Myosin-II inhibition and soft 2D matrix maximize multinucleation and cellular projections typical of platelet-producing megakaryocytes

Shin, Jae Won; Swift, Joe; Spinler, Kyle R.; Discher, Dennis E.

doi:10.1073/pnas.1017474108

Cited by 73 publications

(94 citation statements)

References 35 publications

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“…Collectively, these results demonstrated that MYH10 was involved in the switch from mitosis to endomitosis. This result is in agreement with previous results that have shown that MYH10 is involved in cytokinesis and that its knockdown in cell lines leads to multinucleated polyploid cells 15,20,21 .…”

Section: Myh10 Is Involved In the Switch From Mitosis To Endomitosissupporting

confidence: 94%

“…However, MYH10 silencing does not explain the entire polyploidization process. MYH10 silencing has a central role at the 2N-4N transition in MK by inducing failure of cytokinesis as also observed in MYH10-ablated cardiac myocytes 21 or COS cells 15,20 . However, MKs are not subsequently blocked in 4N.…”

Section: Discussionmentioning

confidence: 59%

“…1c). Blebbistatin, a small molecule inhibitor of myosin 13,14 , has been recently shown to increase MK ploidy 15 . Addition of 50 µM blebbistatin (an enantiomer mixture) 16 in culture led to an increase in MK mean ploidy level that was similar between myh9 f/f(PF4 Cre + ) and control mice (from 10.40N to 14.72N, P = 0.01, t-test, for myh9 f/f(PF4 Cre + ) mice, and from 10.00N to 13.40N, P = 0.01, t-test, for control mice, n = 3, ploidy measured in MK≥8N; Fig.…”

Section: Myh9 Does Not Have An Important Role In Mk Endomitosismentioning

confidence: 99%

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RUNX1-induced silencing of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization

et al. 2012

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megakaryocytes are unique mammalian cells that undergo polyploidization (endomitosis) during differentiation, leading to an increase in cell size and protein production that precedes platelet production. Recent evidence demonstrates that endomitosis is a consequence of a late failure in cytokinesis associated with a contractile ring defect. Here we show that the non-muscle myosin IIB heavy chain (mYH10) is expressed in immature megakaryocytes and specifically localizes in the contractile ring. mYH10 downmodulation by short hairpin RnA increases polyploidization by inhibiting the return of 4n cells to 2n, but other regulators, such as of the G1/s transition, might regulate further polyploidization of the 4n cells. Conversely, re-expression of mYH10 in the megakaryocytes prevents polyploidization and the transition of 2n to 4n cells. During polyploidization, mYH10 expression is repressed by the major megakaryocyte transcription factor RunX1. Thus, RunX1-mediated silencing of mYH10 is required for the switch from mitosis to endomitosis, linking polyploidization with megakaryocyte differentiation.

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Section: Myh10 Is Involved In the Switch From Mitosis To Endomitosissupporting

confidence: 94%

Section: Discussionmentioning

confidence: 59%

Section: Myh9 Does Not Have An Important Role In Mk Endomitosismentioning

confidence: 99%

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RUNX1-induced silencing of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization

et al. 2012

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“…Additional related studies have emphasized the critical role of hydrogel stiffness in skeletal muscle stem cell self-renewal 12 , neural stem cell behavior 13 and megakaryocyte poiesis 14 . 3D mechanical properties are similarly important in controlling adult stem cell fate in alginate hydrogels incorporating adhesive ligands 4 .…”

Section: Static Hydrogels That Mimic Biophysical Cuesmentioning

confidence: 99%

Moving from static to dynamic complexity in hydrogel design

2012

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Hydrogels are water-swollen polymer networks that have found a range of applications from biological scaffolds to contact lenses. Historically, their design has consisted primarily of static systems and those that exhibit simple degradation. However, advances in polymer synthesis and processing have led to a new generation of dynamic systems that are capable of responding to artificial triggers and biological signals with spatial precision. These systems will open up new possibilities for the use of hydrogels as model biological structures and in tissue regeneration.H ydrogels are water-swollen polymer networks that have been used for many decades, with applications as varied as contact lenses and super-absorbant materials. As the field of biomedical engineering has developed, hydrogels have become a prime candidate for application as molecule delivery vehicles and as carriers for cells in tissue engineering, owing to their ability to mimic many aspects of the native cellular environment (for example, high water content, mechanical properties that match soft tissues). Traditional hydrogels, formed through the covalent and non-covalent crosslinking of polymer chains, were regarded as relatively inert materials, providing a simple biomimetic three-dimensional (3D) environment, either for tissue production by local resident cells or for positioning of cells delivered in vivo. However, the simplicity of these materials may have in fact hindered their application, restricting cellular interactions with the environment and preventing uniform extracellular matrix (ECM) production and proper tissue development. In addition, these materials were limited to modelling static environments and lacked the spatiotemporal dynamic properties relevant for complex tissue processes. Fortunately, during the last decade the concepts of hydrogel design and cellular interaction have evolved, shedding light on how they may control cell behaviour, particularly for tissue engineering applications.Hydrogels with a range of mechanical properties, and capable of incorporating a wide range of biologically relevant molecules, from individual functional groups to multidomain proteins, are currently in development 1 . In addition, hydrogels are being designed with spatial heterogeneity, to either replicate properties in native tissue structures or to produce constructs with distinct regionally specific cell behavior 2 . As a result, studies to date have clearly demonstrated the possibility of creating well-defined microenvironments with control over the 3D presentation of signals to cells 3 . However, recently there has been a focus on the concept of hydrogels that exhibit dynamic complexity. These materials should evolve with time and in response to

