Dr. Franklin West, Leader: RBC Fellows Program Dear Students, Faculty and Guest, It is my great pleasure to welcome you to the first Annual RBC Undergraduate Research Fellows Symposium. This symposium gives all of our fellows the opportunity to showcase their hard work, to present their exciting findings and to experience the thrill of exchanging scientific ideas.
The program is sponsored and administered by the Regenerative Bioscience Center at the University of Georgia. The Fellows Program incorporates faculty from the Departments of Franklin West, a University of Georgia Assistant Professor of
Animal and Dairy Sciences, Engineering, and Veterinary Medicine, as well as faculty from
Animal and Dairy Science in the College of Agricultural and Environmental Sciences, sits on the steering committee for the UGA Regenerative Bioscience Center and was named one of the nation's top scholars under 40 by Diverse: Issues in Higher Education magazine. He was recently nominated for
Georgia Tech and Emory
the 2015 Blavatnik National Awards for Young Scientists.
What these students have learned in the lab will not only make them better scientist, but better people — lifelong learners that question and strive to better understand the world around them.
University.
Students gain research experience under the mentorship of internationally recognized academic scientists, graduate students, postdoctoral researchers, and industrial collaborators. Thus, our students
The RBC Undergraduate Research Fellows program is a new initiative and has been wildly successful with 43 fellows in its’ inaugural year. These students have been trained in laboratories across the university spanning material science, exercise science, stem cell biology, embryology, genetic engineering and beyond. Many of these students will be taking the research techniques, problem solving and critical thinking skills that they have honed in the laboratory this year and applying them in research externships and internships at human and veterinary hospitals across the world.
RBC Selection Committee Dr. Steven Stice Director Regenerative Bioscience Center D.W. Brooks Distinguished Professor GRA Eminent Scholar
As faculty, we have had the honor and privilege to work with some truly exceptional students this year and we look forward to watching as they develop into the scientist, veterinarians, medical doctors, engineers and dentist of tomorrow. Sincerely, Franklin West PhD
Dr. Shanta Dhar Assistant Professor Department of Chemistry
will be uniquely positioned to apply for graduate and professional schools with the opportunity to have a very strong foundation on which to build a successful career.
Dr. Lohitash Karumbaiah Assistant Professor College of Agricultural and Environmental Science Dr. Jarrod Call Assistant Professor College of Kinesiology Dr. Luke Mortensen Assistant Professor College of AES and Engineering
RBC CORE RESEARCH Neuroscience Research Bone Tissue Regeneration Cancer Therapies Toxicology Advanced Imaging
For more information please visit us online: www.RBC.uga.edu
Dr. John Peroni Associate Professor College of Veterinary Medicine Dr. Hongxiang Liu Assistant Professor College of Agricultural and Environmental Science
425 River Road Athens, GA 30602 www.rbc.uga.edu
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symposium agenda Wednesday April 22, 2015
12:30 – 1:30: Public Poster Viewing 1:45 – 2:00: Opening Remarks with Dr. Jarrod Call, assistant professor in the College of Education, distinguished member of the RBC, PhD University of Minnesota. Dr. Call received an American Heart Association Postdoctoral Fellowship for his work in skeletal muscle-heart crosstalk in prevention of diabetic cardiomyopathy. ORAL PRESENTATIONS (5 minutes allotted between each speaker for question and response)
2:00 – 2:10: Caroline Coleman; Social Recognition Cognitive Testing in a Piglet Model.
2:15 – 2:25: Christina Ethridge; Characterization of Leigh’s Syndrome Patient Human Induced Pluripotent Stem Cells for Clinical Research.
2:30 – 2:40: Sarah Shaver; Plus T-Maze Testing of Pigs’ Ability to Learn and Create Spatial Memories.
2:45 – 2:55: Piyush Joshi; A Chicken Embryo Culture System for Injections and Imaging.
1110 Minute Break
A donation to the RBC fund could help change the life of a student. With your donation
3:10 – 3:20: Mary Kate Mehegan; Development and Characterization of a Novel Landrace Piglet Cortical Impact Traumatic Brain Injury Model.
3:25 – 3:35: Jennifer Roveto; Assessment of Spontaneous Trial-Unique Memory using a Piglet Model.
3:40 – 3:50: Kayla Hargrove; Utilizing stride velocity, swing, stance, and break-over time to assess motor function and efficacy of iNSCs in a porcine ischemic stroke model.
3:55 – 4:05: Catherine (Cali) Callaway; Development of a Chimeric Chick Neural Tube Injury Model Incorporating 3D, mESC-Derived Neural Aggregates
students can actively participate in breakthrough technologies. Your generosity and support is viewed more than just a financial contribution, but also an
4:10 – Closing Remarks with RBC Fellows Program Leader, Dr. Franklin West.
opportunity to establish meaningful partnerships with those whose values we share and whose actions truly drive change.
