Nicotinamide Enhances Primary Human Megakaryocytic Differentiation from Hematopoietic Stem Cells: Phenotypic Characterization and Mechanism of ActionPublic Deposited
Megakaryocytic cells (Mks) are derived from hematopoietic stem cells (HSCs) and give rise to platelets. Ex vivo culture of HSCs under conditions that promote Mk differentiation has been proposed as a method for producing Mks and platelets for cell therapies. Mk maturation involves the development of polyploid cells via endomitosis, and the number of platelets produced increases with Mk DNA content. However, ploidy levels in cultured human Mks are much lower than those observed in vivo. We have found that adding the water-soluble vitamin nicotinamide (NIC) to mobilized peripheral blood CD34+ cells cultured with thrombopoietin (Tpo) more than doubles the percentage of high-ploidy (?8N) Mks. This was observed regardless of donor-dependent differences in Mk differentiation. Furthermore, Mks in cultures with NIC are larger, have more highly lobated nuclei, reach a maximum DNA content of 64N (vs. 16N with Tpo only), and exhibit more frequent and more elaborate cytoplasmic extensions - an indication of greater platelet producing capacity. However, NIC does not alter Mk commitment, apoptosis, or the time at which endomitosis is initiated. Despite the dramatic phenotypic differences observed with NIC addition, gene expression microarray analysis revealed similar overall transcriptional patterns in primary human Mk cultures with or without NIC, indicating that NIC does not disrupt the normal Mk transcriptional program. As a result, we investigated the hypothesis that changes in post-translational modifications, as well as cellular programs such as metabolic regulation, were largely responsible for the observed effects of NIC on Mks. NIC was found to increase Tpo-mediated ERK activation and activation of the downstream ERK target RSK1. NIC was also found to increase the levels of intracellular NAD(H) in a dose-dependent manner. This work demonstrates that NIC is useful as a supplement to increase the productivity of ex vivo Mk cultures. Moreover, NIC-treated Mks could serve as a novel model for studying Mk polyploidization where cells reach ploidy levels closer to that observed in vivo. Further elucidation of the mechanisms by which NIC increases Mk maturation could lead to a greater understanding of Mk differentiation and may lead to advances in the treatment of Mk and platelet disorders.