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

Authorship contributions

I.B. designed and performed the experiments, analyzed the data, wrote the manuscript; A.B. performed part of the experiments; A.J.B. conceived and designed the research, acquired and analyzed the data, wrote the manuscript, supervised and handled funding.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the Croatian Science Foundation (CSF), grant no. UIP-2014-09-2400, CSF grants for Ph.D. students (CSF-09-2016, I.B.,CSF-ESF-01-2018, A.B.), ICGEB starting grant no. International Centre for Genetic Engineering and Biotechnology CRP/HRV15-04_EC, University of Rijeka supportgrant no. 18-188-1343, American Society of Hematology Global Research Award (A.J.B), and Research Infrastructure for Campus-based Laboratories at the University of Rijeka project, (RC.2.2.06-0001) co-funded by the European Fund for Regional Development (ERDF) and Ministry of Science, Education, and Sports of the Republic of Croatia.