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Oral Presentations Listed in Alphabetical Order
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Catherine “Cali” Callaway Mentor : Ste ven S tice
Oral Presen ta ti on Class o f 2017
Development of a Chimeric Chick Neural Tube Injury Model Incorporating 3D, mESCDerived Neural Aggregates Authors: Catherine Callaway, Elizabeth Wilkins
Due to low cost, availability and ex utero development of the chicken embryo, it serves as the oldest vertebrate developmental model. During development, the primitive spinal cord, termed the neural tube, is patterned caudally by retinoid signaling, and ventrally by a gradient of Sonic Hedgehog (Shh), yielding five specific progenitor domains. The pMN domain gives rise to motor neurons (MN), which synapse on muscles. The other domains yield interneurons, which compose the premotor control circuit. This neural network is cumulatively responsible for controlling all muscle actuation. We generated three-dimensional aggregates of mouse embryonic stem cells (mESC) from a cell line expressing a green fluorescent protein behind a MN-specific promoter. The aggregates were patterned with retinoic acid and a Shh pathway agonist to mimic organotypic ventral neural tube development. The line also expresses channel rhodopsin-2, a lightgated cation channel, which triggers depolarization in neurons when exposed to blue light. We hypothesize that embryos will survive 72 hours post-injection, and that the aggregates, including optically active motor neurons, will integrate into the injured spinal cord. A portion of the neural tube of developing chicks was removed in ovo, followed by microinjection of neural aggregates. After three days, embryos were inspected for survival and integration. Ultimately, we expect a light stimulus to MN will result in a visible motor response within the embryo. As seen in paralytic injuries, adult spinal tissue has limited regenerative properties, making cell replacement an attractive therapy. Here, we provide the first steps towards making that a reality.
Caroline Coleman Mentor : Fran klin Wes t
Oral Presen ta ti on Class o f 2016
Social Recognition Cognitive Testing in a Piglet Mode Authors: Caroline Coleman
According to the CDC, traumatic brain injuries (TBI) are a contributing factor to over 30% of all injury-related deaths in the United States every year, with children aged 0-4 at the highest risk. In addition, there are limited therapy options available for the treatment of TBI and any resulting functional cognitive deficits. Behavioral tests are a useful tool in quantified these deficits following a TBI, giving researchers a representative assessment of the subject’s cognitive state. Due to similarities to the human toddler (age 0-4) in brain structure and development, we used a piglet model to develop a set of accurate, repeatable behavioral tests that would quantify the piglet’s cognitive state. One behavioral test that was developed is the 3-chamber social recognition test. The social recognition test measures sociability, or an inclination towards social interaction, and social memory, or the ability to distinguish between familiar and unfamiliar piglets. The experiment involved four piglets, aged five weeks old, and was conducted twice, five days apart. We found that the test piglets preferred social interaction with the stimulus piglet to interaction with the novel object, thus exhibiting sociability. We also found that test piglets were able to distinguish familiar pigs from unfamiliar piglets and preferentially interacted more with the unfamiliar piglet, demonstrating social memory. The successful development of this behavioral test will allow us, in the future, to assess both cognitive deficits in our piglet model following TBI and potential improvements following human induced pluripotent stem cell-derived neural stem cell treatment. 5|P a g e
Christina Ethridge Mentor : Fran klin Wes t
Oral Presen ta ti on Class o f 2016
Characterization of Leigh’s Syndrome Patient Human Induced Pluripotent Stem Cells for Clinical Research Authors: Christina Ethridge
Background: Leigh’s Syndrome is an inherited disorder of mitochondrial energy metabolism that affects the central nervous system. Symptoms include muscular and neurological degradation leading to death within the first few years of life. No curative treatments are currently available. The reprogramming of mitochondrial diseased fibroblasts into induced pluripotent stem cells will allow for their differentiation into multiple cell types and tissues. If reprogramming is possible, this will function as an invaluable tool for studying disease pathophysiology in vitro with the intention of designing future gene therapies and pharmacological treatments for affected patients. Objective: Our goal in this study was to generate and characterize induced pluripotent stem cells (iPSCs) derived from skin fibroblasts of Leigh’s Syndrome patients. Methods: Generated LS-iPSCs using a non-viral, non-integrating mRNA and microRNA system, then characterized iPSCs using immunofluorescence staining for pluripotency markers (Oct4, Sox2, Nanog, Tra-181, Tra-160, and SSEA4). iPSCs were differentiated and stained for endoderm (VIM, AFP), ectoderm (MAP2, TUBB3), and mesoderm (ACTA2, Desmin) germ layer markers. Results: The generated LS-iPSCs expressed markers of pluripotency as seen by immunofluorescence staining. Differentiated iPSCs staining showed expression of tissue markers from all three germ layers. Conclusion: Characterization of the iPSCs derived from Leigh’s Syndrome patient fibroblasts demonstrated that the LS-iPSCs possess characteristics of true stem cells.
