O'Brien, Susanne; (2018) Induced Human Pluripotent Stem Cell – derived NK Cells as an Alternative Source of Lymphocytes for Anti-Cancer Immunotherapy. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Human induced pluripotent stem cells (hiPSCs) hold great promise in regenerative medicine as they have the potential to differentiate into any specialized cell type and retain unlimited self-renewal capacity in vitro. The use of hiPSC-derived lymphocytes for adoptive cell transfer therapies is appealing as it provides a potentially indefinite cell source and a platform for genetically modifying cells. Natural killer (NK) cell-based therapy has shown promising results in the treatment of haematological malignancies and the unlimited derivation of NK cells from hiPSCs could overcome the current obstacle of insufficient NK cell numbers for cellular anti-cancer immunotherapy. In this study, we initially used a two-stage culture system to differentiate human pluripotent stem cells (hPSCs) into NK cells. First, haematopoietic stem cells (HSCs) were derived by co-culture with murine OP9 stromal cells. The CD34-expressing fraction was enriched with magnetic activated cell sorting (MACS) and phenotypically characterised by cell surface marker expression. Colony forming unit (CFU) assay confirmed that hPSC-derived CD34+ cells had multi-lineage differentiation potential. Subsequently, HSCs were co-cultured with murine EL08.1D2 feeder cells and under feeder-free conditions with GBGM medium to generate NK like cells. Interestingly, we observed a 100-fold lower cell expansion of hiPSC-derived CD34+ cells in NK cell differentiation conditions compared to umbilical cord blood (UCB)-derived CD34+ cells. Despite this observation the phenotype of hiPSC-derived NK like cells was comparable to UCB CD34+-derived NK cells and freshly isolated peripheral blood natural killer cells (PBNKs). As an alternative to the two-stage culture system, we aimed to drive NK cell differentiation by overexpressing the NK cell-specific transcription factor E4bp4 during in vitro derivation. Our data suggest that the overexpression of E4bp4 does not improve the efficiency of hiPSC-derived NK like cell differentiation, nor can E4bp4 alone induce the NK cell-specific transcriptional network in fibroblasts.

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