Origin of Hematopoietic Stem Cells

Abstract Abstract SCI-40 During development, the embryo needs to rapidly produce differentiated blood cells to provide oxygen for its survival and growth, as well as establish a pool of undifferentiated HSCs for lifelong hematopoiesis. These opposing goals are achieved by segregating fetal hematopoiesis into multiple waves that occur in distinct anatomical niches that promote either differentiation or “stemness”. However, it has been unclear which anatomical sites and cellular precursors give rise to the different hematopoietic waves, impeding our ability to study the regulatory mechanisms dictating the fate of these cells. Understanding the development of self-renewing HSCs in the embryo will be crucial for generating these cells in vitro from pluripotent cells for therapeutic purposes. Recent studies have verified that HSCs develop through a hemogenic endothelial intermediate. Using Ncx1−/− (sodium/calcium exchanger) mouse embryos that are unable to initiate heartbeat and circulation, we showed that multipotential myelo-lymphoid HSPCs can be generated in the embryo proper, the yolk sac and the placenta. This implies that hemogenic endothelium extends more broadly than previously thought, spanning from the major intra-embryonic arteries to extra-embryonic hematopoietic tissues. In order to understand how hemogenic endothelium is established, we defined genomewide target genes for Scl/Tal1, a bHLH transcription factor that initiates hematopoietic specification from mesoderm. Our studies indicate that Scl governs the divergence of multiple mesodermal fates, activating major hemato-vascular transcription factor networks required for the establishment of hemogenic endothelium and generation of HSCs, as well as repressing regulators of competing mesodermal fates. Imbalance in these mesodermal networks in Scl-deficient embryos results in complete loss of hematopoietic cells, impaired establishment of hemogenic endothelium and profound cardiac defects with disorganized endocardium and ectopic activation of myocardial and mesenchymal transcriptional networks. These studies reveal a much broader role for Scl than previously anticipated and delineates Scl as a master regulator of mesoderm specification that coordinates proper development of both the blood and circulatory systems. The intimate relationship between the development of these mesodermal organ systems was also evidenced through studies using the heartbeat deficient Ncx1−/− embryos, which revealed that blood flow is essential for the emergence of HSCs from hemogenic endothelium in the placenta and in the embryo. Interestingly, the development of the lineage-restricted progenitors in the yolk sac was not affected, thus providing a unique opportunity to investigate the mechanisms that regulate HSC development specifically. We show that in the absence blood flow, hemogenic precursors are unable to be released from hemogenic endothelium to circulation and accumulate in placental vasculature. Our studies suggest that shear forces and changes in oxygen tension prompted by circulating red cells are required for suppressing endothelial signaling pathways, releasing adherens junctions in hemogenic endothelium and completing the emergence of HSCs. These studies emphasize the highly dynamic nature of the embryonic hematopoietic microenvironments and pinpoint the requirement of the earliest embryonic blood cells for proper HSC development. Disclosures: No relevant conflicts of interest to declare.

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Blood Flow Is Required for the Release of Hematopoietic Stem- and Progenitor Cells From Hemogenic Endothelium in the Placenta.

Blood ◽

10.1182/blood.v114.22.698.698 ◽

2009 ◽

Vol 114 (22) ◽

pp. 698-698

Author(s):

Katrin E Rhodes ◽

Ben Van Handel ◽

Michele Wang ◽

Yanling Wang ◽

Akanksha Chhabra ◽

...

Keyword(s):

