Lisa M. Giammona1, Eleftherios T. Papoutsakis2, and William M. Miller1. (1) Chemical and Biological Engineering, Northwestern University, 2145 N. Sheridan Rd., Tech E136, Evanston, IL 60208, (2) Dept. of Chemical Engineering, Delaware Biotechnology Institute, 15 Innovation Way, Newark, DE 19711
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) more than doubles the percentage of high-ploidy (≥8N) Mks obtained from mobilized peripheral blood CD34+ cells cultured with thrombopoietin (Tpo). 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. Despite the dramatic phenotypic differences observed with NIC addition, gene expression microarray analysis reveals similar overall transcriptional patterns in primary human Mk cultures with or without NIC, which indicates that NIC does not disrupt the normal Mk transcriptional program. Therefore, 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. Although NIC increased Mk ploidy even in the absence of Tpo, the full effects of NIC on Mk polyploidization were only obtained in cultures with Tpo. Moreover, NIC increased Tpo-mediated activation of ERK and the downstream ERK target RSK1, but did not alter Tpo-mediated Akt signaling. In addition, NIC dramatically increased the levels of intracellular NAD(H) (by as much as 5-fold) in a dose-dependent manner, and more than doubled the cell-volume-specific (g/µm3/day) glucose consumption rate. 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.