HSC Development Research Articles

The zebrafish homologue of the murine EVI1 gene critically regulates hsc development

The zebrafish homologue of the murine EVI1 gene critically regulates hsc development

Cannabinoid Receptor-2 Regulates Embryonic Hematopoietic Stem Cell Development via Prostaglandin E2 and P-Selectin Activity.

Cannabinoids (CB) modulate adult hematopoietic stem and progenitor cell (HSPCs) function, however, impact on the production, expansion, or migration of embryonic HSCs is currently uncharacterized. Here, using chemical and genetic approaches targeting CB-signaling in zebrafish, we show that CB receptor (CNR) 2, but not CNR1, regulates embryonic HSC development. During HSC specification in the aorta-gonad-mesonephros (AGM) region, CNR2 stimulation by AM1241 increased runx1;cmyb(+) HSPCs, through heightened proliferation, whereas CNR2 antagonism decreased HSPC number; FACS analysis and absolute HSC counts confirmed and quantified these effects. Epistatic investigations showed AM1241 significantly upregulated PGE2 synthesis in a Ptgs2-dependent manner to increase AGM HSCs. During the phases of HSC production and colonization of secondary niches, AM1241 accelerated migration to the caudal hematopoietic tissue (CHT), the site of embryonic HSC expansion, and the thymus; however these effects occurred independently of PGE2. Using a candidate approach for HSC migration and retention factors, P-selectin was identified as the functional target of CNR2 regulation. Epistatic analyses confirmed migration of HSCs into the CHT and thymus was dependent on CNR2-regulated P-selectin activity. Together, these data suggest CNR2-signaling optimizes the production, expansion, and migration of embryonic HSCs by modulating multiple downstream signaling pathways.

Adult Donor-Derived Human CD34+ Cell Engraftment and Hemato-Lymphoid System Development in 3rd Generation Humanized Mice

Transplantation of human CD34+ hematopoietic stem and progenitor cells into severe immunocompromised newborn mice allows the development of a human hemato-lymphoid system (HHLS) in vivo (Rongvaux et al. Ann. Rev. Immunol. 2013). While fetal liver- or cord blood- derived CD34+ cells lead to high levels of engraftment, adult donor-derived CD34+ cell transplantation usually led to low levels of engraftment in existing humanized mice models. We recently generated novel mouse strains called 3rd generation humanized mice (3rd gen. huMice) in which human versions of cytokines (M-CSF and TPO with or without IL-3/GM-CSF) are knocked into Balb/c Rag2-/-γC-/- strains (MISTRG or MSTRG, respectively). In addition, human Sirpα, which is a critical factor to prevent donor cell to be eliminated by host macrophages, is expressed as transgene in both strains (Rongvaux et al., Nat. Biotechnol. 2014).To evaluate human adult CD34+ cell engraftment in 3rd gen. huMice, CD34+ cells obtained from peripheral blood after G-CSF administration (3.0 – 5.5 x105 cells) were i.h. injected into sub-lethally irradiated newborn MISTRG or MSTRG and NOD/scid/γC-/- (NSG) mice or Rag2-/-γC-/-hSirpαTg (RGS) mice as controls. Seventeen of 18 (94%) MISTRG/MSTRG mice showed human CD45+ cell engraftment (>1% of total CD45+ cells in BM) 10-16 weeks after injection, whereas 4 of 11 (36%) NSG/RGS mice supported human cell engraftment. Percentages of human cells in the BM of the engrafted MISTRG/MSTRG were 7- to 8 fold higher than in the BM of engrafted NSG/RGS mice (30.2% ± 6.9 vs 4.1% ± 0.9, respectively). MISTRG/MSTRG mice supported significantly increased numbers of non-classical monocytes and NKp46+ cells in BM compared with NSG/RGS mice. Moreover, we observed significantly increased numbers of CD34+ and CD34+CD38- cells, a population enriched for human early progenitor cells and HSCs, in the BM of MISTRG/MSTRG mice. In addition, MISTRG/MSTRG mice supported higher level of human thymocyte development compared to NSG/RGS mice. Besides lymphoid organs, we further observed increased human CD45+ cells, mostly myeloid lineage cells, in the liver and lung of MISTRG/MSTRG mice compared to NSG/RGS mice.Taken together, this study demonstrates that our 3rd gen. huMice models support adult donor-derived HSC engraftment and development of myeloid as well as lymphoid lineage cells at high levels in primary lymphoid and non-lymphoid organs. These models thus have the potential for personalized studies of healthy hematopoiesis as well as hemato-immune system diseases from adult individuals. DisclosuresNo relevant conflicts of interest to declare.

