Thesis (Ph. D.)--Duke University, 2007. / Includes bibliographical references.

Thesis (M. S.)--Computer Science, Georgia Institute of Technology, 2009. / Committee Chair: Dr. Samuel Dudley; Committee Member: Dr. Micheal Davis; Committee Member: Dr. Robert Butera

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 206-224).

Current treatment options are limited for patients with myocardial infarction or heart failure. Cellular cardiomyoplasty is a promising therapeutic strategy being investigated as a potential treatment, which aims to deliver exogenous cells to the infarcted heart, for the purpose of restoring healthy myocardial mass and mechanical cardiac function. While several cell types have been studied for this application, only bone marrow cells and human mesenchymal stem cells (hMSCs) have been shown to be safe and effective for improving cardiac function in clinical trials. In both human and animal studies, the delivery of hMSCs to infarcted myocardium decreased inflammatory response, promoted cardiomyocyte survival, and improved cardiac functional indices. While the benefits of using hMSCs as a cell therapy for cardiac repair are encouraging, the desired expectation of cardiomyoplasty is to increase cardiomyocyte content that will contribute to active cardiac mechanical function. Delivered cells may increase myocyte content by several different mechanisms such as differentiating to a cardiomyocyte lineage, secreting paracrine factors that increase native stem cell differentiation, or secreting factors that increase native myocyte proliferation. Considerable work suggests that hMSCs can differentiate towards a cardiomyocyte lineage based on measured milestones such as cardiac-specific marker expression, sarcomere formation, ion current propagation, and gap junction formation. However, current methods for cardiac differentiation of hMSCs have significant limitations. Current differentiation techniques are complicated and tedious, signaling pathways and mechanisms are largely unknown, and only a small percentage of hMSCs appear to exhibit cardiogenic traits. In this body of work, we developed a simple strategy to initiate cardiac differentiation of hMSCs in vitro. Incorporating environmental cues typically found in a myocardial infarct (e.g. decreased oxygen tension and increased concentrations of cell-signaling factors), our novel in vitro conditioning regimen combines reduced-O2 culture and hepatocyte growth factor (HGF) treatment. Reduced-O2 culturing of hMSCs has shown to enhance differentiation, tissue formation, and the release of cardioprotective signaling factors. HGF is a pleiotropic cytokine involved in several biological processes including developmental cardiomyogenesis, through its interaction with the tyrosine kinase receptor c-Met. We hypothesize that applying a combined conditioning treatment of reduced-O2 and HGF to hMSCs in vitro will enhance cardiac-specific gene and protein expression. Additionally, the transplantation of conditioned hMSCs into an in vivo infarct model will result in differentiation of delivered hMSCs and improved cardiac mechanical function. In testing our hypothesis, we show that reduced-O2 culturing can enhance hMSC growth kinetics and total c-Met expression. Combining reduced-O2 culturing with HGF treatment, hMSCs can be conditioned to express cardiac-specific genes and proteins in vitro. Using small-molecule inhibitors to target specific effector proteins in a proposed HGF/c-Met signaling pathway, treated reduced-O2/HGF hMSCs show a decrease in cardiac gene expression. When implanted into rat infarcts in vivo, reduced-O2/HGF conditioned hMSCs increase regional cardiac mechanics within the infarct region at 1 week and 1 month. Further analysis from the in vivo study showed a significant increase in the retention of reduced-O2/HGF conditioned hMSCs. Immunohistochemistry showed that some of the reduced-O2/HGF conditioned hMSCs express cardiac-specific proteins in vivo. These results suggest that a combined regimen of reduced-O2 and HGF conditioning increases cardiac-specific marker expression in hMSCs in vitro. In addition, the implantation of reduced-O2/HGF conditioned hMSCs into an infarct significantly improves cardiac function, with contributing factors of improved cell retention and possible increases in myocyte content. Overall, we developed a simple in vitro conditioning regimen to improve cardiac differentiation capabilities in hMSCs, in order to enhance the outcomes of using hMSCs as a cell therapy for the diseased heart.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 101-109).

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 109-124).

Background: A 73-year-old man with a history of myocardial infarction and hypertension for 5 years suffered heart failure (NYHA III-IV). Methods: 2D echo indicated hypokinesia at septal, left ventricular anterior wall and apical regions. Coronary angiograms demonstrated 60% stenosis in distal left main and 99% stenosis in proximal and distal left anterior descending coronary arteries (LAD). Both proximal artery and middle left circumflex coronary artery (LC) had 90% stenosis, and diffuse stenosis of right coronary artery (RC) was found. Myocardial perfusion imaging using 99mTc-MIBI indicated defective perfusion of left ventricular apex, anterior wall and septal region and severe reduced perfusion of posterior inferior wall. Myocardial metabolic activities (18F-deoxyglucose) also showed comparable reductions. After exposing the heart, LAD, LC, and RC were all completely occluded and bypass procedure could not be completed. Autologous satellite cells were implanted without any complication and the patient had an uneventful recovery. Results: During the first 2 months, he remained in heart failure, and by the third month, he gradually improved and reached NYHA II. At fifth month after the procedure, significant increased ejection fraction (37.1-48.6%) and wall movement with modest reduction of left ventricular systolic diameter (48-45 mm) were observed. Imaging with 18F-deoxyglucose showed dramatic improvement in myocardial metabolic activity with similar improvement in myocardial perfusion (99mTc-MIBI). Conclusion: This is the first successful case of cellular cardiomyoplasty without any conjunctional procedure for patient with severe coronary heart disease and heart failure.

