Cardiomyopathy is currently the leading cause of death for patients with Duchenne muscular dystrophy (DMD), a severe neuromuscular disease affecting young boys. With no current cure, gene therapy is a promising solution, but supplementation with drug therapies is likely inevitable to fully address the pathology seen in older patients. The use of human-induced pluripotent stem cell (hiPSC) models for drug studies is beneficial due to the direct relevance to human physiology and the potential development of personalized care. Dystrophic hiPSC cardiomyocytes have been shown to exhibit calcium reuptake delays, higher resting calcium levels, and frequent arrhythmias. The Mack Lab previously conducted a preliminary drug screen on healthy and DMD-affected cardiomyocytes and found that certain L-type calcium channel blockers (CCBs) indicated a cardioprotective effect. These drug compounds (namely Nitrendipine and Nimodipine) have been shown to alleviate cardiac fibrosis in patients through vasodilation. In this project, I am validating the beneficial aspects of the drug compounds. I initially hypothesized that treatment of DMD hiPSC cardiomyocytes with L-type CCBs will rescue resting calcium levels and normalize relaxation kinetics. To assess this, I cultured mature hiPSC cardiomyocytes on a Microelectrode Array (MEA) system capable of maintaining physiological conditions while measuring properties of cardiac electrophysiology. The cells were then matured using lactate enrichment to attain further maturity and grown in culture prior to MEA experimentation. Using the MEA, I have found that the QT interval for DMD hiPSC cardiomyocytes was significantly longer than isogenic controls. In current experiments, I am using this platform to validate the effect of L-type CCB compounds of interest in relation to DMD cardiomyopathy. The development of this novel platform may not only have broader implications for DMD drug discoveries and targeted therapies, but it can potentially serve as a powerful preclinical model for other neuromuscular disorders.

Melusin, a chaperone protein expressed in cardiac tissue, induces a protective hypertrophic response in response to chronic mechanical stress. This protective hypertrophic response prevents the progression of cardiomyopathy into heart failure. In previous work done in wild-type (WT) and melusin knockout (melKO) mice, the absence of melusin was correlated with a hypertrophic response indicative of heart failure. I plan to investigate the biomechanical role of melusin in humans using human-engineered heart tissues (EHTs) created from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) that lack melusin and their isogenic controls. EHTs are more representative of the human heart, making them an ideal model for studying the role of melusin in humans. I hypothesize that WT EHTs subjected to mechanical stress, i.e., high afterload, will outperform the melKO EHTs. To measure this, I will increase the stiffness of the EHT posts and measure contractile force. I have successfully differentiated high-purity WT and melKO cardiomyocytes from iPSCs, essential for creating healthy EHTs. I will cast both WT and melKO tissues on a bed of silicone posts that can be stiffened to varying extents to induce different amounts of mechanical stress on the cells. I will conduct a western blot on EHTs from all treatment groups to determine the level of melusin expression and examine the expression of Heat Shock Proteins (Hsp) 70 and 90, due to their coregulation with melusin. The EHTs that undergo mechanical stress are expected to express melusin and these results will work to establish whether melusin expression in humans is activated by mechanical stress. I will measure and compare the contractile force between the WT and melKO tissues. Improving our understanding of the role of melusin in humans can lead to further research into therapies and treatments for heart failure.

The retina is made of mostly neurons and glia. In mammals when neurons degenerate in the brain or retina, they are not replaced. Studying other species, the Reh lab discovered a way to stimulate Muller glia in the retina, using the pro-neural Ascl1 transcription factor, to regenerate retinal neurons. My project focuses on another glial cell in the human retina: the astrocyte. Retinal astrocytes come from different progenitors in the brain and migrate, through the optic nerve, to the retina during development. There are two questions about astrocytes that are addressed in this project. When do astrocytes enter the retina? And can human retinal astrocytes be reprogrammed into neurons using the Ascl1 transcription factor? For the first question, based on immunohistochemistry(IHC) stainings done on sections of the human fetal retina I conclude that astrocytes enter the retina between 62-72 days of fetal development. For the second question, I hypothesize the Ascl1 transcription factor can reprogram human retinal astrocytes and the unique development and migration will affect the types of neurons they regenerate. My IHC stainings on cultures of retinal cells confirms that a lenti-virus I added to the culture with an astrocyte marker(Pax2) sufficiently targets astrocytes. I am currently working on isolating astrocytes from other cells in culture. Afterwards a lentivirus with the pro-neural Ascl1 transcription factor will be added to the astrocytes to begin reprogramming trials. Human retinal astrocytes have not been reprogrammed successfully before. If we can reprogram retinal astrocytes into neurons, it would potentially have implications for neural replacement in the retina. This would contribute to research in gene therapies for neurodegenerative retinal diseases such as: glaucoma, AMD and retinitis pigmentosa.

