Progeny Of Hematopoietic Stem Cells Research Articles

The role of microglia in human disease: therapeutic tool or target?

Microglia have long been the focus of much attention due to their strong proliferative response (microgliosis) to essentially any kind of damage to the CNS. More recently, we reached the realization that these cells play specific roles in determining progression and outcomes of essentially all CNS disease. Thus, microglia has ceased to be viewed as an accessory to underlying pathologies and has now taken center stage as a therapeutic target. Here, we review how our understanding of microglia’s involvement in promoting or limiting the pathogenesis of diseases such as amyotrophic lateral sclerosis, Alzheimer’s disease, Huntington’s disease, multiple sclerosis, X-linked adrenoleukodystrophy (X-ALD) and lysosomal storage diseases (LSD) has changed over time. While strategies to suppress the deleterious and promote the virtuous functions of microglia will undoubtedly be forthcoming, replacement of these cells has already proven its usefulness in a clinical setting. Over the past few years, we have reached the realization that microglia have a developmental origin that is distinct from that of bone marrow-derived myelomonocytic cells. Nevertheless, microglia can be replaced, in specific situations, by the progeny of hematopoietic stem cells (HSCs), pointing to a strategy to engineer the CNS environment through the transplantation of modified HSCs. Thus, microglia replacement has been successfully exploited to deliver therapeutics to the CNS in human diseases such as X-ALD and LSD. With this outlook in mind, we will discuss the evidence existing so far for microglial involvement in the pathogenesis and the therapy of specific CNS disease.

Megakaryocytes regulate the quiescence of hematopoietic stem cells through PF4: 2013 ASH meeting highlights.

Hematopoietic stem cells (HSCs) can take one of the three different pathways: quiescence, self-renewal and differentiation. Mechanisms that control the tight balance to maintain lifelong hematopoietic homeostasis have been a major interest of research. Platelet factor-4 (PF4), a weak chemokine, is synthesized exclusively by megakaryocytes and sequestered in platelets. This meeting report highlights a novel study presented at 2013 ASH annual meeting. This study found that megakaryocyte, a progeny of HSC, was involved in maintaining quiescence of HSCs via PF4 in a feedback loop.

