A Gold Standard Protocol for Human Megakaryocyte Culture Based on the Analysis of 1,500 Umbilical Cord Blood SamplesFunding This paper was supported by Cariplo Foundation (2013–0717 and 2017–0920) and ‘Progetti Di Ricerca Di Rilevante Interesse Nazionale’ (PRIN 2017Z5LR5Z). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Three decades after the ground breaking proposal of using human umbilical cord blood (UCB) as a source of transplantable hematopoietic stem cells (HSCs), over 40,000 transplants have been performed worldwide. The hematopoietic reconstitution capacity of UCB resides in the high concentration of CD34+ cells, enriched for hematopoietic stem and progenitors cells. 
Since 1993, public UCB banks have been established worldwide. In Italy, UCB is stored, free of charge, when (1) it is altruistically donated for HSC transplantation and (2) it is for a family affected with, or at risk of, a disease that is treatable with transplantation.
UCBs that do not meet the requirements for banking based on the international standards became invaluable sources for stem cell research. The study of human hematopoiesis is one of the major applications as this human model can overcome drawbacks related (1) to the use of animals, which can be poor predictors of human physiology, (2) to the high cost and expertise necessary for generating the embryonic and induced pluripotent cell lines,   or (3) to the limited supply of adult HSCs, which usually become available when discarded after clinical procedures. 
Different protocols have been established for in vitro megakaryocyte (Mk) differentiation from CD34+ cells. All entail the usage of various concentrations of recombinant human thrombopoietin (TPO) in combination with a variety of hematopoietic cytokines but with contrasting results in terms of Mk and proplatelet phenotypes.   
We report a retrospective analysis of our 15-year experience in UCB processing, with a focus on UCB features and experimental procedures that are basic for a reproducible culture of functional Mks without the need for serum supplementation or coculture with feeder cells.
C.A.D.B. designed research studies, conducted experiments, acquired data, analyzed data, and wrote the manuscript. P.M.S. conducted experiments, acquired data, analyzed data, and wrote the manuscript. C.P.M. analyzed data and wrote the manuscript. C.P. and C.D.F. provided cord blood samples, analyzed data, and edited the manuscript. A.B. designed and supervised the research project, acquired data, analyzed data and wrote the manuscript.
Eingereicht: 18. August 2020
Angenommen: 08. September 2020
07. November 2020 (online)
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