Kayla Hargrove Mentor : Fran klin Wes t
Oral Presen ta ti on Class o f 2016
Utilizing stride velocity, swing, stance, and break-over time to assess motor function and efficacy of iNSCs in a porcine ischemic stroke model Authors: Kayla Hargrove, Dr. Franklin West, Dr. Kylee Jo Duberstein, Trinh Nguyen
A cerebrovascular accident, also known as a stroke, is one of the current leading causes of death and long term disability in the United States. Stroke survivors are forced to undergo long-term rehabilitation in an attempt to regain lost motility and motor function, but are often unsuccessful. Although effective treatment options are limited, induced pluripotent stem cell-derived neural stem cells (iNSCs) have shown promise as a potential regenerative therapy. While previously conducted studies have utilized rodent models, the white/gray matter composition, size, and gyrencephalicism of the porcine brain offer a more translational model to humans. Yucatan pigs were utilized in this study to establish a baseline for normal gait characteristics, assess the deficits in motor function following middle cerebral artery (MCA) occlusion, and test the efficacy of iNSCs by assessing changes in motor function after administration. Pigs were trained to navigate a semicircular track and then recorded at 3 time points prior to stroke, 1, 3, and 5 days post infarction, as well as 1 day, 3 days, 1 week, 2 weeks, 4 weeks, 6 weeks, 9 weeks, and 12 weeks after iNSC or vehicle only (PBS) injection. MRI diffusion weighted imaging (DWI) and apparent diffusion coefficient (ADC) maps confirmed ischemia in each occluded pig. Evaluation of the velocity, hind limb swing, stance, and break-over time revealed marked differences in symmetry after occlusion. Shorter swing time and longer stance time were noted in the paretic limb contralateral to stroked side.
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Piyush Joshi Mentor : Ste ven S tice
Oral Presen ta ti on Class o f 2015
A Chicken Embryo Culture System for Injections and Imaging Authors: Piyush Joshi
The goal of this project is to establish an efficient method of Stage X chicken embryo injections to investigate the dynamics of the early CNS development. Human neural progenitor stem cells (hNPs) will be injected into the developing chicken embryo at Stage X and tracked with fluorescence microscopy. After injection, we expect the cells will migrate and integrate into the central nervous system (CNS) of the chicken. A reliable method of Stage X chicken injection and imaging will allow the observation of human neural cells participating in primary neurulation. When successful, this will provide a means of studying early stages of CNS development as an aspect of a larger project to investigate development neural toxicology with the chick as a model system.
Mary Kate Mehegan Mentor : Fran klin Wes t
Oral Presen ta ti on Class o f 2016
Development and Characterization of a Novel Landrace Piglet Cortical Impact Traumatic Brain Injury Model Authors: Mary Kate Mehegan, Emily Wyatt, Holly Kinder, Jessica McCabe, and Franklin West
In the year 2010, 2.5 million people suffered from a traumatic brain injury (TBI). In the United States alone, approximately 50,000 deaths result from TBIs annually. At this time, there is no adequate TBI treatment available. Recently, the West Laboratory developed induced pluripotent stem cell-derived neural stem cells (iPSC-NSCs). These iPSC-NSCs may potentially serve as a regenerative cell replacement therapy as they are capable of differentiating into neurons, astrocytes and oligodendrocytes while also producing regenerative factors such as VEGF. Although these cells have been shown to lead to significant structural and functional improvement in rodent models, treatments that have been developed in rodent models have regularly failed in clinical trials. Thus, more predictive large animal models are needed. The pig serves as an excellent large animal model with a large gyrencephalic brain that has gray-white matter composition similar to humans, unlike rodent models. We have developed a model with four treatment groups; 2 m/s and 4 m/s at 6 mm impact velocity, as well as 4 m/s at 12 mm and 15 mm depth. This study serves as a model for future iPSC-NSC therapy studies. We hypothesize piglets receiving a cortical impact will develop brain lesions, show changes in inflammatory response, macrophage infiltration, glial scaring and changes in motor function deficits ranging from mild to severe based on impact speed. Development of this model will allow for the testing of efficacy and safety of novel stem cell therapies as well as traditional pharmacological and device approaches. 7|P a g e
Jennifer Roveto Mentor : Fran klin Wes t
Oral Presen ta ti on Class o f 2015
Assessment of Spontaneous Trial-Unique Memory using a Piglet Model Authors: Roveto, J., Kinder, H., Wyatt, E., West, F.
People throughout the world, especially children ages 0-4, suffer from traumatic brain injuries (TBIs) that often result in death or permanent disabilities. No treatments are currently available to address the short and long term neurological deficits that often result from a TBI. In an effort to better assess the cognitive changes that take place after TBI and the effectiveness of different types of TBI treatments, we designed an object recognition test to allow us to evaluate and quantify spontaneous trial-unique memory in a more translatable piglet model. We hypothesized that the normal piglets would be able to differentiate between familiar and novel objects and show a preference toward the novel objects. Four landrace piglets underwent behavioral testing on two separate days, seven days apart. In this test, one piglet was placed in an arena with two similar objects and was allowed to explore the arena for 10 minutes. The piglet was removed from the arena for 10 minutes and then returned to explore the two objects, one familiar and one novel, for another 10 minutes. Time spent with each of the objects was recorded. The piglets initially spent equal time with the two similar objects, but then spent significantly more time with the novel object than the familiar object. This demonstrates a high level of spontaneous trial-unique memory. The results of this test will allow us to quantitatively assess cognitive changes in memory a piglet with a TBI and monitor the effectiveness of different treatments.
