Assembly of lung progenitors into developmentally-inspired geometry drives differentiation via cellular tension

During organogenesis groups of differentiating cells self-organize into a series of structural intermediates with defined architectural forms. Evidence is emerging that such architectural forms are important in guiding cell fate, yet in vitro methods to guide cell fate have focused primarily on un-patterned exposure of stems cells to developmentally relevant chemical cues. We set out to ask if organizing differentiating lung progenitors into developmentally relevant structures could be used to influence differentiation status. Specifically, we use elastomeric substrates to guide self-assembly of human pluripotent stem cell-derived lung progenitors into developmentally-relevant sized tubes and assess the impact on differentiation. Culture in 100 μm tubes reduced the percentage of SOX2⁺SOX9⁺ cells and reduced proximal fate potential compared to culture in 400 μm tubes or on flat surfaces. Cells in 100 μm tubes curved to conform to the tube surface and experienced increased cellular tension and reduced elongation. Pharmacologic disruption of tension through inhibition of ROCK, myosin II activity and actin polymerization in tubes resulted in maintenance of SOX2⁺SOX9⁺ populations. Furthermore, this effect required canonical WNT signaling. This data suggests that structural forms, when developmentally relevant, can drive fate choice during directed differentiation via a tension-based canonical WNT dependent mechanism.