Endothelial Cells ◽

Blood Flow ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Yolk Sac ◽

Positive Staining ◽

Hemogenic Endothelium ◽

Hematopoietic Stem ◽

Notch1 Signaling ◽

Stem And Progenitor Cells

Abstract Abstract 698 Hematopoietic stem cells (HSCs) are required for continuous blood cell production throughout life. HSCs emerge only within a short developmental time window during embryogenesis. Mounting evidence posits that HSCs arise directly from hemogenic endothelial cells during midgestation within the large arteries of the conceptus, which include the dorsal aorta, the umbilical and vitelline arteries and the chorioallantoic vessels of the placenta. However, the microenvironmental signals that mediate this temporally regulated process remain unclear. Here we examine, by using Ncx1−/− embryos that lack heartbeat and circulation, how blood flow imparts instructive cues that ensure proper HSC development. Immunostaining revealed that CD41+ hematopoietic cells, although present, were markedly decreased in Ncx1-/-placentas as compared to wild-type controls. Furthermore, mutant placentas evidenced large clusters of round CD31+ cells protruding into the lumens of the chorioallantoic vessels. Based on these data, we hypothesized that lack of blood flow may impede the generation of hematopoietic stem and progenitor cells (HS/PCs) and that the endothelial clusters represent hemogenic intermediates. FACS analysis and colony forming assays confirmed a dramatic reduction in the number of clonogenic progenitors in the placenta and the embryo proper of Ncx mutants, while the yolk sac was unaffected. However, HS/PC generation in the placenta and embryo could be rescued by culturing explants on OP9 stroma before plating in colony forming assays, verifying intact hematopoietic potential. To determine if the rescue observed was due to expansion of existing progenitors or generation of new HS/PCs, we sorted CD41medckit+hematopoietic progenitors and CD31+CD41− endothelial cells from hematopoietic tissues and co-cultured them on stroma. These experiments demonstrated that endothelial cells from placenta, embryo proper and yolk sac can generate HS/PCs following stroma stimulation, confirming the presence of hemogenic endothelium in these organs. Immunostaining of Ncx−/− placentas revealed that although the development of the arterio-venous vascular network was impaired, Notch1 signaling, required for both arterial specification and HSC development, was robust in cells of the endothelial clusters. Furthermore, positive staining for Runx1 and c-myb indicated that cells in the clusters had activated the hematopoietic program. Interestingly, electron microscopy demonstrated that cells in the clusters were tethered to each other via adherens junctions, a characteristic of endothelial cells. In addition, they also maintained high levels of Flk1, expressed VEGF and were actively proliferating, consistent with exposure to extended hypoxia. These data suggest that although cells in the clusters have initiated hematopoietic commitment, they are unable to down-regulate their endothelial identity and complete hematopoietic emergence, resulting in the formation of clusters of hemogenic intermediates. These results imply that cues imparted via circulation are required to complete the commitment to a hematopoietic fate from hemogenic endothelium. Data from co-culture experiments suggest that prolonged Notch1 signaling impairs hematopoietic emergence from hemogenic endothelial cells, and may account for the HSC emergence defect in the absence of blood flow. Overall, these data suggest that blood flow and circulating primitive red blood cells are critical components of the dynamic microenvironment necessary to both relieve the hypoxia required for the specification and proliferation of hemogenic endothelium and provide important mechanical and/or molecular signals required by HSCs to fully commit to the hematopoietic fate and complete emergence. Disclosures: No relevant conflicts of interest to declare.

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Latent Cardiogenic Potential in Endocardium and Hemogenic Endothelium Revealed in the Absence of Scl/tal1

Blood ◽

10.1182/blood.v118.21.2362.2362 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2362-2362

Author(s):

Amelie Montel-Hagen ◽

Ben Van Handel ◽

Roberto Ferrari ◽

Rajkumar Sasidharan ◽

Tonis Org ◽

...

Keyword(s):

Endothelial Cells ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Lineage Tracing ◽

Bhlh Transcription Factor ◽

Wild Type ◽

Transcriptional Program ◽

Hemogenic Endothelium ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Abstract Abstract 2362 The endothelium in embryonic and extraembryonic hematopoietic tissues has the capacity to generate hematopoietic stem and progenitor cells (HS/PC). However, it is unknown how this unique endothelium is specified. Microarray analysis of endothelial cells from hematopoietic tissues of embryos deficient for the bHLH transcription factor Scl/tal1 revealed that Scl establishes a robust hematopoietic transcriptional program in the endothelium. Surprisingly, lack of Scl also induced an unexpected fate switching of the prospective hemogenic endothelium to the cardiac lineage. Scl deficient embryos displayed a dramatic upregulation of cardiac transcription factors and structural proteins within the yolk sac vasculature, resulting in the generation of spontaneously beating cardiomyocytes. Ectopic cardiac potential in Scl deficient embryos arose from endothelial-derived CD31+Pdgfrα+ cardiogenic progenitor cells (CPCs), which were present in all sites of HS/PC generation. Analysis of Runx1-deficient embryos revealed, that although Runx1 acts downstream of Scl during the emergence of definitive HS/PCs, it is not required for the suppression of the cardiac fate in the endothelium. The only wild type tissue that contained CD31+Pdgfrα+ putative CPCs was the heart, and this population was greatly expanded in Scl deficient embryos. Strikingly, endocardium in Scl−/− hearts also activated a robust cardiomyogenic transcriptional program and generated Troponin T+ cardiomyocytes both in vivo and in vitro. Although CD31+Pdgfrα+ CPCs from wild type hearts did not generate readily beating cells in culture, they produced cells expressing endothelial, smooth muscle and cardiomyocyte specific genes, implying multipotentiality of this novel CPC population. Furthermore, CD31+Pdgfrα+ CPCs were greatly reduced in Isl1−/− hearts, which fail to generate functional, multipotential CPCs. Lineage tracing using VE-cadherin Cre Rosa-YFP mouse strain demonstrated that, in addition to generating HS/PCs in hematopoietic tissues, endothelial cells are also the cell of origin for CD31+Pdgfrα+ CPCs in the heart. Together, these data suggest a broader role for embryonic endothelium as a potential source of tissue-specific stem and progenitor cells and implicate Scl/tal1 as an important regulator of endothelial fate choice. Disclosures: No relevant conflicts of interest to declare.

