Generation and maintenance of mesoderm-biased human pluripotent stem cells

Enver T

UCL Cancer Institute, London.

Human Pluripotent Stem Cells (hPSC) exist in heterogeneous populations when grown in standard culture. We utilised an hPSC reporter line for MIXL1 coupled with stem cell surface antigen, SSEA-3, to identify MIXL1(+)/SSEA-3(+) substate. While the substate is apparent when grown on a mouse embryonic feeder layer, it is virtually non-existent in defined culture systems. We developed "Primo" medium, balancing pro-self renewal and pro-differentiation factors to promote the presence of a MIXL1(+)/SSEA-3(+) substate. The cells grown in Primo exhibit a functional bias in terms of their differentiation potential, in particular, "neutral" Embryoid Bodies produced enhanced mesoderm populations. Bulk and single cell transcriptomics demonstrated that cells correlated with the populations seen in standard culture. Cells could be maintained in this lineage biased state for multiple passages, with a normal karyotype and normal pluripotency associated marker expression. Cells can also be transitioned back, with transcriptional changes reverting back to normal hPSC expression. Here we show the first long term maintenance of a lineage biased pluripotent state which could provide a tool to improve translational medicine for mesodermal derivatives.