leukodepletion-filters-derived-cd34-cells-as-cell-source-to-study

Leukodepletion Filters-Derived CD34+ Cells As a Cell Source to Study Megakaryocyte Differentiation and Platelet Formation

The in vitro expansion and differentiation of human hematopoietic progenitors into megakaryocytes capable of elongating proplatelets and releasing ...

Université de Strasbourg, INSERM, EFS Grand Est

Platelets are produced upon profound reorganization of mature megakaryocytes (MK) leading to proplatelet elongation and release into the blood stream, a process termed thrombopoiesis. This highly dynamic process requires microtubules (MT) reorganization by mechanisms that are still incompletely understood. Adenomatous polyposis coli (APC) is a microtubule plus-end tracking protein involved in the regulation of MT in a number of cell systems and its inactivation has been reported to alter hematopoiesis. The aim of our study was to investigate the role of APC in megakaryopoiesis and the final steps of platelet formation. Down-regulation of APC in cultured human MK by RNA interference increased endomitosis and the proportion of cells able to extend proplatelets (68.8% (shAPC1) and 52.5% (shAPC2) vs 28.1% in the control). Similarly an increased ploidy and amplification of the proplatelet network were observed in MK differentiated from Lin- cells of mice with APC-deficiency in the MK lineage. In accordance, these mice exhibited increased platelet counts when compared to wild type mice (1,323 ± 111 vs 919 ± 52 platelets/µL; n = 12 p 0.0033**). Their platelets had a normal size, ultrastructure and number of microtubules coils and their main functions were also preserved. Loss of APC resulted in lower levels of acetylated tubulin and decreased activation of the Wnt signaling pathway. Thus, APC appears as an important regulator of proplatelet formation and overall thrombopoiesis.

Microtubule plus-end tracking Adenopolyposis Coli negatively regulates proplatelet formation.

The in vitro production of blood platelets for transfusion purposes is an important goal in the context of a sustained demand for controlled products free of infectious, immune and inflammatory risks. The aim of this study was to characterize human platelets derived from CD34 progenitors and to evaluate their hemostatic properties. These cultured platelets exhibited a typical discoid morphology despite an enlarged size and expressed normal levels of the major surface glycoproteins. They aggregated in response to ADP and a thrombin receptor agonist peptide (TRAP). After infusion into NSG mice, cultured and native platelets circulated with a similar 24 h half-life. Notably, the level of circulating cultured platelets remained constant during the first two hours following infusion. During this period of time their size decreased to reach normal values, probably due to their remodeling in the pulmonary circulation, as evidenced by the presence of numerous twisted platelet elements in the lungs. Finally, cultured platelets were capable of limiting blood loss in a bleeding assay performed in thrombocytopenic mice. In conclusion, we show here that cultured platelets derived from human CD34 cells display the properties required for use in transfusion, opening the way to clinical trials.

Catherine Strassel

BPPS UMR-S 1255,
FMTS,
BPPS UMR-S 1255, FMTS

Université de Strasbourg, INSERM, EFS Grand Est

Leukodepletion Filters-Derived CD34+ Cells As a Cell Source to Study Megakaryocyte Differentiation and Platelet Formation

Catherine Strassel

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Université de Strasbourg, INSERM, EFS Grand Est

Leukodepletion Filters-Derived CD34+ Cells As a Cell Source to Study Megakaryocyte Differentiation and Platelet Formation

BPPS UMR-S 1255,
FMTS,
BPPS UMR-S 1255, FMTS