Cardiac cells and tissue are ideal targets for regenerative medicine and fundamental studies of the interplay of cellular and biomolecular level signaling and response for several reasons. First, observation of successful stem cell differentiation to cardiac myocytes is facilitated by readily identifiable immunohistochemical markers as well as characteristic electrical action potentials and mechanical contractions. Second, explanted cells in culture lose their morphology and organization in the absence of drugs or electromechanical stimulation, suggesting that cellular organization is dependent on these cues. Last, myocardium damaged during a heart attack does not regenerate and the weakened muscle results in heart failure. Fundamental understanding of how cardiac myocytes and heart tissue can be regenerated is essential to creating successful therapies for patients with heart disease (affecting 71 million Americans). Recently, several studies have shown that stem cells may offer regenerative potential through direct injection of cells into the damaged myocardium or in situ repair using engineered tissue grafts.
The Intellectual Merit of this project lies in the development of basic knowledge and models for cell response to environmental cues. Pluripotent cell responses to changes in environment offer a testbed for characterizing the thresholds and mechanisms of environmental adaptation and remodeling. The outcomes of the baseline and coupled experiments will be made available as a database for other researchers. Models and results will be disseminated by publication and seminars for researchers in the field as well as public seminar forums.
The Broader Impacts of this work lie in the enhanced knowledge of cell signaling and differentiation, the role of culture environmental parameters in tissue engineering, and the enhanced design guidance and technology developed which will ultimately enable regenerative therapies for victims of heart disease. Topics of this research will be incorporated in modules for teaching basic engineering and materials courses and the Principal Investigators (PIs) will recruit undergraduates for research experiences in their labs. The PIs actively participate in outreach, undergraduate research opportunities, and research experience for teacher programs and will expand these efforts related to this project. A workshop on the research topics will be held in the final year of the project.
We have developed new technologies and analyses approaches for studying differentiation of pluripotent cells to cardiac tissues. This development spans engineering and medicine labs and created knowledge that is transferrable to other areas of inquiry in medicine and biology. For example, optogenetic tools for pacing cells with light signals, electrically activated cell sorting (EACS), mechanically active cell culture arrays, computational approaches that capture growth/remodeling/electrical-? mechanical coupling from cells to tissues, and synthethic biomaterials with engineered stiffness, cell binding, and degradation. These technologies and models are being tested in vitro and in vivo to elucidate the signaling effect on pluripotent cell development. In vivo imaging allows us to track the fate of these cells in the body. Under this project, we developed several new technologies, models and methods for fundamental biological research at a cellular and tissue level. Using these tools, we studied how pluripotent cell types respond and develop toward cardiovascular cell types in the presence of mechanical, electrical, topographical, and biochemical inputs, developed new biomaterials for culturing pluripotent-derived heart cells, and demonstrated coupled computational models of the heart. We developed new platforms for biological cell culture including mechanical strain array devices to evaluate how calibrated stretch levels affect cell alignment, contractility, and proliferation of cells during differentiation and development. We also demonstrated bioreactors that allow us to study how blood vessels develop and respond to mechanical stimulation. Using these systems, we observed that muscle cell alignment is promoted by stretch in both 2D and 3D. We also demonstrated that cell alignment can be promoted during differentiation by surface patterning of proteins or topography molded into cell culture substrates. Further, differentiation toward terminal cell types was promoted by mechanical stretch, thus decreasing the risk of cancer-like growth from pluripotent cells. We also studied the effects of electrical stimulation on heart cells development and maintenance in culture and learned that biphasic stimulation promotes a more normal behavior in culture. Using optogenetics, we also engineered pluripotent cells to express ion channels that are activated by light and demonstrated the ability to pace cultures of heart cells using light flashes. This approach allows us to study communication between heart cells and development under physiological contractile conditions. We also demonstrated a new class of artificial biomaterials with tunable stiffness and biochemical activity. These hydrogels are synthesized and purified using bacteria and have the potential to replace the current standard culture materials derived from animals and tumor cells. We have demonstrated equal or superior performance of these engineered biomaterials in culturing pluripotent cell types and heart cells. For pluripotent cell therapies to become a therapeutic possibility, we also sought to enhance proliferation and yield of heart cells from differentiation protocols. Using protein and gene analysis, we learned several new pathways that responsible for heart cell differentiation. Finally, we developed several new computational tools to guide the design and placement of future tissue engineered heart scaffolds. These models have predictive power to understand how electrocardiograms are disrupted by heart attacks and could be affected by injections or surgical interventions. These fully coupled models of electromechanical coupling in the heart rely on non-invasive imaging studies of heart structures in patients and animal models and were validated against measured electrocardiograms. The Intellectual Merit of the work lay in the development of basic knowledge and models for cell response to environmental cues. Pluripotent cell responses to changes in environment offer a testbed for characterizing the thresholds and mechanisms of environmental adaptation and remodeling. The outcomes of the baseline and coupled experiments will be made available for other researchers. Models and results were disseminated by publication and seminars for researchers in the field as well as public seminar forums. The Broad Impact of this work lay in the enhanced knowledge of cell signaling and differentiation, the role of culture environmental parameters in tissue engineering, and the enhanced design guidance and technology developed which will ultimately enable regenerative therapies for victims of heart disease. Topics of this research were incorporated into modules for teaching basic engineering and materials courses and we will recruit undergraduates for research experiences with our labs. The PIs actively included undergraduates, high school teachers, high school students in this project through mentored summer research projects, and outreach activities for K-12.
