"Recent data show that 7.6 million Americans have survived a myocardial infarction (MI), and 5.1 million Americans suffer from severe heart failure. Stem cell therapy has the potential to improve cardiac function after MI. Two promising cells for cardiovascular regeneration therapies include human mesenchymal stem cells (hMSCs) and pluripotent stem cell derived cardiomyocytes (hPS-CM) each with their own unique method for improving cardiac function post-infarct. However, a limiting factor to cell therapies is that the methods currently used to deliver cells to the myocardium, including intramyocardial injection (considered the gold standard), suffer from low retention rates. To promote localization of delivered cells to the infarct and increase retention rates, our lab has developed a fibrin biological suture that can deliver human mesenchymal stem cells (hMSCs) with an efficiency of 64% compared to just 11% with intramyocardial injection in the normal rat heart. In this dissertation we sought to examine the functionality of hMSC and hPS-CM seeded sutures and their impact on cardiovascular regeneration applications. We began by delivering hMSC seeded fibrin sutures to an infarcted rat heart and found that the sutures are an effective method to deliver cells to the infarcted myocardium and demonstrated a trend towards improved regional mechanical function in the infarct region over infarct alone. Next, we transitioned to using hPS-CM and developed methods to seed the sutures, as well as a method to measure hPS-CM contractility with high spatial and temporal resolution, while concurrently capturing calcium transients. This technique allowed us to examine the contractile behavior in terms of contractile strain and conduction velocity of hPS-CM seeded on fibrin microthreads over 21 days in culture. We found that the fibrin microthread is a suitable scaffold for hPS-CM attachment and contraction and that extended culture promotes cell alignment along the length of the suture as well as improvements in contractile function in terms of increases in contractile strain and conduction velocity. Finally, we delivered the hPS-CM seeded microthreads to an uninjured rat heart and found a delivery efficiency of 67%. Overall, we further demonstrated the technology of the fibrin suture to deliver cells to an infarct as well as the ability to support the attachment, contraction and delivery of hPS-CM to cardiac tissue. "

abstract: An in vitro model of Alzheimer’s disease (AD) is required to study the poorly understood molecular mechanisms involved in the familial and sporadic forms of the disease. Animal models have previously proven to be useful in studying familial Alzheimer’s disease (AD) by the introduction of AD related mutations in the animal genome and by the overexpression of AD related proteins. The genetics of sporadic Alzheimer’s is however too complex to model in an animal model. More recently, AD human induced pluripotent stem cells (hiPSCs) have been used to study the disease in a dish. However, AD hiPSC derived neurons do not faithfully reflect all the molecular characteristics and phenotypes observed in the aged cells with neurodegenerative disease. The truncated form of nuclear protein Lamin-A, progerin, has been implicated in premature aging and is found in increasing concentrations as normal cells age. We hypothesized that by overexpressing progerin, we can cause cells to ‘age’ and display the neurodegenerative effects observed with aging in both diseased and normal cells. To answer this hypothesis, we first generated a retrovirus that allows for the overexpression of progerin in AD and non-demented control (NDC) hiPSC derived neural progenitor cells(NPCs). Subsequently, we generated a pure population of hNPCs that overexpress progerin and wild type lamin. Finally, we analyzed the presence of various age related phenotypes such as abnormal nuclear structure and the loss of nuclear lamina associated proteins to characterize ‘aging’ in these cells. / Dissertation/Thesis / Masters Thesis Bioengineering 2017

No description available.

Drug- and cell-based strategies for treating heart disease, including myocardial infarction, face significant roadblocks on the path to the clinic, a primary obstacle being the lack of information-rich in vitro human model systems. Conventional model systems are hampered by at least one of three fundamental limitations which include a) the lack of an in vivo-like microenvironment specifically engineered for the input cell population, b) a relatively low-throughput assays, and c) the low-content nature of output parameters. We describe an integrated computational, design, and experimental strategy for the rational design of a microfabricated high-content screening platform which we term the Cardiac MicroWire (CMW) system. Within this system, we recapitulate the basic microenvironment found in the heart, one which integrates cardiomyocytes, non-myocytes, the extracellular matrix, and dynamic electromechanical forces. Our results highlight the CMW system’s potential as a powerful discovery tool for screening small molecules and transplantable cells toward heart regeneration therapies.

Drug- and cell-based strategies for treating heart disease, including myocardial infarction, face significant roadblocks on the path to the clinic, a primary obstacle being the lack of information-rich in vitro human model systems. Conventional model systems are hampered by at least one of three fundamental limitations which include a) the lack of an in vivo-like microenvironment specifically engineered for the input cell population, b) a relatively low-throughput assays, and c) the low-content nature of output parameters. We describe an integrated computational, design, and experimental strategy for the rational design of a microfabricated high-content screening platform which we term the Cardiac MicroWire (CMW) system. Within this system, we recapitulate the basic microenvironment found in the heart, one which integrates cardiomyocytes, non-myocytes, the extracellular matrix, and dynamic electromechanical forces. Our results highlight the CMW system’s potential as a powerful discovery tool for screening small molecules and transplantable cells toward heart regeneration therapies.

