S52 Poster Presentations/ Experimental Hematology 42 (2014) S23–S68

P1116 - PROPERTIES OF C-MPL EXPRESSION IN THROMBOPOIETIN-

DERIVED HEPATIC HEMATOPOIETIC PROGENITORS OF XENOPUS

LAEVIS

Yoko Mochizuki1, Yuta Tanizaki2, Takato Otani1, Kei Sato1, Megumi Ichisugi1,

Ayaka Murase1, Shunji Sakai1, and Takashi Kato1,2

1Graduate School of Advanced Science and Engineering, Waseda University, Tokyo,

Japan; 2Department of Biology, School of Education, Waseda University, Tokyo,

Tokyo, Japan

Thrombopoietin (TPO) and its receptor, c-Mpl, are involved in megakaryocytes matu-

ration and the maintenance of hematopoietic stem cells (HSC) in mammals. The evo-

lution of the roles of TPO/c-Mpl remain poorly understood. We cultured whole liver

cells of African clawed frogs (Xenopus laevis) in semi-solid cultures in the presence

of 10 ng/ml of X. laevis TPO (xlTPO). The 0.06% of the seeded cells survived and

proliferated over 3 months. The culture consisted of heterogeneous cells exhibiting

various different properties, and expressed gata-1, myeloperoxidase and c-kit

mRNA. In order to characterize and purify megakaryocytic or earlier hematopoietic

progenitors, we generated antibodies to X. laevis c-Mpl (xlMpl). Firstly, xlTPO-depen-

dent cells maintained over 24 dayswere characterized by polyclonal antibody to xlMpl.

The binding of antibody to xlMpl was clearly detected in xlTPO-dependent cells, and

we observed different relativities among them. Therefore, we considered cell profiling

based on the combinations of c-Mpl expression and megakaryocytic markers should be

useful for characterization. A monoclonal antibody to X. laevis thrombocytes (T12)

was chosen, and the cells were analyzed and sorted by flow cytometry using FACSAria.

The xlTPO-dependent cells were sorted to 4 subpopulations of xlMpllow/T12+

(23.7%), xlMplhigh/T12+ (23.3%), xlMpllow/T12- (45.4%), and xlMplhigh/T12

(6.9%). The xlMpllow/T12- cells exhibited the highest nucleus/cytoplasm ratio

(84.8%, p!0.05) and contained basophilic cytoplasm, suggesting xlMpllow/T12+

cells exhibitedmature thrombocytic properties;whereas xlMpllow/T12- cells consisted

of earlier hematopoietic progenitors.We then tracked the appearance of xlMpllow/T12-

cells in the culture over time, and compared to the initial xlMpllow/T12- proportion in

thewhole liver.We discuss the appearance of xlMpl expressing cells and the enrichment

of very few hematopoietic progenitors in the liver under xlTPO stimulation.

P1117 - DECODING THE TRANSCRIPTIONAL PROGRAM FOR BLOOD

DEVELOPMENT FROM WHOLE TISSUE SINGLE-CELL GENE

EXPRESSION MEASUREMENTS

Victoria Moignard1,5, Steven Woodhouse1,5, Laleh Haghverdi2, Josh Lilly6,

Yosuke Tanaka1,5, Adam Wilkinson1,5, Florian Buettner2, Shin-Ichi Nishikawa3,

Nir Piterman7, Valerie Kouskoff6, Fabian Theis2, Jasmin Fisher4,1, and

Berthold Gottgens1,5

1Haematology, University of Cambridge, Cambridge, United Kingdom; 2Institute of

Bioinformatics and Systems Biology, Helmholtz Zentrum M€unchen, Munich,

Germany; 3RIKEN Institute for Developmental Biology, Kobe, Japan; 4Department

of Biochemistry, University of Cambridge, Cambridge, United Kingdom; 5WT-MRC

Cambridge Stem Cell Institute, Cambridge, United Kingdom; 6Paterson Institute for

Cancer Research, University of Manchester, Manchester, United Kingdom;7Department of Computer Science, University of Leicester, Leicester, United

Kingdom

Blood and cardiovascular development represent paradigms of embryonic tissue for-

mation, with increasingly specialized cells generated from early mesoderm. Here, we

map the progression of mesoderm towards blood by single cell expression analysis of

3,934 cells, aiming to capture an entire embryo equivalent of cells with blood-form-

ing potential at four sequential developmental stages. Using novel computational ap-

proaches, we reconstruct the developmental journey at single cell resolution, which

reveals asynchrony of maturation and sequential waves of expression for major reg-

ulators. Considering transitions between individual cellular states, we synthesize

executable regulatory network models that recapitulate blood development. Using

mouse embryos and embryonic stem cells, we validate model predictions by showing

that Sox7 inhibits primitive erythropoiesis, and that Sox and Hox factors control early

expression of Erg. We therefore demonstrate that single cell analysis of entire devel-

oping tissues coupled with novel computational approaches can reveal the transcrip-

tional programs that control organogenesis.

