Properties Of Hematopoietic Stem Cells Research Articles

Regulation of β1-Integrin by Mir-134 in Mesenchymal Stromal Cells – Implications for Mesenchymal Stromal Cell Adherence and Hematopoietic Stem Cell Interaction

AbstractAbstract 3459The different intra- and extracellular constituents of the hematopoietic stem cell (HSC) niche in the human bone marrow are tightly regulated and of momentous importance for various properties of HSCs. Some of these are regulated through β1-Integrins (CD29) which therefore dramatically influence HSC and mesenchymal stromal cell (MSC) interaction in the niche. Important regulators within these cells are microRNAs (miRNAs). These small, non-coding RNAs control the expression of around two-thirds of the human protein-coding genes. One of these miRNAs, miR-134, previously referred to be a “brain-specific” miRNA was shown to be highly expressed in MSCs in tissue-studies conducted by our group. Since the central nervous system was recently shown to be closely connected to the regulation of HSCs and MSCs, we asked whether miR-134 which has several conserved binding seed-match sequences within the 3’UTR of β1-Integrin, regulates MSC mediated properties in the bone marrow niche.Screening of human MSC cell lines (n=4) by western blotting revealed highest β1-Integrin expression in SCP-1 cells. Transfection of SCP-1 with either siRNA directed against β1-Integrin (siCD29) or pre-miRNA-134 (pre134) revealed a downregulation of β1-Integrin at the mRNA level only in siRNA transfected cells, p=0.01. In contrast, at the protein level, as measured by western blot and FACS analysis, p=0.002, β1-Integrin was downregulated by siCD29 as well as by pre134, indicating a miRNA-specific action of repression. Confirmatory, the 3’UTR of β1-Integrin, which contains several putative binding sites for miR-134, was cloned into a pMiRReporter vector and luciferase activity was measured after cotransfection with pre134. The luciferase activity was significantly reduced in pre134 transfected cells [1.80 ± 0.46 (preCo) vs. 0.99 ± 0.49 (pre134); p<0.001]. To evaluate whether pre134 mediated reduction of β1-Integrin can modulate the adhesion potential of SCP-1, atomic force microscopy (AFM)-based single-cell force spectroscopy (SCFS) was performed. Indeed, transfection of SCP-1 with siCD29 or pre134 resulted in a significantly reduced adherence as compared to their respective controls, p<0.001 and p<0.01. Furthermore, using AFM-based SCFS we investigated the interaction between 32D-cells, which have a high surface expression of the natural interaction partner of β1-Integrin VCAM-1, and SCP-1 cells. Here again, we were able to show, that 32D show a significantly lower adhesion potential to siCD29 and pre134-transfected SCP-1, p<0.001 and p<0.001, respectively. In a translational approach MSCs from healthy bone marrow donors (n=30) and from MDS patients (n=17) were screened for miRNA-expression. This analysis revealed 50% higher miR-134 transcript levels in MSCs from MDS patients [0.0057 ± 0.0021 (healthy) vs. 0.0127 ± 0.0045 (MDS); p<0.001], suggesting a potential role of this miRNA in regulating its MSC adhesion.Regulation of adhesion of MSCs and to MSCs is important for various components of the bone marrow niche. Here, we demonstrate for the first time that β1-Integrin mediated adhesion of MSCs themselves and other cell types onto MSCs via β1-Integrin receptors can be inhibited via miR-134 overexpression. Furthermore, this newly characterized mechanism provides evidence for a potential anti-adhesive influence of miR-134. While this might not only influence adhesion, other mechanisms such as homing of HSCs as well as other cell types, might be affected by modification of miR-134 expression in the stromal niche. Disclosures:Platzbecker:Amgen: Consultancy; GlaxoSmithKline: Consultancy; Celgene: Consultancy; Novartis: Consultancy.

Impact of substrate elasticity on human hematopoietic stem and progenitor cell adhesion and motility

In the bone marrow, hematopoietic stem cells (HSCs) reside in endosteal and vascular niches. The interactions with the niches are essential for the maintenance of HSC number and properties. Although the molecular nature of these interactions is well understood, little is known about the role of physical parameters such as matrix elasticity. Osteoblasts, the major cellular component of the endosteal HSC niche, flatten during HSC mobilization. We show that this process is accompanied by osteoblast stiffening, demonstrating that not only biochemical signals but also mechanical properties of the niche are modulated. HSCs react to stiffer substrates with increased cell adhesion and migration, which could facilitate the exit of HSCs from the niche. These results indicate that matrix elasticity is an important factor in regulating the retention of HSCs in the endosteal niche and should be considered in attempts to propagate HSCs in vitro for clinical applications.

