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Alzheimer’s Disease (AD) affects over 5 million individuals in the U.S. and has a direct cost estimated in excess of $200 billion per year. Broadly speaking, there are two forms of AD—early-onset, familial AD (FAD) and late-onset-sporadic AD (SAD). Animal models of AD, which rely on the overexpression of FAD-related

Alzheimer’s Disease (AD) affects over 5 million individuals in the U.S. and has a direct cost estimated in excess of $200 billion per year. Broadly speaking, there are two forms of AD—early-onset, familial AD (FAD) and late-onset-sporadic AD (SAD). Animal models of AD, which rely on the overexpression of FAD-related mutations, have provided important insights into the disease. However, these models do not display important disease-related pathologies and have been limited in their ability to model the complex genetics associated with SAD.

Advances in cellular reprogramming, have enabled the generation of in vitro disease models that can be used to dissect disease mechanisms and evaluate potential therapeutics. To that end, efforts by many groups, including the Brafman laboratory, to generated patient-specific hiPSCs have demonstrated the promise of studying AD in a simplified and accessible system. However, neurons generated from these hiPSCs have shown some, but not all, of the early molecular and cellular hallmarks associated with the disease. Additionally, phenotypes and pathological hallmarks associated with later stages of the human disease have not been observed with current hiPSC-based systems. Further, disease relevant phenotypes in neurons generated from SAD hiPSCs have been highly variable or largely absent. Finally, the reprogramming process erases phenotypes associated with cellular aging and, as a result, iPSC-derived neurons more closely resemble fetal brain rather than adult brain.

It is well-established that in vivo cells reside within a complex 3-D microenvironment that plays a significant role in regulating cell behavior. Signaling and other cellular functions, such as gene expression and differentiation potential, differ in 3-D cultures compared with 2-D substrates. Nonetheless, previous studies using AD hiPSCs have relied on 2-D neuronal culture models that do not reflect the 3-D complexity of native brain tissue, and therefore, are unable to replicate all aspects of AD pathogenesis. Further, the reprogramming process erases cellular aging phenotypes. To address these limitations, this project aimed to develop bioengineering methods for the generation of 3-D organoid-based cultures that mimic in vivo cortical tissue, and to generate an inducible gene repression system to recapitulate cellular aging hallmarks.
ContributorsBounds, Lexi Rose (Author) / Brafman, David (Thesis director) / Wang, Xiao (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
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Description
This thesis explores and analyzes the emergence of for-profit stem cell clinics in the United States, specifically in the Phoenix metropolitan area. Stem cell therapy is an emerging field that has great potential in preventing or treating a number of diseases. Certain companies are currently researching the application of stem

This thesis explores and analyzes the emergence of for-profit stem cell clinics in the United States, specifically in the Phoenix metropolitan area. Stem cell therapy is an emerging field that has great potential in preventing or treating a number of diseases. Certain companies are currently researching the application of stem cells as therapeutics. At present the FDA has only approved one stem cell-based product; however, there are a number of companies currently offering stem cell therapies. In the past five years, most news articles discussing these companies offering stem cell treatments talk of clinics in other countries. Recently, there seems to be a number of stem cell clinics appearing in the United States. Using a web search engine, fourteen stem cell clinics were identified and analyzed in the Phoenix metropolitan area. Each clinic was analyzed by their four key characteristics: business operations, stem cell types, stem cell isolation methods, and their position with the FDA. Based off my analysis, most of the identified clinics are located in Scottsdale or Phoenix. Some of these clinics even share the same location as another medical practice. Each of the fourteen clinics treat more than one type of health condition. The stem clinics make use of four stem cell types and three different isolation methods to obtain the stem cells. The doctors running these clinics almost always treat health conditions outside of their expertise. Some of these clinics even claim they are not subject to FDA regulation.
ContributorsAmrelia, Divya Vikas (Author) / Brafman, David (Thesis director) / Frow, Emma (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
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Cholangiocytes, the epithelial cells of the bile duct, are the origin of cholangiopathies which often necessitate liver transplants. Current progress in generating functional biliary organoids show potential for modelling cholangiopathies and validating therapeutic drugs. Organoids by groups Ogawa et al. and Sampaziotis et al. utilize soluble molecule induction, OP9 co-culture,

