Developmental differences of in vitro cultured murine bone marrow- and fetal liver-derived megakaryocytes

Abstract

Multiple lines of evidence support differences in the megakaryopoiesis during development. Murine in vitro models to study megakaryopoiesis employ cultured megakaryocytes MKs derived from adult bone marrow (BM) or fetal livers (FL) of mouse embryos. Mouse models allow to study the molecular basis for cellular changes utilizing conditional or knock-out models and permit further in vitro genetic or pharmacological manipulations. Despite being extensively used, MKs cultured from these two sources have not been systematically compared. In the present study, we compared BM- and FL-derived MKs, assessing their size, proplatelet production capacity, expression of common MK markers (αIIb, β3, GPIb α, β) and cytoskeletal proteins (filamin A, β1-tubulin, actin), the subcellular appearance of α-granules (VWF), membranes (GPIbβ) and cytoskeleton (F-actin) throughout in vitro development. We demonstrate that FL MKs although smaller in size, spontaneously produce more proplatelets than BM MKs and at earlier stages express more β1-tubulin. In addition, early FL MKs show increased internal GPIbβ staining and present higher GPIbβ (early and late) and VWF (late stages) total fluorescence intensity (TFI)/cell size than BM MKs. BM MKs have up-regulated TPO signaling corresponding to their bigger size and ploidy, without changes in c-Mpl. Expressing endogenous β1-tubulin or the presence of heparin improves BM MKs ability to produce proplatelets. These data suggest that FL MKs undergo cytoplasmic maturation earlier than BM MKs and that this, in addition to higher β1-tubulin levels and GPIb, supported with an extensive F-actin network, could contribute to more efficient proplatelet formation in vitro.

Keywords

• bone marrow
• fetal liver
• megakaryocytes
• proplatelets