Tissue Engineering

Poster

F-2088 - GUIDED SELF-ASSEMBLY OF SOX2+SOX9+ HUMAN LUNG PROGENITORS INTO SIMPLIFIED DEVELOPMENTALLY INSPIRED TUBULAR ARCHITECTURE

6/22/2018
19:00 - 20:00

Self-assembling organoids from human pluripotent stem cells mimic aspects of lung organogenesis. These systems suggest that cellular architecture exerts an effect on cellular differentiation. We used simplified but developmentally relevant architecture to probe this affect on cellular differentiation. The tubular architecture of lung during the pseudoglandular stage of development was simulated using soft lithography to create 100µm diameter tubes with a depth of 127±11.2µm in a silicon polymer, PDMS. Lung progenitors (LPs) characterized via expression of NKX2.1, FOXA2, SOX2 and SOX9 formed single-cell lined tubes that were mostly SOX9+ SOX2- (74.6±2.9%). In contrast, cells cultured for the same period on flat PDMS were predominantly SOX2+SOX9+ (82.6±2.8%). Cells in larger 400µm tubes were similarly mostly SOX2+SOX9+ (85.7±3.7%). To ensure that decreased SOX2 expression was not due to architecture-created concentration effects LPs were grown on 100µm PDMS posts and were still primarily SOX9+SOX2-(78.1±7.2%). Compared to flat, cells in tubes displayed significantly altered cell morphology by reduction in cell elongation (4.5±0.3µm to 3.3±0.2µm). Furthermore, in 400µm tubes, cells were found to be like flat with respect to cell elongation (4.6±0.2µm). We speculate that there was increased cellular tension because there was increased phospho-myosin light chain in 100µm tubes. Pharmacologic manipulation of tension within cells through inhibition of ROCK1 resulted in an increase of SOX2+SOX9+ lung progenitors in 100µm tubes (85.5±10.8) as did myosin II activity (blebbistatin) and actin polymerization (cytochalasin D). These data suggest a role in cellular tension driven by ROCK1 and/or actomyosin in driving the loss of SOX2+SOX9+ in tubular cultures. Cells exposed first to tubular architecture and then to proximal or distal airway inducing conditions in transwells gained expression of distal epithelial markers (SP-C, SP-B) but not proximal markers (SOX2, p63, MUC5AC). In contrast, cells from flat culture can express both proximal and distal markers. Simplified, developmentally relevant architecture impacts fate choice during directed differentiation. The role that shape plays requires further evaluation in more complex architecture to further assess its role in differentiation.


 

John P. Soleas

University of Toronto, Ontario, Canada

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    F-2088 - GUIDED SELF-ASSEMBLY OF SOX2+SOX9+ HUMAN LUNG PROGENITORS INTO SIMPLIFIED DEVELOPMENTALLY INSPIRED TUBULAR ARCHITECTURE



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