Sarah Shaver Mentor : Fran klin Wes t
Oral Presen ta ti on Class o f 2016
Plus T-Maze Testing of Pigs’ Ability to Learn and Create Spatial Memories Authors: Shaver, S.; Kinder, H.; Wyatt, E.; West, F.
Traumatic Brain Injury is a leading cause of death and disability in the United States most often affecting children ages 04. The results of this injury may range from temporary pain to permanent impairment in cognition and physical functions. There is an urgent need to develop behavioral tests in large animal models to assess changes in cognition after a TBI and subsequent treatment. Our group has developed a Plus T-maze that can be used to assess learning and spatial memory in a pig model. We hypothesized that normal piglets could learn to navigate a Plus T-maze and form spatial memories using extra-visual cues to locate a food reward. The T-maze test was performed over two weeks and data was gathered on four pigs’ ability to learn and choose the correct reward arm of the T-maze as well as the time it takes to make that choice. In order to promote the formation of spatial memories, we randomized the start arm for each trial and placed visual cues around the testing arena. A reversal trial was performed to test the pigs’ ability to make and apply new spatial memories. Results supported our hypothesis. Throughout the study, the pigs had a statically significant increase in the proportion of time the correct reward arm was chosen as well a statistically significant decrease in the latency to choice. This data suggests that normal piglets can be tested in a Plus T-maze test to assess learning and spatial memory.
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Poster Presentations Listed in Alphabetical Order
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Mark Atuan M e n to r : Ja rro d C a l l
Po ste r P re se n t a ti o n C l a ss o f 2 0 1 5
The impact of mitochondrial quality and quantity on functional muscle recovery after traumatic injury Authors: Mark Atuan, Alexandra Flemington, Jarrod Call
The primary objective of this study is to determine if the quality and quantity of mitochondria within skeletal muscle affects muscle remodeling after traumatic muscle injury. We hypothesize that an accumulation of dysfunctional mitochondria will impair functional muscle recovery, and that enhancing mitochondrial content in skeletal muscle will improve functional muscle recovery. To test this hypothesis we separated C57Bl6 wildtype mice into three groups: normal chow (n=5), 1% beta- guanidinopropionic acid (GPA: n=10), and (15mg/kg body mass) 3-methyladenine (3-MA: n=7). GPA is a dietary supplement and creatine kinase analog, which enhances oxidative capacity and mitochondrial content in skeletal muscle. 3-MA is a broad-range autophagy inhibitor, and is used herein to impair skeletal muscle mitochondrial removal. Maximal dorsiflexion torque will be assessed following 6 weeks of intervention, along with fatigue resistance and glucose tolerance tests. The left hindlimbs (tibialis anterior, gastrocnemius muscles) will then be injured via cardiotoxin injection. At 14 days post-injury, maximal dorsiflexion torque will be reassessed to determine functional muscle recovery. Isolated mitochondria from injured and non-injured tibialis anterior muscles will be tested for maximal oxygen consumption, an index of mitochondrial function. We will analyze cell signaling pathways associated with mitochondrial biogenesis and autophagy in injured and non-injured gastrocnemius muscles. Overall, we suspect that 3MA will impair mitochondrial removal, glucose tolerance, and functional recovery of muscle strength and mitochondrial oxygen consumption compared to normal chow mice. We also suspect that GPA mice will have greater mitochondrial content, fatigue resistance, and mitochondrial function at 14-days post-injury.
Nicole Bisel M e n to r : L o h i ta sh Ka ru m b a i a h
Po ste r P re se n t a ti o n C l a ss o f 2 0 1 7
The Role of Chondroitin Sulfate Glycosaminoglycans in Glioma Cell Progression Authors: Nicole Bisel, Meghan Logun, Lohitash Karumbaiah
Glioblastoma multiforme is an aggressive, devastating type of brain tumor characterized by a highly invasive nature. Chondroitin sulfate proteoglycans (CSPGs) and their associated glycosaminoglycan (GAG) side chains have often been implicated in promoting tumor invasiveness. However, conclusive evidence to suggest that CSPGs or their associated CS-GAGs induce brain tumor invasion is currently lacking. We aim to provide evidence that suggests that tumor cell proliferation, migration, and invasion can be affected by the level of sulfation of CS-GAGs in the tumor extracellular matrix. Our goal is to demonstrate that the sulfation of the CS-GAG chains (and not the core protein itself) directly effects tumor cell progression. This will be tested in vitro by encapsulating the invasive glioma cell line U87MG-EGFP into differently sulfated CS-GAG hydrogels. We hypothesize that hydrogels containing the oversulfated GAG CS-E will inhibit tumor invasion while hydrogels containing monosulfated GAGs (CS-A and CS-C) will enhance invasion. Here we will establish a baseline for brain tumor growth in a neutral environment by encapsulating the U87MG-EGFP cells in 4% CSA/C GAG gels and 1% agarose gels. The nature of cell progression in these hydrogels will act as a reference point from which we will compare tumor growth in the sulfated CS-GAG hydrogels. If glioma malignancy is indeed influenced by the level of sulfation of CS-GAGs, this work could contribute to the development of novel therapies for brain cancer and potentially lead to better patient prognoses in clinical medicine. 10 | P a g e
