The research in the CETE group has been focused on the three interrelated themes:
1) Studies of Structural and Functional Interactions between Cardiomyocytes and Other Cells
Paracrine and contact-mediated interactions between cardiomyocytes and endogenous or exogenously added stem cells in the heart are becoming recognized as highly important in normal and abnormal heart development and function as well as in novel cell therapies for myocardial infarction and arrhythmias. Unfortunately, systematic studies of these interactions in situ are complicated by a number of confounding factors including the complex cellular composition and structure of the heart tissue, the lack of direct experimental access to interacting cells inside the heart, and the presence of endogenous paracrine and neurohumoral factors. To address this issue, my group has been the first (and to our knowledge currently the only one) in the field to undertake a significant effort over the last few years to develop standardized in vitro assays for exploring how heterocellular interactions affect cardiac electrical and mechanical function at different spatial scales. Specifically, we have designed several specialized 2- and 3-dimensional co-culture systems where the cell type of interest (e.g., stem cell, genetically modified cell, primary non-myocyte, etc.) can be simply “plugged” into a geometrically and biochemically reproducible cardiac environment and studied in a relatively high-throughput fashion.
This unique in vitro platform has been utilized by our group as: 1) a reproducible cardiomimetic screen for comparing the potential of different candidate stem cells, soluble factors, genetic manipulations, and combinations thereof to safely and efficiently improve compromised cardiac function and 2) a well-controlled system for mechanistic studies of electrophysiological interactions between cardiomyocytes and different unmodified and genetically modified cells. In particular, we have been interested in the use of unexcitable somatic cells with genetically engineered electrical properties for the repair of cardiac damage and improvement of abnormal electrical conduction in the heart. While the potential of different cell-based therapies is exciting, it is imperative that we first determine the best cellular populations before we contemplate clinical translations. We believe that these and similar in vitro assays will have a significant impact in the field by guiding the rational design of novel cell and tissue engineering therapies for safe and efficient treatment of cardiac infarction and arrhythmias.
2) Development of Novel Experimental and Computational Tools for Studies of Cardiac Electrophysiology
Intricate 3D tissue structure in the heart represents an independent and important determinant of cardiac electrophysiological function and malfunction. In particular, the complex roles of healthy or remodeled microstructure in the heart (e.g. cardiac fiber directions, acellular and heterocellular regions) in macroscopic impulse conduction and reentrant arrhythmias remain to be studied. While significant advances in tissue imaging have enabled detailed assessment of cardiac micro and macrostructure, direct experimental studies of cardiac structure-function relationships to date have been hindered by the intrinsic complexity and variability of in vivo models and the overly simplistic structure of in vitro cardiac monolayers. To bridge the gap between current cell culture models and the whole heart preparations, we have introduced a novel and exciting paradigm in cardiac cell culture design where cell micropatterning techniques and a clinical imaging modality (diffusion tensor MRI, DTMRI) are combined to create cardiac monolayers that accurately replicate the realistic microstructure of native tissue sections.
In addition, in collaboration with Dr. Henriquez from Duke BME, we have utilized continuous computational cardiac media to enhance the mechanistic understanding of our experimental results, and have contributed to the development and validation of novel microstructurally-matched models of cardiac monolayers. By developing novel cardiac cell cultures to match the realistic structure and function of native tissues and novel computer models to match the realistic structure and function of cell cultures, we intend to combine different spatial scales and research settings in order to provide detailed mechanistic insights into cardiac structure-function relationships in health and disease. By enhancing the understanding of the microstructural determinants of normal and abnormal cardiac conduction, we aim to promote the design of novel cell and pharmacological therapies for cardiac arrhythmias in the setting of structural disease.
3) Tissue Engineering of Functional Cardiac and Skeletal Muscle for Basic Studies and Therapeutic Applications
Compared to the injection of single cells, the implantation of engineered muscle tissues may further advance cell-based therapies for muscle disease by enabling: 1) better cell retention and survival at the injury site, 2) more concentrated paracrine actions from the implanted cells, and 3) more efficient structural and functional repair of tissue damage. Since the initial work in the field, a number of different approaches have been proposed for the fabrication of engineered muscle tissues in vitro. However, the methods to induce and reproducibly control the direction of 3D muscle cell alignment (an essential structural feature of native muscle) in relatively large tissue constructs were lacking. Therefore, our group has undertaken significant efforts to address this important issue and recently reported a significant advancement in our ability to control engineered muscle tissue structure and function in 3D.
Importantly, while differentiated cardiomyocytes dissociated from heart tissue have been initially used by others and us to establish a number of useful design rules for functional cardiac tissue engineering, it is well recognized that these cells will remain limited to in vitro model systems and proof-of-concept in vivo studies. The use of cardiogenic stem cells, on the other hand, offers a potential for future clinical translation. We have therefore established in vitro techniques for advanced functional differentiation of mouse embryonic stem cell-derived cardiac progenitors and have recently developed methods for their use in functional cardiac tissue engineering. We are continuously advancing our understanding of cardiac developmental processes with the goal of recapitulating developmental cardiogenesis in vitro. Recently, we have made a significant progress in engineering large, electrically and mechanically functional (i.e., "fast conducting and strong contracting") tissue patches consisting of aligned, well-coupled, and differentiated mouse stem cell derived cardiomyocytes. In collaboration with Dr. Rockman from Duke Cardiology, we are in the process of testing these patches in animal models of cardiac infarction. Furthermore, in collaboration with Drs. Christoforou and Leong from Duke BME this research is being expanded towards the use of induced pluripotent stem cells as well as stem cells of human origin. Finally, in collaboration with Dr. Kirby from Duke pediatrics and Dr. Malouf from UNC Pathology, we are investigating mechanisms of transdifferentiation of human bone marrow-derived mesenchymal stem cells into functional cardiomyocytes.
In collaboration with Drs. Truskey, Leong, Yuan, and Izatt from Duke BME, our group has also been actively pursuing the engineering of functional skeletal muscle tissues using skeletal myoblasts (muscle progenitors) isolated from neonatal rat soleus muscle. In general, our ability to perform comparative and complementary research on these two very similar, yet very different, muscle tissues (cardiac and skeletal) is a unique opportunity to elucidate specific and common morphogenic and biophysical mechanisms that govern 3D self-assembly and differentiation of various myogenic progenitors into functional muscle tissues.
Research models and methodologies used:
Micropatterning and microfludic techniques, polymer and hydrogel scaffolds, and bioreactors with computer-controlled mechanical and electrical stimulation are employed to fabricate 2-dimensional and 3-dimensional cardiac and skeletal tissues with controllable architecture and function starting from dissociated primary cells or committed stem cell-derived muscle progenitors. Genetic engineering techniques and lentiviral vectors are additionally used to modify and monitor proliferation, function, and differentiation of somatic and stem cells. Immunostaining, protein and gene expression analysis, optical recordings of electrical propagation with voltage and calcium sensitive dyes, single cell patch-clamp studies, and standard viscoelastic biomechanical tests used in our cell and tissue engineering systems allow for precise correlation between structure and electromechanical function at microscopic and macroscopic spatial scales, as well as functional evaluation before and after animal implantation. Computer models that incorporate cell-specific ion channels, cardiac cell geometry, distribution of intercellular connections and discrete tissue microarchitecture are used to aid the experimental design and the interpretation of results. Zebrafish models of cardiac regeneration and Drosophila models of cardiac specific ion channel mutations are also employed in our collaborative studies.
Our research is currently supported by NHLBI, NIAMS, and AHA.