Development of a liquid culture system for megakaryocyte terminal differentiation: fibrinogen promotes megakaryocytopoiesis but not thrombopoiesis

Abstract

Summary. Megakaryocyte differentiation is composed of three distinct stages: formation of erythromegakaryocytic progenitor cells, maturation of megakaryocytes and production of platelets. We have developed a liquid culture system for megakaryocyte terminal differentiation from haematopoietic stem cells into proplatelets. In this system, CD34 + cells isolated from human cord blood, differentiated to CD41 + cells, were classified either as propidium iodide (PI) + cells (large) or PI -cells (small) by fluorescence-activated cell sorting analysis on the late-stage CD41 + cells. Transmission electron microscopy showed that the cultured small cells were morphologically identical to platelets isolated from normal peripheral blood. Moreover, the number of differentiated cells that were CD42b-positive attained an approximately 60-fold expansion over that of the primary CD34 + cells in this culture system. Furthermore, gene expression of megakaryocytopoietic transcriptional factors, GATA-1 and NF-E2, and several megakaryocytic markers such as glycoprotein (GP)IIb and thromboxane synthase was observed in the individual differentiation stage. Treatment with fibrinogen, a ligand of GPIIb/IIIa, increased the number of CD41 + /PI + cells, but treatment in the late stage suppressed CD41 + /PI -cell formation, suggesting that fibrinogen promotes megakaryocytopoiesis, but not thrombopoiesis. We conclude that this liquid culture system using human CD34 + cells may be used to mimic the physiological development from haematopoietic stem cells into megakaryocytes, as well as promote subsequent thrombopoiesis.

Autoantibody against integrin αvβ3 contributes to thrombocytopenia by blocking the migration and adhesion of megakaryocytes

“…These findings further confirm the impact of the anti-a v b 3 antibody on the interaction between MKs and ECs. In fact, the adhesion of MKs to Fg, the main component of the EC matrix, is conducive to proplatelet formation [40,43,44]. In the present study, we also found that antia v b 3 antibody pretreatment could significantly inhibit the proplatelet formation in MKs cultured on Fg-coated plates, which was consistent with the result of a previous study showing that the anti-a v b 3 antibody could markedly decrease proplatelet formation in vitro [45].…”

Regulation of Down Syndrome Critical Region 1 expression by Nuclear Factor of Activated T cells in megakaryocytes

“…In general, experimental research in megakaryocytes is complicated by the fact that this rare population of bone marrow cells is difficult to access directly, but has been facilitated since identification of the megakaryocytic growth factor TPO enabled the generation of megakaryocytes from their CD34 + precursor cells ex vivo (Bartley et al , 1994). Meanwhile, this system of ‘culture‐derived megakaryocytes’ has been validated by several groups and successfully employed to study megakaryocytic function at comparably large numbers of cells (Choi et al , 1995; den Dekker et al , 2001; Majka et al , 2001; Mattia et al , 2002; Sato et al , 2003; Bonci et al , 2004; Ungerer et al , 2004). Notably, all findings in our study are based on experiments with these primary culture‐derived megakaryocytes, which represent a better approximation of megakaryocyte physiology than cell line models.…”

Homeodomain proteins MEIS1 and PBXs regulate the lineage-specific transcription of the platelet factor 4 gene

“…Isolation of CD34 ϩ progenitor cells from cord blood and subsequent cell culture for megakaryocytopoiesis was performed according to the method of Sato et al 33 Umbilical cord blood samples from healthy full-term newborn infants were obtained from Tokyo Metropolitan Bokutou Hospital, after informed consents of the mothers. Cord blood samples were diluted with phosphate-buffered saline (PBS) and then centrifuged on Ficoll-Paque (d ϭ 1.077 g/mL) (Pharmacia Biotech).…”