Abstract
Heart contraction is essential for organismal life, as it supplies oxygen to all organs via blood circulation. Normal heart rate is regulated by a group of specialized pacemaking cardiomyocytes in the upper right chamber of the heart, or right atrium, that make up a small region of tissue called the sinoatrial node (SAN). Cardiomyocytes (CMs) in the human heart can be functionally classified into two different cell subtypes, contractile CMs, whose primary function is generation of contractions (atrial and ventricular subtypes) and pacemaker cells, which are specialized CMs that have the intrinsic ability to generate action potentials without an external electrical stimulus. Age, physical damage, and inflammation of the sinoatrial node can lead to a disease known as SAN dysfunction. Current therapies for SAN dysfunction usually include the implementation of an electronic pacemaker, which comes with its own challenges. Electronic pacemakers have a battery life of only 5-15 years, have a sensitivity to magnetic interference, and lack an autonomic response. One solution is to develop a replacement for the electronic pacemaker via a stem cell generated biopacemaker. Currently, stem cell therapies are attempting to generate a population of stem cell induced pacemakers that can replace the damaged cells to treat SAN dysfunction. Fibroblasts occupy over 50% of the SAN, suggesting that they are an important component of the pacemaking tissue. It is essential that we understand the secretome of the growth factors and extracellular matrix in a microenvironment that is shaped by the fibroblasts. The primary goal we wished to accomplish during this study was to define the transcriptomic profile of suitable fibroblasts to support pacemaking phenotype in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Recreating this optimal environment with the addition of support fibroblasts may allow for increased retention of the pacemaking cardiomyocytes. Once we are able to generate a more stable pacemaking phenotype, we will be able to implement these cells into stem cell therapies designed for patients suffering from a SAN dysfunction. This biopacemaker may eventually be able to replace traditional electronic pacemaker. To optimize the culture conditions for porcine SAN fibroblasts, cultured human fetal ventricular fibroblasts were subjected to various media, Matrigel types, and substrates and then assessed for viability and proliferation to determine the most suitable condition for the growth of cardiomyocytes and fibroblasts, while not changing fibroblast gene expression from its native environment. qPCR analysis was done to determine changes in gene expression between serum-containing and serum-free media. After optimizing culture conditions, left ventricular and SAN fibroblasts were isolated from porcine hearts, cultured in the optimized condition, and prepared for transcript analysis. Due to low proliferation of the SAN fibroblasts, the resulting RNA concentration was insufficient for analysis by qPCR or sequencing analysis, as originally planned. Cardiac fibroblasts needed for the engineered hiPSC-derived biopacemaker were successfully differentiated via a protocol established in the lab of Joseph Wu. Although the hiPSC-fibroblasts were unable to be compared alongside SAN fibroblasts to determine their suitability for sustaining pacemaking function in the biopacemaker, these fibroblasts were assessed by qPCR, immunostaining, and flow cytometry for markers identified in the SAN fibroblasts to determine the success of the differentiation into cardiac fibroblasts. The successful differentiation of hiPSC-fibroblasts expressing transcription factor T-box 18 (Tbx18) known to be present in the SAN fibroblasts is one step forward in engineering hiPSC-based biopacemakers. The development of stem cell generated biopacemakers would mean a method of treatment for patients suffering from SAN dysfunction without the disadvantages of electronic devices. All research was conducted in the lab of Dr. Deborah Lieu in the Institute of Regenerative Cures and the Genome and Biomedical Sciences Facility at the University of California, Davis.