Natalie Bishop Mentor : Fran klin Wes t
Poster P resen tation Class o f 2016
Using Gait Analysis to Determine the Effect of iPSC-NPCs on Motor Function in a MCAO Porcine Model Authors: Natalie Bishop, Franklin D. West, Dr. Kylee Jo J. Duberstein
Background: Ischemic stroke is one of the leading causes of death and disability worldwide. Much of research has focused on the neurological and physical deficits of strokes in small animals, which have inherent differences to the human brain including size, white matter volume, and brain structure. The Yucatan pig model for ischemic stroke successfully emulates the cephalic anatomy found in humans. With a congruent animal model, we can target an optimal treatment for motor and gait function for patients who have been affected by a life-altering stroke. Stem cells can provide a clinical treatment for the regrowth of brain cells lost in the area of the infarct. Objective: We hypothesize the total time spent on two versus three limbs in the pig model will demonstrate lowered stability, hence more time spent on three limbs, in stroked pigs. Methodology: Eight male Yucatan pigs were selected to model ischemic stroke in humans by surgical induction. Before middle cerebral artery occlusion (MCAO), the pigs were recorded using the EquineTec to record their normal gait. After MCAO, the pigs were recorded again for observation using Kinovea video analysis at multiple time points before receiving stem cell treatment or vehicle-only injection. Analysis post-injection was compared to the pigs normal gait as well as stroked. Results and Conclusion: Stem-cell treatment of ischemic stroked pigs provided strong evidence of enhancing the motor function and gait stability. With positive results, we are closer to improving stroke treatment in human patients affected by motor deficits due to ischemic stroke.
Joe Calpin Mentor : Fran klin Wes t
Poster P resen tation Class o f 2016
Induced Pluripotent Stem Cells Lead to Significant Regeneration of Motor Function in MCAO Ischemic Stroke Pig Models Authors: Joe Calpin, Dr. Franklin West, Dr. Kylee Duberstein
Ischemic stroke accounts for over eighty-five percent of all stroke cases today. In 2014, stroke was the fifth leading cause of death in the United States, according to the Centers for Disease Control and Prevention. One of the major effects that stroke has is on gait. Consequently, gait analysis is frequently used to determine the severity of stroke and the extent to which it has affected one’s overall gait. In our research, stroke was induced in 200-pound male Yucatan pigs. Some of the pigs were treated with induced pluripotent stem cells (iPSCs) and others received vehicle-only injections (PBS), to determine if these cells could help the pigs regain motor function after the stroke was induced. In order to determine the extent of the effect of the stem cell treatment on the pig subjects, gait analysis was performed using Kinovea software at fourteen time points: three pre-stroke, three post-stroke, and eight post-stem cell treatment. For this project, two and three limb support, meaning the amount of time the animal spent supporting its weight on two or three limbs, was measured. It was determined from looking at the data that the stroked pigs on average spent more time in three-limb support. In regards to the pigs treated with the iPSCs, improvement in gait/motor function was observed as early as one week after injection of the iPSCs. The results of this research suggest that treating stroke patients with iPSCs can help patients regain significant motor function after stroke or traumatic brain injury.
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Sanam Desai Mentor : Hong xi ang Liu
Poster P resen tation Class o f 2017
Distribution and Roles of Podoplanin (PDPN) in the Development of Mouse Tongue Authors: Sanam Mihir Desai, Nandakumar Venkatesan, Masako Toda, Yuji Mishina, Hong-Xiang Liu
Podoplanin (PDPN) is a type 1 integral membrane glycoprotein, rich in sialic acid and is expressed in major vital organs. Recent studies have claimed the PDPN plays an important role in the nervous system support and development. Recently we characterized the distribution of PDPN in the developing mouse tongue and its regulatory role in the development of taste organs. Immuno-histochemical analysis of the mid-late embryonic (E13.5-18.5) tongue tissues showed that PDPN was expressed in the basal cells of lingual epithelium and co-localized with Sox2. However, in taste bud cells Sox2 was highly expressed but PDPN immunoproducts were not seen. In the tongue mesenchyme, Sox2 was co-localized with β-III-tubulin in the lingual intrinsic neurons and fibers of nerve trunk. In addition, PDPN immunoproducts were also seen in the tongue muscle cells. In the Sox2-Cre driven podoplanin (PDPN-) mutant tissues, PDPN immunoproducts were not found in any cells in the tongue indicating the successful deletion of PDPN. Hematoxylin and eosin staining of the mutant tongue tissues showed that the lingual epithelium and muscle were highly disorganized, which suggests that PDPN is important in the development of tongue organs. We are planning on using a Cre driven conditional knockout to determine the function of PDPN in different cell types in the taste organs in embryonic and adult mice.
