Use of stem cell-derived macrophages and gene editing to investigate viral pathology and innate immunity

Induced Pluripotent Stem Cell (iPSC)-derived macrophages are a rapidly emerging model of tissue resident macrophages for investigating human disease. As potentially infinitely replicating cells, iPSC lend themselves for genetic manipulation by CRISPR-Cas9 editing before terminal differentiation into a vast array of cell types. As such, long standing challenges in exploration of macrophage gene functions during inflammatory diseases and infections, such as HIV-1, caused by donor variability and immortalised cell line deficiencies can finally be overcome. With over 30 different protocols for macrophage differentiation from iPSC now described, culturing of these cells is gradually becoming more accessible to researchers, and essential processes for differentiation better understood. However, with this progress comes the ever-greater need for clear definition of culturing conditions of these cells, so that we can compare results between methodologies, and eliminate undesirable sources of variability. This thesis sought to capitalise on this new system to explore the complicated and often contradictory role of TRIM5α in HIV-1 infection, and to explore with a precision only achievable through CRISPR-Cas9 genome editing the function of the cyclophilin family of peptidyl-prolyl isomerases. I demonstrate protection of HIV-1 from TRIM5α restriction by cyclophilin A, as well as regulation of the innate immune inflammatory response more generally by the cyclophilin family. However, prior to exploring this, this thesis sets out to address some of current issues in iPSC-derived macrophage culture and the ill-defined components of growth media and cellular environment. I describe a new macrophage differentiation medium using serum free, defined, and open-sourced components to bring needed transparency into the field. I also address the variability in macrophage phenotype and function brought about by the fundamental, but largely overlooked, impact of cell crowding in ex vivo culture. This revealed evidence for a potential plating density-dependent anti-inflammatory feedback loop relevant for human diseases like Alzheimer’s disease.