Evolving a collaborative matrix for teaching informatics online to health sciences students at the Massachusetts College of Pharmacy and Health Sciences

Introduction – The study of informatics is multidisciplinary in nature. The informatics course, HSC 310: Health Care Informatics (HSC 310), for undergraduate health sciences students at the Massachusetts College of Pharmacy and Health Sciences (MCPHS) is an example of a librarian-led, multidisciplinary team functioning within a totally online environment. Description – The development and design of the course HSC 310 is reviewed. Issues and challenges are discussed, as well as the benefits of interdisciplinary expertise in the learning environment. Outcomes – Because informatics is fundamentally interdisciplinary in nature, librarians, instructional designers, statisticians, nurses, pharmacists, and other professionals can learn from each other and strengthen the learning experience of students. The “matrix” of informatics explains how diverse types of information interact with, and impact on, each other. This knowledge is crucial to the understanding of information and its role in one's professional life. There was a journey taken in the design of the course, its evolution cumulating in its final online form. Our unique design was an informatics exercise in itself. Student feedback confirmed that, in addition to the course content, the online environment was a positive educational experience. Discussion – Librarian involvement in teaching informatics at MCPHS began with a National Library of Medicine (NLM) fellowship in informatics at Woods Hole, Massachusetts. This resulted in the library's first for-credit course offered totally in-class and included the participation of faculty from other disciplines. Successful collaboration with the College instructional designer was essential in moving this librarian-led course to a totally virtual environment. In addition, the teaming of librarians with other faculty resulted in two nursing-led, hybrid courses on our Boston and Manchester campuses. During the past two years, interdisciplinary teams have provided informatics instruction to the Boston campus within a totally online environment. This paper discusses one of those courses, HSC 310: Health Care Informatics. The topics discussed in the course and the virtual platform have both provided a valuable learning experience for faculty and students. Informatics is an area where varied disciplines can join to achieve common goals.

Human T cell development in HSC-engrafted NSG mice is dependent on age of the recipient (HEM4P.247)

Abstract Humanized mouse models are an exciting alternative for the investigation of human hematopoiesis, immunobiology, autoimmunity, infectious diseases, cancer biology, transplantation biology and regenerative medicine. However the efficient generation of humanized mice is complicated by the availability of numerous mouse strains and the large diversity of engraftment protocols. NOD-scid IL2rγnull (NSG) mice injected with human HSC support high levels of engraftment and develop a functional human immune system including both innate and adaptive immune cells. Previous studies have shown that injection of human HSC into newborn NSG mice results in more efficient and rapid T cell development as compared to adult NSG (>6 wk) mice. Here we compared engraftment of human HSC and immune system development following intracardiac injection into irradiated newborn NSG mice or intravenous injection into irradiated 3-wk old NSG mice. Total human cell chimerism was monitored in the blood between 6 and 18 wk after transplantation. We also evaluated the development of human T cells, B cells, monocytes, NK cells, NKT cells, conventional and plasmacytoid DC. Our results show that newborn NSG mice have higher levels of T cell reconstitution at earlier time points as compared to 3-wk old NSG recipients but that the older mice achieved comparable T cell levels at later times. Together these findings show that T cell development in humanized mice occurs with slower kinetics in older NSG mice.

Mouse extraembryonic arterial vessels harbor precursors capable of maturing into definitive HSCs

AbstractDuring mouse development, definitive hematopoietic stem cells (dHSCs) emerge by late E10.5 to E11 in several hematopoietic sites. Of them, the aorta-gonad-mesonephros (AGM) region drew particular attention owing to its capacity to autonomously initiate and expand dHSCs in culture, indicating its key role in HSC development. The dorsal aorta contains characteristic hematopoietic clusters and is the initial site of dHSC emergence, where they mature through vascular endothelial (VE)-cadherin+CD45–CD41low (type 1 pre-HSCs) and VE-cadherin+CD45+ (type 2 pre-HSCs) intermediates. Although dHSCs were also found in other embryonic niches (placenta, yolk sac, and extraembryonic vessels), attempts to detect their HSC initiating potential have been unsuccessful to date. Extraembryonic arterial vessels contain hematopoietic clusters, suggesting that they develop HSCs, but functional evidence for this has been lacking. Here we show that umbilical cord and vitelline arteries (VAs), but not veins, contain pre-HSCs capable of maturing into dHSCs in the presence of exogenous interleukin 3, although in fewer numbers than the AGM region, and that pre-HSC activity in VAs increases with proximity to the embryo proper. Our functional data strongly suggest that extraembryonic arteries can actively contribute to adult hematopoiesis.

