Oral Presentation ASSCR, AGCTS, ISCT ANZ and Friends Joint Scientific Conference 2019

An AGM-on-a-chip model for study definitive haematopoiesis and vascular development from hPSCs. (#3)

Osmond Lao 1 , Freya Faith Bruveris 2 , Elizabeth Ng 2 , Ed Stanley 3 , Richard Harvey 4 , Ralph Patrick 4 , Robert Nordon 1 , Jingjing Li 1
  1. Graduate school of biomedical engineering, University of New South Wales , Sydney, NSW, Austrlia
  2. Blood Cell Development and Disease , Murdoch Childrens Research Institute, melbourne, Vic, Australia
  3. Stem Cell Technology laboratory , Murdoch Childrens research institute, Parkville, victoria, Australia
  4. developmental and stem cell biology division, Victor Change Cardiac Research institute, Sydney, NSW, Australia

This study aims to understand the hemodynamic control of human embryonic stem cell (hESC)-derived haematopoiesis and vasculogenesis by applying microfluidics, live cell imaging and single cell RNA sequencing. AGM-like haematopoietic cultures developed by Ng et al. were generated by manipulating WNT and TGFß signalling during mesoderm specification. The effect of pulsatile circulatory flow was studied in a microfluidic dynamic culture system, using SOX17 (Cherry)/RUNX1c(GFP) hESC line to read out arterial endothelial and haematopoietic differentiation, respectively. Microdevices generated cardiac-like, pulsatile flow in a circulatory culture system with a volume of 2–3µL. AGM-like haematopoietic development was observed by time-lapse imaging on chip for more than two weeks. We observed cells entering the circulation from the adherent layer, and the release of lightly tethered SOX17+ cells into the circulation. In parallel bulk differentiation culture using an orbital mixer to mimic the effect of wall shear stress, single cell RNA seq was performed on 8800 single cells at day 18 of culture. Control treatments were static culture and drug vehicle. Hierarchical cluster analysis identified 18 clusters belonging to erythroid-megakaryocytic, cardiovascular and myeloid differentiation pathways. We identified HOXA expressing mesenchymal cells in AGM cultures. Shear treatment promoted proliferative MYB, RUNX1, CD31 expressing blood progenitors, a reduced proportion of unipotent erythroid and megakaryocytic lineages, and increase numbers of myeloid and bipotent megakaryocyte-erythroid progenitors. The production of endocardium and cardiomyocytes was promoted by shear in control culture. This study demonstrates the feasibility of modelling human embryonic blood formation using microfluidic technology. The single cell transcriptome data provide an entry point for deeper analysis of embryonic haematopoiesis, haematopoietic and cardiac lineage development, as well as the effects of shear stress on them.

  • Have you presented your abstract at another international meeting?: Yes