Engineering YAP1/TAZ reporter human ES cell lines to study endothelial-to-haematopoietic transition

Abstract

Haematopoietic stem cell (HSC) transplants are a powerful tool in treatment of certain blood borne diseases. However, access to HSC is outweighed by patient demand, affecting some patient groups disproportionately. One potential solution is the generation of HSCs de novo from pluripotent stem cells, such as human embryonic stem (hES) cells. Current in vitro haematopoietic differentiation protocols, however, fail in generating HSC efficiently in the absence of genetic alteration due to not recapitulation of key developmental cues. In the embryo, a small subset of endothelial cells (ECs) lining the dorsal aorta (DA), called the haematogenic endothelium (HE), can undergo an endothelialto- haematopoietic transition (EHT) culminating in the emergence of HSCs. The molecular mechanisms that underpin HE formation and EHT remain poorly understood, partly due to lacking marker combination distinguishing between haematogenic and nonhaematogenic EC. Recent publications suggest that blood-flow related physical cues exerted on the DA during HSC emergence contribute to EHT. Thus, a lack of adequate mechanical cues in current protocols may explain difficulties in HSC generation. Additionally, unpublished data from our lab suggests that central mechanotransducers YAP1 (Yes-associated protein 1) and TAZ (transcriptional co-activator with PDZ-binding motif) are downregulated as ECs undergo EHT. Therefore, we hypothesised that YAP1 and TAZ mechanoregulation needs to be downregulated for EHT to occur. We decided to pursue this hypothesis using human ES cells as model as it is a tractable system that allows imaging during EHT and manipulation of physical microenvironment through altering the culture conditions. First, to facilitate continuous imaging during in vitro EHT, I established a novel in vitro EHT protocol, which enables the haematopoietic maturation of phenotypically defined HE in a monolayer, which can be readily imaged. Then, to measure endogenous expression of YAP1 and TAZ during in vitro EHT and allow for functional analysis of relevant populations, I generated two independent hES reporter cell lines H9ʸᴬᴾ¹⁻ᵖ²ᴬ⁻ᵐᶜʰᵉʳʳʸ and H9ᵀᴬᶻ⁻ᵍˡʸ.²⁻ᵐᴺᵉᵒⁿ. In vitro haematopoietic differentiation of H9ʸᴬᴾ¹⁻ᵖ²ᴬ⁻ᵐᶜʰᵉʳʳʸ and H9ᵀᴬᶻ⁻ᵍˡʸ.²⁻ᵐᴺᵉᵒⁿ cell lines recapitulates downregulation of YAP1-mCherry and TAZ-mNeon with progressing EHT, respectively. Furthermore, chemical inhibition of YAP1/TAZ-signalling in ECs increases the numbers of generated haematopoietic progenitors, which suggests that YAP1/TAZ downregulation indeed contributes to EHT.