Megakaryocytes Regulate Hematopoietic Stem Cell Quiescence Via PF4 Secretion

Hematopoietic stem cells (HSCs) adapt to the varying needs for the replenishment of terminally differentiated blood cells by switching between quiescence, self-renewal and differentiation. A tight regulation of this balance is crucial to ensure hematopoietic homeostasis over a lifetime. In the bone marrow (BM), HSCs reside in specialized microenvironments, or niches, that regulate HSC behavior. Although all niche cells identified thus far have a non-hematopoietic origin (e.g., mesenchymal stem cells, osteoprogenitors, endothelial cells and sympathetic nerves), less is known about HSC regulation by its progeny. During confocal imaging studies of whole-mount BM, we have noted an association between HSCs and megakaryocytes (Mk); indeed up to 50% of Lin-CD41-CD48-CD150+ HSC were in direct contact with Mk. HSC were located at an average of 15.4 µm-distance to Mk. This distribution was not random (p=6x10-8) as demonstrated by two-sample Kolmogorov-Smirnov test. To assess the functional role of Mk in HSC regulation in vivo, we bred PF4-cre mice (in which the Cre recombinase gene is under the control of the Mk-specific platelet factor 4 (PF4) promoter) with iDTR mice (where cre-mediated recombination induces expression of the diphtheria toxin (DT) receptor). After 7 days of DT treatment PF4-cre:iDTR mice showed specific BM Mk depletion (5.3-fold reduction; P<0.001), followed by a concomitant reduction in platelets (7.0-fold reduction; P<0.0001). Strikingly, Mk depletion led to a 11.5-fold increase (P<0.001) in the number of Lin-c-Kit+Sca-1+CD105+CD150+ HSC due to a 5.5-fold increase in proliferation (determined by BrdU incorporation; P<0.001). Since loss of quiescence has been frequently associated with reduced HSC potential, we tested whether the increase in HSC proliferation after Mk depletion impaired HSC function by competitive transplantations in limiting dilutions. Extreme limiting dilution analysis (ELDA) revealed 625 HSCs with repopulating capacity per femur in DT-treated PF4-cre:iDTR mice which is 4.8-fold more (P<0.01) than in DT-treated PF4-cre transgenic control mice. Importantly, Mk depletion did not result in aberrant hematopoiesis as reflected by WBC and RBC counts in the peripheral blood and progenitor cell (CFU-E, GMP, Pre-GM, PreMegE, Pre CFU-E ) numbers in the BM that were indistinguishable from WT mice. To ascertain whether the effect was mediated by circulating platelets rather BM Mk, we injected mice with neuraminidase, which selectively depletes platelets. Neuraminidase treatment led to a 13.9-fold reduction in blood platelets (P<0.0001), but neither BM Mk nor HSC were significantly altered. Thus, Mk and not platelets regulate BM HSC quiescence. To investigate the mechanisms through which Mk regulate HSC, we screened for factors that have been shown to maintain cell quiescence in several cell types. Of those, PF4 itself, which is expressed exclusively by Mk, had the greatest reduction in the bone marrow extracellular fluid (BMEF) upon Mk depletion as determined by ELISA analyses. We therefore hypothesized that PF4 contributed to Mk-mediated maintenance of HSC quiescence. We first carried out in vitro culture experiments which revealed that recombinant murine (rm) PF4 restrained HSC proliferation in serum-free culture conditions (3.1 fold reduction; P<0.01). This effect was abrogated when PF4 was neutralized by heparin. Following 7 days of rmPF4 injections, mice exhibited a 1.5 fold reduction in HSC numbers (P<0.0002). We confirmed the reduction of functional HSCs by competitive transplantations. Sixteen weeks after transplantation recipient mice transplanted with BM cells from PF4-treated mice showed a significant reduction in the percentage of donor-derived cells (p<0.01). Next, we analyzed HSC numbers in Pf4-/- mice or in huPF4-Tg mice (which overexpress total PF4) in comparison to WT controls. Consistent with its role in HSC quiescence, Pf4-/- showed increased HSC numbers (1.5 fold; P<0.01) and enhanced proliferation (1.6 fold, P<0.05). By contrast, overexpression of PF4 in huPF4-Tg mice led to 1.5-fold (P<0.01) and 2.3-fold (P<0.05) reductions in HSC numbers and proliferation, respectively. These studies demonstrate a feedback loop where an HSC progeny, the megakaryocyte, can directly regulate HSC pool size and proliferation via PF4. Disclosures: Lambert:Amgen: Research Funding; Nestle: Consultancy; GSK: Research Funding.

Genetic Basis For Hematopoietic Stem Cell Generation In The Mammalian Embryo

The generation of hematopoietic stem cells (HSCs) via endothelial-to-hematopoietic transition within the aorta-gonad-mesonephros (AGM) region of the mammalian embryo is crucial for development of the adult hematopoietic system. Many questions remain unanswered regarding the molecular program in hemogenic endothelium that promotes the budding of hematopoietic cell clusters containing HSCs. Previously, we described a deletion of a Gata2 cis-element (+9.5) that depletes fetal liver HSCs, is lethal at E13-14 of embryogenesis, and is mutated in an immunodeficiency that progresses to myelodysplasia (MDS)/leukemia. In contrast to Gata2 knockout mice, which die around E10.5 because of anemia, the prolonged embryonic development of +9.5 site knockout mice provides a unique model system to investigate the potential roles for GATA-2 in HSC production, migration and function, and more specifically, the requirement for the +9.5 element to regulate Gata2 expression during these processes. Using an ex vivo system involving culturing intact AGM, or AGM dissociated into single cells and then reaggregated into an organoid, we demonstrated that the +9.5 deletion reduced Gata2 expression in uncultured AGM (1.4 fold, p<0.05), cultured intact AGM (4 fold, p<0.001) and cultured AGM reaggregates (3.4 fold, p<0.001). The importance of the +9.5 element for Gata2 expression in the AGM suggested that it might control the function of hemogenic endothelium and/or the HSC progeny. The homozygous +9.5 mutation resulted in a complete loss of progenitors and long-term repopulating HSCs in the AGM, as determined by quantitative colony assays and competitive transplantation assays, respectively. To determine whether the ablation of HSC repopulating activity in the +9.5-/- mutant AGM reflects a +9.5 element requirement for HSC genesis from hemogenic endothelium, we used a whole-mount three-dimensional embryo immunostaining assay to visualize HSC genesis in +9.5+/+ and +9.5-/- AGMs. Imaging of E10.5 embryos revealed CD31+c-Kit+ hematopoietic clusters in +9.5+/+ dorsal aorta, while clusters were absent from the +9.5-/- embryos. The absence of hematopoietic clusters in the +9.5-/- dorsal aorta, and the ablation of HSC repopulating activity, demonstrated that the +9.5 element is required for hemogenic endothelium to generate HSCs in the AGM. In principle, the +9.5-dependent genetic network should reveal clues regarding the molecular mechanisms underlying the defective HSC generation in +9.5-/- AGMs. We conducted RNA-seq to define +9.5+/+ and +9.5-/- AGM explant transcriptomes, and this genomic analysis indicated that the +9.5 element instigates a stem cell-regulatory genetic network consisting of genes encoding established regulators of hemogenic endothelium and HSCs, and genes not implicated previously in hematopoiesis. We investigated whether the +9.5 element contributes to the transcriptome of AGM endothelium. Quantitative RT-PCR analysis revealed a similar impact of +9.5 deletion on representative genes in the fraction enriched in endothelial cells (CD31+c-Kit-) from the AGM. These studies establish a new model whereby a composite cis-regulatory element induces Gata2 expression and instigates a complex genetic network in the AGM, which controls the transition of hemogenic endothelium to HSCs in the AGM. Studies are ongoing to establish the genetic network in hemogenic endothelium that mediates the development of the adult hematopoietic system and the applicability of the respective mechanisms to distinct biological and pathological contexts. Disclosures:No relevant conflicts of interest to declare.