Cholangiocytes, the epithelial cells of the bile duct, are the origin of cholangiopathies which often necessitate liver transplants. Current progress in generating functional biliary organoids show potential for modelling cholangiopathies and validating therapeutic drugs. Organoids by groups Ogawa et al. and Sampaziotis et al. utilize soluble molecule induction, OP9 co-culture, and three-dimensional culture to achieve self-organizing tissues which express mature cholangiocyte markers and show cholangiocyte functionality. This thesis describes our efforts to establish a standard for functional PSC-derived bile duct tissues. By directing cell fate and patterning through external cues alone, we were able to produce CK19+ALB+ hepatoblast-like cells. These soluble molecule-induced cells also expressed EpCAM and CEBPA, suggesting the presence of early liver epithelial cells. However, inconsistent results and high levels of cell death with soluble molecule induction in early stages of differentiation prompted the development of a combinatory differentiation method which utilized multiple differentiation tools. We opted to combine transcription-factor triggered differentiation with soluble molecule-mediated differentiation to produce early biliary cells with the potential to develop into mature cholangiocytes. By combining genetic engineering through the activation of doxycycline-inducible GATA6 switch and microbead-mediated CXCR4 separation, we generated patterned tissues which expressed early biliary markers, CD146, CK19, and SOX9. In the future, three-dimensional cell culture and OP9 co-culture could improve our current results by facilitating 3D cellular self-organization and promoting NOTCH signaling for cholangiocyte maturation. Next steps for this research include optimizing media formulations, tracking gene expression over time, and testing the functionality of generated tissues.
ContributorsGo, Suyen Chantal (Author) / Ebrahimkhani, Mohammad (Thesis director) / Kiani, Samira (Committee member) / Harrington Bioengineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
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Description
Several debilitating neurological disorders, such as Alzheimer's disease, stroke, and spinal cord injury, are characterized by the damage or loss of neuronal cell types in the central nervous system (CNS). Human neural progenitor cells (hNPCs) derived from human pluripotent stem cells (hPSCs) can proliferate extensively and differentiate into the various

Several debilitating neurological disorders, such as Alzheimer's disease, stroke, and spinal cord injury, are characterized by the damage or loss of neuronal cell types in the central nervous system (CNS). Human neural progenitor cells (hNPCs) derived from human pluripotent stem cells (hPSCs) can proliferate extensively and differentiate into the various neuronal subtypes and supporting cells that comprise the CNS. As such, hNPCs have tremendous potential for disease modeling, drug screening, and regenerative medicine applications. However, the use hNPCs for the study and treatment of neurological diseases requires the development of defined, robust, and scalable methods for their expansion and neuronal differentiation. To that end a rational design process was used to develop a vitronectin-derived peptide (VDP)-based substrate to support the growth and neuronal differentiation of hNPCs in conventional two-dimensional (2-D) culture and large-scale microcarrier (MC)-based suspension culture. Compared to hNPCs cultured on ECMP-based substrates, hNPCs grown on VDP-coated surfaces displayed similar morphologies, growth rates, and high expression levels of hNPC multipotency markers. Furthermore, VDP surfaces supported the directed differentiation of hNPCs to neurons at similar levels to cells differentiated on ECMP substrates. Here it has been demonstrated that VDP is a robust growth and differentiation matrix, as demonstrated by its ability to support the expansions and neuronal differentiation of hNPCs derived from three hESC (H9, HUES9, and HSF4) and one hiPSC (RiPSC) cell lines. Finally, it has been shown that VDP allows for the expansion or neuronal differentiation of hNPCs to quantities (>1010) necessary for drug screening or regenerative medicine purposes. In the future, the use of VDP as a defined culture substrate will significantly advance the clinical application of hNPCs and their derivatives as it will enable the large-scale expansion and neuronal differentiation of hNPCs in quantities necessary for disease modeling, drug screening, and regenerative medicine applications.
ContributorsVarun, Divya (Author) / Brafman, David (Thesis advisor) / Nikkhah, Mehdi (Committee member) / Stabenfeldt, Sarah (Committee member) / Arizona State University (Publisher)
Created2016
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Description
Stromal cell-derived factor-1α (SDF-1α) and its key receptor, CXCR4 are ubiquitously expressed in systems across the body (e.g. liver, skin, lung, etc.). This signaling axis regulates a myriad of physiological processes that range from maintaining of organ homeostasis in adults to, chemotaxis of stem/progenitor and immune cell types after injury.

Stromal cell-derived factor-1α (SDF-1α) and its key receptor, CXCR4 are ubiquitously expressed in systems across the body (e.g. liver, skin, lung, etc.). This signaling axis regulates a myriad of physiological processes that range from maintaining of organ homeostasis in adults to, chemotaxis of stem/progenitor and immune cell types after injury. Given its potential role as a therapeutic target for diverse applications, surprisingly little is known about how SDF-1α mediated signaling propagates through native tissues. This limitation ultimately constrains rational design of interventional biomaterials that aim to target the SDF-1α/CXCR4 signaling axis. One application of particular interest is traumatic brain injury (TBI) for which, there are currently no means of targeting the underlying biochemical pathology to improve prognosis.

