Title: Development of an in vitro culture system generating haematopoietic dendritic cells (DCs) from mouse embryonic stem cells
Funder: National Centre for the Replacement, Refinement and Reduction of Animals in Research

Abstract: ANIMAL EXPERIMENT IN DC RESEARCH : Dendritic cells (DCs) is a major cell type of the immune system coordinating the development of immune responses or the maintenance of immune tolerance. DCs arise from Flt3R/Flk2+ haematopoietic progenitors and differentiate to control lymphocyte activation by presenting antigens to CD4+ and CD8+ T lymphocytes during immune responses. For this reason, understanding DCs function is of paramount importance in order to maximize protective immune response (vaccination, immunotherapy) and minimize harmful immune responses (autoimmunity). Genes expressed by DCs have been widely characterized in both mouse and human DCs subsets. These studies stimulate the production of numerous genetically modified (GM) mice strains for the functional analysis of these genes. However, this type of research is highly mouse consuming given the rarity of the DCs in the mouse system. DC expansion using Flt3L in vitro (on bone marrow cultures) or in vivo (after injection) still requires a lot of mice. GOAL : In order to reduce animal experimentation in DC research, this proposal intends to implement a new in vitro culture system to generate DCs out of murine ES cells and their genetically modified derivatives. STRATEGY : The strategy underlying this proposal is to take advantage of genetic engineering to conditionally overexpress transcription factors expanding haematopoietic progenitors generated during the spontaneous differentiation of ES cells into embryoid bodies. The approach is to achieve conditional immortalization of progenitors to support quantitative, growth-factor independent expansion while conserving Flt3/Flk2 expression. Various transcription factors (including homeobox genes) will be evaluated in their ability to drive the expansion of Flt3/Flk2+ progenitors. Practically, we will use poly-cistronic lentiviral vectors driving regulated expression of these transcription factors upon doxycycline activation of rtTA transactivatordependent transcription. Then, by relieving the expression of these homeobox transcription factors, DCs will be terminally differentiated in growth factor dependent cultures using FLt3L as an agonist for the Flt3/Flk2 signaling pathway. APPLICATIONS : This method will be suitable to study a wide variety of genetic modification generated at the level of ES cells : gene inactivation (homozygous targeting by homologous recombination using adapted selection processes or stepwise removal of selection cassette), gene silencing (achieved by lentiviral transduction), gene overexpression, fluorescently tagged gene products. Practically, this culture system will be applied to various genes studied in the lab thus generating a major replacement of mouse experiment and breeding (hundreds of mice generated from more than 20 breedings of the GM mice that would need to be generated otherwise). If adopted by the scientific community, this method may have a broad impact on DC studies and may even stimulate developments in the study of other haematopoietic cell types. WHO, WHERE ? The recently opened Guermonprez lab belongs to the King’s College London Guy’s Hospital campus conveniently located in central London. The lab is located within the multidisciplinary Center for Molecular and Cellular Biology of Inflammation headed by Pfr Geissmann and belongs to the Division of Immunology, Infectious and Inflammatory Diseases headed by Pfr Hayday. The PhD candidate will have access to a highly diverse and prestigious scientific environment at the crossroads of cell biology, immunology and inflammation research as well as high-end and state-of-the art facilities providing a stimulating work environment to achieve innovative and productive research.


Research activity codes: 2.6 RESOURCES AND INFRASTRUCTURE (AETIOLOGY);1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: INFLAMMATORY AND IMMUNE SYSTEM