Impact of substrate elasticity on human hematopoietic stem and progenitor cell adhesion and motility

In the bone marrow, hematopoietic stem cells (HSCs) reside in endosteal and vascular niches. The interactions with the niches are essential for the maintenance of HSC number and properties. Although the molecular nature of these interactions is well understood, little is known about the role of physical parameters such as matrix elasticity. Osteoblasts, the major cellular component of the endosteal HSC niche, flatten during HSC mobilization. We show that this process is accompanied by osteoblast stiffening, demonstrating that not only biochemical signals but also mechanical properties of the niche are modulated. HSCs react to stiffer substrates with increased cell adhesion and migration, which could facilitate the exit of HSCs from the niche. These results indicate that matrix elasticity is an important factor in regulating the retention of HSCs in the endosteal niche and should be considered in attempts to propagate HSCs in vitro for clinical applications.

Ex vivo expansion of hematopoietic stem cells: mission accomplished?

A small number of hematopoietic stem cells (HSCs) with self-renewal and multi-lineage repopulation capacity maintain hematopoiesis during the lifetime of an individual. Moreover, HSCs and their potential exist in excess as one individual can share its HSCs with another leading to creation of a genetically identical hematopoietic system. For over half a century this property of HSCs has been utilised by successful allogeneic clinical HSC transplantation for treatment of patients with inherited or acquired genetic and neoplastic diseases of the hematopoietic and immune system. There are now more than twenty thousand allogeneic HSC transplants per year worldwide [1]. However, although more than 17.5 million potential HSC donors are registered and additional 500,000 cord bloods are stored for potential allogeneic HSC transplantation [2], timely availability of appropriately human leukocyte antigen (HLA)-compatible HSCs with sufficient quality for patients still poses a problem in the field. Even if a donor is available, toxicity of the procedure could be reduced by increasing HSC numbers in transplants. One way to solve these issues would be by generation of quality-controlled, off the shelf HSC products via in vitro HSC expansion, a "holy grail" procedure many have been hunting for. Here, we discuss accumulating knowledge on signalling pathways involved in HSC maintenance as well as recent achievements to apply the findings to ex vivo HSC expansion for clinical use. Although the specific issue concerns only highly specialised medicine today, newly generated knowledge will be critical for the whole field of stem cell transplantation and regenerative medicine in the future.

Aortic Tissue as a Niche for Hematopoiesis

The stem cell theory of hematopoiesis predicts that all mature blood cell lineages circulating in the bloodstream or resident in tissues arise via a hierarchy of hematopoietic progenitor cell (HPC) intermediates that are ultimately derived from hematopoietic stem cells (HSC). In the adult human, bone marrow–resident HSC and HPC generate billions of circulating blood cells daily, ranging from the short-lived neutrophils and platelets to long-lived B and T lymphocytes and monocyte macrophages. Recent interest in the roles of macrophages as modulators of angiogenesis1 and as participants in atheroma formation2 highlight the need to better understand the mechanisms of recruitment and retention of macrophages and their progenitor precursors within tissues. In this issue of Circulation , Psaltis and colleagues3 identify clonogenic HPC activity in aortic adventitia (that is enriched in HPC compared with peripheral blood or muscle tissue), which appears skewed toward colony-forming unit-macrophage (CFU-M) differentiation. In addition, stem cell antigen-1–expressing cells in the aortic adventitia coexpressed a variety of HSC antigens, and adoptive transfer of these cells into irradiated recipient mice led to low but sustained multilineage reconstitution, a property of HSC. Although bone marrow (BM)–derived donor hematopoietic cells reconstituted the aorta of lethally irradiated animals, the majority of aorta-derived CFU-M appeared to be constitutive or long term residents of the vessel wall (with sustained but only partial replacement from donor BM cells), suggesting that either the aortic adventitial microenvironment imparts specific differentiation properties to marrow-derived precursors or that a local origin for HSC/HPC exists in the adult vasculature. Is there any evidence to suggest that HSC/HPC may originate and/or migrate through the aortic adventitia? Article see p 592 Hematopoiesis occurs in a site- and tissue-specific pattern that varies throughout mammalian development. The first blood cells (including macrophages) arise in the extraembryonic yolk sac, and the yolk …

Clonal analysis reveals multiple functional defects of aged murine hematopoietic stem cells

Hematopoietic stem cell (HSC) populations change with aging, but the extent to which this is caused by qualitative versus quantitative alterations in HSC subtypes is unclear. Using clonal assays, in this study we show that the aging HSC compartment undergoes both quantitative and qualitative changes. We observed a variable increase of HSC pool size with age, accompanied by the accumulation of predominantly myeloid-biased HSCs that regenerate substantially fewer mature progeny than young myeloid-biased HSCs and exhibit reduced self-renewal activity as measured by long-term secondary transplantation. Old HSCs had a twofold reduction in marrow-homing efficiency and a similar decrease in functional frequency as measured using long-term transplantation assays. Similarly, old HSCs had a twofold reduced seeding efficiency and a significantly delayed proliferative response compared with young HSCs in long-term stromal cell co-cultures but were indistinguishable in suspension cultures. We show that these functional defects are characteristics of most or all old HSCs and are not indicative of a nonfunctional subset of cells that express HSC markers. Furthermore, we demonstrate that cells with functional properties of old HSCs can be generated directly from young HSCs by extended serial transplantation, which is consistent with the possibility that they arise through a process of cellular aging.