Zebraflash transgenic lines for in vivo bioluminescence imaging of stem cells and regeneration in adult zebrafish

The zebrafish has become a standard model system for stem cell and tissue regeneration research, based on powerful genetics, high tissue regenerative capacity and low maintenance costs. Yet, these studies can be challenged by current limitations of tissue visualization techniques in adult animals. Here we describe new imaging methodology and present several ubiquitous and tissue-specific luciferase-based transgenic lines, which we have termed zebraflash, that facilitate the assessment of regeneration and engraftment in freely moving adult zebrafish. We show that luciferase-based live imaging reliably estimates muscle quantity in an internal organ, the heart, and can longitudinally follow cardiac regeneration in individual animals after major injury. Furthermore, luciferase-based detection enables visualization and quantification of engraftment in live recipients of transplanted hematopoietic stem cell progeny, with advantages in sensitivity and gross spatial resolution over fluorescence detection. Our findings present a versatile resource for monitoring and dissecting vertebrate stem cell and regeneration biology.

Proliferation-Dependent Alterations of the DNA Methylation Landscape Underlie Hematopoietic Stem Cell Aging

The functional potential of hematopoietic stem cells (HSCs) declines during aging, and in doing so, significantly contributes to hematopoietic pathophysiology in the elderly. To explore the relationship between age-associated HSC decline and the epigenome, we examined global DNA methylation of HSCs during ontogeny in combination with functional analysis. Although the DNA methylome is generally stable during aging, site-specific alterations of DNA methylation occur at genomic regions associated with hematopoietic lineage potential and selectively target genes expressed in downstream progenitor and effector cells. We found that age-associated HSC decline, replicative limits, and DNA methylation are largely dependent on the proliferative history of HSCs, yet appear to be telomere-length independent. Physiological aging and experimentally enforced proliferation of HSCs both led to DNA hypermethylation of genes regulated by Polycomb Repressive Complex 2. Our results provide evidence that epigenomic alterations of the DNA methylation landscape contribute to the functional decline of HSCs during aging.

Implication of the Distinct Differentiation Pathway of Megakaryocytes From Hematopoietic Stem Cells in the Mouse Bone Marrow

