Recently launched clinical trials aim to use human pluripotent stem cell (hPSC) mesendodermal (ME) derivatives to treat patients with heart failure and type 1 diabetes. As individual hPSC lines can significantly vary in their ME differentiation efficiencies, acquiring a deeper understanding about this phenomenon and the development of tools to rapidly screen hPSC differentiation bias is of great value to the field of regenerative medicine. In this study, we used five karyotypically normal human embryonic stem cell (hESC) lines to investigate molecular mechanisms of hESC differentiation bias towards definitive endoderm (DE). We quantified DE differentiation efficiency using both a classic adherent differentiation and an in vitro model of early gastrulation-associated fate patterning in geometrically-confined micropatterned colonies, and identified VUB04 as a hESC line with significantly lower DE differentiation efficiency when compared to the other lines. As our DE differentiation protocol employs a strong activator of WNT signalling, we hypothesize that differentially expressed genes in VUB04 prevent its proper activation for endodermal specification. Bulk mRNA-sequencing at the undifferentiated stage showed that the main pluripotency genes were expressed at comparable levels between hESC lines, however, pathway enrichment analysis of differentially expressed genes in VUB04 pointed at MAPK/ERK signalling. GO-term analysis of mRNA-sequencing samples 6 and 24 hours after the onset of DE differentiation showed that VUB04 fails to upregulate genes responsible for gastrulation, endoderm formation and BMP signalling. We are currently modifying expression levels of candidate genes selected from a list of 120 genes which were differentially expressed in VUB04 at all three mRNA-sequencing timepoints, and investigating if they have regulatory effects on early lineage specification to DE. Our work provides preliminary insight into the molecular mechanisms of how hESC may manifest DE differentiation bias and provides experimental validation of an in vitro platform that can be employed for high-throughput screens of hPSC differentiation propensities.
|Conference||ISSCR International Symposium - Stem Cells & Organoids in Development & Disease|
|Periode||20/02/19 → 22/02/19|