Towards Ex Vivo Platelet Production: Mechanistic Studies on Megakaryocyte Lineage Commitment and Maturation, and New Tools for Studying the Emerging Importance of Shear Stress on Platelet Release

Ex vivo production of blood cells for transfusion has been a long-standing goal of both the biotechnology and hematology communities. Our lab has specifically focused on ex vivo production of platelets, as donated platelets require room temperature shortage, resulting in a mere 4-5 day shelf-life.;In the body, large polyploid cells called megakaryocytes (MKs) are the source of platelets. Mature MKs associate with blood vessels within the bone marrow and there shed new platelets. Although significant progress has been made on generating large numbers of culture-derived MKs from hematopoietic stem and progenitor cells (HSPCs), there are still several key inefficiencies in the process, including i) low levels of commitment to the MK lineage, ii) deficient MK maturation in culture, and iii) inefficient generation and release of proplatelets (the precursor to platelets) from mature MKs.;In this work, we have taken a mechanistic approach to better understand the aforementioned inefficiencies. Along the way, we have applied and developed new tools to the study of ex vivo MK culture and platelet release. In particular, we studied the dynamic activities of key transcription factors (TFs) during commitment to the MK lineage using a novel assay developed in Professor Lonnie Shea's lab. We initially applied this assay to K562 cells (a cell line that is bipotent for the erythroid (E) and MK lineages) and Ms. Jia Wu (Bagheri lab) subsequently used our data to construct a network for functional interactions between TFs. From this network, we could identify numerous known and novel TF-TF interactions. Additionally, the crucial TF GATA-1 was found to be a network hub for both E and MK differentiation. We also performed preliminary experiments showing that the activity assay can be adapted to the study of primary cell differentiation. We expect that understanding TF activities and interactions in primary MK differentiation will offer us new insights useful for developing culture conditions to optimize MK commitment.;We also examined the benefits of nicotinamide (Vitamin B3) on the maturation of culture-derived MKs, starting with the hypothesis that this was due to inhibition of the deacetylase enzymes sirtuin 1/2 (SIRT1/2). This was found to be true in part: SIRT1 did indeed oppose MK polyploidization, but we are unable to make a firm conclusion about the role (if any) of SIRT2. In addition, we identified a SIRT1-independent mechanism by which nicotinamide enhances MK ploidy. Last, we also made the unexpected observation that SIRT1 is a crucial regulator of differentiation, phenotype and survival for K562 cells. We note that this has potential implications for researchers studying bcr-abl+ myeloid leukemias.;Finally, we sought to address the most obvious deficiency in ex vivo MK cultures: the lack of proplatelet generation and release. Based on previous work by Dr. A.C. Schlinker, we hypothesized that culturing MKs on an ultra-low attachment (ULA) surface may prime them for proplatelet production prior to transferring them to a regular tissue-culture (TC) surface. Culture on ULA surfaces prior to transfer to TC surfaces did indeed result in a rapid burst in proplatelet formation (PPF), suggesting the possible utility of this strategy in promoting PPF synchronization. We also considered the potential benefits of utilizing shear stress to accelerate PPF and release. For this purpose, we developed two microfluidic bioreactor systems --- one designed for production of large numbers of platelets from mature MKs, and the other designed for time-lapse imaging studies on the single-cell level. Although we have conducted preliminary experiments with each of these systems, this work is on-going in our lab. To this point, we have been unable to produce large numbers of platelets from the "production" system, but have been able to capture MKs and image PPT elongation and release in the "single-cell" system.