45th Annual Scientific Meeting Updates
Annual Meeting Program Addendum - Schedule Updates
(confirmed after 28 July)
SESSION 12: NEW INVESTIGATORS AWARD SESSION
Sunday, 28 August 2016
Time change: Revised to 10:45 - 12:45
Lunch on own
Sunday, 28 August 2016
Time change: 12:45 - 14:00
SESSION 13: PRESIDENTIAL SYMPOSIUM
Sunday, 28 August 2016
Time change: Revised to 14:00 - 15:45
1018 – Session Speaker Cancelled
Richard Van Etten, UC Irvine
Central role for ikaros in pre-b cell differentiation and pathogenesis of high-risk b-cell acute lymphoblastic leukemia
1033 – Session Speaker Added
Danica Chen, University of California, Berkeley, USA
Mitochondrial UPR-mediated metabolic checkpoint, hematopoietic stem cell aging and rejuvenation
Cell cycle checkpoints are surveillance mechanisms in eukaryotic cells that monitor the condition of the cell, repair cellular damages, and allow the cell to progress through the various phases of the cell cycle when conditions become favorable. Recent advances in hematopoietic stem cell (HSC) biology highlight a mitochondrial UPR-mediated metabolic checkpoint that is essential for HSCs to return to the quiescent state. As quiescent HSCs enter the cell cycle, mitochondrial biogenesis is induced and mitochondrial oxidative stress is increased, which is associated with increased mitochondrial protein folding stress. Mitochondrial unfolded protein response and mitochondrial oxidative stress response are activated to alleviate stresses and allow HSCs to exit the cell cycle and return to quiescence. Other mitochondrial maintenance mechanisms include mitophagy and asymmetric segregation of aged mitochondria. Because loss of HSC quiescence results in the depletion of the HSC pool and compromised tissue regeneration, deciphering the molecular mechanisms that regulate the mitochondrial metabolic checkpoint in HSCs will increase our understanding of hematopoiesis and how it becomes dysregulated under pathological conditions and during aging. More broadly, this knowledge is instrumental for understanding the maintenance of cells that convert between quiescence and proliferation to support their physiological functions.
NEW INVESTIGATORS SESSIONS
New Investigators Technology Session and Lunch*
Bertie Gottgens, University of Cambridge Suffolk, UK
Doug Hinerfeld, Fluidigm, USA
New Investigators Career Session and Lunch*
Mervin C. Yoder, Indiana University - Wells Center for Pediatric Research Indianapolis, IN
Sheila Chari, Cell Stem Cell, Cambridge, MA, USA
Keith Humphries, Experimental Hematology, Vancouver, BC, Canada
Terry Bishop, NIDDK/NIH Bethesda, MD, USA
POSTERS (confirmed after 28 July)
3001 has declined the presentation invitation.
3002 has declined the presentation invitation.
3004 has declined the presentation invitation.
3014 has declined the presentation invitation.
3038 has declined the presentation invitation.
3047 has declined the presentation invitation.
3053 has declined the presentation invitation.
3075 has declined the presentation invitation.
3079 has declined the presentation invitation.
3097 has declined the presentation invitation.
3108 has declined the presentation invitation.
3111 has declined the presentation invitation.
3112 has declined the presentation invitation.
3114 additional authors are Allison Superneau and Glen Lowery.
3116 has declined the presentation invitation.
3123 has declined the presentation invitation.
3130 has declined the presentation invitation.
3132 has declined the presentation invitation.
3139 has declined the presentation invitation.
3140 has declined the presentation invitation.
3159 has declined the presentation invitation.
3170 has declined the presentation invitation.
3179 presenter has changed from Samanta A. Mariani to Chris Vink
3181 has declined the presentation invitation.
3193 has declined the presentation invitation.
3093 author order has changed to Gudmundur Norddahl, Tobias Maetzig, Patricia Rosten
Annual Meeting Program Addendum - Late-Breaking Abstracts
Saturday, 27 August 2016
13:00 - 14:45
SESSION 9A: LEUKEMIA II
2015 – The spliceosomal SRSF2P95H mutation promotes myeloid biased
Carl Walkley, St. Vincent Institute, Australia
2015 – THE SPLICEOSOMAL SRSF2P95H MUTATION PROMOTES MYELOID BIASED DIFFERENTIATION AND IS SUFFICIENT TO INITIATE MYELODYSPLASTIC SYNDROME (MDS)
Carl Walkley, Monique Smeets, Shuh Ying Tan, Jane Xu, Alistair Chalk, Meaghan Wall, Louise Purton
St Vincent's Institute
Myelodysplastic syndrome (MDS) and chronic myelomonocytic leukaemia (CMML) are hematological malignancies with limited treatment options. In MDS and CMML there are high rates of mutations in components of the RNA splicing machinery. Of the spliceosomal genes, 12-15% of MDS and 28-52% of CMML patients carry mutations in SRSF2, predominantly involving proline 95 (P95). Mutation of SRSF2 is an independent poor risk marker for overall survival and for progression to AML. It is largely uncharacterised how SRSF2P95 mutations impact on hematopoiesis and lead to both MDS and CMML. A recently reported conditional Srsf2P95H mutant mouse model did not develop features of advanced MDS (Kim et al, Cancer Cell 2015), nor did it display myeloid bias or positive selection for SRSF2 mutant cells, as has been reported in humans. We have generated a conditionally activatable Srsf2P95H model where the mutation is expressed from the endogenous locus in a heterozygous manner. Using ubiquitous (R26-CreER) or HSC specific (hScl-CreER) models to activate expression, we see macrocytic anemia and a mild leucocytosis resulting from elevated neutrophils. Analysis of bone marrow demonstrates an increase in frequencies of myeloid cells at the expense of erythroid- and B-cells. There is reduced expression of CD45RB, direct evidence of altered splicing in vivo. Bone marrow transplantation 20-24 weeks after Srsf2P95H expression resulted in a profound myeloid biased differentiation of the mutant cells. There is progressive loss of chimerism with cells isolated from the R26-CreER model, which is not seen with the HSC specific activation model, suggestive of differences in the impact of Srsf2P95H mutation depending on where it is expressed. At ~12 months post-tamoxifen, macrocytic anemia and myeloid dysplasia are seen in the PB of Srsf2P95H/+ animals, core characteristics of MDS. BM cellularity is largely preserved but with a profound myeloid bias (>70% BM granulocytes). In summary, these animals display a profound disruption in BM hematopoiesis. Abnormal phenotypes are seen in both the R26-CreER and hScl-CreER models. The changes are consistent with human MDS with SRSF2P95 mutation. Our results demonstrate that expression of SRSF2P95H from its endogenous locus is sufficient to initiate MDS and establishes a model that recapitulates fundamental features of human.
Saturday, 27 August 2016
13:00 - 14:45
SESSION 9B: TRANSCRIPTIONAL AND EPIGENETIC CONTROL
2017 – Using chromatin profiling to identify signaling-responsive transcriptional regulators of hematopoietic specification
Constanze Bonifer, University of Birmingham, USA
2017 – USING CHROMATIN PROFILING TO IDENTIFY SIGNALING-RESPONSIVE TRANSCRIPTIONAL REGULATORS OF HEMATOPOIETIC SPECIFICATION
Constanze Bonifer1, Nadine Obier1 , Pierre Cauchy1, Jane Gilmour1, Salam Assi1, Michael Lie-A-Ling2, Debbie Goode3, Berthold Goettgens3, Georges Lacaud2, Valerie Kouskoff2
1University of Birmingham, 2University of Manchester, 3University of Cambridge
It is unclear how specific transcriptional networks respond to outside signals to direct developmental stage-specific gene expression. The reason for this is the fact that signaling pathways consist of a myriad of components operating in a cell type-specific fashion and displaying multiple types of cross-talk making this process difficult to study. However, the response to signals is mediated by inducible transcription factors which bind to their cognate cis-regulatory elements, and thus the response to signaling is hard-wired into DNA. By identifying and studying the function of such sequences and the factors binding to them we can obtain first insights into how signal transduction processes are coordinated at the genomic level and then work our way backwards. We have previously identified transcriptional networks driving hematopoietic specification in differentiating embryonic stem cells (Goode et al., Dev Cell 2016). To identify signaling responsive factors interacting with this network, we used DNaseI hypersensitive site mapping and digital footprinting. This approach identified motifs for such factors enriched in cis-regulatory elements playing a role at specific stages of hematopoietic specification and highlighted motifs for TEAD factors as mediators of Hippo signaling, and AP-1 family members as mediators of MAP-Kinase signaling. By targeting these transcription factors directly combined with the identification of their global binding sites in cells representing different stages of hematopoietic specification, we show that (i) TEAD and YAP interaction is essential for hematopoietic development, (ii) AP-1 regulates the balance between the vascular and the hematopoietic program, (iii) AP-1 is required for the binding and expression of TEAD4 at a sub-set of genes in the hemogenic endothelium involved in vasculogenesis, (iv) that after the endothelial-hematopoietic transition cis-regulatory element activity is uncoupled from Hippo signaling and finally, that (v) AP-1 activity is required for myelopoiesis, adding to the activity of cis-elements binding a different set of transcription factors. Our work provides a global view of how signaling responses are integrated at the genomic level and how signaling responsive transcription factors interact with constitutive factors to program the chromatin landscape in response to the environment.
