Abstract
Megakaryocytes are the precursor cells of platelets and are bona fide resident cells in the bone marrow but extremely low in numbers (~1% of total nucleated cells). Upon terminal differentiation, megakaryocytes increase their size, become polyploid and develop a demarcation membrane system. Mature megakaryocytes form proplatelets, which are cytoplasmic extensions that protrude through the endothelial cell layer of venous sinusoids within the bone marrow, entering into the blood circulation and, subsequently, releasing platelets. Despite limited in numbers, megakaryocytes have been successfully isolated from bone marrow (Tolhurst et al., 2012), adult peripheral blood (Mazur et al., 1990; Thornton et al., 1999), cord blood (Sun et al., 2004) and also from embryonic stem cells (Pick et al., 2013; Eto et al., 2002). These procedures rely on immunostaining using antibodies against megakaryocyte surface markers (i.e. CD41 or CD42b) to isolate an enriched population of megakaryocytes. Here, we describe a culture method wherein megakaryocytes can be grown and differentiated in vitro from human peripheral blood mononuclear cells (PBMCs) directly without the need of initial isolation of CD34+ cells. This method is based on a previously published culture method of human erythroid progenitor cells from PBMCs (Borg et al., 2010; Leberbauer et al., 2005). Although the purity of megakaryocytes is not 100% in this culture method, an enriched fraction of megakaryocytes can be further isolated using BSA gradient or cell-sorting techniques. In addition, our method offers the possibility to freeze the cultures after minimal expansion of yet undifferentiated megakaryocytes, which will yield equal megakaryocyte cultures after thawing when compared to fresh uninterrupted cultures. As this has been proven difficult with CD34+ sorted pluripotent cells, it allows managing samples and to perform downstream analysis when human material is not always available.
Materials and Reagents
Note: *These materials/reagents can be replaced by similar alternatives.
Equipment
Note: *Indicated equipment can be replaced by similar alternatives.
Procedure
Optional complementary techniques
Figure 3. PBMC-derived megakaryocyte culture variations. A. Fresh vs. Phase I-frozen PBMC-derived megakaryocyte cultures: Flow cytometry analysis. A cocktail of antibodies against megakaryocyte surface markers is used to distinguish differentiation stages in human megakaryocyte cultures. The profiles from both conditions (Fresh vs. Phase I-Frozen) look similar and suggest that megakaryocytes can be successfully grown with this culture method, which allows freezing of cultures during the first days of culture Phase I. B. Cell morphology of cultures derived fromCD34+ sorted cells instead of PBMCs. Cells were observed under the bright field microscope and pictures were taken. The morphology of megakaryocytes from cultured CD34+ sorted cells is similar to that from cultured total PBMCs.
Recipes
Acknowledgments
This protocol was adapted from previous culture method (Borg et al., 2010; Leberbauer et al., 2005) on human erythroid progenitor cells and was developed by V.S., P.P., and L.G. Blood samples were obtained under informed consent, after approval by our institute medical ethics committee in accordance with the 1964 Declaration of Helsinki. This work was partly supported by an I+D Excelencia 2014 grant (SAF2014-55231-P; Ministerio de Economía y Competitividad -Spain- y Fondos Feder, L.G. and P.P.) and a Ramón y Cajal Fellowship (RYC-2013-12587; Ministerio de Economía y Competitividad -Spain-, L.G.).
References
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Dear Giacomo, in principle you should keep the same conc, yes. You can either replate the cells OR just refresh them on the same plate. Replating is better in the first 3-4 days of the culture, later on its better to keep the culture with less manipulation possible, just add medium.Best.
Dear Petros,Thank you very much for your explanation.One more question:you suggested to manipulate cells as less as possible after 3/4 days. Therefore, in order to start phase 2 or phase 3, It is better to add phase 2/3 medium to old medium without pelleting cells, isn't it?Thank you. Best, Giacomo
Yes, that’s correct.