The RUNX1b Isoform Defines Hemogenic Competency in Developing Human Endothelial Cells.

Sara Menegatti,Bethany Potts,Michael Lie-A-Ling,Valerie Kouskoff,Roberto Paredes,Eva Garcia-Alegria,Georges Lacaud

doi:10.3389/fcell.2021.812639

Abstract

The transcription factor RUNX1 is a master regulator of blood cell specification. During embryogenesis, hematopoietic progenitors are initially generated from hemogenic endothelium through an endothelium-to-hematopoietic transition controlled by RUNX1. Several studies have dissected the expression pattern and role of RUNX1 isoforms at the onset of mouse hematopoiesis, however the precise pattern of RUNX1 isoform expression and biological output of RUNX1-expressing cells at the onset of human hematopoiesis is still not fully understood. Here, we investigated these questions using a RUNX1b:VENUS RUNX1c:TOMATO human embryonic stem cell line which allows multi-parameter single cell resolution via flow cytometry and isolation of RUNX1b-expressing cells for further analysis. Our data reveal the sequential expression of the two RUNX1 isoforms with RUNX1b expressed first in a subset of endothelial cells and during the endothelial to hematopoietic transition while RUNX1c only becomes expressed in fully specified blood cells. Furthermore, our data show that RUNX1b marks endothelial cells endowed with hemogenic potential and that RUNX1b expression level determines hemogenic competency in a dose-dependent manner. Together our data reveal the dynamic of RUNX1 isoforms expression at the onset of human blood specification and establish RUNX1b isoform as the earliest known marker for hemogenic competency.

Highlights

In all species studied to date, the hematopoietic system is established during embryonic life in successive waves, each characterized by their timing of emergence, anatomical location and type of progenitors generated (Lacaud and Kouskoff, 2017; Dzierzak and Bigas, 2018)
Using a double reporter human embryonic stem cells (hESCs) line which allows the simultaneous detection of RUNX1b and RUNX1c, we establish that these two RUNX1 isoforms are sequentially expressed during human blood cell emergence as was previously shown in the mouse system
RUNX1c is not expressed in endothelial cells or during the transition from endothelium to hematopoiesis as the first blood cells emerging at day 6 of differentiation are still RUNX1c:TOMATO negative

Summary

Introduction

In all species studied to date, the hematopoietic system is established during embryonic life in successive waves, each characterized by their timing of emergence, anatomical location and type of progenitors generated (Lacaud and Kouskoff, 2017; Dzierzak and Bigas, 2018). Using a variety of experimental approaches, it was shown that in vertebrate species all blood progenitors initially emerge from endothelial cells with hemogenic properties (Jaffredo et al, 1998; Zovein et al, 2008; Boisset et al, 2010). This endothelial cell population termed hemogenic endothelium (HE) gives rise to blood cells through an endothelial to hematopoietic transition (EHT) during which the endothelial program is shutdown while the hematopoietic program is activated (Gritz and Hirschi, 2016). Narrowing down the differentiation potential, this negative selection is not sufficient to distinguish solely hemogenic endothelium

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Results

Conclusion