84 Azithromycin resistance in Prevotella species isolated from CF patients

Objective: The use of chronic azithromycin treatment has been linked with increased macrolide resistance. Although, Prevotella spp. are one of the most common anaerobes detected in CF pulmonary samples, the effect of chronic azithromycin exposure on this genus is unknown. The aims of this study were to i. investigate if resistance is associated with azithromycin prescription ii. compare azithromycin and clindamycin MICs between Prevotella isolates cultured from CF patients and healthy control subjects. Methods: Isolates were grouped according to source and patient prescription of azithromycin: CF (currently prescribed), n = 27; CF (not currently prescribed), n = 30; healthy controls (none), n = 17. Susceptibility was determined by Etest® and MICs compared between groups using the Mann-Whitney test. Results: CF isolates had significantly higher azithromycin and clindamycin MICs compared to the healthy control isolates (P < 0.001). Current prescription of azithromycin was associated with significantly higher azithromycin MICs (P= 0.016) in the CF isolates. CF isolates (not currently prescribed) had significantly higher azithromycin MICs (P= 0.009) compared to isolates from healthy control subjects. Isolates from the 2 CF groups had equal resistance to clindamycin (P= 0.228). Conclusions: CF patients harbour isolates with increased resistance to azithromycin and clindamycin. Azithromycin resistance is associated with current azithromycin prescription. Work supported by a Department of Employment and Learning, NI (DEL) studentship to L. Sherrard and by HSC Research and Development, Public Health Agency, NI and the Medical Research Council through a US-Ireland Partnership Grant.

85 Investigation into the antibiotic resistome of the Streptococcus genus from adult cystic fibrosis patients

Objective: The use of chronic azithromycin treatment has been linked with increased macrolide resistance. Although, Prevotella spp. are one of the most common anaerobes detected in CF pulmonary samples, the effect of chronic azithromycin exposure on this genus is unknown. The aims of this study were to i. investigate if resistance is associated with azithromycin prescription ii. compare azithromycin and clindamycin MICs between Prevotella isolates cultured from CF patients and healthy control subjects. Methods: Isolates were grouped according to source and patient prescription of azithromycin: CF (currently prescribed), n = 27; CF (not currently prescribed), n = 30; healthy controls (none), n = 17. Susceptibility was determined by Etest® and MICs compared between groups using the Mann-Whitney test. Results: CF isolates had significantly higher azithromycin and clindamycin MICs compared to the healthy control isolates (P < 0.001). Current prescription of azithromycin was associated with significantly higher azithromycin MICs (P= 0.016) in the CF isolates. CF isolates (not currently prescribed) had significantly higher azithromycin MICs (P= 0.009) compared to isolates from healthy control subjects. Isolates from the 2 CF groups had equal resistance to clindamycin (P= 0.228). Conclusions: CF patients harbour isolates with increased resistance to azithromycin and clindamycin. Azithromycin resistance is associated with current azithromycin prescription. Work supported by a Department of Employment and Learning, NI (DEL) studentship to L. Sherrard and by HSC Research and Development, Public Health Agency, NI and the Medical Research Council through a US-Ireland Partnership Grant.

Identification of Hemogenic Endothelium and Its Direct Precursor in Human Embryonic Stem Cell Differentiation Cultures