Sonal Dugar Mentor : Fran klin Wes t
Poster P resen tation Class o f 2017
Determining the Effectiveness of iNPC Treatment in a Stroke Pig Model through Gait Analysis Authors: Sonal Dugar
With a ranking amongst the top common causes of mortality in humans, stroke has become known as a disorder with widespread and debilitating effects. Although the location and causes of the disorder have been recognized, there are still many developments that need to be made in the area of possible treatments. The objective of the study was to determine whether iNPC treatment could regenerate damaged brain tissue after stroke and in turn improve the functional outcome of stroke. In order to effectively determine and quantify the potential success of this treatment, this study analyzed changes in motor function as an indicator of the extent of stroke damage. We induced ischemic stroke in pig models, chosen because of their anatomical similarity to human brain, and then measured motor function with gait analysis across various parameters. In this specific study, the total time spent on each the 2-limb and 3-limb support within one full stride was measured. A series of eight pigs were measured at the set points of before stroke, 1, 3, and 5 days post stroke, and 1 day, 3 days, 1, 2, 4, 6, 9, and 12 weeks post injection. The treatment groups involved were either injection with the pluripotent stem cells or with the PBS vehicle only. Interpretation of the results will provide the necessary data to determine whether the injection of stem cells is a safe option to lessen the damage of stroke.
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Maria Gaynier M e n to r : Fra n kl i n We s t
Po ste r P re se n t a ti o n C l a ss o f 2 0 1 5
Gait analysis of Pig MCAO Ischemic Stroke Model after Treatment with Human Induced Pluripotent Stem Cell Derived Neural Stem Cells Authors: Maria Gaynier, Dr. Franklin West, Dr. Kylee Jo Duberstein
Stroke is the leading cause of long-term disability and fifth leading cause of death. An urgent need to develop novel stroke treatments remains, as only one approved medical treatment exists with limited efficacy. Studies using rodent models have shown induced pluripotent stem cell derived neural stem cells (iNSCs) to proliferate and differentiate in the host brain and improve stroke recovery, but the effect of iNSCs in a large animal stroke model has not been previously studied. Gait analysis has been used in porcine models to evaluate stroke severity and recovery by measuring asymmetry and changes in gait parameters. This study's objective was to utilize gait analysis to quantify the effects of human iNSCs on post-stroke recovery in a pig model. Ischemic stroke was surgically induced by permanent occlusion of the middle cerebral artery. Four pigs received iNSC injections, and control pigs received vehicle-only injections poststroke. Pigs were taught to walk through a track where high-speed cameras recorded their gait. Video analysis software was used to measure pre-stroke, post-stroke, and post-injection gait parameters. Pigs were recorded before stroke (1,2,3 days), post-MCAO (1,2,5 days), and post-iNSC injection (1,3 days; 1,2,4,6,9,12 weeks). Data showed a decrease in swing time and an increase in stance time in front legs following stroke. Several days following injection, swing time of front legs increased, and gait became more symmetrical. Changes in gait parameters may be used to evaluate the effectiveness of stroke treatments. The results support that human iNSCs may improve stroke recovery in a pig model.
Cristina Guzzetti M e n to r : Fra n kl i n We s t
Po ste r P re se n t a ti o n C l a ss o f 2 0 1 5
Gait Analysis of Induced Pluripotent Stem Cell Derived Neural Progenitor Therapy in a MCAO Ischemic Brain Injury Pig Model Authors: Cristina Guzzetti, Dr. Franklin D. West, Dr. Kylee Jo Duberstein
Stroke is the fourth leading cause of mortality and disability in the United States with no effective treatment available. Stem cell therapies have shown significant promise in rodent models of ischemic brain injury but have proved limited in clinical settings. A larger animal model, such as a pig, would be more relevant since their brain structure and size corresponds more to humans. Induced pluripotent stem cells (iPSCs) have the ability to differentiate into neural progenitor cells, can be easily expanded, overcome immune rejection and have no ethical concerns. We proposed to assess human induced neural progenitor cell (hiNPC) function in a middle cerebral artery occlusion (MCAO) pig model by analyzing changes in gait patterns that quantify motor function deficits. Eight pigs underwent surgery in which MCAO confirmed stroke damage on MRI images. Four of the pigs then received hiNPC injections while the other four pigs received vehicle-only injections. Pigs were subjected to gait analysis at three time points before stroke; 1, 3, 5 days after stroke; 1, 3, 5 day and 1, 2, 4, 6, 9, 12 weeks after injections. Pigs were trained to walk through a semi-circular track then recorded with high-speed cameras. Gait analysis of pigs before stroke showed overall symmetry in hindlimbs swing and stance times. After stroke, notable asymmetries in temporal parameters were observed with pigs exhibiting shorter swing time and longer stance time on hindlimbs. Results support gait analysis of stroke injury as a highly sensitive detection method for changes in motor function in pigs. 13 | P a g e
Keira Hall Mentor : Fran klin Wes t
Poster P resen tation Class o f 2015
The Effect of Stroke and the Regenerative Potential of Stem Cells Through Gait Analysis in Pre- and Post- Ischemic Stroke Biomedical Pig Model Authors: Keira Hall, Franklin D. West, Dr. Kylee Jo J. Duberstein
Background: Ischemic stroke accounts for about 88 percent of all strokes. The vast majority of stroke therapies are developed using rodent models. Pigs have a gyrencephalic brain that is larger in size with a more similar gray-white matter composition to humans than traditional stroke rodent models. Objective: The current study was undertaken to determine the effects of stroke and the regenerative potential of stem cells through gait analysis. Ischemic injury was surgically induced by performing a middle cerebral artery occlusion in Yucatan miniature pigs. Pigs were trained to walk through a semi-circular track and recorded on high speed cameras to determine key gait parameters. The computer program Kinovea was used for gait analysis. Pigs were recorded three times prior to MCA occlusion, on days 1, 3, and 5 post surgery and on weeks 1, 2,4,6,9 and 12 post surgery. Data was collected for 2, 3-limb support on left front and right front for five pigs without knowledge of which pigs received stem cells. Results and Conclusions: Gait analysis indicated that stroke resulted in notable delayed temporal variables. Prior to surgery the pigs had higher two limb support times and low three limb support times. After surgery pigs exhibited higher three limb support times than two limb support times. A subset of pigs demonstrated quicker recovery after surgery due to having lower three limb support times in a shorter period compared to other pigs. These results indicate that stem cell treatment can help recover motor functions.