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MixL1 Expression Identifies Putative Hematopoietic Stem Cells In the Murine Allantois.

Blood ◽

10.1182/blood.v116.21.1591.1591 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1591-1591

Author(s):

Adam D Wolfe ◽

Karen Downs

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Blood Cells ◽

Conflicts Of Interest ◽

Preliminary Evidence ◽

Yolk Sac ◽

Cell Populations ◽

Hematopoietic Stem ◽

Poorly Differentiated ◽

Stem Cell Populations

Abstract Abstract 1591 MixL1, a paired-type homeodomain transcription factor, is implicated in pre-hematopoietic commitment of stem cell populations. In poorly-differentiated human lymphoma and leukemia lines, MixL1 is inappropriately over-expressed (Drakos et al., Human Pathol 2007:38:500). When constitutively expressed in mice, MixL1 is sufficient to induce Acute Myeloid Leukemia (Glaser et al., PNAS 2006:103:16460). On the basis of these observations, we hypothesize that MixL1 plays an important role in gating the cellular decision to remain in a poorly differentiated and proliferative phase rather than proceeding to definitive hematopoietic stem cell (HSC) identity. Several years ago, the placenta was shown to be a major site of hematopoiesis (Gekas et al., Dev Cell 2005:8:365; Ottersbach and Dzierzak, Dev Cell 2005:8:377). The placenta is composed of the chorionic disc and the allantois, the latter of which matures into the umbilical component of the placenta. The allantois exhibits definitive hematopoietic potential (Ziegler et al., Development 2006:133:4183; Corbel et al., Dev Biol 2007:301:478), and has recently been demonstrated to contain a core of stem cells referred to as the Allantoic Core Domain, or ACD, where potential placental hematopoietic activity may originate (Downs et al., Dev Dyn 2009:238:532). Our immediate goal is to evaluate whether MixL1 is expressed in the allantois, and to establish its precise spatiotemporal whereabouts with respect to early markers of hematopoietic cells, such as Runx1 (Chen et al., Nature 2009:457:887). Using immunohistochemistry in conjunction with the LacZ/Runx1 reporter mouse (North et al., Development 1999:126:2563), we have demonstrated that MixL1 is strongly expressed in the blood islands of the yolk sac, as well as in a broad, contiguous posterior domain of the embryo that extends to include the ACD stem cell core of the allantois. This domain does not include Runx1, and MixL1 expression temporally precedes that of Runx1 in the allantois. As development proceeds, the MixL1 signal becomes most prominent in putative nascent blood cells budding off from the poorly-described blood vessel common to the allantois, yolk sac and dorsal aortae, which we have called the “Vessel of Confluence” (VOC). Here, fetal blood is shuttled into the umbilical cord to the chorion for exchange with the mother. Shortly thereafter, Runx1 begins to appear within VOC, and is co-expressed with MixL1. These findings provide preliminary evidence that MixL1 is expressed within the allantois, and within nascent blood cells derived from a specific arterial site common to the allantois, yolk sac, and fetus. Moreover, MixL1 expression appears to precede that of Runx1. Thus, MixL1 may identify one of the earliest hematopoietic precursor cell populations thus far known in mammals. Further, these data provide additional evidence that the allantois is a promising model system for the study of definitive hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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Temporal-Spatial Mapping Of Hematopoietic Progenitors In The Embryo Reveals a Differentially Regulated Program Of Endothelial-To-Hematopoietic Transition In The Yolk Sac

Blood ◽

10.1182/blood.v122.21.1178.1178 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1178-1178 ◽

Cited By ~ 1

Author(s):

Jenna M Frame ◽

Kathleen E McGrath ◽

Katherine H Fegan ◽

James Palis

Keyword(s):

