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Creating Biochemical Gradients via Photoconjugation and an In-House Designed Sliding Photomask

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Tissue engineering is an emerging field focused on the repair, replacement, and regeneration of damaged tissue. Engineered tissue consists of three factors: cells, biomolecular signals, and a scaffold. Cell-free scaffolds present a unique opportunity to develop highly specific microenvironments with

Tissue engineering is an emerging field focused on the repair, replacement, and regeneration of damaged tissue. Engineered tissue consists of three factors: cells, biomolecular signals, and a scaffold. Cell-free scaffolds present a unique opportunity to develop highly specific microenvironments with tunable properties. Norbornene-functionalized hyaluronic acid (NorHA) hydrogels provide spatial control over biomolecule binding through a photopolymerization process. With this, biomimetic gradients can be produced to model a variety of tissue interfaces. To produce these patterns, a gradient mechanism was developed to function in tandem with a syringe pump. A conversion equation was derived to calculate a panel speed from the volumetric flow rate setting on the pump. Seven speeds were used to produce fluorophore gradients on the surface of NorHA hydrogels to assess changes in the length and slope of the gradient. The results indicated a strong positive linear correlation between the speed of the panel and the length of the gradient as well as a strong negative correlation between the speed of the panel and the slope of the gradient. Additionally, the mechanism was able to successfully produce several other types of gradients including multiregional, dual, and triregional.

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2019-05

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Pharmacologic Modulation of the Blood-Brain Barrier

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One of the most prominent biological challenges for the field of drug delivery is the blood-brain barrier. This physiological system blocks the entry of or actively removes almost all small molecules into the central nervous system (CNS), including many drugs

One of the most prominent biological challenges for the field of drug delivery is the blood-brain barrier. This physiological system blocks the entry of or actively removes almost all small molecules into the central nervous system (CNS), including many drugs that could be used to treat diseases in the CNS. Previous studies have shown that activation of the adenosine receptor signaling pathway through the use of agonists has been demonstrated to increase BBB permeability. For example, regadenoson is an adenosine A2A receptor agonist that has been shown to disrupt the BBB and allow for increased drug uptake in the CNS. The goal of this study was to verify this property of regadenoson. We hypothesized that co-administration of regadenoson with a non-brain penetrant macromolecule would facilitate its entry into the central nervous system. To test this hypothesis, healthy mice were administered regadenoson or saline concomitantly with a fluorescent dextran solution. The brain tissue was either homogenized to measure quantity of fluorescent molecule, or cryosectioned for imaging with confocal fluorescence microscopy. These experiments did not identify any significant difference in the amount of fluorescence detected in the brain after regadenoson treatment. These results contradict those of previous studies and highlight potential differences in injection methodology, time windows, and properties of brain impermeant molecules.

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2015-05

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The Effect of Nanoparticle Diameter on TAT-mediated Delivery to the CNS In Vivo

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Neurological disorders are difficult to treat with current drug delivery methods due to their inefficiency and the lack of knowledge of the mechanisms behind drug delivery across the blood brain barrier (BBB). Nanoparticles (NPs) are a promising drug delivery method

Neurological disorders are difficult to treat with current drug delivery methods due to their inefficiency and the lack of knowledge of the mechanisms behind drug delivery across the blood brain barrier (BBB). Nanoparticles (NPs) are a promising drug delivery method due to their biocompatibility and ability to be modified by cell penetrating peptides, such as transactivating transciptor (TAT) peptide, which has been shown to increase efficiency of delivery. There are multiple proposed mechanisms of TAT-mediated delivery that also have size restrictions on the molecules that can undergo each BBB crossing mechanism. The effect of nanoparticle size on TAT-mediated delivery in vivo is an important aspect to research in order to better understand the delivery mechanisms and to create more efficient NPs. NPs called FluoSpheres are used because they come in defined diameters unlike polymeric NPs that have a broad distribution of diameters. Both modified and unmodified 100nm and 200nm NPs were able to bypass the BBB and were seen in the brain, spinal cord, liver, and spleen using confocal microscopy and a biodistribution study. Statistically significant differences in delivery rate of the different sized NPs or between TAT-modified and unmodified NPs were not found. Therefore in future work a larger range of diameter size will be evaluated. Also the unmodified NPs will be conjugated with scrambled peptide to ensure that both unmodified and TAT-modified NPs are prepared in identical fashion to better understand the role of size on TAT targeting. Although all the NPs were able to bypass the BBB, future work will hopefully provide a better representation of how NP size effects the rate of TAT-mediated delivery to the CNS.

