S19Oral Short Talk Presentations/ Experimental Hematology 42 (2014) S13–S21

O1027 - LABEL RETENTION SELECTS FOR HEMATOPOIETIC STEM

CELLS IN AGED BONE MARROWAND DISTINGUISHES HSCS FROM

MYELOID RESTRICTED PROGENITORS

Jeffrey Bernitz and Kateri Moore

Development and Regenerative Biology, Icahn School of Medicine at Mt. Sinai, New

York, New York, USA

Loss of immune cell function, increased myeloid cell output, and increased incidence

of myeloproliferative neoplasms all characterize aged hematopoiesis. Studies have

shown that many of these characteristics are due to the aging hematopoietic stem

cell (HSC) compartment. The HSC compartment consists of discreet subsets of

HSCs that differ in their differentiation potential, and HSCs with myeloid-biased

output accumulate in aged individuals contributing to increased myeloid cell produc-

tion. Additionally, phenotypic HSCs are known to accumulate in the aged but demon-

strate functional defects in homing and repopulation after transplantation. To clarify

this apparent contradiction of increased number yet decreased function, we investi-

gated the aged HSC compartment using a tet-off double transgenic pulse-chase sys-

tem that allows us to homeostatically assess HSC divisional history over time (Qiu et

al, Stem Cell Reports 2014). We placed mice on doxycycline for over a year and

observed a small yet significant population of label-retaining HSCs (LR-HSC; Line-

age-Sca1+cKit+Flk2-CD48-CD150+) in aged bone marrow (5.8962.21%, n513).

When transplanted, these aged LR-HSCs maintain the most robust repopulation po-

tential within the aged phenotypic HSC compartment in comparison to non-LR-HSCs

and the entire population. Furthermore, combining CD41 staining with the above

HSC markers demonstrates that the majority of the aged phenotypic HSC compart-

ment is comprised of CD41+ myeloid restricted progenitors (60.2367.31%, n53).

However, when assessing LR-HSCs the majority of cells are CD41-

(64.2365.72%, n53), consistent with reported phenotypes of long-term HSCs.

Transplantation experiments of these populations are ongoing. These findings suggest

that within aged bone marrow HSCs do not increase in number and frequency. Rather

there is an increase in the number of myeloid restricted progenitors with a classical

HSC cell surface phenotype while functional long-term repopulating HSCs slowly

deplete in a proliferation dependent manner.

O1028 - NOTCH3 IS REQUIRED IN THE SOMITIC

MICROENVIRONMENT FOR THE SPECIFICATION OF

HEMATOPOIETIC STEM CELLS

Albert Kim1, Chase Melick1, Wilson Clements1,2, David Stachura1, Martin Distel1,

and David Traver1

1University of California-San Diego, La Jolla, California, USA; 2St. Jude, Memphis,

Tennessee, USA

Hematopoietic stem cells (HSCs) are generated during embryonic development and

possess the ability to reconstitute all adult blood lineages and self-renew for the life

of an organism. Notch signaling is essential for HSC formation, as evidenced by the

fact that many Notch pathway proteins are required for this process including the spe-

cific Notch1 receptor that functions cell-autonomously in mouse. Additionally, recent

data from the zebrafish indicate that Notch signaling downstream of the non-canon-

ical Wnt protein Wnt16 is also required in the somitic environment for HSC and scle-

rotome specification, suggesting that these two developmental events are related. We

asked if the remaining Notch receptors perform any role in HSC emergence, here we

present evidence that Notch3 is required in zebrafish. Tissue and temporal-specific

activation of Notch intracellular domain (NICD) rescue experiments in Notch3

knockdown embryos indicate that Notch3 is required in somitic tissues for the gen-

eration of HSCs and sclerotome well before establishment of the HSC program,

conversely Notch1 homologues Notch1a and Notch1b are required in the endothe-

lium for dorsal aorta (DA) and HSC specification just before HSCs are generated.

Pharmacological inhibition of Notch signaling during somitogenesis affects both

sclerotome formation and HSCs, while inhibition just before HSC emergence affect

DA and HSCs respectively, supporting our findings for specific Notch receptor

knockdown phenotypes. Collectively, these findings demonstrate that multiple inputs

from the Notch pathway are required to specify HSCs, and that Notch3 performs a

novel role within the somite to regulate the neighboring precursors of hemogenic

endothelium.