Stem Cells ◽

Blood Flow ◽

Spatial Distribution ◽

Stem Cell ◽

Conflicts Of Interest ◽

Yolk Sac ◽

Dependent Manner ◽

Erythroid Cells ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem

Abstract It is established that hemogenic endothelium (HE) in major embryonic arteries is the source of hematopoietic stem cells (HSC), and the Runx1-mediated endothelial-to-hematopoietic transition in these sites depends both on arterial specification and on the establishment of blood flow. However, two waves of hematopoietic progenitors emerge prior to HSC specification in the mammalian embryo, yielding primitive and erythro-myeloid(EMP)-definitive hematopoiesis. Each has distinct lineage potential and temporal regulation, suggesting that each wave has a required, non-redundant function. Indeed, both are required for embryonic survival (Fujiwara et al., PNAS, 1996; Chen et al., Cell Stem Cell, 2011), and can sustain hematopoiesis in the murine embryo until birth in the absence of functional HSC. Commencing with the onset of cardiac function at embryonic day (E)8.25, EMP emerge in a Runx1-dependent manner contemporaneous with vasculogenesis and angiogenesis in the yolk sac. Moreover, EMP emergence temporally overlaps both with primitive hematopoietic emergence and HSC specification in the adjacent embryonic arteries (Palis et al., Development, 1999). Given this temporal overlap, we reasoned that identifying the spatial localization and cellular origin of EMP emergence would provide important insight into their regulation and function. We purified the temporally overlapping hematopoietic progenitor populations in the yolk sac, and confirmed their lineage identity by in vitro culture. Although previous studies associated CD41hi cells with emerging EMP in the E8 blood islands (Ferkowicz et al., Development, 2003; Li et al., Stem Cells Dev., 2005), we now demonstrate that CD41hi cells at E9 represent maturing Gp1bβ+ megakaryocytes, while Kithi EMP remain CD41Mid. Furthermore, both CD45 and CD16/32 are co-expressed on primitive macrophages and EMP. These findings suggest kit is the most precise single marker to identify emerging EMP in the yolk sac. Notably, despite the loss of KithiCD41+CD16/32+ EMP in Runx1-/- yolk sacs, specification of single-positive CD41hi and CD16/32+ cells is intact, confirming the developmental origin of the megakaryocyte and macrophage lineages from primitive hematopoietic progenitors. As EMP and primitive hematopoietic emergence temporally overlap but differ in lineage potential, we expected these populations to arise within spatially distinct regions of the yolk sac. Surprisingly, we detect kit+ EMP at E8.25 in the proximal yolk sac, both within and adjacent to the blood islands, where primitive erythroid cells emerge. Many Kit+ cells co-express Runx1 and CD31, have a polygonal, endothelial morphology, and appear integrated into the vascular network, consistent with the hemogenic endothelial origin of EMP. The close association of polygonal and round Kit+ cells further support EMP emergence via an endothelial-to-hematopoietic transition. Prior to E9.5, clusters of Kit+Runx1+ EMP remain in the proximal yolk sac, despite the broad distribution of primitive erythroid cells throughout the yolk sac vasculature following the onset of circulation. This is consistent with our observation that EMP clusters are associated with the vessel walls and may be unable to freely circulate. Between E9-10, the spatial distribution of HE broadens, as both small and large clusters of EMP form more distally in the yolk sac. However, unlike HSC, EMP emerge in both small and large vessels. To address the influence of arterial specification and blood flow on EMP emergence, we analyzed yolk sacs from Ncx1-/- mouse embryos, and found normal cluster morphology and spatial distribution, as well as numbers of immunophenotypic EMP, despite the lack of circulation and vascular remodeling. We next asked whether emerging EMP are preferentially associated with arterial vessels, which are predetermined to arise in the posterior region of the yolk sac (Wang et al., Cell, 1998). However, no anterior-posterior asymmetry of EMP clusters was evident in wildtype or Ncx1-/- yolk sacs. We conclude that EMP emergence from HE is initially restricted to the proximal region of the yolk sac that also generates primitive hematopoiesis, but subsequently extends throughout the yolk sac in both arterial and venous vasculature. Our results highlight that distinct populations of HE exist in the conceptus, and are subject to diverse regulatory mechanisms, which may control progenitor versus stem cell fate. Disclosures: No relevant conflicts of interest to declare.

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EMP Emergence from Hemogenic Endothelium in the Mammalian Yolk Sac Is Independent of Flow and Arterial Identity, but Is Regulated By Canonical Wnt Signaling

Blood ◽

10.1182/blood.v124.21.768.768 ◽

2014 ◽

Vol 124 (21) ◽

pp. 768-768

Author(s):

Jenna M. Frame ◽

Kathleen E McGrath ◽

Katherine H. Fegan ◽

James Palis

Keyword(s):

