Abstract
Because of a lack of platelet supply and a U.S. Food and Drug Administration‐approved platelet growth factor, megakaryocytes have emerged as an effective substitute for alleviating thrombocytopenia. Here, we report the development of an efficient two‐stage culture system that is free of stroma, animal components, and genetic manipulations for the production of functional megakaryocytes from hematopoietic stem cells. Safety and functional studies were performed in murine and nonhuman primate models. One human cryopreserved cord blood CD34+ cell could be induced ex vivo to produce up to 1.0 × 104 megakaryocytes that included CD41a+ and CD42b+ cells at 82.4% ± 6.1% and 73.3% ± 8.5% (mean ± SD), respectively, yielding approximately 650‐fold higher cell numbers than reported previously. Induced human megakaryocytic cells were capable of engrafting and producing functional platelets in the murine xenotransplantation model. In the nonhuman primate model, transplantation of primate megakaryocytic progenitors increased platelet count nadir and enhanced hemostatic function with no adverse effects. In addition, primate platelets were released in vivo as early as 3 hours after transplantation with autologous or allogeneic mature megakaryocytes and lasted for more than 48 hours. These results strongly suggest that large‐scale induction of functional megakaryocytic cells is applicable for treating thrombocytopenic blood diseases in the clinic. Stem Cells Translational Medicine 2017;6:897–909
Highlights
Platelets play a crucial role in physiological hemostasis, and reduced platelet numbers lead to coagulation defects and uncontrollable bleeding [1,2,3]
During the first 3 days, cells expanded at a slow pace, especially in the cryopreserved human cord blood (hCB) group, whereas from day 3 to day 6, the cells cultured with CC1 showed a sharp increase in total and CD34+ cells in both groups (Fig. 1A, 1B)
On day 6, CD34+ cells in fresh and cryopreserved hCB groups showed a 48-fold and a 43-fold increase, respectively (Fig. 1D). These results indicated that CC1 could promote the expansion of both fresh and cryopreserved hCB CD34+ cells
Summary
Platelets play a crucial role in physiological hemostasis, and reduced platelet numbers lead to coagulation defects and uncontrollable bleeding [1,2,3]. Severe and prolonged thrombocytopenia frequently occurs in patients receiving high-dose chemotherapy or in those who undergo hematopoietic stem cell (HSC) transplantation [4, 5]. The delay in platelet recovery after chemotherapy or autologous/allogeneic HSC transplantation may be partly associated with an insufficiency of megakaryocyte (MK)-committed cells [7]. One strategy to accelerate platelet recovery is to implant a sufficient number of MKcommitted repopulating cells until hematopoietic reconstitution occurs in the patient [8]. It has been reported that a higher proportion of megakaryocytic progenitors (MKPs) in transplanted grafts positively affects the platelet recovery [9, 10]. A sufficient number of ex vivo expanded MKs is difficult to obtain for clinical applications. Some research groups have obtained immortalized MKP cell lines from induced pluripotent stem (iPS)
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