O1029 - DUCTUS VENOSUS-ASSOCIATED PERICYTES FORM A NICHE

REGULATING HEMATOPOIETIC STEM CELL PROLIFERATION IN THE

FETAL LIVER

Jalal Ahmed1,2, Yuya Kunisake2, Sandra Pinho2, Anna Arnal2, Miriam Merad1, and

Paul Frenette2

1Mount Sinai School of Medicine, New York, New York, USA; 2Albert Einstein

College of Medicine, Bronx, New York, USA

In placentalmammalian development, oxygenated blood returns to the fetus via the um-

bilical vein (UV). This vessel becomes the ductus venosus (DV), a physiological shunt

that gives branches into the fetal liver (FL). We have identified a population of Nestin+

pericytes lining DV branches and not the hepatic veins. FL Nestin+ pericytes were en-

riched for niche genes and were required to maintain HSC in cytokine-free, serum-free,

FL reaggregate organ cultures.Wegenetically targetedNestin+ cells by diptheria toxin-

mediated depletion in vivo using Cre-recombinase under the NG2 promoter. FL HSC

numbers were reduced by 40% in NG2-Cre;DTA embryos compared to control litter-

mates (p ! 0.05). However, FL HSCs from NG2-Cre;DTA embryos gave 20%

increased engraftment compared to littermate controls in competitive repopulation as-

says (p! 0.05) . Since it is known that HSCs that have progressed into the S-phase of

the cell cycle have an engraftment defect,we hypothesized that the paradoxical compet-

itive repopulation activity after NG2+ cell depletionmay be due to halted cell cycle pro-

gression. HSCs from NG2-Cre;DTA mice indeed had a 50% reduction in BrdU uptake

and a drastic reduction in S-G2 phase HSCs concomitant with an increase in engraft-

ment competent G0-G1 phase HSCs, compared to control littermates. We quantified

HSC activity in the non-repopulation based long-term culture-initiating cell (LTC-

IC) assay. LTC-ICs were decreased by 44% in NG2-Cre;DTA compared to controls

(p ! 0.05). These data suggest that Nestin+ pericytes are required to drive HSCs

through the cell cycle. During E12-14.5, the DV vascular tree exhibits an exponential

increase in total surface area alongwhichNestin+ cells proliferate. HSCnumbers corre-

lated with Nestin+ cell numbers (R250.8). These data indicate that HSC expansion is

titrated against DV angiogenesis. Parturition and ligation of the UV caused a drastic

change in fetal hemodynamics and a reduction in Nestin+ PDGFRb+ cells and HSC

numbers. These data thus raise the possibility that perinatal alterations in the DV

may drive HSC emigration from the FL.

O1030 - LMO1/2 REGULATES DNA REPLICATION IN HEMATOPOIETIC

CELLS

Magali Humbert, Marie-Claude Sincennes, Benoı̂t Grondin, Andr�e Haman,

Mathieu Tremblay, Alain Verreault, and Trang Hoang

IRIC, l’Universit�e de Montr�eal, Montr�eal, Quebec, Canada

Major oncogenes that are activated by recurring chromosomal translocations in T-cell

acute lymphoblastic leukemia (T-ALL) involve transcription factors that are master

regulators of cell fate such as LMO1/2. How LMO1/2 transforms cells remain

elusive. LMO1/2 does not bind DNA and has a well-documented transcriptional func-

tion through interactions with other oncogenic transcription factors. Using a prote-

ome-wide screen for LMO2 interaction partners in hematopoietic progenitors, we

identified 3 DNA replication proteins as new LMO2 binding partners: MCM6,

POLD1 and PRIM1. We show that LMO2 interacts with endogenous replication pro-

teins in Kit+ hematopoietic progenitors and localizes to known origins of replication

together with MCM5. Next, we optimized a synthetic DNA replication assay in

mammalian cells to precisely address the role of LMO2. Using LMO2 fused to the

GAL4-DNA binding domain, we show that LMO2 tethering to DNA is sufficient

to stimulate DNA replication and to transform GAL4 sites into origins of replication.

Indeed, we show that LMO2 tethering recruits DNA replication proteins to GAL4

binding sites in mammalian cells and drives a dose-dependent increase in newly syn-

thesized DNA. Interestingly, we observed that LMO1/2 overexpression specifically

influences cell cycle of thymic progenitors, which are targets of cell transformation

in T-ALL. Indeed, ectopic LMO1/2 expression increases MCM loading on the chro-

matin in G1 phase and recruitment to origins of replication, resulting in accelerated

cell cycle progression in S phase of DN1-DN3 thymocyte subsets. This effect of

LMO1/2 causes a replicative stress as assessed by increased sensitivity to hydroxy-

urea and increased DNA damage, potentially conducive to mutations. In summary,

our data show a direct control of DNA replication by LMO1/2. Therefore, in addition

to its role in transcription activation and thymocyte reprogramming, our study pro-

vides new insights into the process of LMO1/2-driven clonal evolution, indicating

the same oncogene has a dual function in leukemia initiation and evolution.