Abstract
We postulate that exit from pluripotency involves intermediates that retain pluripotency while simultaneously exhibiting lineage-bias. Using a MIXL1 reporter, we explore mesoderm lineage-bias within the human pluripotent stem cell compartment. We identify a substate, which at the single cell level coexpresses pluripotent and mesodermal gene expression programmes. Functionally these cells initiate stem cell cultures and exhibit mesodermal bias in differentiation assays. By promoting mesodermal identity through manipulation of WNT signalling while preventing exit from pluripotency using lysophosphatidic acid, we ‘trap’ and maintain cells in a lineage-biased stem cell state through multiple passages. These cells correspond to a normal state on the differentiation trajectory, the plasticity of which is evidenced by their reacquisition of an unbiased state upon removal of differentiation cues. The use of ‘cross-antagonistic’ signalling to trap pluripotent stem cell intermediates with different lineage-bias may have general applicability in the efficient production of cells for regenerative medicine.
Highlights
We postulate that exit from pluripotency involves intermediates that retain pluripotency while simultaneously exhibiting lineage-bias
We utilised the MIXL1 reporter line engineered by Davis et al.[11], which has an enhanced GFP sequence inserted into the first exon of one of the MIXL1 alleles of the HES3 human pluripotent stem cells (PSC) line
To assess how closely the ‘trapped’ MIXL1-GFP(+)/SSEA-3(+) subset from cells growing in PRIMO medium resembled the similar subset from mouse embryo fibroblast (MEF)/KOSR cultures, we compared the transcriptomes of MIXL1-GFP(−)/SSEA-3(+) and MIXL1-GFP (+)/SSEA-3(+) cells grown in PRIMO conditions for 3 days, as well as MIXL1-GFP(−)/SSEA-3(+) cells from cultures in E8, with the previous data from cells growing in MEF/KOSR conditions
Summary
We postulate that exit from pluripotency involves intermediates that retain pluripotency while simultaneously exhibiting lineage-bias. We identify a substate, which at the single cell level coexpresses pluripotent and mesodermal gene expression programmes These cells initiate stem cell cultures and exhibit mesodermal bias in differentiation assays. By promoting mesodermal identity through manipulation of WNT signalling while preventing exit from pluripotency using lysophosphatidic acid, we ‘trap’ and maintain cells in a lineage-biased stem cell state through multiple passages These cells correspond to a normal state on the differentiation trajectory, the plasticity of which is evidenced by their reacquisition of an unbiased state upon removal of differentiation cues. We use a MIXL1 reporter[11] to delineate the cellular trajectory from pluripotency to committed mesoderm progenitors On this trajectory, we identify within the stem cell compartment a developmental intermediate that exhibits mesoderm lineage bias. The identification and characterisation of mesodermal biased pluripotent intermediates informs our understanding of how mesoderm lineage specification occurs and could provide an attractive starting point for directed differentiation towards mesodermal derivatives
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