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2016-05

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Investigating the efficacy of novel TrkB agonists to augment stroke recovery

Description

Stroke remains the leading cause of adult disability in developed countries. Most survivors live with residual motor impairments that severely diminish independence and quality of life. After stroke, the only accepted treatment for these patients is motor rehabilitation. However, the

Stroke remains the leading cause of adult disability in developed countries. Most survivors live with residual motor impairments that severely diminish independence and quality of life. After stroke, the only accepted treatment for these patients is motor rehabilitation. However, the amount and kind of rehabilitation required to induce clinically significant improvements in motor function is rarely given due to the constraints of our current health care system. Research reported in this dissertation contributes towards developing adjuvant therapies that may augment the impact of motor rehabilitation and improve functional outcome. These studies have demonstrated reorganization of maps within motor cortex as a function of experience in both healthy and brain-injured animals by using intracortical microstimulation technique. Furthermore, synaptic plasticity has been identified as a key neural mechanism in directing motor map plasticity, evidenced by restoration of movement representations within the spared cortical tissue accompanied by increase in synapse number translating into motor improvement after stroke. There is increasing evidence that brain-derived neurotrophic factor (BDNF) modulates synaptic and morphological plasticity in the developing and mature nervous system. Unfortunately, BDNF itself is a poor candidate because of its short half-life, low penetration through the blood brain barrier, and activating multiple receptor units, p75 and TrkB on the neuronal membrane. In order to circumvent this problem efficacy of two recently developed novel TrkB agonists, LM22A-4 and 7,8-dihydroxyflavone, that actively penetrate the blood brain barrier and enhance functional recovery. Findings from these dissertation studies indicate that administration of these pharmacological compounds, accompanied by motor rehabilitation provide a powerful therapeutic tool for stroke recovery.

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2013

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A robust vitronectin-derived peptide substrate for the scalable long-term expansion and neuronal differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (hNPCs)

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

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.

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Date Created
2016

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Using bioengineering approaches to generate a three-dimensional (3D) human pluripotent stem cell (hPSC)-based model for neurodegenerative diseases

Description

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

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.

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Date Created
2016

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Using Molecular, Cellular and Bioengineering Approaches Towards Understanding Muscle Stem Cell Biology

Description

Satellite cells are adult muscle stem cells that activate, proliferate, and differentiate into myofibers upon muscle damage. Satellite cells can be cultured and manipulated in vitro, and thus represent an accessible model for studying skeletal muscle biology, and a potential

Satellite cells are adult muscle stem cells that activate, proliferate, and differentiate into myofibers upon muscle damage. Satellite cells can be cultured and manipulated in vitro, and thus represent an accessible model for studying skeletal muscle biology, and a potential source of autologous stem cells for regenerative medicine. This work summarizes efforts to further understanding of satellite cell biology, using novel model organisms, bioengineering, and molecular and cellular approaches. Lizards are evolutionarily the closest vertebrates to humans that regenerate entire appendages. An analysis of lizard myoprogenitor cell transcriptome determined they were most transcriptionally similar to mammalian satellite cells. Further examination showed that among genes with the highest level of expression in lizard satellite cells were an increased number of regulators of chondrogenesis. In micromass culture, lizard satellite cells formed nodules that expressed chondrogenic regulatory genes, thus demonstrating increased musculoskeletal plasticity. However, to exploit satellite cells for therapeutics, development of an ex vivo culture is necessary. This work investigates whether substrates composed of extracellular matrix (ECM) proteins, as either coatings or hydrogels, can support expansion of this population whilst maintaining their myogenic potency. Stiffer substrates are necessary for in vitro proliferation and differentiation of satellite cells, while the ECM composition was not significantly important. Additionally, satellite cells on hydrogels entered a quiescent state that could be reversed when the cells were subsequently cultured on Matrigel. Proliferation and gene expression data further indicated that C2C12 cells are not a good proxy for satellite cells. To further understand how different signaling pathways control satellite cell behavior, an investigation of the Notch inhibitor protein Numb was carried out. Numb deficient satellite cells fail to activate, proliferate and participate in muscle repair. Examination of Numb isoform expression in satellite cells and embryonic tissues revealed that while developing limb bud, neural tube, and heart express the long and short isoforms of NUMB, satellite cells predominantly express the short isoforms. A preliminary immunoprecipitation- proteomics experiment suggested that the roles of NUMB in satellite cells are related to cell cycle modulation, cytoskeleton dynamics, and regulation of transcription factors necessary for satellite cell function.

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2020

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Investigating the Mechanism of a Multi-State Model of WNT Signaling

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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

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.

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2019