Sunday, 28 August 2016
09:00 - 10:30
Session 11B: MOLECULAR CONTROL III
2033 - Eicosanoid activation of GPCR signaling enhances hematopoiesis and marrow transplant
Jamie L. Lahvic, Harvard Medical School, USA
2033 – EICOSANOID ACTIVATION OF GPCR SIGNALING ENHANCES HEMATOPOIESIS AND MARROW TRANSPLANT
Jamie L. Lahvic1, Michelle Ammerman2, Pulin Li3, Song Yang2, Nan Chiang1, 4, Paul Norris1, 4, Michael Chase2, Olivia Weis2, Yi Zhou1,2, 5, Charles Serhan1, 4, Leonard Zon1, 2,4,5
1Harvard Stem Cell Institute, 2Boston Children’s Hospital, 3California Institute of Technology, 4Brigham and Women’s Hospital, 5Howard Hughes Medical Institute
Bioactive lipids play an important role as signaling molecules both during embryo development and adult tissue homeostasis. However, due to their small-molecule nature, identifying their receptors biochemically has been a long-standing challenge which impedes the understanding of the biological processes they regulate. Previously, we discovered that 11,12-epoxyeicosatrienoic acid (EET) could promote hematopoietic stem and progenitor cells (HSPC) engraftment in adult marrow transplant, as well as specification of HSPC from hemogenic endothelium. However, the identity of its receptor remains unknown despite more than a decade of research. Here, we utilized a novel bioinformatic approach to identify candidate EET receptors and identified a candidate functional in cull culture, zebrafish and mouse assays. We performed transcriptomic profiling on three human cell lines with clear EET-responsive phenotypes and found 37 GPCR expressed in common. 27 of these GPCR were also expressed in a non-EET-responsive cell line, leaving 10 candidate GPCR. Of these, only GPR132 exhibited EET-dependent recruitment of β-arrestin. GPR132 was previously identified as a receptor for a variety of small oxygenated fatty acids, which we confirmed by β-arrestin assay. Like EET, these GPR132 ligands increased HSPC numbers in the zebrafish aorta-gonad- mesonephros (AGM) and caused ectopic expression of the HSPC marker runx1 in the zebrafish tail, a phenotype that was previously seen only with EET treatment. GPR132 is required for EET phenotypes; morpholino knockdown of GPR132 prevented the EET-induced increase of runx1 in both the AGM and tail. Finally, we performed competitive whole bone marrow transplant using wildtype and GPR132-/- mice as donors and found that while treatment with EET increases engraftment of WT donor cells, no such improvement is seen in GPR132-/- cells. Combining bioinformatic, biochemical, and genetic approaches, we identified GPR132 as a receptor for EET and other small oxygenated fatty acids responsible for regulating hematopoiesis and marrow transplant. Genetic manipulation of GPR132 could help illuminate the endogenous roles of its fatty acid ligands.
3170 – IN VIVO ANALYSIS OF PROLIFERATION DURING HAEMATOPOIETIC STEM CELL DEVELOPMENT
Antoniana Batsivari1; Stanislav Rybtsov2; Celine Souilhol2; Anahi Binagui-Casas2; Lucia Morgado-Palacin2; David Hills2; Suling Zhao2; Alexander Medvinsky2
1MRC Centre for Regenerative Medicine, University of Edinburgh, United Kingdom; 2MRC Centre for Regenerative Medicine, University of Edinburgh, UK
Definitive haematopoietic stem cells (dHSC), capable of giving rise to all blood cell types, are generated intra-embryonically in the aorta-gonad-mesonephros (AGM) region of the mid-gestation embryo. Recent reports revealed that HSCs emerge through multistep maturation of precursors: proHSC--> preHSC I--> preHSC II--> dHSC. These haematopoietic precursors are found in the intra-aortic clusters, which bud from the aortic endothelium. Proliferation is a critical process for haematopoiesis as its regulation influences the balance between HSC self-renewal and differentiation. While proliferation of foetal and adult HSCs has been intensely studied, the cell cycle status of embryonic HSCs and their precursors is poorly characterised.
Here we used Fucci reporter mice that enable in vivo visualisation of cell cycle in combination with in vivo functional assays to assess the cell cycle status of preHSCs at different developmental stages. Our analysis shows that at E9.5 proHSCs are slowly cycling (G0/G1), while at E10.5 the emerging preHSC I are actively cycling, suggesting an expansion phase at this stage. Interestingly, one day later, the cell cycle of preHSC I slow down, while the emerging preHSC II are actively cycling. Finally, at E11.5, the dHSCs are found in G0/G1phases, like their foetal liver counterparts. Furthermore, we demonstrate by phenotypic analysis that the slowly cycling precursors show low levels of c-Kit expression and are predominantly closely attached to the endothelium, whereas the mature actively cycling cells express c-Kit highly and are at the top of the cluster.
Collectively these data suggest that transition from one HSC precursor type to another is concomitant with changes in cell cycle profile as well as localisation. Moreover, our data reveal important heterogeneity in cell cycle of preHSCs and suggest a role of c-Kit/SCF signalling pathway in expansion of haematopoietic cells.
3171 – DNA DAMAGE AND REPAIR MECHANISMS IN HUMAN HAEMATOPOIESIS AND THEIR ROLE IN CHEMORESISTANCE OF ACUTE MYELOID LEUKAEMIA
Amy Bradburn1; Kevin Rouault-Pierre2; Dominique Bonnet2
1Francis Crick Institute, London, United Kingdom; 2Francis Crick Institute
Chemoresistance and relapse are currently the biggest problems facing treatment of AML, with retention of leukaemia initiating cells (LIC) post therapy believed to be a main contributor. Standard AML chemotherapy regimes utilize cytarabine, a cytotoxic compound inflicting DNA damage in proliferating cells and inducing cell death. Cancer cells often have a deficiency in DNA repair pathways, which sensitize them to such treatments. Comparing normal and malignant haematopoietic cells in their response to cytarabine will determine whether differing responses to DNA damage and activation of DNA repair mechanisms contribute to chemoresistance. Moreover, DNA repair in primitive human haematopoiesis and LIC’s is still not well established.
Using AML cell lines, cord blood and CD34+ AML patient samples, a characterization of the DNA damage response post cytarabine treatment was performed. In vitro, cell lines were shown to have a range of sensitivities. Analysis of γH2AX foci formationshowed a significant increase in DNA damage after cytarabine exposure, however cell lines resistant to cytarabine, were quicker to recover their DNA damage, as shown by comet assay. As well, there was a faster revival of cell numbers and reduction in apoptosis. Similar findings were also observed when using ionizing radiation as a source of DNA damage. Analysis of the DNA repair mechanisms non-homologous end joining (NHEJ) and homologous recombination (HR) by 53BP1 and RAD51 localization respectively, showed NHEJ was preferentially used in response to cytarabine, with low rates of HR.