Down-Regulation of Homeobox Gene Ventx Promotes Expansion of Human Bone Marrow Hematopoietic Stem Cells (HSC)

Abstract 1272Hematopoietic stem cells (HSC) give rise to mature cells of all lineages of blood and immune systems. HSC transplantation has shown great promise in the treatment of malignancies, reconstitution of hematopoietic systems and HSC-based gene therapy. Cell intrinsic factors/pathways have been the targets of intensive investigation for its potential application in HSC expansion. Over the past decades, several critical cell fate determination pathways, such as the Wnt signaling pathways and senescence pathways have been implicated in the proliferation and differentiation of HSC. Moreover, overexpression of HoxB4 and BMI1 was found to be able to expand human HSC 2∼3 folds. Nevertheless, the regulatory mechanisms of HSC proliferation and differentiation remain incompletely understood and safe and efficacious expansion of human HSC remains as a fundamental challenge that limits the clinical application of HSC-based therapy.VentX is a human homologue of the Xenopus homeobox protein Xom of the BMP4 signaling pathway. Using Xenopus model and methods of reverse genetics, our recent work showed that VentX is a LEF/TCF associated Wnt repressor and an activator of senescence pathways. VentX expression is highly regulated and restricted in hematopoietic cells and serves a major regulator of hematopoietic cell differentiation. To explore the potential role of VentX in proliferation and differentiation of HSC during hematopoiesis, we quantified VentX expression during hematopoiesis, using qRT-PCR methods and examined the effects of altered VentX expression on HSC properties in vitro and in vivo.Our data showed that VentX expression is significantly up-regulated during oncogenesis of hematopioetic cells. We demonstrated that lentiviral knockdown of VentX allowed for more than 5 fold ex vivo expansion of human HSC with balanced lineage development. Importantly, transient knockdown of VentX by siRNA also led to expansion of HSC. The effect of VentX down-regulation on the expansion of human HSC was also demonstrated by enhanced engraftment in the SCID/NODγ2null mouse model. Consistent with its role as a novel regulator of HSC, overexpression of VentX significantly inhibited clonal genesis of HSC. Mechanistically, we demonstrated that VentX controls the expression of cell cycle regulators downstream of the Wnt and senescence pathways, such as the C-myc, CyclinD1 and p21.In summary, using methods of reverse genetic and developmental modeling, we identified VentX as a novel regulator for expansion of human BM HSC. The results of our investigations provide novel insight in regulating HSC proliferation and differentiation. In addition, the findings that transient down-regulation of VentX by SiRNA lead to efficient expansion of bone marrow HSC suggests that VentX may serve as a novel target for safe expansion of HSC for its potential clinical applications. Disclosures:No relevant conflicts of interest to declare.

Inhibition of Let-7 Processing in Adult Murine Hematopoietic Stem Cells Induces a Fetal-Like High Self-Renewal Pattern in Their Progeny

Abstract 45Fetal hematopoietic stem cells (HSCs) in mice differ from their adult counterparts in a number of key properties. These include a higher cycling activity, an ability to more rapidly reconstitute the HSC compartment of irradiated recipient mice, a higher output of myeloid as compared to lymphoid progeny, and a greater sensitivity to the self-renewal promoting activity of Steel factor. We have previously shown that most of these features of fetal HSCs are sustained until 3 weeks after birth at which time they are rapidly (within 1 week), completely and permanently replaced with the corresponding properties of adult HSCs. A candidate regulator of this transition, Hmga2, was identified based on its greater expression in highly purified fetal versus adult HSCs (CD45+EPCR+CD48−CD150+; E-SLAM cells) with persistence of this difference in the matching lineage-negative (lin−) compartments. Experiments in which Hmga2 was overexpressed by lentiviral transduction of purified adult HSCs which were then transplanted into irradiated mice provided evidence that this chromatin remodeling factor can activate a fetal-like HSC program in these cells; i.e., more rapidly reconstitute the HSC compartment (increased self-renewal response) and produce clones with a higher proportion of myeloid cells. Based on the known ability of the let-7 family of microRNAs (miRNAs) to target Hmga2 transcripts resulting in their degradation and/or translational repression, we next hypothesized that let-7 miRNAs might be involved in controlling HSC developmental programs. A comparison of the levels of expression of 6 members of the let-7 family in purified fetal and adult HSCs, as well as in lin− hematopoietic cells, showed that transcripts for all of these are higher in the adult subsets, although this difference was significant only for let-7b (p<0.05). Since Lin28 is a natural inhibitor of let-7 miRNA biogenesis we proposed that overexpression of this protein might be used to simultaneously inhibit all let-7 miRNA species and therefore modulate let-7-mediated effects in HSCs. Transduction of BA/F3 cells with a Lin28-YFP lentiviral vector led to an elevated expression of Lin28 and a significant decrease in multiple let-7 miRNAs. To investigate the influence of Lin28 overexpression on adult HSC self-renewal activity in vivo, we used the same Lin28 lentiviral vector (or a control YFP vector) to transduce highly purified HSCs (40 E-SLAM cells, i.e. ∼20 HSCs/group/experiment, 3 experiments) in a 3–4-hour exposure protocol and then transplanted all of the cells directly into irradiated mice (total of 3–4 mice/group). The number of HSCs regenerated 6 weeks later was subsequently measured by performing limiting-dilution transplants in secondary mice (total of 12–16 secondary mice/group/experiment). Interestingly, analysis of the secondary recipients showed that the Lin28-overexpressing adult HSCs had expanded in the primary recipients ∼6-fold more than the control-virus transduced HSCs (p<0.001). These findings support our thesis that alterations in let-7 miRNA levels play a key role in regulating the developmental switch from fetal to adult HSCs programs that occurs between 3 and 4 weeks after birth in mice. Disclosures:No relevant conflicts of interest to declare.