Growing evidence suggests a relationship between SDF-1α/CXCR4 signaling and endogenous neural progenitor/stem cells (NPSC)-mediated regeneration after neural injury. Long-term modulation of the SDF-1α/CXCR4 signaling axis is thus hypothesized as a possible avenue for harnessing and amplifying endogenous regenerative mechanisms after TBI. In order to understand how the SDF-1α/CXCR4 signaling can be modulated in vivo, we first developed and characterized a sustained protein delivery platform in vitro. We were the first, to our knowledge, to demonstrate that protein release profiles from poly(D,L,-lactic-co-glycolic) acid (PLGA) particles can be tuned independent of particle fabrication parameters via centrifugal fractioning. This process of physically separating the particles altered the average diameter of a particle population, which is in turn was correlated to critical release characteristics. Secondly, we demonstrated sustained release of SDF-1α from PLGA/fibrin composites (particles embedded in fibrin) with tunable burst release as a function of fibrin concentration. Finally, we contrasted the spatiotemporal localization of endogenous SDF-1α and CXCR4 expression in response to either bolus or sustained release of exogenous SDF-1α. Sustained release of exogenous SDF-1α induced spatially diffuse endogenous SDF-1/CXCR4 expression relative to bolus SDF-1 administration; however, the observed effects were transient in both cases, persisting only to a maximum of 3 days post injection. These studies will inform future systematic evaluations of strategies that exploit SDF-1α/CXCR4 signaling for diverse applications.
ContributorsDutta, Dipankar (Author) / Stabenfeldt, Sarah E (Thesis advisor) / Kleim, Jeffrey (Committee member) / Nikkhah, Mehdi (Committee member) / Sirianni, Rachael (Committee member) / Vernon, Brent (Committee member) / Arizona State University (Publisher)
Created2016
Description
Cardiovascular disease (CVD) remains the leading cause of mortality, resulting in 1 out of 4 deaths in the United States at the alarming rate of 1 death every 36 seconds, despite great efforts in ongoing research. In vitro research to study CVDs has had limited success, due to lack of

Cardiovascular disease (CVD) remains the leading cause of mortality, resulting in 1 out of 4 deaths in the United States at the alarming rate of 1 death every 36 seconds, despite great efforts in ongoing research. In vitro research to study CVDs has had limited success, due to lack of biomimicry and structural complexity of 2D models. As such, there is a critical need to develop a 3D, biomimetic human cardiac tissue within precisely engineered in vitro platforms. This PhD dissertation involved development of an innovative anisotropic 3D human stem cell-derived cardiac tissue on-a-chip model (i.e., heart on-a-chip), with an enhanced maturation tissue state, as demonstrated through extensive biological assessments. To demonstrate the potential of the platform to study cardiac-specific diseases, the developed heart on-a-chip was used to model myocardial infarction (MI) due to exposure to hypoxia. The successful induction of MI on-a-chip (heart attack-on-a-chip) was evidenced through fibrotic tissue response, contractile dysregulation, and transcriptomic regulation of key pathways.This dissertation also described incorporation of CRISPR/Cas9 gene-editing to create a human induced pluripotent stem cell line (hiPSC) with a mutation in KCNH2, the gene implicated in Long QT Syndrome Type 2 (LQTS2). This novel stem cell line, combined with the developed heart on-a-chip technology, led to creation of a 3D human cardiac on-chip tissue model of LQTS2 disease.. Extensive mechanistic biological and electrophysiological characterizations were performed to elucidate the mechanism of R531W mutation in KCNH2, significantly adding to existing knowledge about LQTS2. In summary, this thesis described creation of a LQTS2 cardiac on-a-chip model, incorporated with gene-edited hiPSC-cardiomyocytes and hiPSC-cardiac fibroblasts, to study mechanisms of LQTS2. Overall, this dissertation provides broad impact for fundamental studies toward cardiac biological studies as well as drug screening applications. Specifically, the developed heart on-a-chip from this dissertation provides a unique alternative platform to animal testing and 2D studies that recapitulates the human myocardium, with capabilities to model critical CVDs to study disease mechanisms, and/or ultimately lead to development of future therapeutic strategies.
ContributorsVeldhuizen, Jaimeson (Author) / Nikkhah, Mehdi (Thesis advisor) / Brafman, David (Committee member) / Ebrahimkhani, Mo (Committee member) / Migrino, Raymond Q (Committee member) / Plaisier, Christopher (Committee member) / Arizona State University (Publisher)
Created2021
Description

Lab-grown food products of animal cell origin, now becoming popularly coined as, ‘Cellular Agriculture’ is a revolutionary breakthrough technology that has the potential to penetrate the lives of every American or citizen of the world. It is important to recognize that the impetus for developing this technology is fueled by