Endothelial Cells ◽

Blood Flow ◽

Wnt Signaling ◽

Ex Vivo ◽

Yolk Sac ◽

Beta Catenin ◽

Hemogenic Endothelium ◽

Canonical Wnt Signaling ◽

Hematopoietic Stem ◽

Canonical Wnt

Abstract Hematopoietic stem cells (HSCs) emerge from arterial vessels of the mouse embryo through a Runx1-dependent process of endothelial-to-hematopoietic transition beginning at embryonic day 10.5 (E10.5). This arterial endothelial-to-hematopoietic transition is known to require embryonic circulation as well as beta-catenin signaling within the endothelial precursor, known as hemogenic endothelium. However, embryonic survival is dependent on the earlier emergence of a robust wave of yolk sac-derived definitive erythro-myeloid progenitors (EMPs), which have unilineage as well as multilineage potential, including high-proliferative potential colony forming cell (HPP-CFC) potential (Palis et al., PNAS, 2001). Like HSCs, EMP specification is dependent on Runx1, suggesting that they also emerge from a hemogenic endothelial precursor. However, the spatial localization of EMPs in the yolk sac and the mechanisms governing their emergence are not well understood. To visualize emerging EMPs in the yolk sac, we performed whole-mount immunohistochemistry for Kit, which we have demonstrated to contain nearly all EMP potential at E9.5. Kit+ cells coexpress Runx1 and CD31, and a subset have a polygonal/endothelial morphology, appear integrated into the vascular network, and are associated with rounded Kit+ cells in clusters, features consistent with an endothelial-to-hematopoietic transition. However, unlike HSCs, which emerge from major embryonic arteries, clusters of EMPs are located in larger and smaller caliber vessels in branches of both the arterial and venous vasculature, which is spatially organized within the yolk sac. To determine if EMP emergence from the vasculature is dependent on embryonic blood flow, which is required for HSC emergence, we analyzed the yolk sacs of Ncx1-null embryos, which fail to initiate heart contractions and subsequently lack embryonic circulation. Despite the lack of vascular remodeling in these circulation-deficient yolk sacs, Ncx1-null EMPs displayed normal cluster morphology, including both polygonal and rounded kit+ cells, indicating the endothelial-to-hematopoietic transition can occur without the mechanical influence of blood flow. To address whether EMP formation is responsive to other developmental signals, we utilized a yolk sac explant culture to evaluate the propensity of hemogenic endothelial cells to commit to hematopoiesis ex vivo. Culture of intact E8.5 yolk sacs for 48 hours with the canonical Wnt ligand Wnt3a resulted in an increase in both day 6-7 colony forming cells and day 13-14 HPP-CFC when compared with control yolk sacs. Preliminary treatment with Dkk1 alone did not adversely affect colony-forming activity when compared with untreated yolk sacs, and potentiation of endogenous canonical Wnt signaling with HLY78 did not augment colony production, suggesting that low levels of endogenous Wnt ligands are produced ex vivo. Despite the positive effect of Wnt3a on whole yolk sacs, treatment of isolated E9.5 Kit+CD41+CD16/32+ EMPs with Wnt3a did not increase colony formation, suggesting that Wnt signaling augments progenitor production at, or prior to, the hemogenic endothelial stage. Preliminary results utilizing imaging flow cytometry demonstrated increased beta-catenin intensity within the nuclear region in E9.5 Kit+VE-Cadherin/AA4.1+ endothelium following Wnt3a treatment, suggesting that hemogenic endothelial cells in the yolk sac are Wnt responsive. Consistent with this finding, in vitro Wnt3a treatment on primary E8.5-9.5 VE-Cadherin/AA4.1+CD16/32- endothelial cells resulted in upregulation of the beta-catenin target gene Axin2. To address whether Wnt signaling is endogenously active in vivo, we analyzed E8.5-E9 yolk sacs of BAT-gal reporter mice (Maretto et al., PNAS, 2003), and visualized a subset of cells with endothelial morphology expressing LacZ. Taken together, these data support the concept that EMPs, like HSCs, emerge from hemogenic endothelium. Surprisingly, this earlier endothelial-to-hematopoietic transition in the yolk sac is not dependent on blood flow or an arterial identity. However, similar to HSC emergence, EMP emergence from hemogenic endothelium is positively regulated by canonical Wnt signaling. These data highlight the presence of spatially, temporally, and functionally heterogeneous populations of hemogenic endothelium in the mammalian conceptus. Disclosures No relevant conflicts of interest to declare.

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Abstract 356: Scl Represses Cardiogenesis via Distant Enhancers during Hemogenic Endothelium Specification

Circulation Research ◽

10.1161/res.113.suppl_1.a356 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Tonis Org ◽

Dan Duan ◽

Roberto Ferrari ◽

Amelie Montel-Hagen ◽

Ben Van Handel ◽

...