Abstract 1277Animal studies demonstrated that the first HSCs are generated in the aorta-gonad-mesonephros from a unique population of endothelial cells, hemogenic endothelium (HE), through endothelial-hematopoietic transition. However, the identity of HE remains obscure and the specific features that distinguish HE from non-HE and the discrete stages of endothelial transition into hematopoietic cells are not characterized. Here we employed hESC differentiation system in coculture with OP9 to define the distinct population of HE, and to develop an experimental system for analysis of endothelial-hematopoietic transition in vitro. Previously, we demonstrated that endothelial cells could be distinguished from hematopoietic progenitors based on the lack of CD43 expression (Vodyanik et al., Blood 2006;108:2095). Here, we analyzed the earliest stages of endothelial development from hESCs before the typical hematopoietic CFCs could be detected. We found that the first CD144+ endothelial cells appear on day 4 of differentiation within a population of APLNR+ cells expressing a high level of KDR. Based on expression of CD235a/CD43, CD73 and CD41a, we identified three major subsets within the emerging CD144+ cells: 1)CD235a/CD43−CD73+, 2)CD235a/CD43−CD73−, and 3)CD235a/CD43+CD41a−. Although all three subsets formed monolayer endothelial cells when grown on fibronectin in endothelial media, only CD235a/CD43+CD41a− cells displayed hematopoietic CFC potential, which required serum-free medium and were dependent on hematopoietic cytokines and FGF2. After culture with OP9, both CD144+CD235a/CD43−CD73− and CD144+CD235a/CD43+CD41a− cells generated CD31+CD43/45− endothelial cells and a significant amount of CD43+ blood cells. In contrast CD144+CD235a/CD43−CD73+ cells formed almost exclusively endothelial cells. Single cell deposition experiments demonstrated that CD144+CD235a/CD43+CD41a− cells formed predominantly hematopoietic cells, while most CD144+CD235a/CD43−CD73− cells gave rise to either hematopoietic or endothelial clusters with approximately 2.5% of cells forming hematoendothelial clusters, indicating the presence of bipotential progenitors within this population. Though all three CD144+ subsets had a very similar molecular signature and expressed typical endothelial genes and genes associated with angiohematopoietic and HSC development, CD235a/CD43+CD41a− showed the higher expression of hematopoietic genes and lower expression of CDH5, CDH2, CAV1, and APOLD1 typical endothelial genes. Based on the functional, phenotypic and molecular properties we designated the identity of subsets as follows: 1) CD144+CD235a/CD43+CD41a− angiogenic blood cells, i.e. cells that possessed primary hematopoietic characteristics but were also capable of generating endothelial cells; 2) CD144+CD235a/CD43−CD73+ endothelial progenitors, i.e. cells that had all functional and molecular features of endothelial cells and endothelial colony-forming potential on OP9; 3) CD144+CD235a/CD43−CD73− HE cells, i.e. cells with primary endothelial characteristics lacking hematopoietic CFC potential and surface markers, but capable of generating blood and endothelial cells after coculture with stromal cells. To find out the direct precursors of HE and blood cells, we analyzed the differentiation potential of major subsets of mesodermal cells isolated on day 4 of hESC coculture with OP9. We found that only APLNR+KDRbrightCD140alow/− mesodermal cells lacking expression of typical hematopoietic (CD43, CD45), endothelial (CD31, CD144), and mesenchymal (CD73, CD105) markers had the potential to generate both blood and endothelium. Moreover, single cell deposition experiments demonstrated that APLNR+KDRbrightCD140alow/− cells formed HE clusters with a high frequency (about 1/10 cells), strongly indicating that these cells represent the direct precursors of HE. Because APLNR+KDRbrightCD140alow/− cells upregulated expression LMO2, TAL1, CBFB, GATA2, and FLI1 transcriptional regulators of hematopoietic and endothelial development, but were lacking expression of primitive streak genes, we designated these cells as angioblasts. Together these studies provide a hESC-based platform for identification of factors required for acquisition of HSC potential by blood cells following transition from endothelium. Disclosures:Thomson:CDI: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Slukvin:CDI: Consultancy, Equity Ownership.

Prostaglandin E2 Signaling Through the EP4 Receptor Regulates the Proliferation of Hematopoietic Stem/Progenitor Cells Under Stress Conditions