Lab-grown food products of animal cell origin, now becoming popularly coined as, ‘Cellular Agriculture’ is a revolutionary breakthrough technology that has the potential to penetrate the lives of every American or citizen of the world. It is important to recognize that the impetus for developing this technology is fueled by environmental concerns with climate change, rising geopolitical instability, and population growth projections, where farm-grown food has now become a growing national security issue. Notwithstanding its potential, in addition to the necessary technological innovation and economic scalability, the market success of cellular agriculture will depend greatly on regulatory oversight by multiple government agencies without which it can cause undue harm to individuals, populations, and the environment. Thus, it is critical for those appropriate United States governing bodies to ensure that the technology being developed is both safe and of an acceptable quality for human consumption and has no adverse environmental impact. As such, animal foods, derived from farms, previously regulated almost exclusively by the United States Department of Agriculture (USDA) are now being regulated under a joint formal agreement between the US Food and Drug Administration (US FDA) and the USDA if derived from the lab, i.e., lab-grown animal foods. The main reason for joint oversight between the FDA and the USDA is that the FDA has developed the in-house expertise to oversee primary cell harvesting and cell storage, as well as, cell growth and differentiation for the development of 3D-engineered tissues intended for tissue and organ replacement for the emerging field of regenerative medicine. As such, the FDA has been given the authority to oversee the ‘front end’ of lab-grown food processes which relies on the very same processes utilized in engineered human tissues to produce food-grade engineered tissues. Oversight then transitions to the USDA-FSIS (Food Safety and Inspection Service) during the harvesting stage of the cell culture process. The USDA-FSIS then oversees the further production and labeling of these products. Included in the agreement is the understanding that both bodies are responsible for communicating necessary information to each other and collaboratively developing new regulatory actions as needed. However, there currently lacks clarity on some topics regarding certain legal, ethical, and scientific issues. Lab-grown meat products require more extensive regulation than farm-grown animal food products to ensure that they are safe and nutritious for consumption. To do this, CFSAN can create new classes of lab-grown foods, such as ‘lab-grown USDA foods,’ ‘lab-grown non-USDA foods,’ ‘lab-grown extinct foods,’ ‘lab-grown human food tissues,’ and ‘medically activated lab-grown foods.’

ContributorsBanen, Samuel (Author) / Pizziconi, Vincent (Thesis director) / Feigal, David (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor) / School of Molecular Sciences (Contributor)
Created2023-05
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Description
The WNT signaling pathway plays numerous roles in development and maintenance of adult homeostasis. In concordance with it’s numerous roles, dysfunction of WNT signaling leads to a variety of human diseases ranging from developmental disorders to cancer. WNT signaling is composed of a family of 19 WNT soluble secreted glycoproteins,

The WNT signaling pathway plays numerous roles in development and maintenance of adult homeostasis. In concordance with it’s numerous roles, dysfunction of WNT signaling leads to a variety of human diseases ranging from developmental disorders to cancer. WNT signaling is composed of a family of 19 WNT soluble secreted glycoproteins, which are evolutionarily conserved across all phyla of the animal kingdom. WNT ligands interact most commonly with a family of receptors known as frizzled (FZ) receptors, composed of 10 independent genes. Specific interactions between WNT proteins and FZ receptors are not well characterized and are known to be promiscuous, Traditionally canonical WNT signaling is described as a binary system in which WNT signaling is either off or on. In the ‘off’ state, in the absence of a WNT ligand, cytoplasmic β-catenin is continuously degraded by the action of the APC/Axin/GSK-3β destruction complex. In the ‘on’ state, when WNT binds to its Frizzled (Fz) receptor and LRP coreceptor, this protein destruction complex is disrupted, allowing β-catenin to translocate into the nucleus where it interacts with the DNA-bound T cell factor/lymphoid factor (TCF/LEF) family of proteins to regulate target gene expression. However in a variety of systems in development and disease canonical WNT signaling acts in a gradient fashion, suggesting more complex regulation of β-catenin transcriptional activity. As such, the traditional ‘binary’ view of WNT signaling does not clearly explain how this graded signal is transmitted intracellularly to control concentration-dependent changes in gene expression and cell identity. I have developed an in vitro human pluripotent stem cell (hPSC)-based model that recapitulates the same in vivo developmental effects of the WNT signaling gradient on the anterior-posterior (A/P) patterning of the neural tube observed during early development. Using RNA-seq and ChIP-seq I have characterized β-catenin binding at different levels of WNT signaling and identified different classes of β-catenin peaks that bind cis-regulatory elements to influence neural cell fate. This work expands the traditional binary view of canonical WNT signaling and illuminates WNT/β-catenin activity in other developmental and diseased contexts.
ContributorsCutts, Joshua Patrick (Author) / Brafman, David A (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Nikkhah, Mehdi (Committee member) / Wang, Xiao (Committee member) / Plaisier, Christopher (Committee member) / Arizona State University (Publisher)
Created2019