Analysis of Human Embryonic Stem Cell Behavior in Control and Experimental Conditions Using Time-Lapse Video Microscopy Endpoints.

Abstract

Since their first derivation in 1998, researchers have used human embryonic stem cells (hESC) for various purposes. However, the current methods for understanding hESC behavior have been problematic, and better technology is needed for efficient evaluation of hESC behavior in vitro. The purpose of this study was to evaluate and use time-lapse video microscopy (TLVM) to analyze hESC dynamics in vitro using various experimental conditions. The endpoints that were evaluated by observing cellular dynamics included pre-attachment "dancing", attachment/spreading, proliferation, and cell death (apoptosis or necrosis). For all experiments, WiCell H9 hESCs were cultured on Matrigel or gelatin-coated Petri dishes, and images were recorded with a Nikon BioStation IM at 1 or 2 minutes intervals for 3 hours using different experimental conditions. Quantification of the endpoints was done by different individuals counting multiple frames from each experiment. Different individuals also evaluated the same data repeatedly, and the coefficients of variation for both an individual's multiple counts as well as counts by different individuals were low (<1.0%), demonstrating the reliability of the method. One phenomenon observed in the control was pre-attachment "dancing" or rapid movements of paddle-like protrusions on cell surfaces in control culture environments. Similar behavior was observed in prostate stromal cells and mouse embryonic fibroblasts, but the movements were less vivacious and were short-lived compared to hESC. A number of protocols have been used to passage hESCs. Here, we utilized TLVM to compare the attachment/spreading ability of hESCS on Matrigel versus gelatin coated dishes after trypsin or Accutase treatment. The time lapse data showed that hESC survived best when removed from plates using trypsinization followed by subsequent plating on Matrigel-coated dishes. Changes in cellular dynamics also enabled analysis of apoptosis and necrosis. The surface of apoptotic cells underwent rapid blebbing, while necrotic cells became swollen. The toxicity of cigarette smoke and alcohol on hESC survival and growth was evaluated with TLVM endpoints. Treatment of hESC with sidestream (SS) smoke (0.1PE, PE=puff equivalent = smoke in 1 puff that dissolves in 1 ml of solution) from traditional (Marlboro Red) and harm reduction (Advance Lights, Marlboro Lights, and Quest) cigarettes decreased the percentage of cell attachment to Matrigel when compared to untreated controls. hESCs treated with 0.5% alcohol were undergoing apoptosis by 2 hours of treatment, and attached colonies treated with 0.1% alcohol (a level found in the blood of human drinkers) also showed apoptotic blebbing. hESC treated with 0.1% alcohol also had activated caspases that were demonstrated using a poly-caspase FLICA inhibitor. In summary, we have demonstrated that TLVM is a valuable and effective method to evaluate hESC behavior allowing close observation and detailed analysis of cellular dynamics under various experimental conditions. (poster)