Normal cord blood haematopoietic stem (CD34+/CD38-) and progenitor (CD34+/CD38+) populations were compared with leukaemia initiating and tumour populations of CD34+ AML patient samples in vivo. All samples gained an increase in γH2AX foci and apoptosis post cytarabine exposure. Progenitor and tumour populations were most profoundly hit with a sparing of the more quiescent stem compartments. AML samples gave a heterogeneous response overall. NHEJ was primarily observed, however some samples showed increased localization of RAD51 compared to normal, which suggests enhanced HR contributes to response of AML to cytarabine.
Overall we see evidence of DNA repair in response to cytarabine treatment with leukaemic cells activating their repair mechanisms at a greater rate than normal haematopoietic cells.
3172 – THE SCALABLE DERIVATION OF OFF-THE-SHELF AND GENETICALLY ENHANCED HEMATOPOIETIC CELLULAR THERAPEUTICS FROM NAÏVE HUMAN INDUCED PLURIPOTENT STEM CELLS
Raedun Clarke1; Matthieu Bauer2; Jeffrey Sasaki2; Brian Groff2; Ryan Bjordahl2; Ramzey Abujarour2; Greg Bonello2; Tom Lee2; Weijie Lan2; Heather Foster2; Chris Lynn2; Dave Robbins2; Betsy Rezner2; Dan Shoemaker2; Bob Valamehr2
1CA, San Diego, United States; 2Fate Therapeutics Inc
Human induced pluripotent stem cell (hiPSC) technology has advanced into clinical trials, garnishing hope and excitement as an unlimited source of universally applicable cellular therapeutics. To advance hiPSCs as “off-the-shelf” hematopoietic cell-based immunotherapies it is essential to demonstrate a hiPSC platform with enhanced potency through genome-engineering of persistence, targeting and safety mechanisms while retaining the capacity to efficiently and reproducibly generate the diverse repertoire of immune effectors in a robust and scalable process. We have previously demonstrated that our reprogramming platform supports efficient and rapid derivation of clonal hiPSC lines with properties indicative of the naïve state of pluripotency. In addition to maintaining a homogeneous population of hiPSCs, our platform enables efficient multi-gene and multi-loci targeted engineering at a single cell level, in both nuclease-dependent and -independent strategies. Through genetic modifications at the single cell hiPSC stage, we demonstrate integration of inducible caspase-mediated and herpes simplex virus thymidine kinase suicide genes each into a unique safe harbor locus and increased effector cell persistence and immune evasion through HLA-related genetic modifications. Additionally we have developed a novel differentiation platform to derive hematopoietic cells from genetically enhanced hiPSCs in a highly scalable manner, void of an embryoid body intermediate, under serum- and feeder-free conditions. This platform represents a well-defined, small molecule-driven, staged protocol that can readily be translated into cGMP (current good manufacturing practice) settings. The derived hematopoietic population is definitive in nature as determine by Notch dependency and exhibits multi-lineage potential, as demonstrated through the formation of functional effector lymphocytes. These derived lymphocytes, including T and NK cells, are under investigation for in vitro and in vivo effector function including target specific recognition and enhanced killing potential. Hematopoietic cells generated can be successfully cryopreserved and banked, serving as a highly-stable feedstock for subsequent derivation of various cell types for therapeutic use. Our study indicates that naive hiPSCs are an ideal source for “off-the-shelf” hematopoietic cell-based immunotherapies.
3173 – ENZYME REPLACEMENT THERAPY AS LONG-TERM TREATMENT FOR CONGENITAL HAEMOLYTIC ANAEMIA, AS SHOWN IN A MURINE MODEL OF TRIOSEPHOSPHATE ISOMERASE DEFICIENCY
Rationale: Congenital haemolytic anaemias comprise of a collection of severe, often fatal, genetic disorders of the red cell. TPI deficiency is a rare red cell disease caused by a genetic mutation in the enzyme TPI-1, resulting in haemolytic anaemia, splenomegaly, paralysis, and ultimately death in early childhood. No treatments exist for TPI deficiency, but animal models such as the Drosophila have previously shown that enzyme replacement therapy is a viable long-term solution. Adapting this therapy to a mammalian model of this haemolytic disease would provide the foundations for new clinical approaches towards TPI deficiency and similar congenital anaemias.
Aim: Using a mutagenised murine model of TPI deficiency, called RBC19, we hope to demonstrate the first mammalian bone marrow-derived enzyme replacement therapy as a plausible clinically applicable treatment for human TPI deficiency.
Methods: The RBC19 mutant mouse line was generated on an SJL/J background using ENU mutagenesis. G1 progeny were interbred to generate recessive G2 mutants, which were screened for changes in red cell volume (MCV). RBC19 mice were subject to whole exome sequencing (WES), which isolated a phenylalanine to serine base-pair mutation in amino acid 57 of the tpi-1 gene. Enzyme-replacement therapy was conducted using 1x106 bone marrow cells harvested from adult SJL/L wildtype femurs, which were intravenously transplanted into lethally irradiated RBC19 homozygous recipients.
Results: RBC19 homozygous mice were found to have an elevated MCV compared to SJL/J wildtypes (62.1fL to 43.3fL), as well as splenomegaly, reticulocytosis, and a reduced red cell half-life (44% of wildtype). The F57S mutation in the tpi-1 gene was shown to cause TPI protein instability and reduced enzyme activity (29% of wildtype). Following 6 weeks recovery from bone marrow transplantation, RBC19 recipients showed normalised MCV (45.3fL), reduced reticulocytes, and a normalised red cell half-life when compared to SJL controls.
Conclusions: ENU mutagenesis has generated a viable homozygous murine model that accurately recapitulates TPI deficiency with genetic, enzymatic, and haematological parallels to the human disease. This is the first instance in which enzyme replacement therapy has been adapted to a mammalian model of TPI deficiency, and has demonstrated that this red cell disease can be effectively treated long-term using bone marrow transplantation. The data obtained from these investigations provide the foundations for clinically translatable treatment of human TPI deficiency and similar congenital haemolytic anaemias.
3174 – OSCILLATORY STATES OF GATA2 IN SINGLE CELLS DRIVE HEMATOPOIETIC FATE DURING EHT
Christina Eich1; Jochen Arlt2; Chris Vink3; Parham Solaimani Kartalaei4; Polynikis Kaimakis4; Wiggert A, van Cappellen5; Reinier van der Linden4; Elaine Dzierzak3
1Erasmus MC, Dep. of Cell Biology; 2University of Edinburgh, School of Physics and Astronomy; 3University of
Edinburgh, Centre for Inflammation Research, Queens Medical Research Institute; 4Erasmus Medical Center, Stem Cell Institute, Department of Cell Biology; 5Erasmus Medical Center, Department of Pathology
Underpinning cell behavior during development and differentiation are fundamental dynamic changes that drive cell fate processes away from a pre-existing equilibrium. The Gata2 transcription factor is an important regulator of blood cell differentiation and development, and is pivotal to hematopoietic stem cell generation (HSC). Within a short window of developmental time, HSCs originate from a natural differentiation of hemogenic endothelial cells through a process called endothelial to hematopoietic transition (EHT). Genetic studies in mice show that Gata2 is required for EHT and HSC generation, and that this process is very sensitive to reductions in Gata2 dosage. Surprisingly, nothing is known about the in vivo behavior of Gata2 expression in single cells during development, and particularly during EHT. In this study, we used a recently published Gata2-Venus reporter system with unperturbed Gata2 expression (Kaimakis et al, Blood, 2016) to follow Gata2 expression in single cells of the mouse midgestation aorta (the site of EHT and HSC generation) by vital confocal time-lapse imaging. EHT subsets that included single hemogenic endothelial cells, bulging cells and cluster cells were imaged over a 10 hours period. To quantify Gata2 levels, oscillation periodicity and amplitude, we developed a novel automated data processing methodology. EHT cell subsets could be distinguished by Gata2 expression levels and oscillation characteristics, with the greatest magnitude of Gata2 oscillation during EHT occurring in bulging cells. In Gata2 haploinsufficient embryos, which have greatly reduced HSC quantity and quality, Gata2 expression levels and oscillation characteristics in EHT cell subsets were profoundly disturbed. These single cell vital imaging results demonstrate a correlation between Gata2 oscillations, cell differentiation and acquisition of hematopoietic function during EHT, strongly suggesting that Gata2 oscillatory states and levels drive hematopoietic fate.