Keyword(s):

Target Genes ◽

Es Cells ◽

Bhlh Transcription Factor ◽

Hemogenic Endothelium ◽

Master Regulator ◽

Critical Function ◽

Hematopoietic Stem ◽

Mesoderm Development ◽

Chip Sequencing ◽

Mouse Es Cells

Understanding the mechanisms directing mesoderm specification holds a great potential to advance the development of cell-based therapies for cardiovascular and blood disorders. The bHLH transcription factor Scl is known as the master regulator of the hematopoietic fate. We recently discovered that, in addition to its critical function in promoting the establishment of hemogenic endothelium during hematopoietic stem/progenitor cell (HS/PC) development, Scl is also required to repress cardiomyogenesis in endothelium in hematopoietic tissues and endocardium in the heart. However, the mechanisms for the cardiac repression have remained unknown. Using ChIP-sequencing and microarray analysis of Flk+ mesoderm differentiated from mouse ES cells, we show that Scl both directly activates a broad gene regulatory network required for hemogenic endothelium and HS/PC development (e.g. Runx1, cMyb, Lyl1, Mef2C, Sox7 etc.), and directly represses transcriptional regulators required for cardiogenesis (e.g. Gata4, Gata6, Myocd, etc.) and mesoderm development (Eomes, Mixl1, Etv2, etc.). Repression of cardiac and mesodermal programs occurs during a short developmental window through Scl binding to distant enhancers, while binding to hematopoietic regulators extends throughout HS/PC and red blood cell development and encompasses both distant and proximal binding sites. We also discovered that, surprisingly, Scl complex partners Gata 1 and 2 are dispensable for hematopoietic vs. cardiac specification and Scl binding to majority of its target genes. Nevertheless, Gata factors co-operate with Scl to activate selected transcription factors that facilitate HS/PC emergence from hemogenic endothelium. These results denote Scl as a true master regulator of hematopoietic vs. cardiac fate choice and suggest a mechanism by which lineage-specific bHLH factors direct the divergence of competing fates.

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Epigenetic Control of C/EBPa by Noncoding RNAs.

Blood ◽

10.1182/blood.v114.22.3644.3644 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3644-3644

Author(s):

Annalisa Di Ruscio ◽

Alexander K Ebralidze ◽

Francesco D'Alò ◽

Maria Teresa Voso ◽

Giuseppe Leone ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Messenger Rna ◽

Noncoding Rna ◽

Gene Locus ◽

Conflicts Of Interest ◽

Lentiviral Vector ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Factor C

Abstract Abstract 3644 Poster Board III-580 Little is currently known about the role of noncoding RNA transcripts (ncRNA) in gene regulation; although most, and perhaps all, gene loci express such transcripts. Our previous results with the PU.1 gene locus showed a shared transcription factor complex and chromatin configuration requirements for biogenesis of both messenger and ncRNAs. These ncRNAs were localized within the nuclear and cytoplasmic compartments. Disrupting ncRNAs in the cytoplasmic cellular fraction results in increased PU.1 mRNA and protein. Recently, we have focused on the C/EBPa gene locus and observed extensive noncoding transcription. The transcription factor C/EBPa plays a pivotal role in hematopoietic stem cell (HSC) commitment and differentiation. Expression of the C/EBPa gene is tightly regulated during normal hematopoietic development, and dysregulation of C/EBPa expression can lead to lung cancer and leukemia. However, little is known about how the C/EBPa gene is regulated in vivo. In this study, we characterize ncRNAs derived from the C/EBPa locus and demonstrate their functional role in regulation of C/EBPa gene expression. First, northern blot analysis and RT PCR determined a predominantly nuclear localization of the C/EBPa ncRNAs. Second, strand-specific quantitative RT PCR demonstrated a concordant expression of coding and noncoding C/EBPa transcripts. Next, we investigated the results of ablation of ncRNAs using a lentiviral vector containing ncRNA-targeting shRNAs on the expression of the C/EBPa gene. We have observed that reduced levels of ncRNAs leads to a significant downregulation of the expression of coding messenger RNA. These data strongly suggest that C/EBPa ncRNAs play an important role in maintaining optimal expression of the C/EBPa gene at different stages of hematopoiesis and makes targeting noncoding transcripts a novel and attractive tool in correcting aberrant gene expression levels. Disclosures: No relevant conflicts of interest to declare.

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The Transcription Factor ELF4 Promotes Survival of Myeloid Leukemic Stem Cells.

Blood ◽

10.1182/blood.v116.21.1209.1209 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1209-1209

Author(s):

Chun Shik Park ◽

Koramit Suppipat ◽

H. Daniel Lacorazza

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Blast Crisis ◽

Philadelphia Chromosome ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Leukemic Stem Cells ◽

