Safety and Efficacy of Megakaryocytes Induced from Hematopoietic Stem Cells in Murine and Nonhuman Primate Models

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

Large-Scale Ex Vivo Generation of Human Red Blood Cells from Cord Blood CD34+ Cells

“…All cells were previously washed, filtered through a deleukocyting filter, and suspended in the 706-hydroxyethyl starch for xenotransfusion. For positive control I, the amount of transfused RBCs was equal to the collected amount of RBCs; for positive control II and experimental group, the amount of transfused cells was half of the collected amount of RBCs, and cells were labeled with FITC-microbeads (Lumigenex, Suzhou, China, http://www.lumigenex.com/) as described previously [19]. Labeling efficiency of FITC-microbeads was approximately 100%, as confirmed by flow cytometry.…”

“…Cells were cultured in modified medium (MM) based on Iscove's Modified Dulbecco's medium (IMDM) (Life Technologies, Shanghai, China https://www.lifetechnology.com/) added with nutrition supplements [14,15], which consisted of putrescine (100 lM; Sigma-Aldrich, shanghai, China, http://www.sigmaaldrich.com/), selenium (5 ng/ml; Sigma-Aldrich,), insulin (25 mg/ml; Sigma-Aldrich), transferrin (200 mg/ml; Sigma-Aldrich), folic acid (10 mg/ml; Sigma-Aldrich). The ex vivo expansion and differentiation protocol was comprised of four steps: step 1 (day 0-5), step 2 (day 6-12), step 3 (day 13-18), and step 4 (day [19][20][21]. For each step, different combinations and concentrations of various growth factors (Table 1) including stem cell factor (SCF), thrombopoietin (TPO), fms-related tyrosine kinase 3 ligand (FL), interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin (EPO), and fetal bovine serum (FBS) (15% vol/vol; Hyclone, Marlborough, MA, https://promo.gelifesciences.com/gl/ hyclone/) were added to MM to determine the optimal conditions for ex vivo generation of human erythroid cells.…”

Large-Scale Cultures and Bioreactors for the Production of Megakaryocytes and Platelets

“…After initially establishing a static, two stage culture systems [9], the group recently presented an augmented, up-scaled device represented by a 2 L turning bottle device [10]. This design allowed the production of ~2 × 10 10 MKs from 1 × 10 6 CD34 + cells after 13 days.…”

“…As yet discussed by Thon et al 2017, in order to produce an equivalent to one PLT apheresis unit (3 × 10 11 PLTs), the implementation of 3 × 10 9 MKs into a bioreactor of thousand-fold scale would be required [24].…”

“…In a mouse model a short time engraftment of MK progenitors (MKPs) in BM for several weeks was demonstrated which lead to a prolonged release of PLTs into the circulation [9]. However, a certain risk of MKs homing to secondary loci of thrombopoiesis such as the lung has likewise to be addressed.…”