megakaryocyte-culture-3d-methylcellulose-based-hydrogel-to-improve

Megakaryocyte Culture in 3D Methylcellulose-Based Hydrogel to Improve Cell Maturation and Study the Impact of Stiffness and Confinement

The 3D environment leading to both confinement and mechanical constraints is increasingly recognized as an important determinant of cell behavior. 3D ...

Université de Strasbourg, INSERM, EFS Grand Est, BPPS UMR-S 1255, FMTS

The differentiation and maturation of megakaryocytes (MKs) occurs in a 3D environment where the cells must constantly adapt to the external physical and mechanical constraints during their development and migration to sinusoid vessels. In this chapter, we present a method for culture of mouse MKs from bone marrow hematopoietic progenitor cells in a methylcellulose 3D medium with a stiffness mimicking that of bone marrow. In addition, we describe how the MKs can be recovered to allow for analysis of their differentiation and maturation state by transmission electron microscopy, immunofluorescence or flow cytometry techniques and to evaluate their ability to form proplatelets. This approach allows (1) generation of MKs with a morphology that more closely resembles the MKs that differentiate in vivo, (2) recovery of megakaryocyte phenotypes sometimes observed in vivo but not found in classical liquid cultures, and (3) study of mechanotransduction pathways induced by the stiffness of the medium.

Three-Dimensional Culture in a Methylcellulose-Based Hydrogel to Study the Impact of Stiffness on Megakaryocyte Differentiation.

Platelets are produced by bone marrow megakaryocytes through cytoplasmic protrusions, named native proplatelets (nPPT), into blood vessels. Proplatelets also refer to protrusions observed in megakaryocyte culture (cPPT) that are morphologically different. Contrary to cPPT, the mechanisms of nPPT formation are poorly understood. We show here in living mice that nPPT elongation is in equilibrium between protrusive and retraction forces mediated by myosin-IIA. We also found, using WT and β1-tubulin-deficient mice, that microtubule behavior differs between cPPT and nPPT, being absolutely required in vitro, while less critical in vivo. Remarkably, microtubule depolymerization in myosin-deficient mice did not affect nPPT elongation. We then calculated that blood Stokes'forces may be sufficient to promote nPPT extension, independently of myosin and microtubules. Together, we propose a new mechanism for nPPT extension that might explain contradictions between severely affected cPPT production and moderate platelet count defects in some patients and animal models.

Julie Boscher

Université de Strasbourg, INSERM, EFS Grand Est, BPPS UMR-S 1255, FMTS

Megakaryocyte Culture in 3D Methylcellulose-Based Hydrogel to Improve Cell Maturation and Study the Impact of Stiffness and Confinement