1. Effects of Substrate Topography on Stem Cell Behavior Elizabeth L. Smith, RET Fellow 2010 Science Teacher, West Aurora High School RET Mentor: Dr. Michael Cho Chicago Science Teacher Research (CSTR) Program – NSF-RET 2010 HypothesisAbstract Conclusion Material and Methods Results  NSF Grant CBET-EEC-0743068  Prof. A. Linninger, RET Program Director  Dr. Michael Cho, Research Mentor  Brandon Lutz and Hannah Wirtshafter, fellow researchers  University of Illinois- Chicago Acknowledgements References 1. Lovmand, J., Foss, M., Lauridsen, R. H., Lovmand, M., F ̐ ưuchtbauer, E. M., F ̐ ưuchtbauer, A., Wertz, K.,... Markert, L. D. (January 01, 2009). Identification of distinct topographical surface microstructures favoring either undifferentiated expansion or differentiation of murine embryonic stem cells. Stem Cells and Development, 18, 9, 1331-42. 2. Sen, S., Sweeney, H. L., Discher, D. E., & Engler, A. J. (January 01, 2006). Matrix Elasticity Directs Stem Cell Lineage Specification. Cell, 126, 4, 677. 3. Ulmer, J., Grater, S., Surrey, T., Spatz, J. P., & Roos, W. (January 01, 2005). Microtubule Gliding and Cross-Linked Microtubule Networks on Micropillar Interfaces. Nano Letters, 5, 12, 2630-2634. Recent research has proven that the physical, extracellular environment of a cell affects cell morphology and, in stem cells, differentiation. 1,2 This is a preliminary study to further explore the effects of various topographical features on stem cell morphology and differentiation. In this study, human mesenchymal stem cells (hMSCs) were grown on a PDMS substrate of three different topographies: control (no features), posts, and pits. (Figure 2) After seeding, cell samples were fixed after one, three, seven, and nine days for each of the topographies. Preliminary results show a significant difference between cells grown on the control substrate and cells grown on the posts or pits features. Differences in cytoskeleton structure and overall morphology may be a sign of cell differentiation or simply the stem cells’ adaptations to the extracellular environment. Further studies will be required to validate our findings and determine specific final cell lineage. Topographical features were designed using CAD, and created using optical lithography. Liquid PDMS was poured over etched chrome masks and cured. (Figure 1) Features have a pitch of 6 μ m; the depth of the pits and the height of the posts were both 200 nm. Cell samples were stained with rhodamine phalloidin for actin fibers and DAPI for nuclei. Cells were viewed using Differential Interface Contrast (DIC) Microscopy and florescent microscopy; a random sampling of cell images were captured, overlaid, and analyzed using Metamorph imaging software. Analysis involved characterizing cells according to: Number of cells in view field – number of nuclei Physical association with other cells – Isolated, Linked, or Clumped Cell morphology – Elongation or Spreading Actin fiber definition – Defined or Random Additional analysis was done in ImageJ to calculate and compare cell area and cell roundness. Preliminary results show a significant difference between cells grown on the control substrate and cells grown on the posts or pits features. Cells grown on the control substrate showed a constant increase in actin definition (Figure 4) and cell proliferation (Figure 5). For cells grown on pits and posts, cells were slow to proliferate on days one and three, and were observed to have a decrease in f-actin definition; on days seven and nine proliferation and f-actin definition increased. (Figures 4 and 5) Cells on control also showed a significantly more circular morphology than cells grown on pits or posts. (Figure 6) Figure 2. Topography of PDMS substrate. Unobstructed view of control (A), pits (B), and posts (C). DIC image of cells growing on control (D), pits (E), and posts (F). We hypothesize that : 1) A change in the topographical features of a PDMS substrate will potentially result in significant changes, measurable in cell morphology 2) hMSCs are likely to differentiate into osteoblasts, given the rigidity of the PDMS substrate Figure 1. Creation of a PDMS substrate with microscale topographical features. 3 ControlPitsPosts Day 1 Day 3 Day 7 Day 9 Figure 3. A representative sampling of the cells analyzed Figure 4. (Top Left) Percent of samples observed with defined actin fibers. Figure 5. (Bottom) Percent of samples observed, according to number of cells. Figure 6. (Top Right) Effect of Topography on Average Cell Circularity. (*) indicates significance. A change in the topographical features of a PDMS substrate results in significant changes, measurable in cell morphology -Control samples showed a steady increase in defined actin and proliferation -Both post and pit samples showed an initial drop in actin definition and proliferation, a sign that the cell is either being forced to adapt to the environment or differentiating. -In hMSCs, high circularity is possibly a precursor to apoptosis, evidence of proliferation, or a sign of differentiation. The difference between the circularity measured between the cells with and without features shows that cells grown on the pits and post may decrease proliferation, likely differentiating at an increased rate Further testing is needed to validate our results and to test our second hypothesis. A larger, more thorough sampling would hopefully provide greater statistical significance to the results already collected. In addition, more specific testing is needed to measure the direct interaction between the cells and the topographical features (analyzing focal adhesions, etc.) as well as confirm the possible differentiation of the cells and identify the lineage.