Early Appearance and Rapid, Selective Expansion of Lymphoid-Deficient Fetal HSCs Associated with Their Preferential Localization in the Marrow

Abstract 1271Recent tracking of the primitive and mature progeny of more than 500 single cell transplants of highly purified hematopoietic stem cells (HSCs) in cell suspensions isolated from the bone marrow of young adult mice (8–12 weeks old) has shown that this pool is intrinsically heterogeneous. This heterogeneity is manifested as differences both in the durability (beyond 6 months) of the self-renewal activity of the HSCs detected in irradiated recipients as well as in the specific lineage contributions maintained in secondary and tertiary recipients of cells generated within each clone. Interestingly, durable HSC self-renewal activity (serial transplantability) was found to be exclusively associated with a robust and sustained ability to produce myeloid cells, regardless of their ability to produce lymphoid progeny at any stage of lymphoid progenitor development. Thus 2 subtypes of HSCs with durable self-renewal activity could be distinguished depending on whether the sustained myelopoietic activity was accompanied by an equivalent robust lymphoid differentiation activity (β-HSCs), or not (α-HSCs). Preliminary examination of fetal liver HSCs with durable self-renewal activity showed that this compartment is dominated by β-HSCs in contrast to the marrow of young adult mice where ∼30% of this HSC pool are α-HSCs. Since previous studies have documented a switch between 3 and 4 weeks after birth in several key properties of HSCs (indicative of an abrupt change from a fetal to an adult HSC program), we were interested in the question of whether the appearance of α-HSCs might be another aspect of this switch or, alternatively, whether the onset of hematopoiesis in locations other than the fetal liver (e.g., the marrow and spleen) might be a contributing factor. Accordingly, we initiated experiments to examine the proportional representation and absolute number of cells with an E-SLAM phenotype (CD45+EPCR+CD48−CD150+) and their functional activity (4-month repopulation of irradiated mice) specific for HSCs in tissues where hematopoiesis is seen during different stages of development between E14.5 and 4 weeks after birth. The number of E-SLAM cells increased ∼15 fold in the fetal liver between E14.5 and E18.5 by which time they were also present in the marrow and spleen but altogether comprising only ∼5% of the fetal liver compartment (assuming the femurs, tibiae and pelvis represent ∼30% of the total marrow). Analysis of the clonal outputs obtained in vivo from single E-SLAM cell transplants from each of these sources showed that α-HSCs are already present as a rare subet of all HSCs in the E14.5 liver and amplify equally with β-HSCs maintaining a ratio of 1:10 α-HSCs to β-HSCs in this organ. Interestingly, despite the small numbers of E-SLAM cells in the E18.5 marrow, these contained readily detectable HSCs in transplant assays and showed a ratio of α-HSCs to β-HSCs of 1:2. This ratio was mirrored in the marrow of adult mice and was also similar to that seen in the marrow and spleen of 3 and 4 week-old mice. These findings show that α-HSCs, by comparison to β-HSCs, have a greater ability to localize in the marrow which appears to then selectively promote the expansion of α-HSCs. These results also show that the initial appearance and early selective expansion of α-HSCs during fetal and early post-natal life is unlikely to be regulated by the same mechanisms that impose an adult program on murine HSCs between 3 and 4 weeks after birth. Rather these results favor a separate, intrinsically determined developmentally controlled mechanism that endows α-HSCs with a higher self-renewal probability, a more rapid turnover, a lower apoptotic activity, or a combination of these differences as compared to β-HSCs. This would explain the initial rapid and marked selective expansion of α-HSCs that occurs during the late fetal and early neonatal period of high HSC proliferation which extends to 3 weeks after birth and is then followed by a much slower rate of selective expansion of α-HSCs during adulthood when the turnover of HSCs is minimal. Disclosures:No relevant conflicts of interest to declare.