3175 – GENERATION OF HOXA+ HEMOGENIC VASCULATURE FROM HUMAN PLURIPOTENT STEM CELLS THAT RESEMBLES AORTA-GONAD-MESONEPHROS
Elizabeth Ng1; Lisa Azzola2; Freya Bruveris2; Vincenzo Calvanese3; Belinda Phipson2;Katerina Vlahos2; Claire Hirst4; Vanta Jokubaitis5; Qing Yu6; Jovana Maksimovic2; Simone Liebscher7; Vania Januar2; Zhen Zhang8; Brenda Williams8; Aude Conscience4; Jennifer Durnall2; Steven Jackson9; Magdaline Costa4; David Elliott2; David Haylock8; Susan Nilsson8; Richard Saffery2; Katja Schenke-Layland7; Alicia Oshlack2; Hanna Mikkola10; Edouard Stanley2; Andrew Elefanty2
1Murdoch Childrens Research Institute ; 2Murdoch Childrens Research Institute; 3IUniversity of California, Los Angeles; 4Monash University; 5University of Melbourne; 6Shanghai Institutes for Biological Sciences; 7Eberhard Karls University Tübingen; 8Commonwealth Scientific and Industrial Research Organisation; 9University of Wisconsin-Madison; 10University of California, Los Angeles
The production of definitive hematopoietic stem/progenitor cells from human pluripotent stem cells (hPSCs) remains a significant challenge. Compared to repopulation-competent cord blood CD34+ cells, hPSC-derived CD34+ progenitors lackedHOXA gene expression, indicating incorrect mesoderm patterning. Using reporter lines to track the endothelial (SOX17) to hematopoietic (RUNX1C) transition, we show that simultaneous modulation of WNT and ACTIVIN signaling yields hematopoietic progenitors with HOXA codes that more closely resemble those of cord blood. These cultures generate a network of aorta-like SOX17+ vessels, from which RUNX1C+ blood cells emerge - reminiscent of definitive hemogenesis within the aorta-gonad-mesonephros (AGM). Transcriptional profiles of nascent hematopoietic progenitors and corresponding cells sorted from human AGM displayed striking similarity in expression of cell surface receptors, signaling molecules and transcription factors. Our findings argue that HOXA codes are an indicator of mesoderm patterning, providing a rational and tractable readout for optimizing the generation of definitive hematopoietic lineages from hPSCs.
3176 – CD271+CD90+ MESENCHYMAL STROMAL CELLS DISPLAY FUNCTIONAL CHANGES WITH AGING, AND THEIR FREQUENCY IS POSITIVELY CORRELATED WITH CD34+CD38- HEMATOPOIETIC PROGENITOR/STEM CELL NUMBERS
Eugenia Flores-Figueroa1; Alicia Aguilar-Navarro2; Dita Gratzinger3; A Berenice Meza-León2;Hector Mayani4; Ricardo Esquivel-Gómez5
1National Medical Center, IMSS., Mexico, Mexico; 2National Medical Center, IMSS; 3Stanford; 4National Medical Center, IMSS.; 5Orthopedics Hospital, IMSS.
Clonal hematopoiesis is a precursor to myeloid neoplasia, and aging is a factor that increases the risk of both. To date, we have not fully understood the mechanism that triggers clonal hematopoiesis within the bone marrow and the role of aging in this process, but we hypothesize that mesenchymal stromal cells (MSCs) may play an important role. MSCs contribute to development of myeloid neoplasia in mouse models and harbor chromosomal abnormalities in some hematopoietic neoplasms. The vast majority of CD34+ cells are in intimate contact with CD271 MSC in intact bone marrow core biopsies, and CD271+ MSC density is an independent prognosis factor in MDS, Little data exists regarding the functional and phenotypic properties of human MSCs with aging. The aim of the study was to compare the functional properties of MSCs and frequency of hematopoietic stem and progenitor cells in bone marrow samples from healthy adults (40-60 years) and healthy older adults (61-89 years) in the prime age range at risk for myelodysplasia and acute myeloid leukemia.
Methods: Bone marrow samples were obtained from otherwise healthy adults undergoing hip replacement surgery (n=23)(institutional ethical approval R-2012-785-092), from adults (40 to 60 years) and older adults (61 to 89 years). Flow cytometry was performed using the following markers: CD45, low affinity NGFR (CD271) and CD90, CD34, CD38, CD49f and lineage. MSC clonogenic potential was evaluated by the CFU-F assay. MSC potential to differentiate into adipocytes was evaluated by oil red O staining, and osteogenic differentiation by Von Kossa staining.
Results: The clonogenic capacity of MSC was significantly lower in the older adult group as compared to the adult group (3.1±1 vs 1.5±1.2 p=0.03). Among older adults differentiation potential was intact towards the adipogenic lineage, but increased towards osteoblasts (p=0.03 chi-square test). When we analyzed the adults and older adults groups pooled together, we found a strong correlation between the frequency of CD271+CD90+lin- MSC and CD34+CD38-lin- hematopoietic progenitor/stem cells (HPSC) (p=0.0055) but not to CD34+CD38-CD90+CD49f+lin- hematopoietic stem cells (HSC) or more committed CD34+CD38+ hematopoietic progenitor cells (HPC).
Conclusions: We have found decreases in the clonogenic capacity and changes in the differentiation potential of MSCs among older adults in the age range at increased risk of myeloid neoplasia. Among adults and older adults HPSC but not HSC or committed HPC frequency correlates with MSC frequency. The combination of functional changes in MSC with aging and the correlation of MSC frequency with HPSC frequency suggests that changes in the myeloid compartment among older adults might be related to MSC aging. Funding. Conacyt (CB- 2012-01/19947)
3177 – PROFILING OF N-GLYCAN ALTERATIONS IN CO-CULTURED HEMATOPOIETIC CELLS WITH BONE MARROW DERIVED STROMAL CELLS UNDER HYPOXIA CONDITION
Xiang Li1; Sijie Zhang2; Ganglong Yang2; Zengqi Tan2
1Wuxi Medical School, Jiangnan University, Wuxi, China; 2Jiangnan University
Mesenchymal stromal cells (MSC) are an important constituent of bone marrow microenvironment (so called niche) where they support hematopoiesis via direct cell-cell interactions with hematopoietic stem- and progenitor cells, and by releasing soluble factors. Glycans, such as N-glycans, are involved in numerous biological processes, including inflammation, cell–cell interactions, morphogenesis, and cancer development and progression. Recent information provided by lectin-based microarray has revealed novel opportunities to explore the aberrant expressed N-gylcans and their functions in bone marrow niche. Using stromal/hematopoietic in vitro cell coculture system, we firstly investigated the effects of stroma-derived signals on apoptosis sensitivity in co-cultured hematopoietic cells under hypoxia condition. Our results showed that hematopoietic cells KG1a changed its resistance to apoptosis into sensitive to p53-dependent apoptosis after co-cultured with the human stromal cell line HS27a(derived from normal marrow) in hypoxia (1% O2 ). We next applied an strategy using lectin-based glycomic techniques for global profiling of N-glycans in co-cultured hematopoietic cells with bone marrow derived stromal cells under hypoxia. Specifically, we found the enhanced levels of core-fucosylated N-glycans(catalyzed by FUT8) and terminal GalNAc(catalyzed by MGAT3) and elevated levels of their responded genes. Our understanding of the bone marrow microenvironment will be improved by further studies of core-fucosylated N-glycans and terminal GalNAc.