AbstractAbstract 1204 Background.Hematopoietic progenitor cells are the progeny of hematopoietic stem cells (HSC) that coordinate the production of precise number of mature blood cells of diverse functional lineages. Megakaryocytes (Meg) are mapped at the downstream of bilineage progenitors for erythroid and megakaryocyte (MEP) in the most widely accepted scenarios, although different notions have also been suggested. Thrombopoietin (TPO) is thought to be the master cytokine for megakaryopoiesis. In mice lacking cMpl, the receptor for TPO, production of platelets and Meg is severely impaired. However, Meg are known to be still present in the bone marrow of these mice. These findings suggested that TPO independent signaling for Meg differentiation would exist. Purpose.To clarify the differentiation pathway of the Meg lineage, we focused on GPIb (CD42)-V-IX complex, expression of which has not been characterized in any progenitor cells whereas it is well known to be expressed on mature Meg and platelets. We also investigated how TPO-cMpl signaling would affect at MEP or pure megakaryocyte progenitor (MKP) stage using the cMpl deficient mice. Results and Discussion.GPIb alpha (CD42b) was expressed on 3–6 % of a mouse bone marrow population characterized as common myeloid progenitors (CMP), i.e., Lin-c-Kit+Sca1-CD34+CD16/32low cells. The GPIb alpha+ CMP (thereafter designated 34-alpha) population also expresses CD9, SLAM1, and CD41. These 34-alpha cells showed a restricted differentiation capacity to the mature Meg in in vitro culture. By intravenously infusing 34-alpha cells derived from CAG promoter-driven GFP-expressing mice into sublethally irradiated syngenic mice, GFP-expressing platelets were generated in vivo. Thus, we designate the 34-alpha cells as 34-alpha MKP. Gene expression analysis also supported that 34-alpha MKP has a restricted capacity of megakaryopoiesis.In vitro colony-forming assay and short-term liquid culture assay suggested that they are not derived from MEP but from the SLAM1+Flt3-c-Kit+Sca1+Lin- population, which highly contain HSC. When experimental thrombocytopenia was induced by injecting 5-fluorouracil into mice, the frequency of 34-alpha MKP was rapidly increased compared to that of MEP. These data imply a distinct pathway of Meg differentiation, which originates at the proximity of HSC.We next investigated whether generation of 34-alpha MKP and MEP is differently impaired in cMpl-deficient mice. The frequency of MEP was only mildly reduced. In contrast, 34-alpha MKP were much severely reduced. Notably, in vitro Meg differentiation was markedly impaired from both MEP and 34-alpha MKP derived from cMpl-deficient mice. These data suggested that discordance between Meg and platelet production is caused by the different dependence on TPO-cMpl signaling between the pathways generating MEP and 34-alpha MKP from HSC.We also found that Hes1, a transcription factor that is the best characterized effector functioning downstream of the Notch signaling pathway, is highly expressed in 34-alpha MKP. Conversely, Meg differentiation was abrogated by retroviral transduction of a dominant-negative mutant of Hes1.Taken together, our data imply the presence of two distinct Meg differentiation pathways from HSC and further suggest that the dependency of TPO-cMpl signaling is different in these pathways and Notch-Hes signaling plays an additional role in them. Disclosures:No relevant conflicts of interest to declare.

The Microtubule Plus-End Tracking Protein CLASP2 Is Required for Hematopoiesis and Hematopoietic Stem Cell Maintenance

Mammalian CLASPs are microtubule plus-end tracking proteins whose essential function as regulators ofmicrotubule behavior has been studied mainly incultured cells. We show here that absence of murine CLASP2 invivo results in thrombocytopenia, progressive anemia, and pancytopenia, due to defects in megakaryopoiesis, in erythropoiesis, and in the maintenance of hematopoietic stem cell activity. Furthermore, microtubule stability and organization are affected upon attachment of Clasp2 knockout hematopoietic stem-cell-enriched populations, and these cells do not home efficiently toward their bone marrow niche. Strikingly, CLASP2-deficient hematopoietic stem cells contain severely reduced mRNA levels of c-Mpl, which encodes the thrombopoietin receptor, an essential factor for megakaryopoiesis and hematopoietic stem cell maintenance. Our data suggest that thrombopoietin signaling is impaired in Clasp2 knockout mice. We propose that the CLASP2-mediated stabilization ofmicrotubules is required for proper attachment, homing, and maintenance of hematopoietic stem cells and that this is necessary to sustain c-Mpl transcription.

Characterization of thrombopoietin (TPO)-responsive progenitor cells in adult mouse bone marrow with in vivo megakaryocyte and erythroid potential

Hematopoietic progenitor cells are the progeny of hematopoietic stem cells that coordinate the production of precise numbers of mature blood cells of diverse functional lineages. Identification of cell-surface antigen expression associated with hematopoietic lineage restriction has allowed prospective isolation of progenitor cells with defined hematopoietic potential. To clarify further the cellular origins of megakaryocyte commitment, we assessed the in vitro and in vivo megakaryocyte and platelet potential of defined progenitor populations in the adult mouse bone marrow. We show that megakaryocytes arise from CD150(+) bipotential progenitors that display both platelet- and erythrocyte-producing potential in vivo and that can develop from the Flt3(-) fraction of the pregranulocyte-macrophage population. We define a bipotential erythroid-megakaryocyte progenitor population, the CD150(+)CD9(lo)endoglin(lo) fraction of Lin(-)cKit(+)IL7 receptor alpha(-)FcγRII/III(lo)Sca1(-) cells, which contains the bulk of the megakaryocyte colony-forming capacity of the bone marrow, including bipotential megakaryocyte-erythroid colony-forming capacity, and can generate both erythrocytes and platelets efficiently in vivo. This fraction is distinct from the CD150(+)CD9(hi)endoglin(lo) fraction, which contains bipotential precursors with characteristics of increased megakaryocytic maturation, and the CD150(+)CD9(lo)endoglin(hi) fraction, which contains erythroid lineage-committed cells. Finally, we demonstrate that bipotential erythroid-megakaryocyte progenitor and CD150(+)CD9(hi)endoglin(lo) cells are TPO-responsive and that the latter population specifically expands in the recovery from thrombocytopenia induced by anti-platelet serum.

