Developing a co-culture system for effective megakaryo/thrombopoiesis from umbilical cord blood hematopoietic stem/progenitor cells
Javad Hatami; Pedro Z. Andrade; António Pedro Alves de Matos; Dusan Djokovic; Carla Lilaia; Frederico Castelo Ferreira; Joaquim M.S. Cabral; Cláudia L. da Silva
Cytotherapy, 17(4) (2015), 428–442
http://dx.doi.org/10.1016/j.jcyt.2014.12.010
Background aims
Platelet transfusion can be a life-saving procedure in different medical settings. Thus, there is an increasing demand for platelets, of which shelf-life is only 5 days. The efficient ex vivo biomanufacturing of platelets would allow overcoming the shortages of donated platelets.
Methods
We exploited a two-stage culture protocol aiming to study the effect of different parameters on the megakaryo/thrombopoiesis ex vivo. In the expansion stage, human umbilical cord blood (UCB)-derived CD34+-enriched cells were expanded in co-culture with human bone marrow mesenchymal stromal cells (BM-MSCs). The megakaryocytic commitment and platelet generation were studied, considering the impact of exogenous addition of thrombopoietin (TPO) in the expansion stage and a cytokine cocktail (Cyt) including TPO and interleukin-3 in the differentiation stage, with the use of different culture medium formulations, and in the presence/absence of BM-MSCs (direct versus non-direct cell-cell contact).
Results
Our results suggest that an early megakaryocytic commitment, driven by TPO addition during the expansion stage, further enhanced megakaryopoiesis. Importantly, the results suggest that co-culture with BM-MSCs under serum-free conditions combined with Cyt addition, in the differentiation stage, significantly improved the efficiency yield of megakaryo/thrombopoiesis as well as increasing %CD41, %CD42b and polyploid content; in particular, direct contact of expanded cells with BM-MSCs, in the differentiation stage, enhanced the efficiency yield of megakaryo/thrombopoiesis, despite inhibiting their maturation.
Conclusions
The present study established an in vitro model for the hematopoietic niche that combines different biological factors, namely, the presence of stromal/accessory cells and biochemical cues, which mimics the BM niche and enhances an efficient megakaryo/thrombopoiesis process ex vivo.
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