST-HSCs, while all the other hematopoietic compartments were comparable to mice fed on control diet. Given the dependency on short-term progenitors to rapidly reconstitute hematopoiesis following bone marrow transplantation, we tested the effect of pharmacological mitochondrial activation on the recovery of mice transplanted with a limiting HSC dose. Survival 3 weeks post-transplant was 80% in the treated group compared to 0% in the control group, as predicted by faster recovery of platelet and neutrophil counts.In conclusion, we have found that mitochondrial activation regulates the long-term to short-term HSC transition, unraveling mitochondrial modulation as a valuable drug target for post-transplant therapy. Identification of molecular pathways accountable for the metabolically mediated fate switch is currently ongoing. Disclosures:No relevant conflicts of interest to declare.

Direct Toll-Like Receptor-Mediated Stimulation of Hematopoietic Stem and Progenitor Cells Occurs In Vivo and Promotes Differentiation Toward Macrophages

As Toll-like receptors (TLRs) are expressed by hematopoietic stem and progenitor cells (HSPCs), they may play a role in hematopoiesis in response to pathogens during infection. We show here that TLR2, TLR4, and TLR9 agonists (tripalmitoyl-S-glyceryl-L-Cys-Ser-(Lys)4 [Pam3CSK4], lipopolysaccharide [LPS], and CpG oligodeoxynucleotide [ODN]) induce the in vitro differentiation of purified murine lineage negative cells (Lin(-) ) as well as HSPCs (identified as Lin(-) c-Kit(+) Sca-1(+) IL-7Rα(-) [LKS] cells) toward macrophages (Mph), through a myeloid differentiation factor 88 (MyD88)-dependent pathway. In order to investigate the possible direct interaction of soluble microorganism-associated molecular patterns and TLRs on HSPCs in vivo, we designed a new experimental approach: purified Lin(-) and LKS cells from bone marrow of B6Ly5.1 mice (CD45.1 alloantigen) were transplanted into TLR2(-/-) , TLR4(-/-) , or MyD88(-/-) mice (CD45.2 alloantigen), which were then injected with soluble TLR ligands (Pam3CSK4, LPS, or ODN, respectively). As recipient mouse cells do not recognize the TLR ligands injected, interference by soluble mediators secreted by recipient cells is negligible. Transplanted cells were detected in the spleen and bone marrow of recipient mice, and in response to soluble TLR ligands, cells differentiated preferentially to Mph. These results show, for the first time, that HSPCs may be directly stimulated by TLR agonists in vivo, and that the engagement of these receptors induces differentiation toward Mph. Therefore, HSPCs may sense pathogen or pathogen-derived products directly during infection, inducing a rapid generation of cells of the innate immune system.

A Sensitive and Quantitative Polymerase Chain Reaction-Based Cell Free In Vitro Non-Homologous End Joining Assay for Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are responsible for sustaining hematopoietic homeostasis and regeneration after injury for the entire lifespan of an organism. Maintenance of genomic stability is crucial for the preservation of HSCs, which depends on their efficient repair of DNA damage, particularly DNA double strand breaks (DSBs). Because of the paucity of HSCs and lack of sensitive assays, directly measuring the ability of HSCs to repair DSBs has been difficult. Therefore, we developed a sensitive and quantitative cell free in vitro non-homologous end joining (NHEJ) assay using linearized plasmids as the substrates and quantitative polymerase chain reaction (qPCR) technique. This assay can sensitively detect DSB repair via NHEJ in less than 1 µg 293T cell nuclear proteins or nuclear extracts from about 5,000 to 10,000 human BM CD34+ hematopoietic cells. Using this assay, we confirmed that human bone marrow HSCs (CD34+CD38− cells) are less proficient in the repair of DSBs by NHEJ than HPCs (CD34+CD38+ cells). In contrast, mouse quiescent HSCs (Pyronin-Ylow LKS+ cells) and cycling HSCs (Pyronin-Yhi LKS+ cells) repaired the damage more efficiently than HPCs (LKS− cells). The difference in the abilities of human and mouse HSCs and HPCs to repair DSBs through NHEJ is likely attributed to their differential expression of key NHEJ DNA damage repair genes such as LIG4. These findings suggest that the qPCR-based cell free in vitro NHEJ assay can be used to sensitively measure the ability of human and mouse HSCs to repair DSBs.

