Invited Speaker Presentation Eradicate Cancer 2020

Modelling T-cell development using pluripotent stem cells (70483)

Ali Motazedian 1 2 , Freya F Bruveris 1 2 , Santhosh V Kumar 1 2 , Jacqueline V Schiesser 1 2 , Tyrone Chen 3 , Elizabeth S Ng 1 , Ann P Chidgey 4 , Christine A Wells 3 5 , Andrew G Elefanty 1 2 4 , Ed Stanley 1 2 4
  1. Murdoch Childrens Research Institute, Parkville, VIC, Australia
  2. Department of Pediatrics, University of Melbourne, Melbourne, VIC, Australia
  3. Centre for Stem Cell Systems, Anatomy and Neuroscience, University of Melbourne, Melbourne, VIC, Australia
  4. Department of Anatomy and Developmental Biology, Monash University, Melbourne, VIC, Australia
  5. The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia

The generation of T-cells from pluripotent stem cells (PSCs) can provide insights into aspects of early T-cell development and has potential for biotechnology applications where defined and reproducible T-cell products might be required. We have developed genetically modified reporter lines, chemically defined differentiation media and robust differentiation protocols to probe aspects of early human haematopoiesis, including the emergence of lymphoid progenitors.

 

Developmental studies indicate that T-cell progenitors can arise at several extra- and intra- embryonic sites, including sites that pre-date the appearance of haematopoietic stem cells, the cell type that gives rise to lymphoid cells within the adult blood system. This observation suggests that lymphoid progenitor generation can occur independently of HSC development and, in some cases, precedes the development of organs such as the Thymus that are required for ongoing maturation. Using RAG1:GFP reporter cells, we were able to observe the direct emergence of cells with T-cell potential from haemogenic endothelium generated in haematopoietic organoids. These RAG1+ progenitors were multipotent, able to give rise to cells representing the erythro-myeloid and lymphoid lineages, the latter including T, B and NK like cells. Under conditions favourable for ongoing T-cell development, organoids supported the further differentiation of a small fraction of cells to the CD4+CD8+ stage. Analysis of organoids suggested that arrest at this stage may have occurred due to the limited availability of NOTCH ligands, whose expression was restricted to a small population of endothelial cells within the organoids. As such, we have sought to devise new differentiation protocols aimed at increasing the frequency and persistence of NOTCH expressing cells, with aim of promoting ongoing T-cell development. These newer methods support the generation of small populations of CD3+ cells, suggesting that even late stage events of T-cell development can be successfully recapitulated in a completely defined in vitro system.

 

These authors contributed equally: Andrew G. Elefanty, Edouard G. Stanley.