In vitro electrophysiologic host-graft model for cardiac stem cell integration [electronic resource]

The limited ability of the human heart to regenerate has made myocardial infarction and heart failure debilitating conditions. Recently, an approach using pluri- or multi-potent stem cells to repair damaged heart tissue is being explored for its potential to regenerate tissue as a tailored, patient-specific treatment. However, the mechanisms of integration remain unclear, and many cardiac grafting procedures utilizing both embryonic and adult stem cells have been met with limited success. While current evidence suggests that grafts are likely viable in host myocardium, clinical studies have reported pro arrhythmic side-effects following transplantation, which arise from disrupted propagation patterns. These issues may be attributed to grafts lacking cardiac differentiation, or possessing conduction properties inconsistent with the host tissue. Consequently, understanding the role of the electrical environment throughout the engraftment process is necessary, but infeasible due to a lack of proper tools. Elucidating the electrical aspects of stem cell transplantation aims to ensure proper integration of the transplanted cells to prevent aberrant electrical pathways in the heart. In this work, a set of in vitro tools were developed to study the potential mechanisms underlying the risk of arrhythmia following stem cell transplantation. A planar microelectrode array was first used to investigate the possibility of conduction block if undifferentiated or non cardiomyocyte stem cells, such as mesenchymal stem cells, are used as grafts. Conduction in murine cardiomyocytes was purposely blocked by co-culture with non-conducting murine fibroblasts, and a novel mathematical transform known as a co occurrence matrix was developed to quantitatively analyze the uniformity of conduction. The observed sensitivity of cardiomyocyte conduction illustrated the risk of grafting non-cardiomyocyte cell types despite any potential of differentiating into muscle-like cells. Unlike non-conducting fibroblasts, stem cell grafts are expected to electrically conduct if proper cardiac differentiation takes place. However, possible differences in the conduction properties of these grafts may still lead to arrhythmia. To perform a controlled study of such conduction mismatch, an in vitro co-culture system coupled to microelectrode arrays was developed. Spatially separated cultures representing the host and the graft were allowed to gradually merge above the microelectrode array, allowing the measurement of conduction throughout the integration process. Modeled host and graft cell populations were evaluated by analyzing the co occurrence matrix and conduction velocity for the quality and speed of conduction over time. Co cultures between murine cardiomyocytes (host) and murine skeletal myoblasts (graft) exhibited significant differences in conduction despite synchronous electrical activity. In contrast, conduction was well matched when the same host cells were co cultured with murine embryonic stem cells (mESC). A model using murine cardiomyocytes (host) and differentiating human embryonic stem cells (graft) allowed the characterization of conduction properties relevant to current trans-species animal models, and demonstrate the co-culture device as a screening platform for candidate graft cells. The limited region of the graft that supported conduction exhibited differences in the co-occurrence matrix as well as conduction velocity when compared to the host region. In an effort to improve the effects of conduction mismatch, both host and graft cell populations were electrically paced over the length of time the cultures remained viable (4-5 days). Although a difference between conduction velocities between host and graft was still observed, the overall uniformity of conduction improved in paced co-cultures, implying increased cardiac differentiation. A preliminary study of genomic changes due to paced mESCs resulted in a significant upregulation of several important cardiac genes and a significant downregulation of many embryonic genes. Further efforts are currently underway to examine gene expression with paced hESCs to optimize integration in the host-graft model, and ultimately to understand how the electrical environment influences stem cell transplantation.

Keywords: Bioengineering.
Advisor: Kovacs Gregory T. A. primary advisor. Thesis advisor
Smolke Christina D. advisor. Thesis advisor
Wu Joseph Ching-Ming