Hematopoietic Stem

Abstract Abstract 1209 Chronic myeloid leukemia (CML) is a myeloproliferative disease that originate in hematopoietic stem cells (HSCs) as a result of the t(9;22) translocation, giving rise to the Ph (Philadelphia chromosome) and BCR-ABL oncoprotein. Although treatment of CML patients with tyrosine kinase inhibitor can efficiently eliminate most leukemic cells, chemoresistant leukemic stem cells (LSCs) can survive and drive recurrence of CML in these patients. A number of genes have been described to promote or inhibit proliferation of LSCs. Some of them have similar roles in normal HSCs. The transcription factor ELF4 promotes cell cycle entry of quiescent HSCs during homeostasis (Lacorazza et al., 2006). Thus, to investigate the function of ELF4 in CML initiation and maintenance, we developed a BCR-ABL-induced CML-like disease using retroviral transfer of BCR-ABL in Elf4-null bone marrow (BM) cells. We first investigated whether ELF4 is required for the induction of CML. Recipient mice of BCR-ABL-transduced WT BM cells developed CML and died with a latency 16–23 days, whereas recipient mice of BCR-ABL-transduced Elf4-/- BM cells showed longer latency of 45–47 days (n=20; p<0.0005). Progression of leukemia was monitored in peripheral blood, BM and spleen by flow cytometry. In mice transplanted with BCR-ABL-transduced Elf4-null BM cells, Gr-1+ leukemic cells expanded the first two weeks after BM transplantation followed by a decline at expense of a secondary expansion of B220+ cells. In contrast, Gr-1+ leukemic cells continuously expanded in mice receiving BCR-ABL-transduced WT BM cells. These results suggest that loss of ELF4 causes a profound abrogation in BCR-ABL-induced CML, while allowing progression of B-cell acute lymphocytic leukemia. Since loss of Elf4 led to impaired maintenance of myeloid leukemic cells, we postulated that ELF4 may affect survival of LSCs. Thus, we analyzed the frequency of Lin-c-Kit+Sca-1+ (LSK) cells that are BCR-ABL positive in BM and spleen. We found that BCR-ABL+ LSK cells were significantly reduced in recipients of BCR-ABL-transduced Elf4-/- BM cells. These studies indicate that ELF4 is essential to maintain the LSC pool in CML acting as a molecular switch between myeloid and lymphoid blast crisis. Disclosures: No relevant conflicts of interest to declare.

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A Modified HIV1-Based Lentiviral Vector Can Transduce Rhesus Hematopoietic Repopulating Cells as Efficiently as An SIV Vector in An Autologous Transplantation Model.

Blood ◽

10.1182/blood.v114.22.692.692 ◽

2009 ◽

Vol 114 (22) ◽

pp. 692-692

Author(s):

Naoya Uchida ◽

Phillip W Hargrove ◽

Kareem Washington ◽

Coen J. Lap ◽

Matthew M. Hsieh ◽

...

Keyword(s):

T Cells ◽

Blood Cells ◽

Conflicts Of Interest ◽

Lentiviral Vector ◽

Autologous Transplantation ◽

Vector System ◽

Transduction Efficiency ◽

Large Animal ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Abstract 692 HIV1-based vectors transduce rhesus hematopoietic stem cells poorly due to a species specific block by restriction factors, such as TRIM5αa which target HIV1 capsid proteins. The use of simian immunodeficiency virus (SIV)-based vectors can circumvent this restriction, yet use of this system precludes the ability to directly evaluate HIV1-based lentiviral vectors prior to their use in human clinical trials. To address this issue, we previously developed a chimeric HIV1 vector (χHIV vector) system wherein the HIV1-based lentiviral vector genome is packaged in the context of SIV capsid sequences. We found that this allowed χHIV vector particles to escape the intracellular defense mechanisms operative in rhesus hematopoietic cells as judged by the efficient transduction of both rhesus and human CD34+ cells. Following transplantation of rhesus animals with autologous cell transduced with the χHIV vector, high levels of marking were observed in peripheral blood cells (J Virol. 2009 Jul. in press). To evaluate whether χHIV vectors could transduce rhesus blood cells as efficiently as SIV vectors, we performed a competitive repopulation assay in two rhesus macaques for which half of the CD34+ cells were transduced with the standard SIV vector and the other half with the χHIV vector both at a MOI=50 and under identical transduction conditions. The transduction efficiency for rhesus CD34+ cells before transplantation with the χHIV vector showed lower transduction rates in vitro compared to those of the SIV vector (first rhesus: 41.9±0.83% vs. 71.2±0.46%, p<0.01, second rhesus: 65.0±0.51% vs. 77.0±0.18%, p<0.01, respectively). Following transplantation and reconstitution, however, the χHIV vector showed modestly higher gene marking levels in granulocytes (first rhesus: 12.4% vs. 6.1%, second rhesus: 36.1% vs. 27.2%) and equivalent marking levels in lymphocytes, red blood cells (RBC), and platelets, compared to the SIV vector at one month (Figure). Three to four months after transplantation in the first animal, in vivo marking levels plateaued, and the χHIV achieved 2-3 fold higher marking levels when compared to the SIV vector, in granulocytes (6.9% vs. 2.8%) and RBCs (3.3% vs. 0.9%), and equivalent marking levels in lymphocytes (7.1% vs. 5.1%) and platelets (2.8% vs. 2.5)(Figure). Using cell type specific surface marker analysis, the χHIV vector showed 2-7 fold higher marking levels in CD33+ cells (granulocytes: 5.4% vs. 2.7%), CD56+ cells (NK cells: 6.5% vs. 3.2%), CD71+ cells (reticulocyte: 4.5% vs. 0.6%), and RBC+ cells (3.6% vs. 0.9%), and equivalent marking levels in CD3+ cells (T cells: 4.4% vs. 3.3%), CD4+ cells (T cells: 3.9% vs. 4.6%), CD8+ cells (T cells: 4.2% vs. 3.9%), CD20+ cells (B cells: 7.6% vs. 4.8%), and CD41a+ cells (platelets: 3.5% vs. 2.2%) 4 months after transplantation. The second animal showed a similar pattern with higher overall levels (granulocytes: 32.8% vs. 19.1%, lymphocytes: 24.4% vs. 17.6%, RBCs 13.1% vs. 6.8%, and platelets: 14.8% vs. 16.9%) 2 months after transplantation. These data demonstrate that our χHIV vector can efficiently transduce rhesus long-term progenitors at levels comparable to SIV-based vectors. This χHIV vector system should allow preclinical testing of HIV1-based therapeutic vectors in the large animal model, especially for granulocytic or RBC diseases. Disclosures: No relevant conflicts of interest to declare.