Embryonic stem cells (ESCs) are being considered as a cell source for cardiac regeneration because of their potency and availability. We studied the electrophysiological implications using co-cultures of ESCs and neonatal rat ventricular myocytes (NRVM) grown on a multi-electrode array (MEA). To mimic expected engraftment rates 5% mouse ESCs were co-cultured with NRVMs. Comparing cultures without and with 5% ESCs at 4 days, the mean bipolar field potential duration (FPD) of NRVMs increased from 26.3 ± 2.2 ms (n=10) to 44.3 ± 6.2 ms (n=9; p < 0.05), the interspike interval (ISI) increased from 358.3 ± 62.8 ms (n=10) to 947.8 ± 214.6 ms (n=7; p < 0.01), and conduction velocity (CV) decreased from 14.2 ± 1.3 cm/s (n=8) to 4.6 ± 1.2 cm/s (n=5; p < 0.01). To evaluate whether ESC were having direct or paracrine effects on NRVMs, media conditioned by 3x106 ESCs for 24 hr was diluted 1:1 with fresh media and then introduced to NRVM cultures on the day of plating. Conditioned media was changed daily and altered mean FPD, ISI, and CV to 46.1 ± 7.8 ms, ISI to 682.0 ± 128.5 ms, and 4.2 ± 0.4 cm/s (n=8; p < 0.01 for each measure), respectively at 4 days. However, changes were not seen in media that was incubated for 24hrs and diluted 1:1 with fresh media and introduced to NRVM cultures in a similar fashion (n=7; p > 0.05). Slowed CV is associated with increased arrhythmic risk and reports demonstrate an inverse relationship between CV and nonphosphorylated Cx43(NP-Cx43). Western blots for total Cx43 expression revealed a decrease in ratio of P-Cx43/NP-Cx43 in the 5% mouse ESCs and ESC conditioned media cultures as compared to controls (n=8; p < 0.01 for each). There was not significant increase in the total Cx43 expression (n=6; p > 0.05). Culturing ESCs with NRVMs resulted in a decreased ISI, prolonged FPD, and slowed CV of the co-cultures as compared to controls leading to pro-arrhythmic conditions. Similar effects on NRVMs were observed when applying media conditioned by ESCs, suggesting that the electrophysiological changes were mediated by soluble factors. The increase in NP-Cx43 leads to gap junction uncoupling being a potential mechanism for these arrhythmogenic substrates. Further research into preventing NP-Cx43 in cultures is currently underway.

Background: Cellular cardiomyoplasty is the method of transplanting myogenic cells into injured myocardium to restore the lost heart muscle cells and to improve ventricular function. Method: Three patients, all with a history of coronary heart disease, underwent coronary artery bypass grafting and implantation of autologous satellite cells. A muscle biopsy of 2-4 g from the right vastus lateralis muscle was obtained for satellite cell (myogenic stem cell from skeletal muscle) isolation and proliferation before implanted into the donor's heart. The cells were suspended in serum-free medium and injected into 30-40 sites at and around the ischemic areas just before reversing the hypothermic cardioplegia to eliminate arrhythmia and to improve retention. After recovery, each patient was maintained at the intensive care unit for 3-4 days with ECG monitoring before transferring to the patient floor. Results: All patients survived the procedure with an uneventful recovery and were discharged from the hospital. At 3-4 months follow-up examination, increased left ventricular ejection fraction of 11% (35-46%), 5.4% (40-45.4%) and 1% (40-41%) and decreased left ventricular diastolic diameter of 4, 2 and 9 mm were observed for the patients, respectively. Arrhythmia was not detected during the follow-up evaluation by ECG. Improved perfusion (99mTC-MIBI) and increased metabolic activity (18F-deoxyglucose) were found at the sites of satellite cell implantation. Significant increase of wall thickness and movement at the areas of cell injection was also observed using 2D-echo. Conclusion: Cellular cardiomyoplasty using autologous satellite cells is a safe procedure with encouraging beneficial outcomes in patients.

In this work, gadolinium-based carbon nanocapsules are developed as a novel nanotechnology that addresses the shortcomings of current diagnostic and therapeutic methods of stem cell-based cardiomyoplasty. With cardiovascular disease (CVD) responsible for approximately 30% of deaths worldwide, the growing need for improved cardiomyoplasty has spurred efforts in nanomedicine to develop innovative techniques to enhance the therapeutic retention and diagnostic tracking of transplanted cells. Having previously been demonstrated as a high-performance T1-weighted magnetic resonance imaging (MRI) contrast agent, Gadonanotubes (GNTs) are shown for the first time to intracellularly label pig bone marrow-derived mesenchymal stem cells (MSCs). Without the use of a transfection agent, micromolar concentrations of GNTs deliver up to 10^9 Gd(III) ions per cell, allowing for MSCs to be visualized in a 1.5 T clinical MRI scanner. The cellular response to the intracellular incorporation of GNTs is also assessed, revealing that GNTs do not compromise the viability, differentiation potential, or phenotype characteristics of the MSCs. However, it is also found that GNT-labeled MSCs exhibit a decreased response to select cell adhesion proteins and experience a non-apoptotic, non-proliferative cell cycle arrest, from which the cells recover 48 h after GNT internalization. In tandem with developing GNTs as a new stem cell diagnostic agent, this current work also explores for the first time the therapeutic application of the magnetically-active GNTs as a magnetic facilitator to increase the retention of transplanted stem cells during cardiomyoplasty. In vitro flow chamber assays, ex vivo perfusion experiments, and in vivo porcine injection procedures all demonstrate the increased magnetic-assisted retention of GNT-labeled MSCs in the presence of an external magnetic field. These studies prove that GNTs are a powerful ‘theranostic’ agent that provides a novel platform to simultaneously monitor and improve the therapeutic nature of stem cells for the treatment of CVD. It is expected that this new nanotechnology will further catalyze the development of cellular cardiomyoplasty and other stem cell-based therapies for the prevention, detection, and treatment of human diseases.