Current understanding of stem cell-derived heart tissue has primarily focused on embryonic stem cells, and, recently, induced pluripotent stem cells. These cell populations can form tumors, limiting their therapeutic use. Adult stem cells have shown little ability to differentiate into cardiac muscle. However, recent characterization of a population of amniotic fluid stem cells (AFSC) suggests that these cells have potential similar to embryonic stem cells, but do not form tumors, and some researchers consider AFSC to be the most promising therapeutic stem cells. However, AFSC behave differently than embryonic stem cells, particularly in heart tissue formation. AFSC have shown the potential to express cardiac marker proteins in laboratory studies, and have been shown to differentiate into contractile cells in coculture and animal studies, but laboratory differentiation into contractile cells has not been demonstrated. The differentiation of progenitor cells into functioning heart muscle requires a specific interplay of physical and chemical stimulation. Studies by this PI, as well as other groups, have demonstrated a link between physical cues, particularly substrate stiffness, fluid flow and electrical stimulation, and the maturation of heart muscle, though the effect on stem cell differentiation and the signaling mechanisms involved are poorly understood. This proposed study will investigate the effect of controlling the biophysical environment, and specifically substrate stiffness, fluid flow and electrical fields, on the differentiation of AFSC into heart cells and on the signaling pathways involved. The intellectual merit of this proposal is based on the hypothesis that the use of tuned substrate stiffness, fluid flow and electrical stimulation will produce a mature, functional cardiac phenotype in AFSC. To test this hypothesis, AFSC will be cultured in a series of rationally designed experimental conditions to quantify the effect of chemical protocols, alterations in substrate stiffness over a physiologic range, alterations in fluid flow in a range found in the developing heart, and chronic electrical pacing, as well as combinations of these factors. Expression heart protein markers and functional assays of calcium handling and cell contraction typical of heart tissue will be evaluated. Additionally, the effects of inhibiting certain pathways involved in sensing mechanical cues will be measured, and the activation of specific pathways shown to be involved in mechanical sensing will be quantified. Results will indicate the mechanism of mechanical sensing involved in AFSC response to substrate stiffness, fluid flow and electrical pacing and cooperative effects of these biophysical cues on differentiation into heart cells. In the long term, we envision a novel future therapy that allows collection of amniotic fluid by amniocentesis when a heart defect is detected prior to birth, growth of heart tissue in a laboratory setting, and surgical reconstruction of the newborn heart using functional tissue constructs created from the infants own cells. This technology could allow surgeons to consider transformative changes in heart reconstructions and pave the way for development of a total bioartificial heart. The broader educational and social impacts of this proposal are tightly integrated with the intellectual merit. Specifically, the proposed educational program is leveraged to engage a large number or elementary, middle and high school students in inquiry-based exploration of the cardiopulmonary system and inspire lifelong interest in science and medical technology. Curricula will be developed in cooperation with the John P. McGovern Museum of Health and Medical Science (MMHM) in Houston to be used in field trips to the museum and teacher workshops, in presentations to local high schools, and in teaching Systems Physiology to Rice University undergraduates. The PI, Dr. Jacot, will also serve as a consultant on the planned expansion of the MMHM exhibitions, including a redesign of the Amazing Body exhibit with a large focus on the heart. Educational curricula will be posted to digital libraries and research results will be shared in peer-reviewed literature and at scientific conferences.
