S41Poster Presentations/ Experimental Hematology 42 (2014) S23–S68

P1072 - EX VIVO EXPANSION OF HEMATOPOIETIC STEM CELLS FROM

STEADY STATE PERIPHERAL BLOOD

Christine Jobin2,1, Marc Cloutier2, and Sonia N�eron2,1

1Universit�e Laval, Biochimie, Microbiologie et Bio-informatique, Qu�ebec, Quebec,

Canada; 2H�ema-Qu�ebec, Recherche et D�eveloppement, Qu�ebec, Quebec, Canada

Bone marrow, cord blood and mobilized adult stem cells are the main sources of he-

matopoietic stem cells (HSC) for transplantation. Besides, HSC are present in periph-

eral blood of healthy adults and have been proposed as an alternative with great

therapeutic potential. In this study, we have analyzed the potential of HSC recovered

from leucoreduction systems isolated by positive CD34 selection and cultured using

an ex vivo expansion model. We have determined the phenotype of HSC populations

in blood mononuclear cells (PBMCs) and following their 7-day expansion using a

defined animal protein free medium. Cultures were done at 8% (n57) and 21%

(n56) of oxygen. Flow cytometry data were analyzed by two distinct methods: Span-

ning-Tree Progression Analysis of Density Normalized Events (SPADE) and directed

gating using two-dimension diagrams with FCS Express software. The potential of

committed progenitors was assessed by determining the colony forming efficiency

of the isolated CD34+ cells and the expanded cells. Overall, about 0.10%60.04%

of PBMCs were CD45+CD34+ cells. Among those HSC, 83.2%60.7% were

CD38+ cells, 7.9%61.7% were CD90+ cells and 7.5%61.5% were CD133+ cells.

Seven days of culture in both conditions of oxygen resulted in about 15-fold expan-

sion of the CD34+ cells. On day 7, the distribution of expanded cells was similar for

cells submitted to 8% and 21% O2. At 8% O2, the dominant cells phenotypes were as

follows: 22.3%63.6% of CD45- cells, 6.8%61.7% of CD45+CD34+ cells, 40.3%6

12.0% of CD38+ cells and 2.8%60.4% of CD133+. The frequencies of

CD38+CD90+ cells were highly variable from one sample to another, ranging

from 0.4% to 59%. Our experiments also underline that BFU-E was the predominant

type of committed progenitors in the expansion product, as well as in the freshly iso-

lated CD34+ cells. This study allowed us to establish a simple model that enables

expansion of HSC from steady state peripheral blood. This culture model will be

used to characterize the subtypes of ex vivo generated progenitor cells and thus to

better define their potential for cell therapy.

P1073 - RETINOIC ACID RECEPTOR g REGULATES LYMPHOPOIESIS

VIA NESTIN+ BONE MARROWAND THYMIC MICROENVIRONMENT

CELLS

Chacko Joseph1,2, Celeste Nota1, Racheal Costanzo1, Alanna Green1,2, Tanja Jovic1,

Julie Quach1, David Izon1, and Louise Purton1,2

1St. Vincent’s Institute of Medical Research, Fitzroy, Victoria, Australia;2Department of Medicine, The University of Melbourne, Fitzroy, Victoria, Australia

Vitamin A has essential but largely unexplained roles in regulating lymphopoiesis.

Short-term treatment of mice with the biologically active derivative of vitamin A,

all-trans retinoic acid, resulted in significantly elevated (1.4-fold) numbers of B lym-

phocytes in peripheral blood (PB) and pre-B cells in the bone marrow (BM). Signif-

icantly increased numbers (1.5-fold) of T lymphocytes were also observed in PB and

thymus, accompanied by significantly increased (1.6-fold) thymic size. These effects

were not due to direct effects of ATRA on hematopoietic cells, implying microenvi-

ronment regulation. BM microenvironment cells identified by either their expression

of nestin (Nes) or osterix (Osx) have previously been shown to have roles in regu-

lating lymphoid cells. Furthermore, we have shown that retinoic acid receptor

(RAR)g knockout mice have hematopoietic defects, some of which were microenvi-

ronment-induced. We therefore conditionally deleted RARg in NesCre- or OsxCre-

targeted microenvironment cells. 12 week old OsxCre RARgfl/fl mice had no

hematopoietic defects. In contrast, NesCre RARgfl/fl mice had significant (1.5-

fold) reductions in PB B lymphocytes and 1.4-fold reductions in pre-B lymphocytes

in BM. They also had significant (1.5-fold) reductions in PB CD4+ and CD8+ T lym-

phocytes and a 1.4-fold reduction in thymic size, with reductions in all T cell subsets

in the thymus. In contrast, no alterations in other hematopoietic cells were observed

in NesCre RARgfl/fl mice. In the thymus, nestin expression was restricted to

6C3+EpCam-PDGFRa+ thymic stromal cells. These cells express high levels of

Cxcl12 and Scf. Furthermore, we have identified two distinct populations of BM

Nes-targeted cells by their differential expression of 6C3 and PDGFRa, and their na-

ture is being further investigated. Collectively these studies show that nestin+ cells in

the BM and thymic microenvironments are key regulators of B and T lymphopoiesis,

respectively, and that RARg regulates lymphocyte numbers through these nestin+

microenvironments.