Akil Kalathil Mentor : Shanta Dhar
Poster P resen tation Class o f 2014 FDW
Aspirin conjugated Dendron encapsulated polymeric nanoparticles for Inflammation Authors: Akil A. Kalathil, Shanta Dhar
One of the leading causes of death in the United States is stroke. Despite all the research being done on various stroke treatment options, the FDA has approved only one. As such, there is a need for new therapies for stroke victims. The devastating effects of a stroke arise due to the cell death that occurs when the blood supply to the brain is interrupted. This leads to the loss of certain brain functions. As a response to the dying cells, a cascade effect that causes oxidative and inflammatory stress, leading to an even more toxic environment for neurons, resulting more tissue damage, and ultimately loss of more brain functions. Use of adult stem cells (ASCs) to restore lost brain function can be a unique way of treatment, but the toxic environment caused by the inflammation and the oxidative stress proves to be too much for the ASCs to survive. Our focus is to use a blood brain barrier penetrating biodegradable nanoparticle containing a hydrophobic aspirin analogue, which will be able to be effectively delivered in the brain in a controlled release fashion in the mitochondria of brain cells. This will reduce the toxicity due to inflammation, and will allow the stem cells to differentiate and potentially restore lost brain function.
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Maranda Leary Mentor : Hong xi ang Liu
Poster P resen tation Class o f 2016
Characterization of Brainbow Reporter Transgene and Neural Crest Derived Mesenchymal Cell Types in the Developing Mouse Tongue Authors: Maranda S. Leary, Kristin Boggs, Hong-Xiang Liu
Mammalian taste bud cells, specialized gustatory sensory organs that primarily reside in the epithelium of lingual taste papillae and soft palate, have both epithelial and neuronal features. It has been widely accepted that taste bud cells are derived from the local surrounding epithelium, which is in distinction from many other sensory organs that have a neuronal origin, including neural crest. Recently we have found that brightly labeled P0-Cre/RFP cells are abundantly distributed in both early and mature lingual taste buds in addition to the extensive distribution in the underlying mesenchyme. Given the fact that the epithelial cells that surround taste buds are not labeled in P0-Cre/RFP mouse tongue tissues, our findings strongly support that a significant population of taste bud cells share the same origin as the adjacently underlying mesenchymal cells, which are derived from neural crest. To further understand the number of neural crest derived stem/precursor cells that are involved in each taste bud, we used one (Stock #021011) of the nine Brainbow reporter mouse lines from Jackson Laboratory and characterized the distribution of Brainbow reporter gene expression driven by P0-Cre in embryonic and postnatal tongue tissues. We found that exposure of Brainbow reporter transgene to P0-Cre did not efficiently alter the transgenes. Scattered EYFP+ cells were found in the mesenchyme but not in the tongue epithelium, including taste buds. We are planning on testing another Brainbow mouse model (Stock#007910 and #007901) and on characterizing the neural crest derived cell types in tongue mesenchyme, e.g., fibroblast, intrinsic neurons.
Hannah Mason Mentor : Lohi ta sh Karu mbaiah
Poster P resen tation Class o f 2017
Chondroitin-Sulfate Glycosaminoglycan Based Hydrogel Carriers for Neural Stem Cell Transplantation after Moderate Traumatic Brain Injury Authors: Hannah Mason
Every year in the United States, over a million people are effected by traumatic brain injury (TBI), however, no effective treatments exist. Neural stem cells (NSCs) can potentially retroact the rampant neural tissue loss sustained after TBIs, and promote neuroprotection. However, long term survival and self-renewal of transplanted NSCs is challenging to achieve in the pro-inflammatory environment prevailing after TBI. Chondroitin-sulfate glycosaminoglycans (CS-GAGs) are important components of the neural stem cell niche extracellular matrix that are known to promote NSC self-renewal. In this study we hypothesized that NSCs encapsulated in photocrosslinkable CS-GAG hydrogel carriers can promote neuroprotection and lead to functional recovery after moderate TBI in a rat model. Ongoing studies involve intraparenchymal injections of NSCs encapsulated in CS-GAG hydrogels, followed by the subsequent longitudinal assessment of behavioral recovery using balance beam, rotarod, and open field tests.
