Cytoskeletal mechanics of proplatelet maturation and platelet release

Abstract

New steps in the process of conversion of proplatelet extensions from megakaryocytes into mature platelets are defined.

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

“…S2A), and 40% of treated cells also rapidly fragment to average sizes similar to those of large human platelets (3 to ∼4 μm). Platelets are now known to be generated by shearing of proplatelets (16), and no fragmentation was observed in untreated cells. Projection lengths up to the point of divergent fragmentation vary from 10 to ∼20 μm, which is similar to in vivo fragmented proplatelet lengths of ∼14 μm (8).…”

Platelet bioreactor-on-a-chip

“…Mounting evidence that cell-cell contacts, 22,23 ECM composition 19,20 and stiffness, 21 vascular shear stress, 24,25 pO2/pH, 51,52 soluble factor interactions, 22,53 and temperature 54 contribute to proPLT formation and PLT release have suggested that recapitulating key components of BM and blood vessel microenvironments within a three-dimensional microfluidic culture system is necessary to achieve clinically significant numbers of functional human PLTs. Our microfluidic bioreactor design expands control and resolution of the microenvironment, and has allowed us to drastically improve the time to PLT release (from 18 hours down to 2 hours) and more For personal use only.…”

“…A major limitation of twodimensional liquid cultures has been their inability to account for three-dimensional BM composition and stiffness, directionality of proPLT extension, and proximity to venous endothelium. 15,24,25 Alternatively, intravital microscopy studies have provided physiologically accurate examples of proPLT production, however, poor resolution and limited control of the microenvironment has prohibited detailed study of how the BM microenvironment contributes to PLT release. 11,15 Nakagawa et al 50 recently published 3 bioreactor designs to produce human PLTs at scale from hiPSC-and ESC-derived MKs.…”

“…Early histologic studies in both humans and mice have predicted that whole MKs, as well as MK fragments may be squeezing through 16,39 Large PLT intermediates called prePLTs were recently discovered in blood, 40 and venous infusion of mouse BM-derived MKs, fetal liver culturederived MKs, 42 and prePLTs 25 into mice produced PLTs in vivo. In the present study, $100 mm diameter primary mouse MKs were routinely observed squeezing through 2 mm gaps ( Figure 4A and supplemental Movie 3A-B) or extending large MK fragments ( Figure 4B and supplemental Movie 4), supporting a model of vascular PLT production.…”

“…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.…”