3178 – SPECIFIC INHIBITION OF HEMATOPOIETIC CELL MIGRATION TO ADIPOGENIC MARROW – “NO PLACE TO HOME”
Troy Lund1; Ashley Kramer2; Michael Lehrke3
1University of Minnesota; 2Washington University in St. Louis; 3Mayo Graduate School
The hematopoietic stem cell niche serves to maintain hematopoiesis and recruit cells after hematopoietic cell transplant. Cells comprising the bone marrow niche include: mesenchymal stromal cells, CXCL12-abundant reticular cells, perivascular cells, bone marrow endothelium, adipocytes, chondrocytes, and osteogenic cells. The determinants of homing and engraftment after transplant are incompletely understood, but are mediated by chemokines secreted by niche cells. We determined the transcriptome of both zebrafish and mouse niche cells and found several small candidate proteins that were induced by pre-conditioning radiation. Specifically, dermatopontin (DPT), a 24kDa secreted extracellular matrix protein, was highly expressed in adipocytes (10-fold versus endothelial cells and 7.5-fold versus osteocytes, ANOVA p = 0.02). Immunohistochemistry (IHC) and ELISA confirmed that DPT expression was predominately localized to adipocyte-rich regions of the marrow. After radiation, DPT transcript levels increased 20-fold in the bone marrow (p < 0.01). Homing assays in DPT pre-treated irradiated mice showed a 90% reduction (n = 12, p = 0.02) and a 40% reduction (n = 6, p < 0.05) in whole marrow and lin-sca1+kit+ (LSK) cells that migrated to the marrow 20 hours post-transplant, respectively. This reduction in homing translated to decreases in overall engraftment in primary and secondary transplant recipients.
The bone marrow microenvironment is known to be heterogeneous in its distribution of niche cells. For example, the murine tibia has an adipocyte rich distal end and adipocyte poor proximal end, with the former recruiting 2-fold fewer donor hematopoietic cells versus adipocyte poor regions at 20 hours after adoptive transfer in irradiated recipients (n = 9, p < 0.05). Colony forming unit and apoptosis assays of LSK cells revealed no adverse effects of DPT on hematopoietic progenitor cells, suggesting a specific defect in cell migration as opposed to stem cell toxicity. In vitro adhesion assays demonstrated a 25% decrease in the number of hematopoietic cells that adhered to DPT coated plates (n=6, p = 0.01), and IHC revealed that DPT binds to the endothelial lumen of the vasculature after myeloablative conditioning. CFU assays of circulating cells 20 hours after adoptive transfer showed that DPT led to an increased number of circulating donor derived CFU (149 v 83 CFU/ml, p=0.002), indicating that transplanted cells remained in circulation instead of efficiently homing to the marrow. These data suggest that DPT retards the ability of hematopoietic cells to adhere and transmigrate across the endothelial barrier during the homing process particularly in regions of increase adiposity making DPT a new and novel regulator of hematopoietic cell trafficking.
3179 – THE IMPORTANCE OF BEING A MACROPHAGE DURING HEMATOPOIETIC STEM CELL DEVELOPMENT
Samanta Mariani1; Zhuan Li1; Chris Vink1; Elaine Dzierzak2
1The University of Edinburgh; 2The University of Edinburgh and Erasmus MC
Hematopoietic stem cells (HSC) are responsible for the life-long maintenance and regeneration of the adult vertebrate blood system. The first HSCs arise in the mouse embryonic aorta-gonad-mesonephros (AGM) region at E10.5 but very little is known concerning the microenvironment that supports their generation. Interestingly, macrophages (Mφ) can be found in the embryo before the production of the first HSC, leading to the hypothesis that yolk sac-derived cells could be involved in the HSC niche. Recent studies in zebrafish revealed that Mφ have a role in hematopoiesis by affecting the migration of hematopoietic stem and progenitor cells from the AGM to the caudal hematopoietic tissue. To investigate whether this mechanism is conserved in mice, we took advantage of the MacGreen transgenic mouse model that expresses EGFP under the control of the Csf1r (colony stimulating factor 1 receptor) promoter. Multi color flow cytometry confirmed that all the EGFP+ cells in the embryo (E8-E11) can be phenotypically defined as Mφ (CD45+CD11b+F4/80+MerTK+CD16/32+Ly6C-Ly6G-). Mφ numbers in the AGM increase over time, with a peak between E10 and E10.5 just before the production of the first HSC. The recruitment of Mφ to the AGM may be due to chemokine receptors/ligands expression by these cells. We found that AGM Mφ are positive for CCR2, CCR3, CCR5, CXCR4 and CX3CR1 by flow cytometry. Moreover, semi-quantitative real time PCRs on FACS sorted AGM endothelial cells from both Macgreen and Ly6AGFP mice, showed CX3CL1, CXCL12 and CCL3expression, suggesting that these chemokines can mediate Mφ recruitment to the aorta. Time lapse vital imaging on MacGreen AGM sections (E10-E11) showed that embryonic Mφ are highly motile cells that undergo mitosis, intravasate into the aorta and interact with CD31+cKit+ cells. Moreover, AGM explant experiments revealed a 40% decrease in the number of CFU-GEMM colonies upon treatment with clodronate liposomes that selectively ablate the 80% of Mφ in the AGM.Together, these results suggest that Mφ play a role in regulating the definitive hematopoietic wave in the mouse and future experiments will focus on specific molecular mechanisms so as to further our understanding on the role that extrinsic factors play in the establishment of definitive hematopoiesis.
3180 – INFECTION OF STEM AND PROGENITOR CELLS BY DENGUE VIRUS
Guey Chuen Perng1; Yi-Hui Hsu1; Jih-Jin Tsai2; Po-Lin Chen1; Tzu-Chuan Ho1; Ya-Ping Chen1;Tsai-Yun Chen1; Yu-Chih Lo1; Shang-Rung Wu1
1National Cheng Kung University; 2Kaohsiung Medical University Hospita
Bone marrow suppression resulting in defective hematopoiesis is a known clinical finding in dengue patients. Viral infections may induce damage and/or dampen the potency and capacity of their proliferated and differentiated functions, contributing to imbalance blood components in circulation and to disease development. Yet the physical interactions between dengue virus (DENV) and stem and progenitor cells remain in the shroud of mystery. Experimental designs with multicolor flow cytometry analysis were initiated to document the infection of stem and progenitor cells by DENV. Kinetic results from whole bone marrow cells, isolated stem and progenitor cells, and immune thrombocytopenic purpura donors revealed the importance of stem and progenitor cells in human bone marrow (HBM) for DENV infection. Peripheral blood mononuclear cells (PBMC) from healthy donors also demonstrated that the levels of stem and progenitor cells in the PBMC were a critical component for the DENV infection. Furthermore, results from PBMC of acute dengue patients showed that the levels of stem and progenitor cells were significantly and positively correlated to plasma viral load. Importantly, homing marker investigations on the stem and progenitor cells from PBMC of acute dengue patients revealed that the preferential organs for these cells homing to are gut, regional lymph node, lung, and bone marrow in respective order, suggesting clinical and pathogenic relevant in dengue patients. The cumulative results suggest that DENV infection of stem and progenitor cells in HBM not only lead to destruction of these progenitor cells but also may tailor these cells to serve as viral reservoirs for further dissemination of DENV into distant anatomic sites.
3182 – CLONAL AND QUANTITATIVE IN VIVO ASSESSMENT OF HEMATOPOIETIC STEM AND PROGENITOR CELL DIFFERENTIATION REVEALS STRONG ERYTHROID FATE BIAS OF MULTIPOTENT CELLS
Smrithi Rajendiran1; Scott Boyer2; Anna Beaudin3; Jessica Perez-Cunningham3; Stephanie Smith-Berdan3; Mark Landon3; Camilla Forsberg3
1SOE, UCSC, Santa Cruz, United States; 2Dako, an Agilent Technologies Company; 3UCSC
The lineage potential, heterogeneity, and relationships of hematopoietic stem and progenitor cell populations are currently unclear. To gain new insights on these questions, we performed quantitative analyses of mature cells produced from hematopoietic stem cells (HSCs) and multiple hematopoietic progenitor populations. Although our donor chimerism results supported the previous observations for the lineage potential of the various stem and progenitor populations, assessment of the absolute numbers of mature cell types produced by each progenitor cell revealed a striking erythroid bias. This bias was accompanied by strong platelet production that far exceeded generation of myeloid, B and T cells, even in the myeloid competent progenitors. Clonal analysis by single cell transplantation and spleen colony assays revealed that a significant fraction of HSCs and multipotent progenitors have multilineage potential at the single-cell level. The erythroid-dominant cell production observed by peripheral blood analysis was also evident within individual spleen colonies. Splenectomy differentially reduced erythro- and megakaryopoiesis, but not GM, B and T cell production. However, MegE production was not dependent on the spleen environment, as multilineage reconstitution from transplanted cells was also observed in splenectomized mice. Together, our quantitative comparative assessments of multilineage potential prompt an erythroid-biased model of hematopoietic differentiation.