Myosin-II Is a Major Modulator of Human Hematopoietic Stem Cell Proliferation and Differentiation

Abstract 2343Non-muscle myosin-II (NMM-II) promotes cell division, membrane rigidity and adhesion to a rigid matrix, and so NMM-II activity might be predicted to be low in dormant hematopoietic stem cells (HSCs) and to increase with differentiation. Deletion of NMM-II is known to be embryonic lethal, but its role in adult HSC differentiation is not known. Recently, we showed that sustained pharmacological inhibition of NMM-II together with soft 2D matrices like the perivascular niches in marrow, rather than rigid like bone, maximizes both MK maturation and platelet generation (Shin et al., PNAS, 2011; 108:11458-63). HSCs exhibit some similarities to mature MKs in that long-term HSCs remain undivided in vivo while various progenitors and maturing cells rapidly expand in number. Here, reversible inhibition of NMM-II sustained over several cell cycles enriches long-term HSCs up to 20 fold by selective elimination of proliferating progenitors. CFSE dilution analysis indicates that inhibition of NMM-II eliminates the accumulation phase of hematopoietic progenitors and accelerates natural cell death rate by apoptosis. Interestingly, supplementation of G-CSF significantly enhances HSC survival under NMM-II inhibition and further accelerates progenitor elimination. Molecular profiling and functional analyses indicate that NMM-II isoforms play distinct roles during HSC differentiation. NMM-IIA is a marker for differentiation with significantly lower expression in HSCs than committed progenitors, which is consistent with greater membrane flexibility of HSCs measured by micropipette aspiration. In contrast, NMM-IIB is 5 fold higher in HSCs and progenitors than differentiated CD34− cells. HSC and progenitor numbers are also sensitive to matrix elasticity in a NMM-II dependent manner, with maximal expansion on soft and high-density fibronectin matrices (not collagen). However, upon NMM-II inhibition, the extent of HSC enrichment relative to multipotent progenitors is more sensitive to matrix ligand density than matrix elasticity.To identify physiological mechanisms of regulating NMM-II activity during early HSC differentiation, we investigated post-translational modifications of NMM-IIA, specifically the de-activating and isoform-specific phosphorylation at myosin Ser1943 (pS1943) in HSC and progenitors. In a phospho-specific flow cytometry approach, pS1943 level proves highest in HSCs and decreases during differentiation with Tpo and G-CSF but not SCF alone. TGF-beta inhibits the reduction of pS1943 level, consistent with TGF-beta’s known role in HSC hibernation. Therefore, pS1943 level dictates HSC enrichment and parallels the dose-response to pharmacological NMM-II inhibitors. Furthermore, phospho-mimetic mutation of NMM-IIA at Ser1943 decreases cytoskeletal integrity, increases membrane flexibility, and limits matrix elasticity sensing, indicating that biophysical properties of HSCs can also be regulated by HSC-specific signaling via NMM-IIA heavy chain phosphorylation. Myosin-inhibited CD34+-derived bone marrow cells show reduced colony-forming unit progenitors in vitro, but maintain functional long-term HSCs in vivo in the marrows of xenografted mice with an added benefit to increase platelet circulation simultaneously. Therefore, myosin-II inhibition and soft, high ligand fibronectin constitutes an important ‘microenvironment mimetic' approach to enrichment of long-term HSCs. Myosin-II is clearly a central, matrix-regulated node for HSC proliferation and differentiation. Disclosures:No relevant conflicts of interest to declare.

Low Level of c-Kit Expression Marks Deeply Quiescent Murine Hematopoietic Stem Cells

Although c-kit is expressed highly on murine hematopoietic stem cells (HSCs) and essential for bone marrow (BM) hematopoiesis, the significance of the high level of expression of c-kit on HSCs was not well determined. We show here that CD150(+) CD48(-) Lineage(-) Sca-1(+) c-kit(+) HSCs in adult BM are distributed within the range of roughly a 20-fold difference in the expression level of c-kit, and that c-kit density correlates with the cycling status of the HSC population. This predisposition is more evident in the BM of mice older than 30 weeks. The HSCs in G(0) phase express a lower level of c-kit both on the cell surface and inside the cells, which cannot be explained by ligand receptor binding and internalization. It is more likely that the low level of c-kit expression is a unique property of HSCs in G(0). Despite functional differences in the c-kit gradient, the HSCs are uniformly hypoxic and accessible to blood perfusion. Therefore, our data indicate the possibility that the hypoxic state of the HSCs is actively regulated, rather than them being passively hypoxic through a simple anatomical isolation from the circulation.

Ex Vivo Expanded Hematopoietic Stem Cells Overcome the MHC Barrier in Allogeneic Transplantation

The lack of understanding of the interplay between hematopoietic stem cells (HSCs) and the immune system has severely hampered the stem cell research and practice of transplantation. Major problems for allogeneic transplantation include low levels of donor engraftment and high risks of graft-versus-host disease (GVHD). Transplantation of purified allogeneic HSCs diminishes the risk of GVHD but results in decreased engraftment. Here we show that exvivo expanded mouse HSCs efficiently overcame the major histocompatibility complex barrier and repopulated allogeneic-recipient mice. An 8-day expansion culture led to a 40-fold increase of the allograft ability of HSCs. Both increased numbers of HSCs and culture-induced elevation of expression of the immune inhibitor CD274 (B7-H1 or PD-L1) on the surface of HSCs contributed to the enhancement. Our study indicates the great potential of utilizing exvivo expanded HSCs for allogeneic transplantation and suggests that the immune privilege of HSCs can be modulated.