P1074 - MICROENVIRONMENTAL REGULATION OF FETAL LYMPHO-

MYELOID LINEAGE CHOICE

Ekaterina Kajikhina1, Motokazu Tsuneto2,1, and Fritz Melchers1

1Department of Lymphocyte Development, Max Planck Institute for Infection

Biology, Berlin, Germany; 2Department of Stem Cell and Developmental Biology,

Mie University Graduate School of Medicine, Tsu, Japan

Hematopoiesis describes a process of stepwise differentiation from a pluripotent he-

matopoietic stem cell towards all kinds of blood cells. In the mouse embryo, the fetal

liver is colonized by circulating precursors around embryonic day 12.5. It remains the

major hemato- and B-lymphopoietic site until birth.

The establishment of hematopoietic lineages is supported by niches largely composed

of non-hematopoietic cells. They provide extrinsic factors which induce specific

intracellular programs and act in sequential and parallel fashions. Different stromal

compartments perform discrete functions, and precursors at various developmental

stages are consequently found in changing microenvironments.

We analyse the kinetics of hematopoietic subset development within the fetal liver

from E12.5 until birth and observe a rapid wave of differentiation from pHSCs to

fully VDJ-rearranged B cells. We focus on a subset which might represent the

branching point of early lineage diversification. These cells display pHSC and early

progenitor markers, but also express a mixture of both lymphoid and myeloid genes.

In vitro differentiation assays show their multipotency towards lymphoid and myeloid

lineages.

A broad range of chemokine receptors is transcribed in these progenitors at E13.5,

which ensures their responsiveness to the coordinating microenvironment during

the phase of fetal liver colonization. We observe changes in receptor expression

and decreasing ex vivo chemotactic responsiveness as embryonic development pro-

ceeds and lineage differentiation takes place.

Correspondingly, two non-hematopoietic subsets are identified to support and coordi-

nate migration, differentiation and maintenance of the analysed progenitors. Gene

expression analysis and colocalization studies at E13.5 show that VCAM-1+ Lyve-

1+ endothelial cells coordinate the entry of early progenitors into the fetal liver during

the colonization stage by chemoattraction. After E15.5 the VCAM-1+ALCAM+

mesenchymal stroma compartment is found to attract and support differentiating

and proliferating B-lineage precursors.

P1075 - GATA2 EXPRESSION DYNAMICS CORRELATES WITH DISTINCT

STAGE SPECIFIC WAVES IN ES CELL HEMATOPOIETIC

DIFFERENTIATION

Mari-Liis Kauts2,1, Polynikis Kaimakis1, Emma de Pater1, Reinier van der Linden1,

Marina Gabriel2, and Elaine Dzierzak2,1

1Erasmus MC, Rotterdam, Netherlands; 2University of Edinburgh, Edinburgh, United

Kingdom

In the mouse embryo the production of the hematopoietic system occurs in several

spatiotemporally distinct waves. Adult repopulating hematopoietic stem cells

(HSCs) are generated during the final definitive wave of hematogenesis. HSC produc-

tion begins at E10.5 in the aorta-gonads-mesonephros (AGM) region and is tightly

controlled by a combination of transcription and extrinsic factors.

The Gata2 transcription factor has a pivotal role in hematopoiesis. Germline Gata2

deletion results in severe hematopoietic defects and embryonic lethality at E10.5

(Tsai, 1994). Conditional knockouts reveal that Gata2 is required for HSC generation

in the embryo and HSC survival in the adult (de Pater, 2013; Gao, 2013). We devel-

oped a Gata2-Venus reporter knockin mouse model in which Gata2 expressing cells

can be prospectively isolated and examined for hematopoietic function, and found

that all HSCs in vivo are Gata2 expressing. To study the role of Gata2 in the early

stages of HSC development, we use the novel Gata2-Venus ES cell (ESC) line for

embryoid body (EB) differentiation. EB cultures are known to recapitulate early he-

matopoietic development. Our analysis of Gata2 expression dynamics shows that

ESC hematopoietic differentiation occurs in several waves, producing progeny

with stage-specific potential. These waves are similar to those observed in vivo.

The first wave of Gata2 expressing EBs recapitulates primitive hematopoiesis,

whereas gene expression analysis and hematopoietic progenitor assays suggest that

cells initiating Gata2 expression later serve as precursors for definitive multipotent

progenitors. To initiate further waves/stages of hematogenesis we have developed a

novel chimeric ESC differentiation model where AGM derived stromal cells are ex-

ploited to create a hematopoiesis-supportive niche within the EBs. This results in a

significant increase in Gata2 expressing cells and is accompanied by the generation

of multipotent hematopoietic progenitor cells. Thus, by tracking Gata2 expression we

have established a culture system that enhances definitive hematopoietic progenitor

production and may allow for HSC generation.