Related Experiment Video
Updated: Jul 30, 2025
Megakaryocyte Culture in 3D Methylcellulose-Based Hydrogel to Improve Cell Maturation and Study the Impact of Stiffness and Confinement
Published on: August 26, 2021
Matrix stiffness controls megakaryocyte adhesion, fibronectin fibrillogenesis, and proplatelet formation through
Ines Guinard1, Thao Nguyen1, Noémie Brassard-Jollive1
1UMR_S1255, INSERM, Etablissement Français du Sang-Grand Est, Fédération de Médecine Translationnelle de Strasbourg, Université de Strasbourg, Strasbourg, France.
Abstract:
Megakaryocytes (MKs) are the precursor cells of platelets, located in the bone marrow (BM). Once mature, they extend elongated projections named proplatelets through sinusoid vessels, emerging from the marrow stroma into the circulating blood. Not all signals from the microenvironment that regulate proplatelet formation are understood, particularly those from the BM biomechanics. We sought to investigate how MKs perceive and adapt to modifications of the stiffness of their environment. Although the BM is one of the softest tissue of the body, its rigidification results from excess fibronectin (FN), and other matrix protein deposition occur upon myelofibrosis. Here, we have shown that mouse MKs are able to detect the stiffness of a FN-coated substrate and adapt their morphology accordingly. Using a polydimethylsiloxane substrate with stiffness varying from physiological to pathological marrow, we found that a stiff matrix favors spreading, intracellular contractility, and FN fibrils assembly at the expense of proplatelet formation. Itgb3, but not Itgb1, is required for stiffness sensing, whereas both integrins are involved in fibrils assembly. In contrast, soft substrates promote proplatelet formation in an Itgb3-dependent manner, consistent with the ex vivo decrease in proplatelet formation and the in vivo decrease in platelet number in Itgb3-deficient mice. Our findings demonstrate the importance of environmental stiffness for MK functions with potential pathophysiological implications during pathologies that deregulate FN deposition and modulate stiffness in the marrow.
Insights
Megakaryocytes (MKs) sense and adapt to bone marrow (BM) stiffness. Environmental stiffness regulates MK function and proplatelet formation, impacting platelet production.
Area of Science:
- Hematology
- Biophysics
- Cell Biology
Background:
- Megakaryocytes (MKs) in the bone marrow (BM) produce platelets by forming proplatelets.
- BM biomechanics, specifically matrix stiffness, are understudied regulators of MK function.
- Myelofibrosis can alter BM stiffness due to increased fibronectin (FN) deposition.
Purpose of the Study:
- To investigate how MKs perceive and respond to changes in their microenvironmental stiffness.
- To elucidate the role of matrix stiffness in MK morphology, contractility, and proplatelet formation.
- To identify the integrins involved in MK mechanosensing and matrix interaction.
Main Methods:
- Utilized polydimethylsiloxane (PDMS) substrates with varying stiffness, mimicking physiological and pathological BM conditions.
- Coated substrates with fibronectin (FN) to study MK interactions with matrix proteins.
- Assessed MK morphology, intracellular contractility, FN fibril assembly, and proplatelet formation.
- Investigated the role of integrins Itgb3 and Itgb1 in stiffness sensing and matrix interactions using knockout mice.
Main Results:
- Mouse MKs detect substrate stiffness and alter morphology accordingly.
- Stiff matrices promote MK spreading, contractility, and FN fibril assembly, while inhibiting proplatelet formation.
- Soft substrates enhance proplatelet formation in an Itgb3-dependent manner.
- Itgb3 is essential for stiffness sensing, while both Itgb3 and Itgb1 are involved in FN fibril assembly.
Conclusions:
- Environmental stiffness is a critical regulator of megakaryocyte function and platelet production.
- Altered BM stiffness, particularly in myelofibrosis, can significantly impact platelet counts.
- Integrin-mediated mechanotransduction plays a key role in MK adaptation to their microenvironment.
Related Concept Videos
01:17Intracellular Signaling Affects Focal Adhesions
Some...
01:24Overview of Cell-Matrix Interactions
01:13Cell-matrix's Response to Mechanical Forces
Anchoring junctions mechanically attach a cell to the...
01:22Formation of the Platelet Plug
As the injured blood vessel contracts, endothelial cells undergo contraction, revealing collagen fibers in the basement membrane and underlying connective tissue. Furthermore, the plasma membrane of endothelial cells becomes adhesive, preparing the site for platelet adhesion. Platelets...
01:15Activation of Integrins
In "outside-in signaling," external factors in the extracellular space bind to exposed ligand binding sites on integrins. This causes the inactive protein to undergo a conformational change to become active. Integrins are often clustered on the cell membrane. Repetitive and regularly spaced ligand binding...
01:18Structure and Function of Platelets
Platelets are continually replenished, circulating in the bloodstream for 9-12 days before being removed by phagocytes, primarily in the spleen. A microliter of circulating blood contains between 150,000 and 450,000...