Abstract 2377Hematopoietic stem cells (HSCs) maintain the balance between self-renewal and differentiation in order to replenish all blood cell lineages throughout the lifespan of the individual. Signaling molecules produced by various niche cells regulate the function and fate of HSCs. Prostaglandin E2 (PGE2) is a known mediator of various pathological states and physiological systems. In the hematopoietic system, PGE2 signaling regulates HSC development and enhances repopulation activity through its interactions with the Wnt/β-catenin pathway. However, it is not been clear which receptor is responsible for PGE2 signaling in adult HSCs.Here we analyzed the function of PGE2 receptors, EP2 and EP4, in the regulation of adult bone marrow (BM) hematopoietic stem/progenitor cells (HSPCs). Quantitative real time PCR (Q-PCR) analysis revealed that EP4 was highly expressed in Lin−Sca-1+c-Kit+ (LSK) fractions compared to Lin− or Lin+ fractions. On the other hand, EP2 mRNA expression was detected at a higher level in LSKCD34+Flt3+ fractions than in other fractions. Digital PCR analysis showed that EP2 transcripts were present in much lower numbers than EP4 transcripts in HSCs.We next analyzed the effects of short-term treatment with an EP2 or EP4 agonist on the colony forming activity of LSK cells. Although the EP2 agonist did not affect the colony formation activity of LSK cells, the EP4 agonist increased the number of colony forming unit in culture (CFU-C) and high proliferative potential-colony forming cells (HPP-CFC) in a dose dependent manner, indicating that PGE2/EP4 signaling affects the proliferation of HSPCs. A knockdown of EP4 reduced the engraftment capacity of LSK cells, suggesting that PGE2/EP4 signaling contributes to the BM repopulation activity of HSPCs.Next, we examined the effect of PGE2/EP4 signaling on the recovery of HSCs after myelosuppression. 5-FU-injected mice were treated with PGE2 and the proportion of LSK cells in the mice at various days after 5-FU injection was examined. The frequency of LSK cells at the early phase of post 5-FU treatment was comparable in control and PGE2-treated mice. In the later phase of post 5-FU treatment, mice treated with PGE2 showed a higher frequency of LSK cells, and the LSK cells derived from the PGE2-treated mice had significantly higher expression of Cdk4, Cdk6, Ctnnb1, and Myc compared to the control mice. These data indicate that PGE2 promoted BM recovery by accelerating the proliferation of HSPCs. The EP4 agonist was as efficient as PGE2 in enhancing BM recovery after myelosuppression. In addition, we found that EP4 agonist treatment induced an increase in the nuclear localization of β-catenin, indicating that PGE2 signaling through EP4 impinges on Wnt/β-catenin signaling in HSCs. Altogether, these data suggest that EP4 plays a crucial role in the PGE2 signaling involved in the proliferation of HSCs under stress conditions. This study was conducted under the collaboration with Dr. Narumiya (Kyoto University Graduate School of Medicine) Disclosures:No relevant conflicts of interest to declare.

A blood flow–dependent klf2a-NO signaling cascade is required for stabilization of hematopoietic stem cell programming in zebrafish embryos

AbstractBlood flow has long been thought to be important for vessel development and function, but its role in HSC development is not yet fully understood. Here, we take advantage of zebrafish embryos with circulation defects that retain relatively normal early development to illustrate the combinatorial roles of genetic and hemodynamic forces in HSC development. We show that blood flow is not required for initiation of HSC gene expression, but instead is indispensable for its maintenance. Knockdown of klf2a mimics the silent heart (sih/tnnt2a) phenotype while overexpression of klf2a in tnnt2a morphant embryos can rescue HSC defects, suggesting that klf2a is a downstream mediator of blood flow. Furthermore, the expression of NO synthase (nos) was reduced in klf2a knockdown embryos, and ChIP analysis showed that endogenous Klf2a is bound to the promoters of nos genes in vivo, indicating direct gene regulation. Finally, administration of the NO agonist S-nitroso N-acetylpenicillamine (SNAP) can restore HSC development in tnnt2a and klf2a morphants, suggesting that NO signaling is downstream of Klf2a which is induced by hemodynamic forces. Taken together, we have demonstrated that blood flow is essential for HSC development and is mediated by a klf2a-NO signaling cascade in zebrafish.