Alcohol administration suppresses the proliferative response of marrow LKS cells to E. coli bacteremia

Alcohol administration suppresses the proliferative response of marrow LKS cells to E. coli bacteremia

Hematopoietic Stem Cell Repopulation Modulated by ROS-Detoxifying Enzymes,

Abstract 4172A tightly regulated level of reactive oxygen species (ROS) is critical for proper functioning of hematopoietic stem cells (HSCs) and high levels of ROS were shown to be able to exhaust the HSC compartment under stress conditions. Intracellular excessive ROS are normally scavenged by antioxidant enzymes. Superoxide anion radical (O) converts into hydrogen peroxide (H2O2) by superoxide dismutase (SOD), and then detoxified by catalase and glutathione peroxidase (GPx) into water. We hypothesize that maintaining a low redox status by ectopically expressing these ROS-detoxifying enzymes, namely manganese-containing SOD (MnSOD) and catalse is able to augment HSC regeneration. In this study, we explored the potential usefulness of MnSOD-PL, a drugable MnSOD plasmid and lipfectin complex, in enhancing the efficiency of HSC transplant. We have also investigated the effects of overexprressed MnSOD or catalase via retroviral delivery on the engraftment efficiency of transduced HSCsSCs. Our results showed that the basal mRNA levels of MnSOD and catalase in long-term and short-term HSCs were less than 1–3% of that of the cellular beta-actin. The mRNA levels were 2 to 4 fold higher in the short-term repopulating HSCs than those in the long-term repopulating HSCs. Irradiation did not induce enzymes expression except catalase in megakaryocytic-erythroid progenitors (MEP) and GPx1 in granulocyte-monocytic progenitors (GMP), which were significantly increased after exposure to 800 cGy (p=0.05) and 400 cGy (p=0.046) radiations, respectively. Administration of MnSOD-PL before TBI conferred significant radiation protection for irradiated recipient mice. On the one hand, the recovery of endogenous hematopoietic could be boosted. On the other hand, the function of the donor HSCs was more preserved from the ROS damage and proliferative stress, and those preserved HSCs were able to replenish the BM when a secondary stress occurred. However, the beneficial effects of MnSOD-PL seemed to be largely via the host environment, since our subsequent experiment showed that overexpression of MnSOD in HSCs only provided minimal benefits to the hematopoietic reconstitution. This potential utility of MnSOD-PL suggests an alternative therapeutic strategy to enhance the HSCs engraftment efficiency in bone marrow transplant. In order to further explore the effects of catalase in HSC protection, human catalase was over-expressed in mouse LKS cells via retroviral delivery in comparison with MnSOD. In the colony-forming cell assay, CFU-M colonies were significantly higher in catalase or MnSOD over-expressed group than those of vector control group in the 200 cGy irradiated plates (p=0.01). Within three months after competitive transplantation, the engraftment levels were increased to 2.7–3.4 fold in catalase group than those of vector group. After 200 cGy re-irradiation engraftment levels were significantly increased to 6.4–7.9 fold in the catalase group (p<0.05). The results showed that overexpression of catalase alone could significantly improve the HSC repopulation. Despite no significant difference in the primary transplantation experiments, MnSOD over-expression also showed a positive effect on the HSC repopulation capacity in the serial transplantation. Taken together, these results demonstrate that maintaining low ROS by ectopically expressing the ROS-detoxifying enzymes is a viable approach to improve HSC functions under stress and damaging conditions. Disclosures:No relevant conflicts of interest to declare.

Candida albicans Induces Selective Development of Macrophages and Monocyte Derived Dendritic Cells by a TLR2 Dependent Signalling

As TLRs are expressed by haematopoietic stem and progenitor cells (HSPCs), these receptors may play a role in haematopoiesis in response to pathogens during infection. We have previously demonstrated that in in vitro defined conditions inactivated yeasts and hyphae of Candida albicans induce HSPCs proliferation and differentiation towards the myeloid lineage by a TLR2/MyD88 dependent pathway. In this work, we showed that C. albicans invasive infection with a low virulence strain results in a rapid expansion of HSPCs (identified as LKS cells: Lin− c-Kit+ Sca-1+ IL-7Rα−), that reach the maximum at day 3 post-infection. This in vivo expansion of LKS cells in TLR2−/− mice was delayed until day 7 post- infection. Candidiasis was, as expected, accompanied by an increase in granulopoiesis and decreased lymphopoiesis in the bone marrow. These changes were more pronounced in TLR2−/− mice correlating with their higher fungal burden. Accordingly, emigration of Ly6Chigh monocytes and neutrophils to spleen was increased in TLR2−/− mice, although the increase in macrophages and inflammatory macrophages was completely dependent on TLR2. Similarly, we detected for the first time, in the spleen of C. albicans infected control mice, a newly generated population of dendritic cells that have the phenotype of monocyte derived dendritic cells (moDCs) that were not generated in TLR2−/− infected mice. In addition, C. albicans signalling through TLR2/MyD88 and Dectin-1 promotes in vitro the differentiation of Lin− cells towards moDCs that secrete TNF-α and are able to kill the microorganism. Therefore, our results indicate that during infection C. albicans can directly stimulate progenitor cells through TLR2 and Dectin-1 to generate newly formed inflammatory macrophages and moDCs that may fulfill an essential role in defense mechanisms against the pathogen.