title: Developing a co-culture system for effective megakaryo/thrombopoiesis from umbilical cord blood hematopoietic stem/progenitor cells.
abstract: OBJECTIVE
  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.
authors: Department of Bioengineering and IBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
authors: Javad
authors: J
authors: Hatami
authors: Department of Bioengineering and IBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
authors: Pedro Z
authors: PZ
authors: Andrade
authors: Centro de Estudos do Ambiente e do Mar (CESAM/FCUL)-Faculdade de Ciências da Universidade de Lisboa and Centro de Investigação Interdisciplinar Egas Moniz (CiiEM), Campus Universitário, Quinta da Granja, Monte de Caparica, Caparica, Portugal.
authors: António Pedro
authors: AP
authors: Alves de Matos
authors: Department of Obstetrics, Centro Hospitalar Lisboa Ocidental E.P.E., Hospital São Francisco Xavier, Lisboa, Portugal.
authors: Dusan
authors: D
authors: Djokovic
authors: Department of Obstetrics, Centro Hospitalar Lisboa Ocidental E.P.E., Hospital São Francisco Xavier, Lisboa, Portugal.
authors: Carla
authors: C
authors: Lilaia
authors: Department of Bioengineering and IBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal. Electronic address: frederico.ferreira@ist.utl.pt.
authors: Frederico Castelo
authors: FC
authors: Ferreira
authors: Department of Bioengineering and IBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
authors: Joaquim M S
authors: JM
authors: Cabral
authors: Department of Bioengineering and IBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
authors: Cláudia L
authors: CL
authors: da Silva
mesh_terms: D018952:Antigens, CD34 / Q000378:metabolism; D001792:Blood Platelets / Q000166:cytology; D002450:Cell Communication / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques; D005312:Fetal Blood / Q000166:cytology; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D008533:Megakaryocytes / Q000166:cytology*; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D017713:Platelet Transfusion / Q000379:methods*; D034061:Thrombopoiesis / Q000502:physiology*; D013926:Thrombopoietin / Q000494:pharmacology
chemical_list: D018952:Antigens, CD34; C508596:IL3 protein, human; D007377:Interleukin-3; D013926:Thrombopoietin
keywords: cell-cell contact; hematopoietic stem/progenitor cells; megakaryocyte; platelet; serum-free medium; umbilical cord blood