HOXA9 promotes hematopoietic commitment of human embryonic stem cells

S50

Poster Presentations/ Experimental Hematology 42 (2014) S23–S68

P1108 - HOXA9 PROMOTES HEMATOPOIETIC COMMITMENT OF HUMAN EMBRYONIC STEM CELLS Panlo Menendez1, Veronica Ramos-Mejia2, Veronica Ayllon2, Clara Bueno1, Pedro Real2, and Oscar Navarro-Montero2 1 School of Medicine, Josep Carreras Leukemia Research Institute, Barcelona, Spain; 2 GENyO, Granada, Spain The molecular determinants regulating the specification of human embryonic stem cells (hESCs) into hematopoietic cells remain elusive. HOXA9 plays a relevant role in leukemogenesis and hematopoiesis. It is highly expressed in hematopoietic stem/progenitor cells (HSPCs) and is downregulated upon differentiation. Hoxa9deficient mice display impaired hematopoiesis, and deregulation of HOXA9 expression is frequent in acute leukemia. Analysis of the genes differentially expressed in cord blood (CB)-HSPCs versus hESC-derived HSPCs identified HOXA9 as the most down-regulated gene in hESC-derived HSPCs, suggesting that the expression levels of HOXA9 may be crucial for hematopoietic differentiation of hESC. Here we show that during hematopoietic differentiation of hESCs, HOXA9 expression parallels hematopoietic development but is restricted to hemogenic precursors (HEP, CD31+CD34+CD45-), and diminishes as HEPs differentiate into CD45+ blood cells. Different gain- and loss-of-function studies reveal that HOXA9 enhances hematopoietic differentiation of hESCs by specifically promoting the commitment of HEPs into primitive and total CD45+ blood cells. Gene expression analysis suggests that NF-kB signaling collaborates with HOXA9 to increase hematopoietic commitment. However, HOXA9 on its own is not sufficient to confer in vivo long-term engraftment potential to hESC-hematopoietic derivatives, reinforcing the idea that additional regulators are needed for the generation of definitive in vivo functional HSPCs from hESC.

P1109 - BONE MARROW MESENCHYMAL STEM CELLS FROM APLASTIC ANEMIA PATIENTS PRESERVE FUNCTIONAL AND IMMUNE PROPERTIES AND DO NOT CONTRIBUTE TO THE PATHOGENESIS OF THE DISEASE Panlo Menendez1, Clara Bueno1, Mar Roldan2, Mario Delgado3, Damia Romero-Moya1, Eduardo Anguita1, and Beatriz Martin-Antonio1 1 School of Medicine, Josep Carreras Leukemia Research Institute, Barcelona, Spain; 2 GENyO, Granada, Spain; 3IPB Lopez Neyra, Granada, Spain Aplastic anemia is a life-threatening bone marrow failure disorder characterized by peripheral pancitopenia and marrow hipoplasia. The majority of aplastic anemia cases remain idiopathic, and hematopoietic stem cell deficiency and impaired immune responses are hallmark underlying mechanisms causative for the bone marrow failure in aplastic anemia. Mesenchymal stem/stromal cells constitute an essential component of the bone marrow hematopoietic microenvironment because of their immunomodulatory properties and their ability to support hematopoiesis, and they have been involved in the pathogenesis of several hematological malignances. We have addressed whether bone marrow mesenchymal stem cells contribute, directly or indirectly, to the aplastic anemia pathogenesis. Here, we report that mesenchymal stem cell cultures can be established from the bone marrow of aplastic anemia patients and display the same phenotype and differentiation potential as their counterparts from normal bone marrow. Mesenchymal stem cells from aplastic anemia patients support the in vitro homeostasis and the in vivo repopulating function of CD34+ cells, and preserve their immunosuppressive and anti-inflammatory properties. These data demonstrates that bone marrow mesenchymal stem cells from aplastic anemia do not exert impaired functional and immunological properties, suggesting that they do not seem to contribute to the pathogenesis of the disease.

P1110 - SCL/TAL1-MEDIATED TRANSCRIPTIONAL NETWORK ENHANCES MEGAKARYOCYTIC SPECIFICATION OF HUMAN EMBRYONIC STEM CELLS Panlo Menendez1, Miguel Toscano2, Oscar Navarro-Montero2, Veronica Ayllon2, Veronica Ramos-Mejia2, Clara Bueno1, Marien Cobo2, Francisco Martin2, and Pedro Real2 1 School of Medicine, Josep Carreras Leukemia Research Institute, Barcelona, Spain; 2 GENyO, Granada, Spain Human embryonic stem cells (hESCs) are a unique in vitro model for studying human developmental biology and may represent a potential source for cell replacement strategies. Platelets can be generated from cord blood progenitors and hESCs however, the molecular mechanisms and determinants controlling the in vitro megakaryocytic specification of hESCs remain elusive. We have recently shown that SCL overexpression accelerates the emergence of hemato-endothelial progenitors from hESCs and promotes their subsequent differentiation into blood cells with higher clonogenic potential. Given that SCL participates in megakaryocytic commitment, we hypothesized that it may potentiate megakaryocytic commitment in hESCs. Here, we show that ectopic SCL expression enhances the emergence of megakaryocytic precursors, mature megakaryocytes and platelets in vitro. SCL-overexpressing megakaryocytic cells and their derived platelets respond to different activating stimuli, similarly to their control counterparts. Gene expression profiling of megakaryocytic precursors showed that SCL-overexpression renders a megakaryopoietic molecular signature. The Connectivity Map analysis revealed that trichostatin A (TSA), which is a histone deacetylase (HDAC) inhibitor, functionally mimicked the SCL overexpression-induced effects. Finally, we confirmed that TSA treatment of hESCs promotes the emergence of megakaryocytic precursors. We demonstrated that SCL gain-of-function and HDAC inhibitors are regulators of hESC-derived megakaryocytic cells.

P1111 - DECLINED PRESENTATION TRACKING CLONAL COMPETITION AND EVOLUTION AT THE FUNCTIONAL LEVEL IN MURINE EXPERIMENTAL MODELS OF ACUTE MYELOID LEUKEMIA Francois Mercier1, Jiantao Shi2, David Sykes1, Youmna Kfoury1, and David Scadden1 1 Center for Regenerative Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA; 2Harvard School of Public Health, Boston, Massachusetts, USA Human malignancies are often composed of multiple, related clones that are thought to arise through a process of branching Darwinian evolution. In acute myeloid leukemia (AML), high-throughput DNA sequencing identifies clonal heterogeneity at the mutational level, but the downstream molecular pathways driving clonal fitness, and their impact on response to therapy are still poorly understood. We report on the development of a novel experimental tool that allows prospective tracking of clonal evolution at the functional level. Using a combination of humanized models of AML and fluorescent protein labeling, we can measure clonal evolution in real time, serially isolate live clones competing in the same environment for phenotypic characterization, and correlate these findings with mutational and gene expression profiling. We tracked in vivo the sequence of events leading to the development of winner and loser leukemic clones, and repeatedly sampled DNA and RNA for genetic analysis. Our preliminary data indicates that spontaneously occurring mutations converge in activating a few key signaling pathways promoting cell growth in vivo. We are currently validating the significance of the clonal fitness signature identified in our model using shRNA knockdown, with the goal of identifying which microenvironmental interactions are relevant to cancer growth.