The 3′ Region of the Chicken Hypersensitive Site-4 Insulator Has Properties Similar to Its Core and Is Required for Full Insulator Activity

Chromatin insulators separate active transcriptional domains and block the spread of heterochromatin in the genome. Studies on the chicken hypersensitive site-4 (cHS4) element, a prototypic insulator, have identified CTCF and USF-1/2 motifs in the proximal 250 bp of cHS4, termed the “core”, which provide enhancer blocking activity and reduce position effects. However, the core alone does not insulate viral vectors effectively. The full-length cHS4 has excellent insulating properties, but its large size severely compromises vector titers. We performed a structure-function analysis of cHS4 flanking lentivirus-vectors and analyzed transgene expression in the clonal progeny of hematopoietic stem cells and epigenetic changes in cHS4 and the transgene promoter. We found that the core only reduced the clonal variegation in expression. Unique insulator activity resided in the distal 400 bp cHS4 sequences, which when combined with the core, restored full insulator activity and open chromatin marks over the transgene promoter and the insulator. These data consolidate the known insulating activity of the canonical 5′ core with a novel 3′ 400 bp element with properties similar to the core. Together, they have excellent insulating properties and viral titers. Our data have important implications in understanding the molecular basis of insulator function and design of gene therapy vectors.

Mechanism of Reduction in Titers From Lentivirus Vectors Carrying Large Inserts in the 3′LTR

Self-inactivating (SIN) lentiviruses flanked by the 1.2-kb chicken hypersensitive site-4 (cHS4) insulator element provide consistent, improved expression of transgenes, but have significantly lower titers. The mechanism by which this occurs is unknown. Lengthening the lentiviral (LV) vector transgene cassette by an additional 1.2 kb by an internal cassette caused no further reduction in titers. However, when cHS4 sequences or inert DNA spacers of increasing size were placed in the 3'-long terminal repeat (LTR), infectious titers decreased proportional to the length of the insert. The stage of vector life cycle affected by vectors carrying the large cHS4 3'LTR insert was compared to a control vector: there was no increase in read-through transcription with insertion of the 1.2-kb cHS4 in the 3'LTR. Equal amount of full-length viral mRNA was produced in packaging cells and viral assembly/packaging was unaffected, resulting in comparable amounts of intact vector particles produced by either vectors. However, LV vectors carrying cHS4 in the 3'LTR were inefficiently processed following target-cell entry, with reduced reverse transcription and integration efficiency, and hence lower transduction titers. Therefore, vectors with large insertions in the 3'LTR are transcribed and packaged efficiently, but the LTR insert hinders viral-RNA (vRNA) processing and transduction of target cells. These studies have important implications in design of integrating vectors.

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

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

Solid Tumors Target the HSC Niche to Establish Metastatic Footholds in the Marrow

