Abstract
Megakaryocytes (Mks, CD41a+CD61+ cells) are the progenitor cells of platelets, and they differentiate from CD34+ hematopoietic stem cells (HSCs) and progenitor cells through complex development processes in the bone marrow (BM). Increasing the number of Mk by ex vivo induction and expansion culture for stem cells transplant may provide an approach to accelerate platelet reconstruction in patient after high-dose chemotherapy. In this study, a serum-free, stromal-free and optimal cytokine cocktail containing medium (SF-Mk medium: HIT (8 g/L HAS, 1.8 μg/ml Insulin, 50.5 μg/ml transferrin) + CC-Mk (3.0 ng/ml TPO，2.9 ng/ml IL-3，12.5 ng/ml SCF，0.5 ng/ml IL-6，1.4 ng/ml FL，1.7 ng/ml IL-9以及7.3 ng/ml GM-CSF) in Iscove’s modified Dulbecco’s medium (IMDM)) was established by 2-level factorial design and steepest ascent path methods for Mk generation from serum-free expanded CD34+ cells. After serum-free induction, the maximum expansion for the accumulated Mks increased over 4000-fold. Furthermore, SF-Mk medium was superior and comparable with Panserin 401TM, X-VIVO 10TM, X-VIVO 15TM, X-VIVO 20TM and PRO 293TM commercial serum-free medium in induction of Mks. The serum-free induced Mks were characterized by surface marker expression of CD41a and CD61, gene expression of NF-E2 and GATA-1, polyploidy distribution, and platelet activation ability. Importantly, transplantation of serum-free induced Mks could accelerate human platelet recovery in NOD/SCID mice. In conclusion, we have developed a serum-free Mk induction (SF-Mk) medium, and the combination of SF-Mk and SF-HSC media can generate a large amount of functional Mk efficiently. Our method may represent a promising source of Mk and platelets for future cell therapy.