Direct Activation of STAT5 by TEL-Lyn Fusion Protein Promotes Induction of Myeloproliferative Neoplasms with Myelofibrosis

AbstractAbstract 4114 BackgroundMyeloproliferative neoplasms (MPN), a group of hematopoietic stem cell (HSC) disorders, are often accompanied by myelofibrosis. The V617F somatic mutation in the Janus kinase 2 (JAK2) gene has recently been found in the majority of patients with polycythemia vera (PV) and more than half of patients with essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). The expression of JAK2 V617F causes a PV-like disease with myelofibrosis in a murine bone marrow (BM) transplant model. In addition, a gain-of-function c-MPL W515 mutation was described in nearly 10% of patients with JAK2 V617F-negative IMF. However, the mechanism responsible for MPD and the formation of myelofibrosis in patients without JAK2 or c-MPL mutations is still unclear. We previously identified the fusion of the TEL gene to the Lyn gene (TEL-Lyn) in idiopathic myelofibrosis with ins(12;8)(p13;q11q21). The introduction of TEL-Lyn into HSCs resulted in fatal MPN with massive myelofibrosis in mice, implicating the rearranged Lyn kinase in the pathogenesis of MPN with myelofibrosis. However, the signaling molecules directly downstream from and activated by TEL-Lyn remain unknown. Design and MethodsWe examined the signaling pathways activated by TEL-Lyn by Western blotting, immunoprecipitation, and in vitro kinase assay using a TEL-Lyn kinase-dead mutant as a control. We further characterized the functional properties of Stat5-deficient HSCs transduced with TEL-Lyn by colony-forming assay and bone marrow transplantation to evaluate the role of STAT5 in TEL-Lyn-induced MPN. ResultsTEL-Lyn was demonstrated to be constitutively active as a kinase through autophosphorylation. In TEL-Lyn-expressing cells, STAT5, STAT3, and Akt were constitutively activated. Among these signaling molecules, STAT5 was activated most prominently and this occurred without the activation of Jak2, the major kinase for STAT5. TEL-Lyn was co-immunoprecipitated with STAT5, and STAT5 was phosphorylated when incubated with TEL-Lyn, but not with TEL-Lyn kinase-dead mutant. These results indicate that TEL-Lyn interacts with STAT5 and directly activates STAT5 both in vitro and in vivo. Of note, the capacity of TEL-Lyn to support the formation of hematopoietic colonies under cytokine-free conditions in vitro and to induce MPN with myelofibrosis in vivo was profoundly attenuated in a Stat5-null background. ConclusionsIn this study, we clearly showed that TEL-Lyn directly activates STAT5 and the capacity of TEL-Lyn to induce MPN with myelofibrosis was profoundly attenuated in the absence of STAT5. Our findings of TEL-Lyn in this study support the role of the Src family kinases in the regulation of STAT pathways and implicate active Lyn in the alternative pathway for STAT activation in pathological cytokine signaling. Our mouse model of MPD with myelofibrosis would be beneficial for the analysis of therapeutic approaches for myelofibrosis. Disclosures:No relevant conflicts of interest to declare.

Knockdown of N-cadherin suppresses the long-term engraftment of hematopoietic stem cells

AbstractDuring postnatal life, the bone marrow (BM) supports both self-renewal and differentiation of hematopoietic stem cells (HSCs) in specialized microenvironments termed stem cell niches. Cell-cell and cell-extracellular matrix interactions between HSCs and their niches are critical for the maintenance of HSC properties. Here, we analyzed the function of N-cadherin in the regulation of the proliferation and long-term repopulation activity of hematopoietic stem/progenitor cells (HSPCs) by the transduction of N-cadherin shRNA. Inhibition of N-cadherin expression accelerated cell division in vitro and reduced the lodgment of donor HSPCs to the endosteal surface, resulting in a significant reduction in long-term engraftment. Cotransduction of N-cadherin shRNA and a mutant N-cadherin that introduced the silent mutations to shRNA target sequences rescued the accelerated cell division and reconstitution phenotypes. In addition, the requirement of N-cadherin for HSPC engraftment appears to be niche specific, as shN-cad–transduced lineage−Sca-1+c-Kit+ cells successfully engrafted in spleen, which lacks an osteoblastic niche. These findings suggest that N-cad–mediated cell adhesion is functionally required for the establishment of hematopoiesis in the BM niche after BM transplantation.

Gene transfer by electroporation into hemogenic endothelium in the avian embryo

Hematopoiesis is the dynamic process whereby blood cells are continuously produced in an organism. Blood cell production is sustained by a population of self-renewing multipotent hematopoietic stem cells (HSCs) throughout the life of an organism. Cells with definitive HSC properties appear in the mid-gestation embryo as dense clusters of cells budding from the floor of the aorta, and that of the vitelline and umbilical arteries in the aorta-gonads-mesonephros region. Attempts to genetically modify the aortic floor from which these HSCs arise have been unsuccessful in the mouse, since the regulation of gene expression in the hemogenic endothelium is largely unknown. Here we report the implementation of gene transfer by electroporation into dorsal aortic endothelial cells in the chick embryo. This approach provides a quick and reproducible method of generating gain/loss-of-function models to investigate the function of genes involved in HSC birth.