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Clonal Compensation Between Hematopoietic Stem Cells upon Differentiation Deficiency

Blood ◽

10.1182/blood.v128.22.422.422 ◽

2016 ◽

Vol 128 (22) ◽

pp. 422-422

Author(s):

Rong Lu ◽

Lisa Nguyen

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Blood Cell ◽

Cell Tracking ◽

Conflicts Of Interest ◽

Genetically Modified ◽

Cell Types ◽

Clonal Expansion ◽

Hematopoietic Stem ◽

Organ Systems

Abstract In most organ systems, regeneration is a coordinated effort that involves many stem cells, but little is known about how individual stem cells compensate for the functional deficiencies of other stem cells. Functional coordination between stem cells is critically important during disease progression and treatment when a subset of HSCs fail or become malignant. We hypothesize that individual HSCs heterogeneously compensate for specific deficiencies, as recent work from our group and others suggest that HSCs heterogeneously supply blood. To test this hypothesis, we tracked mouse HSCs in vivo using a single-cell tracking technology that we had previously developed. We found that individual HSCs heterogeneously compensate for the lymphopoiesis deficiencies of other HSCs by increasing individual clonal expansion and altering lineage bias. Clonal expansion refers to the increase in clonal progenies. Lineage bias refers to the preferential production of specific blood cell types. This compensation rescues the overall blood supply and influences blood cell types outside of the deficient lineages in distinct patterns. We identified the molecular regulators and signaling pathways associated with this form of HSC coordination using RNA sequencing. Specifically, the STAT3 pathway and NF-B signalingwere activated, and PTEN signaling was inhibited in HSCs during the compensation process. To investigate the dynamics of HSC coordination, we employed a genetically modified mouse model that expresses simian diphtheria toxin (DT) receptor under the control of the CD11b promoter. Monocytes derived from this mouse line can be ablated upon DT administration. We co-transplanted HSCs derived from normal and the genetically modified mice, then conditionally ablated the monocyte population repeatedly, and tracked the temporal responses of individual normal HSCs. Our time-course analysis revealed that a distinct subset of HSC clones produced rapid and persistent responses to the blood perturbations. These clones had not been highly active in the affected lineages prior to the perturbation. We identified several significant temporal profiles that indicate a remarkable heterogeneity in the responses of HSCs to blood system changes. Together, these data suggest that HSC differentiation is coordinated in a deterministic manner during compensation and is independent of the normal differentiation program. Our findings suggest that stem cells interact with each other and form a coordinated cellular network that is robust enough to withstand minor functional disruptions. Individual HSCs distinctly adapt their differentiation program to compensate for deficient HSCs and specifically overproduce undersupplied cell types. The heterogeneity in the compensation activities of individual HSC clones may be essential for maintaining robustness in blood regeneration and suggests that stem cell coordination is a complex process. A better understanding of the clonal level differences in individual HSCs is critically important for identifying the pathogenesis of blood diseases. Exploiting the innate compensation capacity of stem cell networks may improve the diagnosis and treatment of many diseases. For example, the identification of the molecular regulators and pathways involved in HSC compensation can help develop new therapeutic treatments that enhance the innate compensation capacity of stem cells. Disclosures No relevant conflicts of interest to declare.

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