Cpsf1 is required for definitive HSC survival in zebrafish

AbstractA comprehensive understanding of the genes and pathways regulating hematopoiesis is needed to identify genes causally related to bone marrow failure syndromes, myelodysplastic syndromes, and hematopoietic neoplasms. To identify novel genes involved in hematopoiesis, we performed an ethyl-nitrosourea mutagenesis screen in zebrafish (Danio rerio) to search for mutants with defective definitive hematopoiesis. We report the recovery and analysis of the grechetto mutant, which harbors an inactivating mutation in cleavage and polyadenylation specificity factor 1 (cpsf1), a gene ubiquitously expressed and required for 3′ untranslated region processing of a subset of pre-mRNAs. grechetto mutants undergo normal primitive hematopoiesis and specify appropriate numbers of definitive HSCs at 36 hours postfertilization. However, when HSCs migrate to the caudal hematopoietic tissue at 3 days postfertilization, their numbers start decreasing as a result of apoptotic cell death. Consistent with Cpsf1 function, c-myb:EGFP+ cells in grechetto mutants also show defective polyadenylation of snrnp70, a gene required for HSC development. By 5 days postfertilization, definitive hematopoiesis is compromised and severely decreased blood cell numbers are observed across the myeloid, erythroid, and lymphoid cell lineages. These studies show that cpsf1 is essential for HSC survival and differentiation in caudal hematopoietic tissue.

Origin of Hematopoietic Stem Cells

AbstractAbstract SCI-40During 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.

Sphingosine-1-Phosphate Mediates Proliferation Maintaining the Multipotency of Human Adult Bone Marrow and Adipose Tissue-derived Stem Cells

High renewal and maintenance of multipotency of human adult stem cells (hSCs), are a prerequisite for experimental analysis as well as for potential clinical usages. The most widely used strategy for hSC culture and proliferation is using serum. However, serum is poorly defined and has a considerable degree of inter-batch variation, which makes it difficult for large-scale mesenchymal stem cells (MSCs) expansion in homogeneous culture conditions. Moreover, it is often observed that cells grown in serum-containing media spontaneously differentiate into unknown and/or undesired phenotypes. Another way of maintaining hSC development is using cytokines and/or tissue-specific growth factors; this is a very expensive approach and can lead to early unwanted differentiation. In order to circumvent these issues, we investigated the role of sphingosine-1-phosphate (S1P), in the growth and multipotency maintenance of human bone marrow and adipose tissue-derived MSCs. We show that S1P induces growth, and in combination with reduced serum, or with the growth factors FGF and platelet-derived growth factor-AB, S1P has an enhancing effect on growth. We also show that the MSCs cultured in S1P-supplemented media are able to maintain their differentiation potential for at least as long as that for cells grown in the usual serum-containing media. This is shown by the ability of cells grown in S1P-containing media to be able to undergo osteogenic as well as adipogenic differentiation. This is of interest, since S1P is a relatively inexpensive natural product, which can be obtained in homogeneous high-purity batches: this will minimize costs and potentially reduce the unwanted side effects observed with serum. Taken together, S1P is able to induce proliferation while maintaining the multipotency of different human stem cells, suggesting a potential for S1P in developing serum-free or serum-reduced defined medium for adult stem cell cultures.

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

Abstract 698Hematopoietic 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.

C-Myc Is a Major Target of Rbm15 in the Regulation of Hematopoietic Stem Cell and Megakaryocyte Development