3183 – THE ROLE OF ESSENTIAL TRANSCRIPTION FACTOR BCL11B IN T-CELL COMMITMENT GENE REGULATORY NETWORKS
Maile RomeroWolf1; Maria Quiloan2; Mary Yui2; Jonas Ungerback2; Ellen Rothenberg2
1Caltech, Pasadena, United States; 2Caltech
In order for precursor T cells to progress to a committed state, they must first shut off stem and progenitor programs, and deny access to alternative hematopoietic lineages. Transcription factor Bcl11b is required for T commitment. It comes on sharply at T cell commitment and stays on for the life of the cell. We know that if Bcl11b is deleted before commitment the cells still have access to alternative lineages including myeloid and NK. Our project describes the nature of the changes Bcl11b makes in the gene regulatory network that transition T cells from a progenitor to a committed T cell. Can a “committed” T cell return to a multipotent stem state if a key commitment transcription factor is deleted? Transcription factors like Spi1 (PU.1) that are expressed up until commitment appear to preserve the option of the progenitor cell to become myeloid under permissive conditions. Other genes, like Nfil3 and Zbtb16, enable cells to become natural killers or innate-like lymphocytes. We used Bcl11b conditional knock-out cells to determine whether removal of Bcl11b after commitment can restore access to these alternative cell fates. In vivo, Bcl11b knock out was accomplished using mice that have Bcl11b flanked by lox-P sites and expressing a Cre recombinase driven by a T cell promoter active immediately after commitment (Lck). These Bcl11b KO cells resemble neither pre-commitment nor post-commitment controls. Based on RNAseq analysis from purified from KO cells, some progenitor genes like Kit come back on, but the key myeloid gene Spi1 (PU.1) remains silenced. Others, including Nfil3 andZbtb16, show much higher expression levels in the Bcl11b knock out cells than in wild type cells. When these KO cells are cultured in permissive conditions with or without Notch signaling, some cells divert to NK fate, as indicated by NK1.1 and Dx5 expression by flow analysis. Additionally, the mutant cells without Notch signaling can express markers for alternative cell types including CD11b+Gr1+ putative granulocytes as well asCD11b+CD11c+ putative myeloid dendritic cells. Understanding the action of Bcl11b in this highly defined system helps illuminate how alternative cell fate potentials are extinguished and allows insight into the developing T-cell's gene regulatory network at commitment.
3184 – DIAGNOSIS AND PROGNOSIS OF FOLLICULAR LYMPHOMA ACCORDING TO BLOOD LEYKOCYTE CULTURE
Tamar Saralidze1; Nino Gogokhia2; Tinatin Shvelidze3; Tatia Swanidze4; Tinatin Saralidze5; Lia Mokhevishvili6
1Asiistant Professor, Georgia; 2Tbilisi State Medical University First University Hospital;3Medical Center "IUNONA"; 4Tbilisi State Medical University; 5AIETY Medical School; 6Children's Hospital
Though modern WHO classification of lymphomas are based on the morphological, immunophenotypic and cytogenetic investigations, timely diagnosis and estimation of the degree of malignancy often remains to be a challenging problem that concerns follicular lymphoma (FL) and its atypical forms as well. 30-40 percent of patients with FL eventually transform to more aggressive form and needs high-dose chemotherapy (www.lymphoma.org). Timely diagnosis and prediction of this transformation is also very important. Considering above mentioned problems we searched vein blood leukocyte cultures of patients with lymphoma or suspected on lymphoma according to method worked out by us. This method supports proliferation of malignant cells and reveals proliferative clone responsible for the onset and course of disease [www.e-bookland.ge/ATLAS-of-HEMATOLOGY]. Our research showed lymphocyte proliferation with their atypical forms and lymphocytosis over 3 weeks of cultivation in cases of lymphoma whereas in healthy donors active proliferation of macrophages was observed; We investigated 8 patients with FL. Their lymphocytes were positive for B-cell markers CD10, CD19, CD22, and CD20. In blood leukocyte cultures of these patients atypical lymphoid cells with cleaved nuclei and figures of atypical mitosis were revealed. In vitro FL cells are more cleaved and misshaped than in blood or in lymph node smears that supports to confirm diagnosis especially in cases when due to little amount of lymphoma cells in vivo immunophenotyping is not helpful. In cases of high grade FL abundance of atypical lymphocytes with cleaved nuclei and multiple figures of atypical mitosis in cultures shows high speed of tumor growth or progression and necessity of appropriate treatment. On contrary single atypical lymphocytes with cleaved nuclei in vitro points to low grade FL and indolent course of disease that helps physician to avoid a patient from high-dose chemotherapy and extend "watchful waiting" period. According to our investigations leukocyte culture method can be successfully used for the diagnosis of obscure cases of FL and estimation the degree of malignancy. Culture data not only represents the morphological grade of FL, but reveals tumor progression and predicts clinical course of disease that can help to determine appropriate treatment. Immunophenotyping of in vitro proliferating cells can be held as well.
3185 – MELOXICAM INDUCES AUTOPHAGY IN MEGAKARYOCYTES IN RESPONSE TO DNA DAMAGE
Mary Saunders1; Pratibha Singh2; Khalid Mohammad2; Melissa Kacena2; Christie Orschell2; Louis Pelus2
1Indiana University School of Medicine, Indianapolis, United States; 2Indiana University School of Medicine
DNA repair and damage responses are critical to maintain and restore genomic integrity following exposure to a variety of exogenous factors. Within the bone marrow niche, normal hematopoietic stem cells are relatively quiescent. Recent evidence suggests that megakaryocytes can promote HSC proliferation and differentiation following injury. Cells of the developing megakaryocyte lineage must remain viable, progress through the cell cycle, and undergo extensive remodeling to maintain mesenchymal stromal and endothelial cells of the bone marrow vasculature. Preserving homeostasis and cellular remodeling of the bone marrow niche requires activation of autophagic pathways. We have found that treatment of mice with non-steroidal anti-inflammatory drugs (NSAIDs) following irradiation promotes autophagy in megakaryocytes in young and old C57BL/6 mice. Following 650 cGy TBI, total marrow CD41+ megakaryocytes and CD71+ proliferating megakaryocytes increase significantly in comparison to non-irradiated control mice. The radiation induced megakaryocyte increase is ablated by NSAID treatment which blocks endogenous PGE2 production. However, the number of SLAM LSK and LSK hematopoietic stem cells is increased significantly in comparison to irradiated control and dimethyl-PGE2 treated mice. NSAIDS also promoted autophagy in normal, non-irradiated megakaryocyte lineage cells. In contrast, in an irradiated megakaryoblastic leukemia cell line, Meg-01, selective inhibition of cyclooxygenase pathways by treatment with meloxicam did not result in the loss of autophagy nor suppression of megakaryopoiesis and differentiation. Interestingly, there was greater than 2-fold increase in the number of megakaryoblast cells and 4-fold increase in thrombocytes. This study represents the first in kind of non-steroidal anti-inflammatory drugs influencing autophagy in megakaryocytes and may confer a protective advantage to cells of the hematopoietic niche following radiation induced injuries.
3186 – VASCULAR REGULATION OF HEMATOPOIETIC STEM CELL TRAFFICKING INTO AND OUT OF BONE MARROW NICHES
Stephanie Smith-Berdan; Mark Landon; Leah Kramer; Thessaly Alexander; Camilla Forsberg
Despite the use of hematopoietic stem cells (HSCs) in clinical therapy for over half a century, the mechanisms regulating HSC trafficking, engraftment and life-long persistence after transplantation are unclear. Findings over the past several years have demonstrated that life-long HSC function within bone marrow (BM) niches depends on cells associated with the BM vasculature. We recently showed that the vascular endothelium not only regulates HSC maintenance within BM niches, but also trafficking between the vasculature and BM space. Surprisingly, instead of acting as barriers to cellular entry, we found that vascular endothelial cells, via the guidance molecule ROBO4, actively promote HSC translocation across vessel walls into the BM space. In contrast, we found that the vasculature inhibits the reverse process, as induced vascular permeability led to a rapid increase in HSCs in the blood stream. Thus, the vascular endothelium reinforces HSC localization to BM niches both by promoting HSC extravasation from blood-to-BM and by forming vascular barriers that prevent BM-to-blood escape. Well-established modulators of vascular permeability, such as vascular endothelial growth factor (VEGF) and histamine, had profound effects on HSC location, revealing that vascular integrity is an important regulator of HSC location. In addition, we show that modulation of Robo4 via its putative Slit ligands affects HSC trafficking. Our work aims to uncouple the complex molecular and cellular mechanisms regulating the directionality of HSC migration across vessel walls. These insights will further enhance our ability to regulate HSC trafficking and thereby improve the safety and efficiency of hematopoietic transplantation therapies.
