Current feeder-free culture systems employing undefined Matrigel are still more effective in maintaining human embryonic stem (ES) cells than defined surfaces using extracellular matrix (ECM) proteins. While the role of substrate stiffness in stem cell fate is becoming increasingly evident, all previous culture systems use ECM proteins on rigid polystyrene surfaces. Here, we used factorial designs to screen and evaluate combinations ECM proteins and substrate stiffness for their effect on short-term pluripotency and self-renewal. Using optimal conditions determined from our screening experiments, defined and near xeno-free culture systems maintained CA1 human ES cells for over 10 passages in Essential 8 (E8) medium. Under these conditions, we found that human ES cell self-renewal was greater on soft polydimethylsiloxane (PDMS) substrates than on rigid polystyrene dishes. The culture systems and screening tools developed in this project will help develop robust and defined xeno-free culture systems that incorporate both biochemical and biomechanical factors.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/43297 |
| Date | 09 December 2013 |
| Creators | Pang, Justin Tse Wei |
| Contributors | Simmons, Craig Alexander |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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