Abstract
Megakaryopoiesis and platelet production are complex biological processes that require tight regulation of successive lineage commitment steps and are ultimately responsible for maintaining and renewing the pool of circulating platelets in the blood. Despite major advancements in the understanding of megakaryocytic biology, the detailed mechanisms driving megakaryocytic differentiation have yet to be elucidated. Here we show that automated image analysis algorithms applied to two-photon excited fluorescence (TPEF) images can non-invasively monitor structural and metabolic megakaryocyte behavior changes occurring during differentiation and platelet formation in vitro. Our results demonstrate that high-contrast, label-free two photon imaging holds great potential in studying the underlying physiological processes controlling the intricate process of platelet production.
Highlights
Megakaryopoiesis and platelet production is an intricate biological phenomenon, requiring a tightly regulated sequence of cellular transformations
Hematopoietic Stem Cell (HSC) differentiation into MKs requires several successive lineage commitment steps and is actively driven by biochemical and mechanical signals triggered by multiple cytokines and extracellular matrix components, among which, thrombopoietin (Tpo) and fibronectin, play protagonistic roles [3, 4]
Human MKs were differentiated from cord blood-derived hematopoietic stem cells (HSCs), according to an established protocol [15]
Summary
Megakaryopoiesis and platelet production is an intricate biological phenomenon, requiring a tightly regulated sequence of cellular transformations. Megakaryocytes (MKs) are cells of hematopoietic origin that reside primarily in the bone marrow [1] and are responsible for maintaining and renewing the pool of circulating platelets. Platelets are released from the tip of the proplatelets into the blood stream [5]. Both in vivo and in vitro, MKs in various transformational stages coexist, creating an inherently complex and heterogeneous biological system spatially and temporally. Despite major advancements in the understanding of MK biology, the exact mechanisms activated during MK differentiation that drive or interfere with differentiation progression and with platelet formation remain elusive. Elucidating the metabolic behavior of MKs dynamically during their tightly controlled maturation, could further advance our understanding of platelet generation and be exploited for new therapeutic strategies and the improvement of in vitro platelet production protocols to achieve clinical-grade standards [6, 7]
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