Metastasis is the most deadly aspect of many solid tissue cancers. The majority of metastases utilize pathways established to regulate hematopoietic stem cell (HSC) homing to gain access to the marrow. Whether solid tumors target the niche to establish footholds in the marrow is unknown. Using prostate caner (PCa) as a model, we determined if PCa and HSC compete for the HSC niche. To determine if disseminated PCa competes for the niche and prevents HSC engraftment, sham-operated or tumor-implanted NOD/SCID mice (Ly5.1) were transplanted with marrow cells from Ly5.2 donor animals. To preserve the niche, the transplanted animals received no preconditioning. At 16 weeks, greater HSC engraftment was observed in sham-operated group than in the tumor-bearing group as reflected by mature HSC progeny in the peripheral blood (0.7 ± 0.4% vs. 0.2 ± 0.2%, p = 0.022). Multiphoton microscopy revealed that PCa and SLAM receptor isolated HSCs co-localize at the endosteal bone surfaces in vivo and in vitro, further suggesting that PCa compete for the niche. Since osteoblasts (OBs) are vital components of the niche, it was determined if alterations in OB numbers also regulates metastasis. Here, vertebrae from control or conditional osteoblast knockout (Col2.3Δ-TK) mice were transplanted into SCID animals. Following treatment with ganciclovir which ablated the osteoblasts, metastasis to these tissues was evaluated. Fewer disseminated PCa were recovered from the Col2.3Δ-TK transplants than vehicle treated transplants (41.6 ± 23.7% vs. 100.0 ± 69.9%, p = 0.009). If metastases target the niche, then we should be able to mobilize metastases using HSC mobilizing regimes. Here, PCa tumors were established and removed. Later G-CSF was administered, and PCa numbers were determined in the blood. More PCa cells were seen in the blood after G-CSF treatments vs. the vehicle group (616.9 ± 463.0 cells vs. 143.7 ± 159.1 cells, p = 0.025). Part of the mechanism responsible for the mobilization of PCa was an increase in the production of MMP-2 and MMP-9 in the marrow. Finally, we evaluated if soluble factors produced by PCa alter HSCs activities. Animals were treated with either control or PCa conditioned medium for 3 days, and then HSC were harvested. Alterations of many mRNA transcripts involved in HSC self renewal were observed in animals exposed to the PCa conditioned medium. Our findings indicate that PCa and HSCs compete for the osteoblastic HSC niche, and that PCa utilize the same mechanisms as HSCs to gain access and egress from the niche. Taken together, these data provide the first evidence that the HSC niche serves as pre-metastatic niche for solid tumors and plays a central role in bone metastases.

Stat4 suppresses the proliferation of connective tissue-type mast cells

Mast cells are the progeny of hematopoietic stem cells, and murine mast cells are usually divided into two distinct populations, mucosal mast cells (MMCs) and connective tissue-type mast cells (CTMCs). We previously reported that CTMCs expressed signal transducer and activator of transcription (Stat) 4, but MMCs did not. Stat4 is also expressed in T cells and plays important roles in their homeostasis. In the present study, we show that Stat4 is involved in the homeostasis of CTMCs. The number of skin CTMCs increased in Stat4-deficient Balb/c mice, but that of gastric MMCs did not, when compared to those in control Balb/c+/+ mice. The comparison between cultured Stat4-deficient CTMCs and cultured Balb/c+/+ CTMCs revealed that cell cycle progression and cyclin D3 expression in the cultured Stat4-deficient CTMCs were enhanced in a Stat3 activation-dependent manner. This phenotype was explained by upregulation of KitL-induced interleukin (IL)-6 acting in an autocrine manner in cultured Stat4-deficient CTMCs. These results show that Stat4 suppresses the proliferation of CTMCs by controlling IL-6 via an autocrine mechanism.

E2A Proteins Promote Development of Lymphoid-Primed Multipotent Progenitors

The first lymphoid-restricted progeny of hematopoietic stem cells (HSCs) are lymphoid-primed multipotent progenitors (LMPPs), which have little erythromyeloid potential but retain lymphoid, granulocyte, and macrophage differentiation capacity. Despite recent advances in the identification of LMPPs, the transcription factors essential for their generation remain to be identified. Here, we demonstrated that the E2A transcription factors were required for proper development of LMPPs. Within HSCs and LMPPs, E2A proteins primed expression of a subset of lymphoid-associated genes and prevented expression of genes that are not normally prevalent in these cells, including HSC-associated and nonlymphoid genes. E2A proteins also restricted proliferation of HSCs, MPPs, and LMPPs and antagonized differentiation of LMPPs toward the myeloid fate. Our results reveal that E2A proteins play a critical role in supporting lymphoid specification from HSCs and that the reduced generation of LMPPs underlies the severe lymphocyte deficiencies observed in E2A-deficient mice.