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Jessica Rook Mentor : Fran klin Wes t
Poster P resen tation Class o f 2015
Injection of Human Induced Pluripotent Stem Cells in Pigs Suffering from an Induced Stroke to Reduce Gait Deficits Authors: Jessica Rook, Trinh Nguyen
Stroke is the leading cause of adult disability in the United States. Many stroke survivors suffer neurological impairments that impact their ability to perform daily activities, including gait deficits. Using gait analysis in clinical applications for stroke will allow health centers to better understand gait abnormalities and provide better treatment for post-stroke patients. In order to determine whether iNPC treatment would improve gait deficits in a more translatable stroke model, we induced stroke in eight Yucatan pigs and transplanted human iNPCs. We then performed gait analysis over a course of 12 weeks and analyzed five different gait parameters for all limbs (stance phase time, swing phase time, 2-limb support, 3-limb support, and break-over time). The conclusion of our study will contribute to the promising potential of stem cell based therapies for stroke in terms of a complete functional recovery in stroke patients.
Tau Shelley Mentor : Fran klin Wes t
Poster P resen tation Class o f 2018
Determination of Normal Pig Behavior using an Open Field Behavioral Test Authors: Shelley Tau, Holly Kinder, Emily Wyatt, and Franklin D. West
Approximately 2 million people experience a Traumatic Brain Injury (TBI) each year. TBI occurs when an external force injures the brain and can affect learning, memory, motor functions, and behavior depending on the severity of the damage. It is especially detrimental to normal development in young children whose brains are still maturing. Behavioral tests, such as an open field test, can be used to assess both normal and abnormal behaviors in a more translatable large animal pig model. We hypothesize that in an open field test normal piglets will initially exhibit common behaviors such as high exploratory interest and movement but will become habituated to the testing arena over time. Four piglets were used in the open-field test. They were placed into a 12’ x 14’ arena and observed for 10 minutes twice, seven days apart, to assess their normal open field behaviors such as zones entered, time spent moving, stationary, sniffing/exploring, and trying to escape. As the pigs got used to the arena, they generally entered fewer zones, became less exploratory, less mobile, less anxious, and more stationary. We concluded that these normal piglets spent significantly more time exploring their environment on the first day of testing and overall did not exhibit any unusual behaviors. This data will serve as a comparison to the behavior of piglets with TBI in order to determine the damage extent of the TBI and help us understand what parts of the brain are being affected.
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Karishma Sriram Mentor : Ste ven S tice
Poster P resen tation Class o f 2016
Exosome Imaging Authors: Karishma Sriram, Robin Webb
A common thread emerging from neurodegenerative disease research is that abnormal accumulations of aggregation prone proteins underlie a spectrum of age related neurodegenerative diseases. Increasing evidence indicates that human neural cells are particularly vulnerable to disruption of protein turnover and that degradation pathways become less efficient with aging. Regenerative therapies such as stem cell transplantation holds promise, and can improve cognitive deficits in rodent models, but the cells themselves often do not engraft into native tissue, prompting attribution of benefits to paracrine effects of transplanted cells. A mechanism of paracrine communication converging on multiple processes disrupted in neurodegenerative diseases has emerged that may also account for benefits from stem cell treatments. Accumulating evidence suggests that cells in the central nervous system communicate by releasing small secretory vesicles called exosomes. These exosomes originate from the fusion of intracellular multivesicular bodies with the plasma membrane, and are produced by cortical neurons, astrocytes, microglia, and oligodendrocytes in the central nervous system. We hypothesize that these secreted vesicles may confer the paracrine benefits of stem cell transplantation while negating the need to transfer cells themselves, thereby positively influencing innate regenerative capacity while minimizing the need for immune modulation in the recipient. The objective of the current study was to determine if it was to image exosomes using transmission and scanning EM. The methods include using cells in serum free media and cells in exosome depleted FBS. The exosome depleted media was then used for exosome purification in preparation for the transmission and scanning EM.
Olivia Valente Mentor : Fran klin Wes t
Poster P resen tation Class o f 2017
Gait analysis of stroke induced porcine models before and after iPSC-NPC treatment for motor function recovery Authors: Olivia B. Valente, Franklin D. West, Kylee Jo Duberstein, Trinh Nguyen
Each year, over 795,000 Americans suffer a stroke, killing a yearly average of 130,000 people. Though these numbers are high, stroke therapy and recovery is still limited. Currently, there is only one FDA approved treatment for stroke. With advancements in technology, the future of stroke recovery is turning to induced pluripotent stem (iPS) cells. The idea is that the iPS cells can regenerate brain cells damaged by stroke, which will then improve the motor function and gait deficit often found in stroke victims. These impairments have also been observed in stroke induced animal models, making them eligible candidate for stroke recovery research. The objective of this study was to perform gait analysis on a stroke induced porcine model, which has a brain structure similar to that of humans, both before and after iPSC-NPC treatments, to determine the effects of the treatment. Pigs were trained to walk through a track and were recorded using high-speed cameras to detect changes in gait parameters before and after the induced strokes, then again after the cell treatments. Analysis of stance time, break over time, and swing time of the front limbs were then completed using Kinovea software. The data collected during this study has not been existent up to this point. The results of this study can provide useful information about the efficacy of iPSC-NPC treatments for motor function recovery after stroke.
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