The development of a combinatorial matrix microarray to study the effect of extracellular matrix and substrate stiffness on cell fate

Ireland, Ronald 1, 2 ; Kibschull, Mark 3 ; Lye, Stephen 3, 4, 5 ; Simmons, Craig 1, 2, 6;

 1.  Institute of Biomaterials and Biomedical Engineering, University of Toronto; 2.  Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research, University of Toronto; 3.  Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital ; 4.  Departments of Obstetrics and Gynaecology, Physiology, and Medicine, University of Toronto; 5.  Fraser Mustard Institute for Human Development, University of Toronto; 6.  Department of Mechanical and Industrial Engineering, University of Toronto;

 In vivo, stem cells are subjected to a complex interplay of biochemical cues and biophysical forces that act in concert to orchestrate cell fate. Interrogating the role of each factor in the cell microenvironment, however, remains difficult due to the inability to study microenvironmental cues and tease apart their interactions in high throughput. To address this need, we have developed an extracellular matrix (ECM) microarray screening platform capable of tightly controlling substrate stiffness and ECM protein composition to screen the effect of these cues and their interactions on cell fate. As a proof of principle study, we used this platform to screen for optimal conditions that can maintain human pluripotent stem cell (hPSC) pluripotency in defined, xeno-free conditions. While xeno-free culture media are generally effective, xeno-free culture substrates are poorly-defined and/or not effective for all hPSC sources. Combinations of ECM proteins (fibronectin, vitronectin, laminin-521, and collagen IV) were deposited on PDMS substrates with elastic moduli ranging from 3-40 kPa using a high throughput protein plotter. hPSCs (CA2, PB110, H9) were seeded on the arrays and grown in E8 media for four days prior to evaluating cells for Oct4 expression. The number of Oct4+ cells per condition was modeled as a function of matrix composition and substrate stiffness to investigate single factor effects and multifactorial interactions. For all cell lines tested, fibronectin, laminin-521, and vitronectin had the most prominent positive effect on Oct4+ cell counts. Interestingly, we observed a synergistic interaction with fibronectin and laminin-521, whereas vitronectin and laminin-521 had a pronounced antagonistic interaction. Stiffness also played a significant role, with stiffer substrates predicted to be better for cell attachment and pluripotency. Cells grown on optimized PDMS substrates remained pluripotent for over 25 passages and expressed high levels (>90% cells positive) of Nanog, Oct4, and Sox2. Cells grown on these substrates also retained the capacity to differentiate into the three germ layers. This proof of principle study shows the promise of our matrix microarray platform and its unique ability to tease out single factor and combinatorial effects of ECM protein and substrate stiffness on cell fate.