Direct Reprogramming of Fibroblasts to Dendritic Cells for Immunotherapy
July 16, 2016
MSc. Thesis - University of Coimbra
Fábio Alexandre Fiúza Rosa
The maintenance of cellular identity relies on stable and complex gene regulatory networks. However, several studies have shown that cell fate can be reversed or modified by defined sets of lineage specific Transcription Factors (TFs). The process of direct cellular reprogramming holds promise for the generation of autologous cells for regenerative medicine. In the immunotherapy field, the use of immune modulatory cells enables the manipulation of patients ‘own immune system to target cancer cells. In this context we aim to apply direct cellular reprogramming for the generation of Dendritic Cells (DCs) as ideal antigen-presenting cells to kick-start adaptive immune responses. Here, TF-mediated direct reprogramming approach was established to generate DCs from fibroblasts. First, we employ a combination of literature mining and computational analysis to identify candidate TFs to induce DC fate in vitro. Candidate TFs were selectively expressed in DC populations in both mice and humans and their disruption caused abnormal adaptive immune phenotypes in mice. This analysis generated 19 candidate TFs with key developmental roles in the DC lineage. We have expressed a set of these TFs using a reprogramming proven Doxycycline-inducible lentivirus in mouse embryonic fibroblasts (MEFs). Employing transgenic MEFs harbouring the DC-specific reporter Clec9a-Cre X R26-stop-Tomato, a minimal combination of 4 TFs was identified. This set of 4 TFs activated the DC-specific reporter and generated tdTomato+ cells. TdTomato+ cells acquired DC-like morphology with increased size and complexity. Moreover, a percentage of tdTomato+ cells expressed Major Histocompatibility Complex (MHC) Class II at the cell surface, a critical molecule for antigen presenting function. Finally, overexpression of the 4TFs in Human Dermal Fibroblasts generated cells with DC-like morphology. These morphological changes emerged with similar timing and efficiency in mouse and Human, supporting species conservation of transcriptional regulators underlying DC commitment. Collectively, DC-like cells were generated via a TF-mediated direct reprogramming approach. The results presented in this study highlight the potential of direct reprogramming to a better understanding of transcriptional events underlying lineage specification and to generate immune modulatory cells for immunotherapy.