MicroRNA regulation directs lineage- and developmental stage-specific transgene expression in hematopoietic stem cell progeny

MicroRNA regulation directs lineage- and developmental stage-specific transgene expression in hematopoietic stem cell progeny

Elements of a Chromatin Insulator Necessary for Consistent, High-Level Globin Gene Expression

Vectors flanked by the chicken b-globin locus hypersensitive site-4 (cHS4) insulator provide consistent, improved expression (chromatin barrier activity) and reduced genotoxicity (enhancer-blocking activity); features important for randomly integrating viral vectors. However, full-length 1.2Kb cHS4 reduces vector titers significantly. Initial studies mapped the enhancer blocking activity of cHS4 to the first 250bp ‘core' in a copy-number dependent manner (Felsenfeld and colleagues). However, elements required for the barrier effect are not well defined. We studied various regions of cHS4 for chromatin barrier effect. Beta-globin-LCR lentivirus vectors were constructed with the core (sBGC), 2 copies of the core (sBG2C), 400bp (sBG400) and 800bp (sBG800) fragments (both including the core) and compared to the un-insulated vector (sBG) and the vector with the full-length cHS4 (sBG-I). Barrier activity (improved probability and consistency of expression from individual integrants) in MEL cells and the clonal progeny of hematopoietic stem cells was analyzed. Single-copy MEL clones showed that sBG-I clones had a significantly higher proportion of hβ+ cells/clone. However, proportion of hβ+ cells from all other vectors carrying various fragments of cHS4 were not significantly different from the un-insulated vector (Table 1). Interestingly, all clones carrying a fragment containing the core showed reduced clonal variegation (coefficient of variation of hβ expression). We next analyzed hβ expression from the sBG, sBGC, sBG2C, sBG400 and sBG-I vectors in primary and secondary thalassemia (Hbb th3/+) mice. In the primary mice and secondary CFU-S analyzed thus far (Table 2), it appears that all vectors with cHS4 insulator fragments containing the core provide some barrier activity, but the full-length insulator is necessary for maximum barrier effect. To corroborate whether elements outside the core contribute to barrier activity, we also tested vectors with inert DNA spacers in addition to the core (sBG400−S, sBG-800−S and sBG1200−S). Single copy MEL clones confirmed that the sBG400−S, sBG800−S and 1200−S vectors had significantly lower proportion of hβ+ cells/clone [41±4.4%, 41±7% and 28±6% respectively, n=36]. These data suggest that regions of 950 bp region of cHS4 outside the core contains elements that cooperate with the core for optimal barrier activity. These studies have important implications for vector design for gene therapy for thalassemia, where variability in expression from uninsulated vectors compromizes efficacy.Table 1β globin expression in MEL cell clones from vectors carrying portions of the cHS4 insulatorsBGsBG csBG 2csBG 400sBG 800sBG-I% hβ+ cells59±549±651±351±349±684±3**CV53±437±3**40±2**42±1.5**42±3**36±2**Values are expressed as Mean±SEM, *P<0.05, **P<0.01 by ANOVA (Dunnet: comparisons of all vectors to the control vector, sBG)Table 2β globin expression in primary thalassemia mice and in the clonal progeny of hematopoietic stem cells of secondary micePrimary mice (16 weeks)MocksBGsBG csBG 2csBG 400sBG-IHCT (%)20±332±336±237±237±339±2Reticulocytes (%)28±216±511±1**13±6**7±2**7±2**%HbA (mαhβ by HPLC)/vector copyNA32±1924±529±331±343±4*% hβ+RBC/ vector copyNA36±1157±8*67±13*76±17*104±18** Secondary CFU-S (Single Copy)% hβ+ cells/cloneNA26±345±3*34±248±9*75±3**CVNA88±968±2*71±2*72±361±2**Values are expressed as Mean ± SEM, *P<0.05, **P<0.01 by ANOVA (Dunnett: comparison of all vectors to the control, sBG)

MicroRNA-Mediated Gene Regulation Effectively Restricts In Vivo Transgene Expression in Hematopoietic Stem Cell Progeny.