With cardiovascular disease being the leading cause of death worldwide, new and improved disease models are required to facilitate the research and production of new treatments. In this research, we are developing improved methods for modeling hypertension in engineered heart tissues (EHTs) to investigate resulting tissue remodeling at the tissue and cellular levels. We developed a model of hypertension using 3D-printed polylactic acid braces that enable stiffness adjustment of the flexible polydimethylsiloxane (PDMS) EHT platform. The braces were validated by mechanical testing to quantify their stiffening effect. Braces were shown to increase stiffness according to beam bending theory, with bracing half of the post’s length resulting in a 7-fold increase in stiffness. Next, we applied braces to tissues that only contain stromal cells, which are responsible for remodeling the extracellular matrix (ECM). The next steps are to quantify how stiffness affects ECM remodeling, including tissue-wide effects such as changes in tissue length and width, and micro-scale effects such as changes in cell migration, apoptosis, and cytoskeletal structure, which are quantified in 3D using IHC and confocal microscopy. We hypothesize that hypertension results in tissue thinning and lengthening, as well as decreased cell density, increased apoptosis, and increased expression of cytoskeletal markers. Next, the braces are applied to EHTs containing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The effect of hypertension on EHT remodeling and hiPSC-CM maturation is then quantified using a custom MATLAB image processing suite. Finally, increasing systolic resistance of novel varieties of EHTs that incorporate hiPSC-derived cardiac fibroblasts (hiPSC-CFs) alongside cardiomyocytes enables evaluation of fibrotic remodeling. We hypothesize that hypertension promotes a hypertrophic response in both hiPSC-CMs and hiPSC-CFs in EHTs, displaying increased sarcomerization and fibrosis respectively. Developing this improved hypertension model will accelerate cardiac regenerative medicine research, and provide new approaches for drug discovery.

In the early stages of pathological hypertrophic cardiomyopathy, stress-induced signal transduction promotes the addition of new contractile units to maintain tensional homeostasis through poorly understood mechanisms. Our previous data shows that tension can be changed by altering the contractions within the cells. Inhibition of contraction by expression of D65A cTnC (point mutation on the calcium-binding site of troponin C) results in complete myofibrillar disarray. The mechanism behind the maintenance of myofibril and passive tension seen in these non-contractile CMs is not explained. Data has shown that microtubules provide resistance in the cell and go through rounds of rapid growth or disassembly based on the cell’s need which could be caused by the change in tensional homeostasis. With this in mind, my goal was to determine the role of microtubules in maintaining tensional homeostasis in response to changes in internal tension in CMs. Here, wild-type human induced pluripotent stem cell-derived cardiomyocytes were transduced to express cardiac troponin C with point mutations L48Q (hyper-contractile), I61Q (hypo-contractile), and D65A to study the effect of varying levels of contractility or internal load on microtubules. It was found that microtubular remodeling occurred where the noncontractile CMs had an increase in microtubule density. These noncontractile CMs were then cultures on nanopatterns that provided external tension via topological cues. In addition to the myofibrillar alignment seen previously, it was observed that microtubule density decreased further confirming that microtubules play a compensatory role in these CMs. The next step is to determine if a similar increase in microtubule density observed in the 2D culture is also seen on a tissue level by developing engineered heart tissues. This new data gives insight into how the microtubule remodeling in non-contractile and dysfunctional cardiomyocytes maintains tension in the early stages and its possible role in myofibril formation.

The retina is a neuronal tissue located on the back of the eye that receives light information, and relays this information to the brain, allowing us to see. The light is received by cells called photoreceptors, which send information through a series of inner-retinal neurons before being relayed to the brain via ganglion cells. My project is investigating the effect of a protein called DICER on retinal cell fate in the developing human retina. DICER is important for the maturation of microRNAs, which influence gene expression of retinal precursors that play a crucial role in the progression from early cell types in the mouse, such as ganglion cells, to later cell types, such as photoreceptors and inner retinal neurons. To understand if DICER is also necessary in human development, I am culturing human retinal organoids derived from embryonic stem cells (ESCs), which recapitulate human development in gene expression and developmental timing. I then preferentially knock out (KO) DICER at different developmental time points depending on the timing of infection. I have knocked out DICER in retinal organoids at an early time point and observed that, like mouse studies, cells without DICER more frequently become the early born cell type, ganglion cells. I am now investigating the necessity of DICER in retinal development at later time points. If DICER plays a similar role in cell fate progression, I expect to see an enrichment of later born cell types in the DICER KO cells. By better understanding human retinal development, we will advance future research of regenerative therapies to mitigate the impacts of retinal degenerative diseases leading to vision loss, such as macular degeneration and glaucoma, which affect photoreceptor cells and ganglion cells, respectively.