444. Large Scale Culture and Differentiation of Induced Pluripotent Stem Cells for Neutrophil Replacement Therapies

Abstract

Neutrophils are a key component in the innate immune system and are crucial in the protection against bacterial and fungal infections. Patients with neutropenias are at high risk to develop serious life threatening bacterial and fungal infections. Transfusion of donor neutrophils into neutropenic patients may help alleviate disease burden, but difficulty in collecting and transfusing sufficient quantities of viable donor neutrophils has limited the clinical adaptation of neutrophil replacement therapies. Induced pluripotent stem cells (iPSC) are an attractive alternative source to donor derived neutrophils due to their ability to self-renew and differentiate into cells of the three embryonic germ layers, including neutrophils. Successful implementation of iPSC for neutrophil replacement therapies will require a) derivation of iPSC, b) large scale culture of iPSC, and c) efficient differentiation of these iPSC into functional neutrophils in xeno-fee, GMP-compliant conditions. First, normal human fibroblasts were reprogrammed in xeno-free, GMP compliant conditions though nucleofection of a non-integrating 3 plasmid system carrying OCT4, SOX2, KLF-4, l-MYC, Lin28 and shp53. The reprogrammed fibroblasts were cultured in defined conditions on Vitronectin XF in TeSR-E7 media until colony formation when they were transitioned to E8 medium. We generated 3 lines under these conditions and all iPSC derived and cultured under these conditions retain a normal karyotype, express pluripotency markers SSEA-4, Tra-1-60, Tra-1-81, and CD9 at >85% as determined by FACs analysis, and differentiate into all three germ layers in vitro. Once iPSC lines were established, cultures were adapted from adherent cultures to non-adherent, aggregate suspension cultures in spinner flask bioreactor systems in mTeSR or E8 medium. Briefly, adherent iPSC are dissociated into single cells and seeded into a spinner flask at a concentration of 2×105 cells/ml and spun at 60 RPM. We evaluated 2 lines in 3 independent experiments and cells readily formed clusters and were able to achieve 4-log expansion (range: 2-4 log) within approximately 40 days (10 passages). All lines retained a normal karyotype, expressed pluripotency markers SSEA-4, Tra-1-60, Tra-1-81, and CD9 (>85%), and displayed a normal morphology when re-plated in adherent culture. We next adapted a neutrophil differentiation system for differentiation from iPSC from the defined media/spinnerflask system. Clusters of cells from spinner flask cultures were induced to form embryoid bodies (EBs) by transitioning the cultures to EB specification medium for 5 days, followed by a neutrophil specification medium containing IL-3 and G-CSF in adherent conditions. After 7-14 days in neutrophil specification conditions, EBs attached to the plate and CD45+CD11b+CD16+ neutrophils were shed into the medium and were harvested twice weekly for 30-60 days. In 10 experiments, we collected an average of 6.5×10^5 CD45+ cells per well of differentiation representing a 6.5 fold expansion (range: 3-10 fold). In conclusion, iPSC have been derived in GMP compliant conditions and can be cultured at a large scale using spinner flask bioreactors, and the iPSC from spinner flask bioreactors can then successfully be differentiated into CD45+ cells. Thus, iPSC represent an attractive, self-renewing resource of neutrophils for neutrophil replacement therapies.