Subproject for Milan Mrksich under Main Project SP0032595

For patients experiencing seriously low platelet counts following cancer treatment or trauma, there is a real need to increase the supply of platelets as a life saving treatment to prevent bleeding. There are more than six million platelet transfusions each year in the US using volunteer donors. Generating platelets from megakaryocytic (MK) cells ex vivo using GMP manufacturing would ensure quality and a readily available source of platelets for transfusions, especially during national emergencies. Umbilical cord blood (CB) is an attractive source of stem and progenitor (CD34+) cells (HSPCs) for ex vivo platelet generation. One of the principal critical barriers in this field is scalability to generate enough platelets from a single CB unit. Typically one apheresis platelet unit contains 3 x 1011 platelets, while a CB unit usually contains 2 - 5 x 106 CD34+ cells. We hypothesize that CB CD34+ cells and/or more primitive CD34+CD90+ cells can be extensively expanded in culture, while still retaining efficient MK potential, via the addition of HDAC inhibitors, alone or in combination with selected small-molecule enzyme inhibitors. To test our hypothesis we are proposing a multistep cell culture system comprised of initial HSPC expansion; intermediate MK commitment, expansion, and maturation; and a final stage of platelet release. Our specific aims are: Aim 1. Optimize expansion of CB CD34+ cells which best retain MK differentiation potential. Expansion of CD34+ cells retaining MK potential will be optimized with the aid of differential HDAC activity assays in Aim 2. Aim 2. Investigate the epigenetic processes that augment MK proliferation and differentiation from expanded CD34+ cells using SAMDI (Self-Assembled Monolayers for matrix-assisted laser Desorption/Ionization mass spectrometry) arrays to determine changes in differential HDAC activity depending on the conditions used in Aim 1. Aim 3. Optimize the production of MK cells and platelets from expanded CD34+ cells. Culture-derived platelets obtained using the optimal culture conditions will be purified and tested in vitro for surrogate functionality and in vivo in thrombocytopenic immunodeficient mice. Northwestern University investigators (Miller & Mrksich labs) will complete the following scope of work: Evaluate expanded CD34+ and CD34+CD90+ cells produced under a wide range of conditions at UIC for MK cell production and differentiation potential. Perform SAMDI assays using lysates from fresh and expanded CD34+ cells from UIC, as well as MK cells subsequently generated from these cells at Northwestern, to identify a limited set of acetylated peptides that is sufficient to discriminate changes in HDAC activity profiles with CD34+ cell expansion and MK differentiation. Use HDAC isoform-specific inhibitors in the SAMDI experiments described above to identify the most active HDAC isoforms as a function of CD34+ cell expansion and MK differentiation. Perform SAMDI assays using lysates from expanded CD34+ cells with good and poor Mk maturation potential (produced at UIC) to identify HDAC activity profiles that correlate with Mk potential, and use the results to suggest HDAC inhibitors to evaluate in CD34+ cell expansion cultures at UIC. Use SAMDI assays on lysates from CD34+ and CD34+CD90+ cells produced at UIC to ensure process consistency in the expanded cells. Optimize MK cell production from expanded CB CD34+ cells (produced at UIC) by modulating culture conditions, and demonstrate that the MK cells produced are able to generate platelets with functional activity (via f