3187 – ZEBRAFISH CAUDAL HEMATOPOIETIC EMBRYONIC STROMAL CELLS SUPPORT HEMATOPOIETIC STEM AND PROGENITOR CELL PROLIFERATION AND DIFFERENTIATION
David Stachura1; Julian Aggio2; Anja Brown2; Francis Wright2; Gabriel Marquez2; Clyde Campbell3; David Traver3
1California State University, Chico, Chico, United States; 2California State University, Chico; 3University of California, San Diego
Zebrafish are an excellent model system for studying hematopoiesis due to their external development, high fecundity, and optical transparency. They are also genetically amenable, allowing large-scale mutagenesis and chemical screens for elucidating genes and molecular pathways involved in hematopoiesis to be easily performed. While forward genetic screens have been utilized to identify genes essential for the generation of primitive blood and the emergence of hematopoietic stem cells (HSCs), they have not elucidated genes essential for hematopoietic stem and progenitor cell (HSPC) proliferation and differentiation, due to a lack of methodologies to functionally assess HSPCs. We previously pioneered techniques to test the developmental potential of HSPCs by culturing them on zebrafish kidney stromal (ZKS) cells, a cell line derived from the main site of hematopoiesis in zebrafish. We have also derived zebrafish embryonic stromal trunk (ZEST) cells, isolated from tissue surrounding the embryonic dorsal aorta, the site of HSC emergence in developing fish. In addition to these cell lines, we now report the generation of caudal hematopoietic embryonic stromal tissue (CHEST) cells from 72-hour post fertilization (hpf) caudal hematopoietic tissue, the site of embryonic HSC expansion. CHEST cells expand two-fold more hematopoietic cells than ZKS or ZEST cells in vitro, and morphological and qRT-PCR analysis of these cultures indicated lymphoid, myeloid, and erythroid differentiation. Transcriptional comparison of ZKS, ZEST, and CHEST cells will allow the discovery of molecular pathways expressed by these cell lines that support HSPC proliferation and differentiation, but are temporally and ontologically distinct. We have performed RNA-sequencing on these cell lines, and these data allow for the first time a comparison of molecular signals critical for HSPC proliferation and differentiation in the zebrafish, and comparison with mammalian-supportive cell lines should further an evolutionary understanding of vertebrate hematopoiesis. Importantly, they allow the testing and validation of bona fide HSPCs in zebrafish, cells commonly dysregulated in hematologic diseases.
3188 – FUNCTIONAL AND TRANSCRIPTIONAL HETEROGENEITY OF HUMAN HAEMOPOIETIC LYMPHO-MYELOID PROGENITORS AT THE SINGLE CELL LEVEL
Bilyana Stoilova1; Dimitris Karamitros2; Zahra Aboukhalil2; Georg Otto2; Andreas Reinisch3;Fiona Hamey4; Eshita Sharma5; Marina Samitsch2; Lynn Quek2; Nicolas Goardon2; Bertie Gottgens4; Ravindra Majeti3; Paresh Vyas2
1Weatherall Institute of Molecular Medicine, University of Oxford, UK, United Kingdom; 2Weatherall Institute of Molecular Medicine, University of Oxford, UK; 3Stanford Institute for Stem Cell Biology and Regenerative Medicine, USA; 4Cambridge Institute for Medical Research, UK; 5Wellcome Trust Centre for Human Genetics, University of Oxford, UK
In steady state murine haemopoiesis most blood cells are generated from progenitors downstream of haemopoietic stem cells. However, early progenitor populations remain incompletely defined. We and others have described three human early lympho-myeloid progenitors: lymphoid-primed multipotent progenitor (LMPP), multi-lymphoid progenitor (MLP) and granulocyte-macrophage progenitor (GMP), but the relationship between these progenitors is unclear. Defining early progenitors is not only important to understand normal haemopoiesis but is relevant to our appreciation of differentiation block in human Acute Myeloid Leukemia (AML).
Here, we focus on the earliest human lympho-myeloid progenitors LMPP, MLP and GMP, and using functional (in vivo transplantation, colony assays, quantitative single cell and limit dilution in vitro liquid culture) and transcriptional assays (bulk and single cell RNA analysis) we show: 1. In vitro liquid culture analysis of 1760 single cells and quantitative limit dilution studies showed that LMPP and GMP are highly clonogenic (55-65%) and heterogeneous. In single cell analysis in positive wells, in most cases both progenitors only gave rise to uni-lineage output, with LMPP being lymphoid-biased and GMP myeloid-biased. MLP had poor cloning efficiency of 10% and was exclusively a lymphoid progenitor. Only 10% of the LMPP and GMP had combined lympho-myeloid potential, whereas MLP was not multi-potential in vitro. 2. Transplantation of LMPP and GMP into humanized ossicles in NSG mice gave 1-3% engraftment per 1000 transplanted human cells. The LMPP gave robust GM and B cell engraftment whereas the GMP gave principally GM engraftment. The MLP engrafted poorly with mainly B cell output. 3. Analysis of the expression of 96 lineage-affiliated genes in 900 single LMPPs, GMPs and MLPs showed that the functional heterogeneity was mirrored transcriptionally at the single cell level and that differentiation probably occurs as a continuum. 4. Comparison of the transcriptional programs of normal progenitors and human progenitor-like AML stem cells shed light on the nature of the differentiation block in AML.These data suggest a new model of lympho-myeloid progenitor specification altering our understanding of the molecular mechanisms regulating normal progenitor cell fate choice during haemopoiesis and provide a base for understanding the differentiation arrest in human AML.
3189 – SUPPRESSION OF LUTEINIZING HORMONE ENHANCES HSC RECOVERY AFTER HEMATOPOIETIC INSULTS
Enrico Velardi1; Jennifer Tsai2; Jarrod Dudakov3; Marcel Van Den Brink2; Kimon Argyropoulos2; Malcolm Moore2; Shieh Jae-Hung2
1Memorial Sloan Kettering Cancer Center, New York, United States; 2Memorial Sloan Kettering Cancer Center; 3Fred Hutchinson Cancer Research Center
A tightly regulated network of intrinsic and extrinsic signaling pathways exists to preserve hematopoietic stem cell (HSC) pool size and function. This is particularly relevant during periods of hematopoietic stress or injury when HSCs are induced to exit their quiescent state and transiently enter into a program of proliferation and differentiation to replenish blood loss.
There has been growing interest in how long-range effects of circulating sex steroids integrate HSC function with overall tissue physiology. Here we describe a novel and unexpected role of luteinizing hormone (LH) in regulating HSC biology independently of downstream sex steroids. Our findings revealed that the most primitive human and murine HSCs highly expressed the LH receptor. LH administration stimulated HSC proliferation whereas LH ablation promoted HSC quiescence after hematopoietic insults, including radiation exposure, Poly I:C treatment and repeated rounds of chemotherapy.
