Konference: 2014 19th Congress of the European Hematology Association - účast ČR

Kategorie: Nádorová biologie/imunologie/genetika a buněčná terapie

Téma: Hematopoiesis, stem cells and microenvironment

Číslo abstraktu: S690

Autoři: Renata Balounová; Mgr. Martina Benešová; Mgr. Tereza Vavrochová; Dr. Andrea Jurisicova, PhD; Hoon-ki Sung; Meritxell Alberich-Jorda, PhD; RNDr. Dominik Filipp


Background: Toll like receptors (Tlrs) are critically important in the pathogen recognition and regulation of innate and adaptive immune responses. In addition, direct pathogen sensing of adult bone marrow hematopoietic stem cells and hematopoietic progenitors via Tlrs seem to play a crucial role in directing the hematopoietic cell fates towards enhanced myelopoiesis under inflammatory conditions. So far, the expression of Tlrs during early stages of embryonic development at the onset of hematopoiesis has not been addressed.

Aims: The aim of this study was to determine the lineage potential of Tlr2 expressing embryonic hematopoietic progenitors.

Methods: Mouse embryos at different stages of development were dissected from time pregnant females, enzymatically dissociated by Dispase (1 mg/ml) and upon antibody staining analyzed by flow cytometry. For qRT-PCR analyses and in vitro assays distinct cellular populations were sorted using Influx cell sorter. Sorted lineageCD45+/– c-kit+/– TLR2+/– cells were grown on OP-9 stroma along with myeloid cytokines (SCF, IL-3, M-CSF, GM-CSF) or TLR2 agonist Pam3CSK4. Alternatively, sorted cells were plated directly to semisolid media supplemented with erythroid and myeloid differentiation promoting cytokines (Methocult M3434). Day 3 colonies grown in semisolid media were subjected to single colony qRT-PCR to detect the presence of embryonic hemoglobin. For microscopic analyses embryonic day 7.5 (E7.5) embryos were fixed, stained with TLR2 antibody and visualized by Zeiss LSM 780 microscope equipped with two photon, argon and helium–neon lasers.

Results: Using a transgenic model to trace cells of embryonic origin we showed that Tlrs are expressed on embryonic myeloid cells as well as hematopoietic precursors. Together with the prototypic marker of hematopoietic progenitors, c-kit, TLR2 is specifically expressed on the surface of hematopoietic precursors in early gastrulation embryos. Our qRT-PCR analyses showed that E8.5 CD45 c-kit+ TLR2+ cells express markers of hematopoietic progenitors as well as endothelial cells and myeloid cells. E7.5-E8.5 TLR2+ c-kit+ cells express CD45 mRNA and gradually differentiate through an intermediate c-kit+ CD45+ stage to c-kit CD45+ myeloid cells. Upon TLR2 triggering, E8.5 TLR2+ c-kit+ cells proliferate and differentiate to CD45+ CD11b+ myeloid cells in a MyD88dependent manner. Pre-circulation E7.5 CD45 c-kit+ TLR2+ cells as well as E6.5 CD45 TLR2+ cells differentiate to myeloid cells when cultured on OP-9 stroma with myeloid cytokine supplements. In addition E7.5 CD45 c-kit+TLR2+ cells give rise not only to myeloid but also to primitive erythroid colonies when cultured in semisolid media supplemented with erythroid and myeloid differentiation promoting cytokines. Using flow cytometry and whole mount fluorescence microscopy we show that E7.5 TLR2+ progenitors are predominantly detected in the yolk sac (YS) region. Nevertheless scarce TLR2+ c-kit+ cells with a distinct surface expression profile can be also found in the embryo proper (EP). This opens the question whether the emergence of hematopoietic precursors in EP is dependent or not on YS primitive hematopoiesis.

Summary/Conclusion: Our results indicate that the expression of TLR2 marks the emergence of a common embryonic hematopoietic progenitor of early erythro-myeloid lineage and endows embryonic hematopoietic progenitors with the capacity to boost the production of myeloid cells. These data suggest a functional link between embryonic hematopoiesis and pattern recognition receptors under inflammatory conditions.

Keywords: Hematopoiesis, Myelopoiesis, Progenitor

Datum přednesení příspěvku: 14. 6. 2014