P1118 - EXOGENOUS NEUROTROPHINS ALTER CELL ADHESION AND

EXPRESSION OF NEURAL MARKERS ON CORD BLOOD

HEMATOPOIETIC STEM AND PROGENITOR CELLS IN VITRO

Ileana Mondragon-Garcia, Patricia Flores-Guzman, and Hector Mayani

IMSS National Medical Center, Mexico City, D.F., Mexico

Umbilical Cord Blood (UCB) is an important source of Hematopoietic Stem and Pro-

genitor Cells (HSPC), which are ontogenetically more primitive than their adult

counterparts. Thus, it has been assumed that UCB cells possess a higher cell plas-

ticity potential. On the other hand, experimental evidence from different laboratories

suggests that neural or neuro-endocrine elements may be capable of regulating the

behavior of non-neural cells and that HSPC are responsive to these stimuli. Based

on these observations, in the present study a cell population enriched for UCB

HSPC (CD45+ CD34+ CD38- Lin-) was used to assess the effects of different com-

binations of neurotrophins (NGF, BDNF, GDNF) and hematopoietic cytokines (SCF,

TPO, FL, IL6, IL3, GM-CSF, G-CSF) using a three-phase culture protocol. For the

first 7 days the enriched cell population was cultured in a serum-free medium supple-

mented with different combinations of cytokines and neurotrophins. After this initial

phase all cultures were treated in the same manner, so as to curtail the differences

displayed by the soluble factor combinations. For the next 7 days all soluble factors

were removed and fetal bovine serum was added (10% v/v), and for the last 10 days

cells were cultured in a neural expansion medium. After 24 days of culture, there

were no significant differences in proliferation or in the content of colony-forming

cells, between cells exposed to neurotrophins and those that were not. However, cells

initially exposed to neurotrophins showed higher adherence to the plastic plates and

increased expression of different neural markers; among these, Nestin and GFAP

were the ones showing the greatest increments. Interestingly, despite the acquisition

of several neural-like characteristics, almost all adherent and non-adherent cells re-

mained CD45+. These results suggest that UCB contains a cell population that is sen-

sitive to extrinsic neural stimuli and that is capable of acquiring neural

characteristics; however, such cells seem to preserve, at least in part, their original

(hematopoietic) identity.

P1119 - DECLINED PRESENTATION

PROMISING CELL THERAPY ACHIEVES IMPROVEMENT OUTCOMES

Laila Montaser and Sherin Fawzy

Clinical Pathology, Menoufia University, College of Medicine, Shebin El-Kom,

Egypt

Stem cell research is perhaps the most exciting medical technology of the twenty-first

century. The stem cell effort consists of technology development aimed at the produc-

tion, expansion, and differentiation of stem cells. Stem cells are progenitor cells that

are capable of self-renewal and differentiation into many different cell lineages. Stem

cells survive well and show stable division in culture, making them ideal targets for in

vitro manipulation. The researchers suggest potential future benefits of stem cells in

many fields of medicine. The potential application of stem cells is to form cells and

tissues for medical therapies. Stem cells offer a viable source of replacement cells to

treat diseases and can potentially reduce the morbidity and mortality for those await-

ing transplants. By directing stem cells to differentiate into specialized cell types,

there is the exciting possibility to provide a renewable source of replacement cells

for those suffering from diseases. Stem cells hold the promise of treatments and cures

for more than 70 major diseases and conditions that affect millions of people,

including diabetes, liver disease, arthritis, nerve disease, Alzheimer’s, cancer, multi-

ple sclerosis, spinal cord injuries, blindness, and HIV, as well as numerous genetic

disorders. This paper presents synopsis draws on some research studies to document

examples of researches on the application of cell therapy in some diseases.