title: Importance of environmental stiffness for megakaryocyte differentiation and proplatelet formation.
abstract: Megakaryocyte (MK) differentiation occurs within the bone marrow (BM), a complex 3-dimensional (3D) environment of low stiffness exerting local external constraints. To evaluate the influence of the 3D mechanical constraints that MKs may encounter in vivo, we differentiated mouse BM progenitors in methylcellulose (MC) hydrogels tuned to mimic BM stiffness. We found that MKs grown in a medium of 30- to 60-Pa stiffness more closely resembled those in the BM in terms of demarcation membrane system (DMS) morphological aspect and exhibited higher ploidy levels, as compared with MKs in liquid culture. Following resuspension in a liquid medium, MC-grown MKs displayed twice as much proplatelet formation as cells grown in liquid culture. Thus, the MC gel, by mimicking external constraints, appeared to positively influence MK differentiation. To determine whether MKs adapt to extracellular stiffness through mechanotransduction involving actomyosin-based modulation of the intracellular tension, myosin-deficient (Myh9-/-) progenitors were grown in MC gels. Absence of myosin resulted in abnormal cell deformation and strongly decreased proplatelet formation, similarly to features observed for Myh9-/-MKs differentiated in situ but not in vitro. Moreover, megakaryoblastic leukemia 1 (MKL1), a well-known actor in mechanotransduction, was found to be preferentially relocated within the nucleus of MC-differentiated MKs, whereas its inhibition prevented MC-mediated increased proplatelet formation. Altogether, these data show that a 3D medium mimicking BM stiffness contributes, through the myosin IIA and MKL1 pathways, to a more favorable in vitro environment for MK differentiation, which ultimately translates into increased proplatelet production.
authors: INSERM, Unité Mixte de Recherche en Santé 949, Strasbourg, France.|Etablissement Français du Sang, Strasbourg, France.|Faculté de Médecine, Université de Strasbourg, Strasbourg, France.|Fédération de Médecine Translationnelle, Strasbourg, France; and.
authors: Alicia
authors: A
authors: Aguilar
authors: INSERM, Unité Mixte de Recherche en Santé 949, Strasbourg, France.|Etablissement Français du Sang, Strasbourg, France.|Faculté de Médecine, Université de Strasbourg, Strasbourg, France.|Fédération de Médecine Translationnelle, Strasbourg, France; and.
authors: Fabien
authors: F
authors: Pertuy
authors: INSERM, Unité Mixte de Recherche en Santé 949, Strasbourg, France.|Etablissement Français du Sang, Strasbourg, France.|Faculté de Médecine, Université de Strasbourg, Strasbourg, France.|Fédération de Médecine Translationnelle, Strasbourg, France; and.
authors: Anita
authors: A
authors: Eckly
authors: INSERM, Unité Mixte de Recherche en Santé 949, Strasbourg, France.|Etablissement Français du Sang, Strasbourg, France.|Faculté de Médecine, Université de Strasbourg, Strasbourg, France.|Fédération de Médecine Translationnelle, Strasbourg, France; and.
authors: Catherine
authors: C
authors: Strassel
authors: Institut Charles Sadron, UPR 22, Strasbourg, France.
authors: Dominique
authors: D
authors: Collin
authors: INSERM, Unité Mixte de Recherche en Santé 949, Strasbourg, France.|Etablissement Français du Sang, Strasbourg, France.|Faculté de Médecine, Université de Strasbourg, Strasbourg, France.|Fédération de Médecine Translationnelle, Strasbourg, France; and.
authors: Christian
authors: C
authors: Gachet
authors: INSERM, Unité Mixte de Recherche en Santé 949, Strasbourg, France.|Etablissement Français du Sang, Strasbourg, France.|Faculté de Médecine, Université de Strasbourg, Strasbourg, France.|Fédération de Médecine Translationnelle, Strasbourg, France; and.
authors: François
authors: F
authors: Lanza
authors: INSERM, Unité Mixte de Recherche en Santé 949, Strasbourg, France.|Etablissement Français du Sang, Strasbourg, France.|Faculté de Médecine, Université de Strasbourg, Strasbourg, France.|Fédération de Médecine Translationnelle, Strasbourg, France; and.
authors: Catherine
authors: 0000-0002-8597-9929
authors: C
authors: Léon
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism*; D001853:Bone Marrow / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D020100:Hydrogels / Q000737:chemistry; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism*; D008747:Methylcellulose / Q000737:chemistry; D051379:Mice; D018345:Mice, Knockout; D018995:Myosin Heavy Chains; D024507:Nonmuscle Myosin Type IIA / Q000235:genetics / Q000378:metabolism; D013500:Surface Tension; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism
chemical_list: D020100:Hydrogels; C511173:Mrtfa protein, mouse; C525088:Myh9 protein, mouse; D015534:Trans-Activators; D008747:Methylcellulose; D024507:Nonmuscle Myosin Type IIA; D018995:Myosin Heavy Chains