(336a) High-Throughput Evolutionary Substrate Screening for Embryonic Stem Cell Attachment and Long-Term Maintenance

Due to their self-renewal capacity and differentiation potential, embryonic stem (ES) cells have great potential in the fields of drug discovery, tissue regeneration and cell therapy.  To achieve the full potential of these cells, strategies must be implemented to maintain them in a pluripotent state.  Cell attachment is essential for maintaining ES cells in an undifferentiated state and preventing them from forming unwanted embryoid bodies.  Fully defined, synthetic substrates are an ideal candidate for the development of a simple, broadly applicable, uniform culture system for pluripotent stem cells. The use of polymeric materials as scaffolds has been well documented and offers several advantages for supporting ES cell maintenance.  However, little is known about how surface chemistry influences stem cell pluripotency. 

In this work, we identify a defined substrate from a comprehensive library of synthetic chemically modified surfaces. It supports long-term cell attachment of mouse ES cells and sustains their pluripotency.  Using an established high-throughput synthesis and screening platform, we have been able to create a library of 66 synthetic chemical surfaces in three 96-well plates.  Mouse ES cells were pre-stained with Cell-Tracker Green and seeded into 96-well plates containing the library of 66 synthetic chemical surfaces. The cell attachment on these chemically modified surfaces was measured by the fluorescence intensity, and a cell attachment index was calculated by normalizing the fluorescence intensity with that of a control group. The surface chemistry with the highest attachment index was our new surface and the lowest attachment was with polyethylene glycol. The cell attachment on the “hit” surface chemistry was further confirmed by scanning electron microscopy (SEM) observation.  The pluripotency of ES cells grown on the “hit” surface was confirmed by retaining pluripotency markers of stem cells, Oct4 and stage-specific embryonic antigen (SSEA-1), the ability to form embryoid bodies, and the maintenance of differentiation potential.  From our initial results, we selected 11 representative monomers and further investigated cell attachment using two different cell lines.  One surface from this set exhibited the highest cell attachment for both cell lines.  We also investigated the potential to use this same chemistry for long-term maintenance of ES cells.  The mechanism of ES cell attachment to the “hit” surface will be discussed.  This monomer-based, chemically defined, scalable, sustainable and controllable, polymeric substrate provides a new avenue for understanding and manipulating surface chemistry for stem cell expansion and differentiation.