Label free monitoring of megakaryocytic development and proplatelet formation in vitro

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. Münger, and I. Georgakoudi, "Endogenous two-photon fluorescence imaging elucidates metabolic changes related to enhanced glycolysis and glutamine consumption in precancerous epithelial tissues," Cancer Res.

High ploidy large cytoplasmic megakaryocytes are hematopoietic stem cells regulators and essential for platelet production

“…In the current study, no significant difference in the mitochondrial networks of 32N LCM and 16N LCM was evident, but both populations had significantly larger mitochondrial networks than 32N SCM, 16N SCM or HSC, despite the mitochondria being fragmented. In addition, previous analyses of cell lines and cultured MK suggests MK are metabolically active, with mitochondria, as an intracellular source of energy and reactive oxygen species, being important for MK maturation, including the processes of polyploidation and proplatelet formation 47,74,75 . The current data suggest that fragmentation is important for packaging mitochondria into platelets.…”

Cytoskeletal-based mechanisms differently regulate <i>in vivo</i> and <i>in vitro</i> proplatelet formation

“…[7][8][9][10][11][12][13][14] The same denomination was also given to the cytoplasmic MK extensions in culture or in bone marrow explants. 8,[15][16][17][18][19] Yet, the morphology of PPT observed in vitro strongly differs from that observed in situ/in vivo. Early in vitro observations of marrow explants 8,15,16,20 and later of progenitor-differentiated MK in culture 17,19,21 similarly recorded PPT presenting branched thin shafts (1-4 mm in diameter) leading to an entanglement of PPT surrounding the MK body.…”

“…Early in vitro observations of marrow explants 8,15,16,20 and later of progenitor-differentiated MK in culture 17,19,21 similarly recorded PPT presenting branched thin shafts (1-4 mm in diameter) leading to an entanglement of PPT surrounding the MK body. [17][18][19] These morphological differences between these two types of MK extensions raise the possibility that the mechanisms at stake could as well differ between in vitro cultured PPT (hereafter referred as cPPT) and in vivo generated native PPTs (referred as nPPT).…”

A novel method for automated assessment of megakaryocyte differentiation and proplatelet formation

“…This may explain the variation in the percentage of proplatelet-forming MKs found in different studies (7-21) (Supplemental Table 1). There have been previous attempts to classify maturing MKs into different stages, and proplatelet-forming MKs have been dissected into two developmental stages (7,(27)(28)(29). Our morphological evaluation revealed five major subtypes, which can be described as follows:…”