Stem cell engineering and targeted in vivo gene delivery increasingly require tight control of transgene expression. Lineage- and differentiation stage-specific gene regulation is classically afforded by pol-II-dependent transcript regulation. A super-imposed layer of post-transcriptional control would be valuable to correct undesirable expression patterns or fine-tune developmentally regulated or inducible gene expression. microRNAs (miRNAs) have recently emerged as potent repressors of gene expression at the post-transcriptional level. In this study, we investigate the potential of using miRNA regulation to provide lineage-restricted expression, exploiting miRNAs with distinctive expression patterns in hematopoietic tissue. miR-223 is preferentially expressed in granulocytes and monocytes (80-and 110-fold, respectively). miR-181a is highly expressed in B-lymphocytes and, particularly, in thymocytes, but down-regulated (∼1000-fold) in post-thymic T cells. We, therefore, constructed lentiviral vectors encoding either green fluorescent protein (GFP) or an antigen-specific receptor, placed under the transcriptional control of the ubiquitous EF1a promoter and tagged with miRNA-recognition elements (MREs) complementary to the mature miR-223 or miR-181a. In a panel of murine and human cell lines expressing varying levels of the two miRNAs we find that GFP knockdown is dependent on the presence of the miRNA and directly proportional to the number of MRE repeats. Four copies of the repeat permit better down-regulation than 2 copies (albeit depending on the level of endogenous miRNA). Two different MREs can be combined in tandem, resulting in additive down-regulation. In vivo, in mouse bone marrow chimeras harboring the miR-223-responsive vector, GFP expression is specifically repressed in myeloid cells (>85% compared to the control vector lacking miRNA target sequences). Reciprocally, chimeras harboring the miR-181a-regulated vector express GFP in myeloid and erythroid lineages, but transgene expression is profoundly repressed in thymocytes and B-cell progenitors. A vector harboring a composite MRE confers GFP expression almost exclusively confined to the erythroid lineage. These results demonstrate for the first time that transgene expression can be selectively regulated at the post-transcriptional level within the hematopoietic tissue. In mouse chimeras expressing a CD19-specific antigen receptor (which we and others are currently using in clinical trials in B cell malignancies), we analyze receptor expression in the 4 double negative subsets (DN1, DN2, DN3 and DN4), the double-positive subset (DP), and the CD4+ and CD8+ single positive (SP) subsets. We detail and quantify receptor knock-down at each developmental stage, and show that miR-181a-mediated regulation prevents receptor expression at critical stages of positive and negative thymic selection. Antigen receptor expression is dramatically repressed in DN and DP cells, while rising in CD4+ and CD8+ SP thymocytes. Importantly, expression is fully restored in post-thymic T cells, and maintained in activated T cells. miRNA-mediated post-transcriptional regulation is thus proving to be a powerful means to direct lineage- and differentiation stage-specific transgene expression in genetically modified stem cells.

Improved Human β-globin Expression from Self-inactivating Lentiviral Vectors Carrying the Chicken Hypersensitive Site-4 (cHS4) Insulator Element

Effective gene therapy for beta-thalassemia major (beta-TM) requires consistent, high expression of human beta-globin (hbeta-globin) in red blood cells (RBCs). Several groups have now shown that lentiviral (LV) vectors stably transmit the hbeta/hgamma-globin genes and large elements of the locus control region, resulting in correction of the murine thalassemia intermedia (TI) phenotype and survival of mice with the TM phenotype. However, current LVs show variable hbeta/hgamma-globin expression and require a high number of vector copies/cell for a therapeutic effect. To address this, we designed LVs flanked by the chicken hypersensitive site-4 (cHS4) chromatin insulator element and compared them with their "un-insulated" counterparts. We observed a consistent twofold-higher hbeta expression from insulated vectors in single-copy mouse erythroleukemia cell clones, an increase that resulted from reduced position effect variegation (PEV) and increased probability of expression from individual integrants. This effect was confirmed in vivo: an approximately twofold increase in hbeta expression was seen in the RBC progeny of murine hematopoietic stem cells, with significantly higher numbers of hbeta-expressing cells in individual secondary spleen colony-forming units. In summary, cHS4-insulated hbeta-globin LVs showed distinct chromatin barrier activity, resulting in higher, consistent hbeta expression. These studies have important implications for vector design for clinical trials for gene therapy for hemoglobinopathies.

Elucidation of the Phenotypic, Functional, and Molecular Topography of a Myeloerythroid Progenitor Cell Hierarchy

The major myeloid blood cell lineages are generated from hematopoietic stem cells by differentiation through a series of increasingly committed progenitor cells. Precise characterization of intermediate progenitors is important for understanding fundamental differentiation processes and a variety of disease states, including leukemia. Here, we evaluated the functional in vitro and in vivo potentials of a range of prospectively isolated myeloid precursors with differential expression of CD150, Endoglin, and CD41. Our studies revealed a hierarchy of myeloerythroid progenitors with distinct lineage potentials. The global gene expression signatures of these subsets were consistent with their functional capacities, and hierarchical clustering analysis suggested likely lineage relationships. These studies provide valuable tools for understanding myeloid lineage commitment, including isolation of an early erythroid-restricted precursor, and add to existing models of hematopoietic differentiation by suggesting that progenitors of the innate and adaptive immune system can separate late, following the divergence of megakaryocytic/erythroid potential.