THE ROLE OF SHEAR STRESS IN THE GENERATION OF DEFINITIVE HAEMATOPOIETIC LINEAGES AND ARTERIAL VASCULATURE FROM HUMAN PLURIPOTENT STEM CELLS AT THE SINGLE-CELL LEVEL

This study aims to characterise the effect of fluid shear stress on human embryonic stem cell (hESC)-derived haematopoiesisand vasculogenesisby applying microfluidics, live cell imagingand single cell RNA sequencing. AGM-like haematopoietic cultures developed by Ng et al. were generated by manipulating WNT and TGFs 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 flowin a circulatory culture systemwith 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. Controltreatments 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, CD31expressing blood progenitors, a reduced proportion of unipotenterythroid and megakaryocyticlineages, and increase numbers of myeloid and bipotent megakaryocyte-erythroid progenitors. The production of smooth muscle and cardiomyocytes was promoted by shear in non-AGM culture. This study demonstrates the feasibility of modelling human embryonic blood formation using microfluidic technology.