Platelet generation in vivo and in vitro.

Platelets play crucial roles in hemostasis, thrombosis, and immunity, but our understanding of their complex biogenesis (thrombopoiesis) is currently incomplete. Deeper insight into the mechanisms of platelet biogenesis inside and outside the body is fundamental for managing hematological disorders as well as development of novel cell-based therapies. Here we address current understanding of in vivo thrombopoiesis, including mechanisms of platelet generation from megakaryocytes (proplatelet formation, cytoplasmic fragmentation and membrane budding) and their physiological location. Progress has been made replicating these processes in vitro for potential therapeutic application, notably in platelet transfusion and bioengineering of platelets for novel targeted therapies. Current platelet-generating systems and their limitations, particularly yield, scalability, and functionality, are discussed. Finally, we highlight current controversies and challenges in the field that need to be addressed to achieve full understanding of these processes, in vivo and in vitro.

Extracellular matrix stiffness controls cardiac fibroblast proliferation via the nuclear factor-Y (NF-Y) transcription factor.

The proliferative expansion of cardiac fibroblasts (CF) contributes towards cardiac fibrosis, which results in myocardial stiffening, cardiac dysfunction, and heart failure. CF sense and respond to increased stiffness of their local extracellular matrix, modulating their phenotype towards increased collagen synthesis and higher proliferation, leading potentially to a vicious circle of positive feedback. Here we describe a novel mechanism that mediates increased CF proliferation in response to a pathologically stiff Exteracellular matrix (ECM). The mechanism we describe is independent of the well-characterised mechano-sensitive transcript factors, YAP-TEAD and MKL1-SRF, which our data indicate are only responsible for part of the genes induced by stiffened ECM. Instead, our data identify Nuclear Factor-Y (NF-Y) as a novel mechanosensitive transcription factor, which mediates enhanced CF proliferation in response to a stiff ECM. We show that levels of NF-YA protein, the major regulatory subunit of NF-Y, and NF-Y transcriptional activity, are increased by a stiff ECM. Indeed, NF-Y activity drives the expression of multiple cell-cycle genes. Furthermore, NF-YA protein levels are dependent on FAK signalling suggesting a mechanistic link to ECM composition. Consistent with its role as a mechano-sensor, inhibition of NF-Y using siRNA or dominant negative mutant blocks CF proliferation on plastic in vitro, which models a stiff ECM, whereas ectopic expression of NF-YA increases the proliferation of cells interacting under conditions that model a physiologically soft ECM. In summary, our data demonstrate that NF-Y is a biomechanically sensitive transcription factor that promotes CF proliferation in a model of pathologically stiffened ECM.

Highly efficient platelet generation in lung vasculature reproduced by microfluidics.

Platelets, small hemostatic blood cells, are derived from megakaryocytes. Both bone marrow and lung are principal sites of thrombopoiesis although underlying mechanisms remain unclear. Outside the body, however, our ability to generate large number of functional platelets is poor. Here we show that perfusion of megakaryocytes ex vivo through the mouse lung vasculature generates substantial platelet numbers, up to 3000 per megakaryocyte. Despite their large size, megakaryocytes are able repeatedly to passage through the lung vasculature, leading to enucleation and subsequent platelet generation intravascularly. Using ex vivo lung and an in vitro microfluidic chamber we determine how oxygenation, ventilation, healthy pulmonary endothelium and the microvascular structure support thrombopoiesis. We also show a critical role for the actin regulator Tropomyosin 4 in the final steps of platelet formation in lung vasculature. This work reveals the mechanisms of thrombopoiesis in lung vasculature and informs approaches to large-scale generation of platelets.