Large animal models of hematopoietic stem cell gene therapy

Large animal models have been instrumental in advancing hematopoietic stem cell (HSC) gene therapy. Here we review the advantages of large animal models, their contributions to the field of HSC gene therapy and recent progress in this field. Several properties of human HSCs including their purification, their cell-cycle characteristics, their response to cytokines and the proliferative demands placed on them after transplantation are more similar in large animal models than in mice. Progress in the development and use of retroviral vectors and ex vivo transduction protocols over the last decade has led to efficient gene transfer in both dogs and nonhuman primates. Importantly, the approaches developed in these models have translated well to the clinic. Large animals continue to be useful to evaluate the efficacy and safety of gene therapy, and dogs with hematopoietic diseases have now been cured by HSC gene therapy. Nonhuman primates allow evaluation of aspects of transplantation as well as disease-specific approaches such as AIDS (acquired immunodeficiency syndrome) gene therapy that can not be modeled well in the dog. Finally, large animal models have been used to evaluate the genotoxicity of viral vectors by comparing integration sites in hematopoietic repopulating cells and monitoring clonality after transplantation.

Aberrant Bone Marrow Perivascular Niches are Involved in Multiple Myeloma Progression: Role of Notch–Delta Pathway.

Abstract 2800Poster Board II-776Bone marrow (BM) angiogenesis is implicated in Multiple Myeloma (MM) progression. In this study, we tested the hypothesis that MM progression occurs when aberrant BM perivascular niches are established. We isolated BM endothelial cells derived from MM patients (MM-BMECs) from BM aspirates using anti-CD31Ab coupled to magnetic beads. FACS analysis showed that of all the cell lines isolated were endothelial: more than 95% expressed Ulex Europaeus Agglutinin-1 and Factor VIII and were negative for monocyte-macrophage (CD14) and plasma cell markers (CD38). To test the hypothesis that in MM patients BM perivascular niches are aberrant we analyzed how MM-BMECs modulate hematopoietic stem cells (HSCs) properties using a BM microvascular endothelial cell line isolated from a healthy donor (BMECs) as control. We co-cultured cord blood cells CD34+ HSCs in the presence of MM-BMECs or BMECs feeder layer and we analyzed the ability of MM-BMECs compared with BMECs to modulate HSCs adhesion, chemotaxis and apoptosis. The results show that MM-BMECs promote CD34+ HSCs adhesion, recruitment and protect them from apoptosis. In detail, we showed that after 24h of co-culture there was a significant increase in the number of adherent HSCs on MM-BMECs than on BMECs: 43±9% versus 25±6%. Moreover, when HSCs were cultured for 48 hours in 1% of serum in the presence of MM-BMECs they were less sensitive to apoptosis (9±11% of Annexin V+ cells) than HSCs cultured in the presence of BMECs (14±1% of Annexin V+ cells) or without a feeder layer, as control (17±3% of Annexin V+ cells). For the migration assay a transwell chamber system, in which the upper and the lower chambers were separated by 5-μm pore-size filter, was used. BMECs, MM-BMECs or nothing was plated in the lower chamber, while HSCs were seeded into the upper chamber. Both chambers were loaded with unsupplemented EBM-2 plus 2% of serum. Cell migration was studied over a 6-8 hours period and evaluated as number of cells migrated into the lower chamber. The results showed a significantly greater migration of HSCs in the presence of MM-BMECs than BMECs: 12±2% versus 5±1% of migrated cells. Taken together, these data showed that MM-BMECs promoted HSCs migration, adhesion and survival. Next we evaluated how MM-BMECs modulate the hemopoiesis recovery after irradiation in a NOD-SCID mouse model. When injected into sub-lethally irradiated (3 Grey) NOD-SCID mice MM-BMECs were detected in the BM integrated within the murine BM vessels and promoted hematopoietic recovery. In detail, MM-BMECs provided signals favoring the commitment towards lymphoid lineage. In fact, 7 days after injection, the BM of mice injected with MM-BMECs showed an increase in the percentage of lymphoblast (2.7%), compared with mice injected with BMECs or PBS, as control (respectively, 1.5% and 1.4%); followed, 14 days after injection, by a significant increase in the percentage of peripheral blood lymphocytes in mice injected with MM-BMECs (75±6%) versus mice injected with BMECS and PBS (respectively 60±0.5% and 47±7%). Since MM is a plasma cells disorder and the Notch-Delta pathway has been shown to play a central role in regulating HSCs properties, including the decisions of HSCs to undergo T- or B-cell differentiation, we investigated the involvement of this pathway in MM-BMECs and HSCs interaction. As determined by FACS and RT-PCR analysis, MM-BMECs, compared to BMECs, over expressed Delta-like Notch ligand 4 (DII4). Thus, we investigated the role of DII4 in the MM-BMECs/BMECs-HSCs adhesion. The first results showed that the expression of DII4 by MM-BMECs is necessary to promote HSCs adhesion. In fact, using a blocking antibody against DII4 (AbαDII4) at 50ug/ml there was an impairment in HSCs adhesion to MM-BMECs (43±9% versus 24±2% of adherent cells without and with AbαDII4 treatment), but not on BMECs (25±6% versus 26±1.4% of adherent cells without and with AbαDII4 treatment). Ongoing experiments are focusing on the role of DII4 in the modulation of HSCs proliferation, protection against apoptosis and in vitro-in vivo B commitment by MM-BMECs. Taken together, all these data suggest that BMECs in MM may function as “aberrant perivascular niches”, modulating HSCs properties. This aberrant phenotype could be due to an alteration of the Notch-Delta pathway in BMECs that favors malignant clonal growth by protecting it from apoptosis, favoring migration, adhesion and providing self-renewing and/or proliferative cues. Disclosures:No relevant conflicts of interest to declare.