<p>Glaucoma is a leading cause of blindness characterized by the progressive loss of retinal ganglion cells (RGCs), essentially severing the connection between the eye and the brain. Among many underlying causes of the disease, mutations in the Optineurin (OPTN) gene result in severe RGC neurodegeneration in the absence of elevated intraocular pressure, providing a novel opportunity to study molecular mechanisms that lead to RGC neurodegeneration associated with glaucoma. Efforts of this study establishing a human pluripotent stem cell (hPSC)-derived in vitro disease model by inserting OPTN(E50K) mutation via CRISPR/Cas9 genome editing and investigate the cellular mechanisms of RGC neurodegeneration associated with glaucoma. OPTN(E50K) RGCs revealed neurodegeneration phenotypes, including downregulation of RGCs transcription factors, neurite retraction, and hyperexcitability, suggesting that OPTN(E50K) RGCs can serve as an appropriate disease model to study glaucoma-associated neurodegeneration. Since OPTN serves a primary role as an autophagy receptor, we further hypothesized that the OPTN(E50K) mutation disrupts autophagy in RGCs, and modulation of autophagy by mammalian target of rapamycin (mTOR)-independent pathways can preserve RGC phenotypes by maintaining mTOR signaling. OPTN(E50K) RGCs exhibited a higher number of OPTN puncta along with an overall reduced expression of OPTN protein, indicating a gain of toxic protein accumulation or loss of protein function. Furthermore, OPTN(E50K) RGCs revealed an accumulation of the autophagosome protein LC3 in a punctal manner as well as increased expression of lysosomal proteins, suggesting a disruption of degradation pathway in autophagosome and lysosome fusion. As mTOR complex 1 (mTORC1) signaling serves as a negative regulator of autophagy, a downregulation of mTORC1 signaling via activation of stress sensor adenosine monophosphate-activated protein kinase (AMPK) was observed as a possible compensatory mechanism for autophagy deficits in OPTN(E50K) RGCs. Pharmacological inhibition of mTOR in wild-type hRGCs resulted in similar disease-related phenotypes, while preservation of the mTOR pathway in OPTN(E50K) RGCs by treatment with the mTOR-independent autophagy modulator trehalose cleared OPTN accumulated puncta, preserving mTORC1 signaling, as well as rescuing neurodegenerative phenotypes. To further validate these associations in an animal model, the microbead occlusion mouse model was established by injection of magnetic microbeads in the anterior chamber to block aqueous outflow resulting ocular hypertension. In agreement with our findings in hRGCs, a decrease in mTOR signaling associated with an increase in the expression of autophagy-associated proteins was observed in RGCs in the microbead occlusion model. Additionally, these disease-related phenotypes were observed specifically within RGCs but not cortical neurons with an underlying OPTN(E50K) mutation, demonstrating that autophagy represents an essential pathway in RGCs to maintain homeostasis, and selective disrupt of autophagy in RGCs leads to neurodegeneration. Taken together, the results of this study highlight an essential balance between autophagy and mTORC1 signaling that is essential for the homeostasis of RGCs, while disruption to these signaling pathways contributes to neurodegenerative features in glaucoma. These results also demonstrated the ability to pharmacologically intervene to experimentally manipulate these pathways and rescue neurodegenerative phenotypes, providing a potential therapeutic target to prevent glaucoma-associated neurodegeneration. </p>

abstract: The pathophysiology of neurodegenerative diseases, such as Alzheimer’s disease (AD), remain difficult to ascertain in part because animal models fail to fully recapitulate the complex pathophysiology of these diseases. In vitro models of neurodegenerative diseases generated with patient derived human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) could provide new insight into disease mechanisms. Although protocols to differentiate hiPSCs and hESCs to neurons have been established, standard practice relies on two dimensional (2D) cell culture systems, which do not accurately mimic the complexity and architecture of the in vivo brain microenvironment. I have developed protocols to generate 3D cultures of neurons from hiPSCs and hESCs, to provide more accurate models of AD. In the first protocol, hiPSC-derived neural progenitor cells (hNPCs) are plated in a suspension of Matrigel™ prior to terminal differentiation of neurons. In the second protocol, hiPSCs are forced into aggregates called embryoid bodies (EBs) in suspension culture and subsequently directed to the neural lineage through dual SMAD inhibition. Culture conditions are then changed to expand putative hNPC populations and finally differentiated to neuronal spheroids through activation of the tyrosine kinase pathway. The gene expression profiles of the 3D hiPSC-derived neural cultures were compared to fetal brain RNA. Our analysis has revealed that 3D neuronal cultures express high levels of mature pan-neuronal markers (e.g. MAP2, β3T) and neural transmitter subtype specific markers. The 3D neuronal spheroids also showed signs of neural patterning, similar to that observed during embryonic development. These 3D culture systems should provide a platform to probe disease mechanisms of AD and enable to generation of more advanced therapeutics. / Dissertation/Thesis / Masters Thesis Bioengineering 2016