Blood donor‐derived buffy coat to produce platelets in vitro

Abstract

Background and objectives Platelet transfusion is a standard medical therapy used to treat several bleeding disorders. However, a critical drawback is the dependency on donor-derived platelets, which leads to concerns like insufficient availability and immunological complications. In vitro platelet production from hematopoietic progenitor cells (CD34) may represent a reasonable solution.Materials and methods CD34+ cells were isolated from either buffy coat or peripheral blood and compared in terms of platelet production in vitro. The number and the quality of magnetically isolated CD34+ cells and their capability to differentiate into mature megakaryocytes were investigated using flow cytometry. Additionally, the functionality of megakaryocytes in term of in vitro platelet production was tested.Results Similar purity and quantity of CD34+ cells was found after their isolation from both cell sources. In contrast, after 6 days of culture, enhanced number of CD34+ cells isolated from buffy coat compared with peripheral blood was observed (5Á3 x 106 vs. 3Á0 x 106, respectively). Interestingly, despite a comparable nuclear maturation phenotype, the yield of platelets released from buffy coatderived megakaryocytes was significantly higher than from peripheral blood cells (platelet yield pro MK: 7Á2 vs. 2Á7, respectively). Importantly, platelets produced from buffy coat-derived cells could be activated by agonists.Conclusion Haematopoietic progenitor cells isolated from buffy coat have increased yield of platelets released from mature megakaryocytes and enhanced in vitro functionality, compared with peripheral blood-derived cells. Our study, suggests that buffy coat, obtained during blood donation processing, might be a promising source of megakaryocytes for in vitro platelet production.

Autoantibody Mediated Desialylation Impairs Human Thrombopoiesis and Platelet Life Span

“…Megakaryocytes were produced from CD34 + cells as previously described with minor modifications. 22,23 In brief, CD34 + cells were magnetically isolated and cultured for differentiation into megakaryocytes in cell medium supplemented with 50 ng/mL of thrombopoietin for 14 days. The megakaryocytes generated in vitro were identified and characterized using a flow cytometry gating strategy based on DNA content, expression of CD41 and CD42a as well as viable markers, as previously described.…”

“…The megakaryocytes generated in vitro were identified and characterized using a flow cytometry gating strategy based on DNA content, expression of CD41 and CD42a as well as viable markers, as previously described. 23,24 In order to investigate the impact of AAb-induced desialylation on thrombopoiesis, megakaryocytes were incubated with IgG from ITP patients or healthy donors and proplatelet generation as well as platelet release were assessed by microscopy and flow cytometry, respectively, as previously described with minor modifications. 25 Furthermore, the ability of megakaryocytes to adhere to human serum albumin, fibrinogen, VWF or collagen (Collagen-Horm, Takeda, Linz, Austria) was verified and quantified after 2 hours (h) of incubation with IgG fractions from ITP patients or healthy donors, taking images from seven different microscope fields (x40, Olympus IX73, Olympus GmbH, Germany).…”

Generation and manipulation of human iPSC-derived platelets

“…HSCs, the origin of all adult-type hematopoietic cells, mostly reside in the bone marrow, but they can be isolated from cord blood without invasiveness or from peripheral blood under specific conditions such as mobilized conditions that reduce the invasiveness compared with the bone marrow (Table 2). Significantly owing to the discovery of the key cytokine TPO [55][56][57][58], HSCs can be differentiated into megakaryocytes in vitro [59][60][61]. However, the number of obtainable HSCs is small (10 6 order), and their complete expansion has not been achieved, thus they cannot provide a sufficient number of megakaryocytes for clinical application [62,63].…”

Platelet storage and functional integrity

“…Furthermore, PAS provide a buffering capacity and allow few allergic reactions or transfusion-associated complications [3]. Very promising efforts have also been made to exploit the possibility of using buffy coats as a source of hematopoietic progenitor cells for platelet production [4].…”