Increasing C/Ebpα Expression Regulates the Transition of Hematopoietic Stem Cells During Development.

Abstract 1506Poster Board I-529During ontogeny, hematopoietic stem cells (HSCs) undergo a change from rapid dividing cells with high reconstitution ability to mainly quiescent cells with lower repopulation capacity. However, little is known about how this switch is regulated. Here we report that levels of C/EBPα, a transcription factor that is frequently disrupted in human acute myeloid leukemia, regulate the proliferation and self-renewal transition of HSCs during development. Loss of C/EBPα in adult mice resulted in a profound expansion of phenotypic HSCs and elevated proliferation rates. Limiting dilution transplantation to measure the frequency of competitive repopulation units (CRU) revealed an increase in the number of functional HSC in C/EBPα-/- mice. Serial transplantation of C/EBPα-/- bone marrow showed advanced reconstitution ability, indicating enhanced self-renewal ability. Interestingly, levels of C/EBPα in HSCs were significantly up-regulated 3 weeks after birth during which HSCs change from an actively cycling state to quiescence in bone marrow. When we conditionally inactivated C/EBPα in mice of different age, we observed a tight correlation between the age-specific levels of C/EBPα expression and the expansion of HSCs. Gene profiling analysis of C/EBPα-/- adult HSCs showed the up-regulation of oncogenes c-myc and n-myc, whose expression can regulate pluripotency and self-renewal of stem cells, as shown by recent induced pluripotent stem cell studies. Knocking down n-myc and c-myc expression in C/EBPα-/- HSCs using shRNA, we observed reduced proliferation and decreased colony formation in serial replating assay, which assesses the preservation of “self-renewal” in the progenitor cell compartment. Consistently, we observed down-regulation of n-myc in HSCs during the transition time, which is reciprocal to C/EBPα expression. Together, our data indicate C/EBPα as a key regulator of HSC self-renewal and proliferation during development, whose levels of expression might control the fetal to adult switch of HSC properties through regulating myc genes. DisclosuresNo relevant conflicts of interest to declare.

Communication between bone marrow niches in normal bone marrow function and during hemopathies progression

Hematopoietic stem cell (HSC) chemotaxis, adhesion, proliferation, quiescence and differentiation are regulated by interactions with bone marrow (BM) niches. Two niches have been identified in the adult BM: the endosteal (close to the bone) and the perivascular niche (close to blood vessels). A vast body of literature has revealed the molecular basis for the interaction of HSCs with the two niches. However, the signals that regulate the communication between the two niches have not been well defined. Taking in consideration several clinical and experimental arguments this review highlights the molecular cues, involved in the communication between the BM niches, which regulate the basic properties of HSCs in physiological and malignant conditions. As such, it aims at clarifying the most important advances in basic and clinical research focusing on the role of different factors in the regulation of the BM microenvironment.

Hematopoietic stem cell aging is associated with functional decline and delayed cell cycle progression

The molecular mechanisms underlying hematopoietic stem cell (HSC) aging remain to be elucidated. In this study, we investigated age-related changes in the functional and phenotypic properties of murine HSCs. Consistent with previous studies, we found that the number and frequency of CD34 −/lowc-Kit +Sca-1 +lineage marker − (CD34 −KSL) cells, a highly enriched HSC population, significantly increased in old mice, though their repopulating ability was reduced. Continuous bromodeoxyuridine labeling revealed a significant delay in the cell cycle progression of CD34 −KSL cells in old mice. This delay was also observed in young recipients transplanted with whole bone marrow cells from old mice. When cultured in vitro, CD34 −KSL cells from old mice showed a greater capacity to give rise to primitive CD48 −KSL cells with reduced HSC activity. Gene expression profiling identified age-related changes in the expression of several cell cycle regulatory genes, including p21/Cdkn1a and p18/Cdkn2c. These results support the notion that HSC aging is largely regulated by an intrinsic genetic program.