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Home > Medical Professionals > Research Initiatives Conference > Poster Abstracts Accepted > Abilez Manipulation of Contractility in P19 Stem Cell

Manipulation of Contractility in P19 Stem Cell Derived Myocytes for Incorporation into Tissue-Engineered Vascular Grafts

Oscar Abilez, Peyman Benharash, Emiko Miyamoto, Adrian Gale, Chengpei Xu, Christopher Zarins
Stanford University, Stanford, CA

Objectives: Contractile properties of myocytes derived from stem cells for use in vascular tissue engineering are poorly understood. Myocytes must exhibit both functional organization and contractility in order to serve as components for tissue-engineered vascular grafts (TEVG). Our objectives were to differentiate P19 stem cells into myocytes and then investigate their contractile response to adrenergic stimulation and external electrical pacing. Methods: We first constructed a custom-made bioreactor with a microscope mounted chamber capable of delivering precise temperature and gas-mixture control. In addition to real-time video capture, time-lapse microscopy was employed to capture image frames every 30 minutes for 14 days. On day 0, we added 1% dimethylsulfoxide (DMSO) (known to differentiate cells into myocytes) to P19 cells and cultured them in a 37°C, 5% CO₂ incubator. On day 5, we exchanged the media containing DMSO with regular media and transferred the cells to the bioreactor chamber for continuous culture and observation. From Days 6-20, we visually assessed the cells for signs of viability and contractility. Adrenergic stimulation was accomplished at 4, 8, 20, 40, and 100 nanomolar concentrations of epinephrine. A Medtronic 5330 pulse generator (current output 0.1-20 mA and frequency 30-180 cycles/min) attached to gold electrodes spaced 2 cm apart was used for external pacing. Results: P19 cells remained viable and grew in the bioreactor as verified by video and time- lapse microscopy (Figure 1). On Day 11 we observed the first spontaneous contractions. For individual colonies, we observed local synchronization and organization. Contractile rate increased up to 240 cycles/min with increasing concentrations of epinephrine. We were also able to synchronize multiple colonies with externally applied electrical fields at a threshold of 18 mA (see Figure 2). Conclusions: We have shown that stem-derived myocytes respond to adrenergic stimulation and electrical pacing similar to native myocytes. Understanding such characteristics will allow manipulation of stem cells into a more vascular smooth muscle phenotype suitable for incorporation into tissue-engineered vascular grafts.