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“…This results in yields of 10 122 PLTs per CD34 1 cord blood-derived or embryonic stem cell-derived MK, 18 which are themselves of limited availability, constituting a significant bottleneck in the ex vivo production of a PLT transfusion unit. Although second-generation cell culture approaches have provided further insight into the physiological drivers of PLT release, they have been unable to recreate the entire BM microenvironment, exhibiting limited individual control of extracellular matrix (ECM) composition, 19,20 BM stiffness, 21 endothelial cell contacts, 22,23 and vascular shear stresses, 24,25 and have been unsuccessful in synchronizing proPLT production, resulting in nonuniform PLT release over a period of 6 to 8 days. 26 Moreover, the inability to reproduce the BM microenvironment ex vivo, and resolve physiological proPLT extension and release by high-resolution live-cell microscopy, has significantly hampered efforts to study the cytoskeletal and signaling mechanics of PLT production.…”

Section: Introductionmentioning

confidence: 99%

Platelet bioreactor-on-a-chip

Thon

Mažutis

et al. 2014

Blood

185

210

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• We have developed a biomimetic microfluidic platelet bioreactor that recapitulates bone marrow and blood vessel microenvironments.• Application of shear stress in this bioreactor triggers physiological proplatelet production, and platelet release.Platelet transfusions total >2.17 million apheresis-equivalent units per year in the United States and are derived entirely from human donors, despite clinically significant immunogenicity, associated risk of sepsis, and inventory shortages due to high demand and 5-day shelf life. To take advantage of known physiological drivers of thrombopoiesis, we have developed a microfluidic human platelet bioreactor that recapitulates bone marrow stiffness, extracellular matrix composition, micro-channel size, hemodynamic vascular shear stress, and endothelial cell contacts, and it supports high-resolution live-cell microscopy and quantification of platelet production. Physiological shear stresses triggered proplatelet initiation, reproduced ex vivo bone marrow proplatelet production, and generated functional platelets. Modeling human bone marrow composition and hemodynamics in vitro obviates risks associated with platelet procurement and storage to help meet growing transfusion needs. (Blood. 2014;124(12):1857-1867) IntroductionAlthough platelets (PLTs) play critical roles in hemostasis, 1 angiogenesis, 2 and innate immunity, 3 PLT production remains poorly understood. Consequently, PLT units are derived entirely from human donors, despite serious clinical concerns owing to their immunogenicity and associated risk of sepsis. 4 More than 2.17 million apheresisequivalent PLT units are transfused yearly in the United States 5,6 at a cost of .$1 billion per year. Although demand for PLT transfusions has increased markedly in the past decade, a near-static pool of donors and a 5-day PLT unit shelf life resulting from bacterial contamination 7 and storage-related PLT deterioration, 8 have resulted in significant PLT shortages. 9 Furthermore, artificial platelet substitutes have failed to replace physiological platelet products. 10 An efficient, donorindependent PLT bioreactor capable of generating clinically significant numbers of functional human PLTs is necessary to obviate risks associated with PLT procurement and storage, and help meet growing transfusion needs. In vivo, megakaryocytes (MKs) PLT progenitors sit outside blood vessels in the bone marrow (BM) and extend long, branching cellular structures designated proPLTs into the circulation from which PLTs are released. 11-15 Nearly 100% of human adult MKs must produce ;10 3 PLTs each to account for circulating PLT counts. 16 Although functional human PLTs were first grown in vitro in 1995, 17 to date only ;10% of human MKs initiate proPLT production in culture. This results in yields of 10 122 PLTs per CD34 1 cord blood-derived or embryonic stem cell-derived MK, 18 which are themselves of limited availability, constituting a significant bottleneck in the ex vivo production of a PLT transfusion unit. Although second-generation c...

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Myosin-II inhibition and soft 2D matrix maximize multinucleation and cellular projections typical of platelet-producing megakaryocytes

Cited by 73 publications

References 35 publications

RUNX1-induced silencing of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization

RUNX1-induced silencing of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization

Moving from static to dynamic complexity in hydrogel design

Platelet bioreactor-on-a-chip

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