Uncontrolled proliferation after hematopoietic injury, such as chemotherapy or irradiation, has been associated with HSC exhaustion, long-term myelosuppression and ultimately death. We found that pharmacological inhibition of LH using a luteinizing hormone-releasing hormone-antagonist (LHRH-Ant) 24h after a lethal dose of irradiation (L-TBI) promoted hematopoietic recovery and mouse survival. We could demonstrated that the regenerative effects were independent from downstream sex steroids given that administration of LHRH-Ant improved survival rates in surgically castrated mice following radiation injury, while surgical castration alone did not. To definitively determine whether the regenerative effects of LHRH-Ant treatment depended on suppression of LH, we administered the LH receptor agonist human chorionic gonadotropin (hCG) to LHRH-Ant treated mice that had been given L-TBI one-day prior. Consistent with our hypothesis, administration of hCG abrogated the beneficial effects of LHRH-Ant on survival after radiation injury. Taken together these data not only reveal an unexpected role of LH in regulating HSC homeostasis, but also shows that inhibition of LH signaling using a single dose of an LHRH-Ant represents a rational and feasible approach to preserve the HSC pool, after high dose radiation, thereby mitigating acute hematopoietic radiation syndrome.
3190 – MICROGLIA COLONIZATION OF THE DEVELOPING ZEBRAFISH MIDBRAIN IS REGULATED BY APOPTOTIC NEURON AND LYSOPHOSPHATIDYLCHOLINE
Tienan Wang1; Yi Wu2
1Hong Kong University of Science and Technology, Hong Kong; 2Hong Kong University of Science and Technology, China
Microglia are central nervous system (CNS)-resident macrophages and play important roles in neural development and function. Yet, how microglial precursors born in peripheral hematopoietic organs colonize the CNS remains undefined. Here, by utilizing in vivoimaging and genetic manipulation of zebrafish, we showed that microglial precursors enter the optic tectum of the midbrain, where the majority of microglia reside during early development, via the lateral periphery between the eyes and brain and the ventral periphery of the brain in a circulation-independent manner. Intriguingly, long-term colonization of the optic tectum by microglial precursors is driven by apoptotic neuronal death in the midbrain and involves homing and settling processes. We further provide evidences indicating that the homing process is mediated in part by lysophosphatidylcholine (LPC), a phospholipid known to be released from apoptotic cells. Our study reveals that microglia colonization of the developing midbrain is triggered by neuronal death naturally occurred during neurogenesis possibly via releasing chemoattractant.
3191 – DISTINCT REGULATORY NETWORKS ORCHESTRATE THE DEVELOPMENT OF MICROGLIA OF DIFFERENT ORIGINS
Tao Yu1; Ye Tian2
1Hong Kong University of Science and Technology; 2Hong Kong University of Science and Technology, China
Microglia are central nervous system (CNS)-resident macrophages and they play pivotal roles in the CNS development and homeostasis.Recent fate mapping studies have revealed that zebrafish has two different microglia populations, which arise from the rostral blood island (RBI) and ventral wall of dorsal aorta (VDA) and manifest distinctive colonization kinetics and potential. Yet, the genetic programs governing their development remain largely undefined. Here we showed that the development of the RBI- and VDA-derived microglia is orchestrated by distinctive regulatory networks, vertical and parallel, formed by the Ets transcription factors Pu.1 and Spila, respectively. Epistatic studies further revealed that the two distinctive regulatory networks control the development of microglia derived from the RBI and VDA largely through regulating a common downstream regulator Irf8. Our study indicates that tissue-specific transcription factors Pu.1, Spila and Irf8 form distinct genetic networks to control the development of microglia of different origins.
3192 – EMBRYO-DERIVED MACROPHAGES REGULATE DENDRITIC CELL POOL SIZE IN THE ADULT SPLEEN
Claudia Waskow1; Gulce Percin2
1Regeneration in Hematopoiesis, TUD, Germany; 2TUD
Dendritic cells (DCs) are potent antigen presenting cells that depend on Flk2-mediated signals for their differentiation. In Flk2-deficient mice, however, a small but functionally active fraction of DCs remains. We show here that the combined deficiency for Flk2 and Csf1r, another member of the same family of receptor tyrosine kinases, results in natural DC null mice. In contrast to Flk2, Csf1r-mediated signals affect DC differentiation by a cell extrinsic, non-hematopoietic mechanism that does not require Csf1r signaling within the entire adult mouse. During embryonic development Csf1r signaling is crucial for the generation of macrophages, which emerge from erythro-myeloid progenitors in the yolk sac before the presence of definitive hematopoietic stem cells (dHSCs). The tissue-resident red pulp macrophages (RPMp) in the spleen are such embryonic macrophages, and we show here that Csf1r signaling is required for their differentiation. In the absence of Flk2-mediated signals, loss of Csf1r expression results in complete ablation of spleen DCs in vivo due to the lack of embryo-derived RPMps. The dependency of a normal DC pool size on the presence of tissue-resident macrophages maintains throughout adulthood of the mice, suggesting that there is a continuous cross-talk between myeloid cells of adult and embryonic origin, and this communication is a crucial regulator for hematopoietic cell pool size control in vivo. RPMps have been implicated in the maintenance of tissue hemostasis, removal of pathogens and clearance of cellular debris. We assign here a novel important role to RPMps in the maintenance of tissue homeostasis by regulating the pool size of dHSC-derived myeloid cells in the spleen.
3194 – CELLULAR BARCODING LINKS B-1A B CELL POTENTIAL TO A FETAL HEMATOPOIETIC STEM CELL STATE AT THE CLONAL LEVEL
Joan Yuan1; Trine Kristiansen2; Elin Jaensson Gyllenbäck1; Alya Zriwil2; Tomas Björklund2;Jeremy Daniel3; Ewa Sitnicka1; Shamit Soneji1; David Bryder1
1Lund University, Lund, Sweden; 2Lund University; 3Copenhagen University Center for protein research, Copenhagen, Denmark
Hematopoietic stem cells (HSCs) undergo a functional switch in neonatal mice hallmarked by a decrease in self-renewing divisions and entry into quiescence. Here, we investigate whether the well-established developmental attenuation of B-1a cell output is a consequence of a shift in stem cell state during ontogeny. In vivo single cell fate mapping was achieved by cellular barcoding to resolve developmental changes in lineage potential. We demonstrate that fetal liver definitive HSCs give rise to both B-1a and B-2 cells at the single cell level. Further, B-1a potential is selectively diminished in all HSCs with time while B-2 output is maintained. Importantly, B-1a / B-2 plasticity can be reinitiated in a subset of adult HSCs by ectopic expression of the fetal RNA regulator Lin28b. Using barcodes as unique cellular identifiers, we establish a direct link between Lin28b induced B-1a plasticity and fetal-like HSC behavior including elevated HSC self-renewal and repopulation potential.
3196 – MACROPHAGE COLONY-STIMULATING FACTOR RECEPTOR MARKS AND REGULATES A FETAL MYELOID-PRIMED B CELL PROGENITOR IN MOUSE
Alya Zriwil1; Charlotta Boiers2; Lilian Wittmann1; Joanna Green3; Petter Woll4; Sten Eirik Jacobsen4; Ewa Sitnicka1
1Lund University, Lund, Sweden; 2Lund University, Sweden; 3University of Oxford, Oxford, UK; 4Karolinska Institutet and Center for Hematology and Regenerative Medicine, Oxford, U.K.
While it is well established that unique B cell lineages develop through distinct regulatory mechanisms during embryonic development, much less is understood about the differences between embryonic and adult B-cell progenitor cells, likely to underpin the genetics and biology of infant and childhood PreB acute lymphoblastic leukemia (PreB-ALL), initiated by distinct leukemia-initiating translocations during embryonic development. Herein, we establish that a distinct subset of the earliest CD19+ B-cell progenitors emerging in the E13.5 mouse fetal liver express the Colony Stimulating Factor-1 receptor (CSF1R), previously thought to be expressed and play a lineage-restricted role in development of myeloid lineages, and macrophages in particular. These early embryonic CSF1R+CD19+ ProB cells also express multiple other myeloid genes, and in line with this possess residual myeloid as well as B cell, but not T cell lineage potential. Notably, these CSF1R+ myeloid-primed ProB cells are uniquely present in a narrow window of embryonic fetal liver hematopoiesis and do not persist in adult bone marrow. Moreover, analysis of CSF1R-deficient mice establishes a distinct role of CSF1R in fetal B-lymphopoiesis. CSF1R+ myeloid-primed embryonic ProB cells are relevant for infant and childhood PreB-ALLs which frequently have a bi-phenotypic B-myeloid phenotype, and in which CSF1R-rearrangements have recently been reported.
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