A novel serum free system for cloning human megakaryocytic progenitors (CFU-Meg). The role of thrombopoietin and other cytokines on bone marrow and cord blood CFU-Meg growth under serum free conditions.

Human megakaryocytic progenitors (CFU-Megs) are usually cloned in plasma clot cultures. Since the medium employed to prepare plasma clot contains animal or human serum, there exists a potential risk that CFU-Megs growing in vitro could be exposed to the serum derived megakaryopoietic inhibitors. To address this issue, we aimed to establish a relatively simple "serum free" cloning model for these progenitors. Accordingly, we found that if human bone marrow or cord blood CD34+ cells are plated in the methylcellulose medium containing serum substitute, and are stimulated with recombinant thrombopoietin (TpO), they exclusively form CFU-Meg colonies. Subsequently these colonies can be easily scored with an inverted microscope based only on their morphological criteria. We found that the cloning efficiency of CFU-Megs was higher in our serum free cloning system than in the traditional plasma clot cultures. Since the model proposed in this paper is relatively simple, and moreover does not require time consuming immunostaining to identify CFU-Meg colonies, it should be widely recommended for studying in vitro human megakaryopoiesis. We also found, that under serum free conditions TpO is crucial for CFU-Meg formation. In absence of TpO, neither gp 130 activating cytokines (IL-6, IL-11, LIF, CNTF) nor the other hematopoietic growth factors or cytokines (KL, IL-3, GM-CSF, EpO) were able, when added alone, to stimulate the growth of human CFU-Meg colonies. Finally, we report also that cord blood CD34+ cells are enriched in megakaryocytic progenitors, and moreover, CFU-Megs from cord blood possess a higher proliferative capacity than CFU-Megs isolated from normal adult bone marrow.