Acute megakaryoblastic leukemia (AMKL, FAB-M7) with the t(1;22)(p13;q13) chromosome translocation is a unique subtype of AMKL, characterized by its early onset, poor response to existing therapies, rapid progression, and poor prognosis. t(1;22) is often the sole abnormality found in infants with this AMKL subtype, suggesting that the RBM15-MKL1 (also known as OTT1-MAL) fusion gene created by the translocation plays a crucial role in the pathogenesis of this leukemia. Here we have explored the normal functions of one of the t(1;22) partner genes, Rbm15, by conditionally knocking it out in the mouse (Rbm15 CKO mice) and by crossing these mice with an Mx1-Cre transgenic line to delete Rbm15 in the hematopoietic system. We found both the percentage and absolute number of long-term hematopoietic stem cells (Lin-Sca1+ckit+/Flk2-, LT-HSCs) to be increased in Rbm15-deleted (Rbm15lx/lx;Mx1-Cre+) mice (0.23 ± 0.02% of total nucleated marrow cells [TNMC], 2.3-fold higher) compared to age- and gender-matched wild-type littermates (Rbm15lx/lx;Mx1-Cre-) (0.10 ± 0.01% TNMC) (P <0.0001, n = 18 mice per group). However, Rbm15-deleted bone marrow (BM) cells were markedly impaired in their ability to reconstitute normal hematopoiesis in competitive repopulation studies, with only 9.73 ± 2.32% donor-derived cells in the peripheral blood (PB) of recipient animals compared to the contribution by donor cells from littermate controls (47.52 ± 7.26%) (P = 0.00015; n = 10 mice per group) when transplanted at a 1:1 ratio of donor vs. wild-type competitor bone marrow. Additional analysis showed that Rbm15-deleted donor-derived cells were also significantly decreased in the recipient BM as a whole; importantly, however, the percentages of donor-derived HSC and LT-HSC populations were markedly increased (8.8-fold and 5.3-fold, respectively) in recipients transplanted with Rbm15 mutant cells compared to those transplanted with wild-type control marrow. This stem cell accumulation was more profound than that observed in the original Rbm15 mutant mice. Additional analysis that will be presented suggested an HSC activation/differentiation defect due to Rbm15 absence rather than abnormalities of HSC homing, survival or proliferation. Collectively, these alterations are highly reminiscent of those seen in c-Myc knockout (KO) mice, in which the expression levels of several adhesion molecules have been shown to be altered and an HSC-niche interaction defect posited to be the underlying mechanism of the HSC abnormalities observed. We found that similar to the c-Myc KO phenotype, the expression of both N-cadherin and beta1-integrin is increased on Rbm15-deficient HSCs and the N-cadherin expression level can be downregulated by ectopic expression of RBM15. Interestingly, in addition to the abnormalities of c-Myc-null HSCs that have been previously reported, we have identified a heretofore unrecognized significant increase of megakaryopoiesis in c-Myc knockout mice as well. We also determined that Rbm15 deficiency predisposes HSCs toward megakaryocyte lineage differentiation – the percentages of CD41-positive megakaryocytes were found to be increased both in vivo in the spleens of Rbm15 mutant mice and after the in vitro culture of Rbm15-deleted HSCs in a megakaryocyte liquid differentiation medium, as compared to wild-type littermate controls – thus uncovering another phenotypic similarity between mice lacking Rbm15 and c-Myc in their hematopoietic systems. Furthermore, c-Myc expression was decreased in Rbm15-deficient HSCs (nearly 2-fold lower compared to littermate control HSCs), as confirmed by TaqMan real-time PCR. Importantly, the megakaryocytic differentiation bias of Rbm15-deficient HSCs could be at least partially rescued by c-Myc overexpression in liquid culture conditions in vitro, suggesting a functional interaction between Rbm15 and c-Myc activity. Our data suggest that c-Myc may be a major target and effector of Rbm15 function in the regulation of HSC and megakaryocyte development. The putative oncoprotein RBM15-MKL1 has been hypothesized to be involved in the pathogenesis of AMKL in part through a dominant-negative mechanism that impairs normal RBM15 function; our data imply that such an inhibition of normal RBM15 function would in turn lead to diminished c-MYC activity, thus contributing to the aberrant megakaryocyte lineage involvement observed in t(1;22)-positive AMKL.

AGM microenvironment: Role of the BMP pathway during HSC development

AGM microenvironment: Role of the BMP pathway during HSC development

Positional information in mouse HSC development

Positional information in mouse HSC development

The Placenta Is a Niche for Hematopoietic Stem Cells

The hematopoietic system develops during embryogenesis at temporally and anatomically restricted sites. The anatomical origin of definitive HSCs is not fully resolved, and little is known about how the different fetal hematopoietic microenvironments direct HSC development. Here, we show that the mouse placenta functions as a hematopoietic organ that harbors a large pool of pluripotent HSCs during midgestation. The onset of HSC activity in the placenta parallels that of the AGM (aorta-gonad-mesonephros) region starting at E10.5-E11.0. However, the placental HSC pool expands until E12.5-E13.5 and contains >15-fold more HSCs than the AGM. The expansion of the CD34(+)c-kit(+) HSC pool in the placenta occurs prior to and during the initial expansion of HSCs in the fetal liver. Importantly, the placental HSC pool is not explained by rare circulating HSCs, which appear later. These data support an important, but unappreciated, role for the placenta in establishing the mammalian definitive hematopoietic system.

Cytokine-mediated expansion of human NOD-SCID-repopulating cells.

There has been great interest in ex vivo expansion of human long-term repopulating hematopoietic stem cells (LTR-HSC) for a variety of developing clinical applications including HSC transplantation, gene therapy, and production of mature blood cells in manufacturing. Because the development of HSC is thought to be regulated, at least in part, by interactions of cytokine receptor signals, many investigators used various combinations of cytokines that have been shown to act on primitive hematopoietic cells to obtain the optimal culture condition for HSC expansion.