!2015%Regenerative%Medicine%and%Cellular%

Therapy%Research%Symposium:%%

“Clinical Translational Issues and Trials” April%24th%2015%

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Sponsored%by:%The%GRU%Institute%for%Regenerative%and%

Reparative%Medicine%Welcome to Members of the Regenerative Medicine, Tissue Engineering and Cellular Therapy Research Community. The Georgia Regents University’s Institute for Regenerative and Reparative Medicine is pleased to host this day long Symposium Friday April 24th 2015. We appreciate your attendance and share your work with regional colleagues. Our focus is on moving translational research into clinical trials. This will include talks from researchers and clinician scientists who have ongoing regenerative medicine based clinical trials. We are targeting researchers and clinicians who are involved in Regenerative Medicine, Tissue Engineering, and Cellular Therapy in Georgia and regionally, including the Georgia Regenerative Engineering and Medicine partnership. We would like to provide an opportunity to highlight the regional translational and clinical trial research with the goal of facilitating collaborations between groups at neighboring institutions and providing links to GRU clinical research programs.

We are pleased to also welcome our Keynote Speakers:

Meeting Keynote Speaker: Arnold I. Caplan, Ph.D. Case Western Reserve University

Arnold I. Caplan is professor of Biology and Director of the Skeletal Research Center at Case Western Reserve University. He is widely regarded as "The Father of the Mesenchymal Stem Cell". Dr. Caplan has

served as Chief Scientific Officer and founder of Osiris Therapeutics,Inc., co-founded Cell Targeting Inc., and has served as Chief Scientific Officer of OrthoCyte Corporation, a subsidiary of BioTime, Inc. He is active in developing clinical translational therapies and advocating pathways for realizing cell therapeutic regenerative medicine. Learn more: http://www.case.edu/artsci/biol/skeletal.

Symposium Keynote Speakers:

Eben Alsberg, Ph.D. Case Western Reserve University

Dr. Alsberg took a faculty position in 2005 at Case Western Reserve University, where he is currently an associate professor of Biomedical Engineering and Orthopaedic Surgery and serves as Director of the Stem Cell and Engineered Novel Therapeutics Laboratory. His lab focuses on the engineering of new technologies to regenerate tissues and treat diseases through the development of novel bio-materials and micro-environments. He’s co-authored over 75 peer-reviewed papers and book chapters and 130 abstracts and conference proceedings. His work has been recognized with the 2008 Ellison Medical Foundation New Scholar

in Aging Award and the Crain’s Cleveland Business 2009 Forty Under 40 Award. The NIH, DOD, NSF, the Ellison Medical Foundation, the Coulter Foundation, the Musculoskeletal Transplant Foundation, the State of Ohio and the AO Foundation have supported his lab’s research. Learn more:http://bme.case.edu/alsberg.

Steve Stice, Ph.D. University of Georgia

Dr. Steve Stice is a Georgia Research Alliance Eminent Scholar endowed chair, Professor and Director of the Regenerative Bioscience Center at the University of Georgia. He serves as the Director of the Regenerative Engineering and Medicine partnership and also serves as Chief Scientific Officer for ArunA Biomedical Inc. Dr. Stice co-founded five biotechnology companies, including Advanced Cell Technology, and CytoGenesis, Inc., which was later purchased by BresaGen. Throughout his career, he has published and lectured on cloning and stem cell technologies. His research focuses on developing innovative animal

cloning and stem cell technologies. He is also developing large animal models for use in neurological and orthopedic transitioning from preclinical studies in rodents to human clinical trials. Learn more:http://stice.uga.edu.

David C. Hess, M.D. Georgia Regents University

David C. Hess holds the Presidential Distinguished Chair and is the Chair of the Department of Neurology at MCG of GRU and Clinical Chair of the Brain and Behavior Discovery Institute at GRU. He has been part of the STEPS 1, 2, and 3 meetings to develop recommendations for clinical

trials of cell therapies in stroke. Dr. Hess has worked in both the pre-clinical arena and in clinical trial design of cell therapies. He has worked closely with Athersys, Inc on the MASTERS clinical trial in stroke. Dr Hess is a specialist in Cerebrovascular Disease and Stroke. He is actively involved in the MCG Stroke Program Stroke Service, and sees outpatients with a wide variety of neurological problems. Dr Hess's research interests include the use of bone marrow-derived stem cells to regenerate the brain after stroke; and the planning and coordination of clinical trials to prevent stroke. Learn more: http://www.gru.edu/mcg/neurology.

Johnna Temenoff, Ph.D. Georgia Institute of Technology

Dr. Temenoff is currently an Associate Professor in the Coulter Department of Biomedical Engineering at Georgia Tech/Emory University in Atlanta, GA. Over the course of her career to date, Dr. Temenoff has published over 40 peer-reviewed papers and 10 book chapters, co-authored a biomaterials textbook published by Pearson-Prentice Hall, and has produced ~90 scientific abstracts for national and international conferences. Dr. Temenoff has received funding from a wide range of sources, including federal agencies (NIH and NSF) and groups such as the Aircast Foundation and National Football League

Charities, and currently serves at the Principle Investigator on a NIH T32 Predoctoral Training grant in Biomaterials. She has been honored with several prestigious awards, such as AIMBE Fellow, the NSF CAREER Award, and the Arthritis Foundation Investigator Award, and was recently named the Co-Director for the Regenerative Engineering and Medicine Center, a statewide initiative encompassing Georgia Tech, U. Georgia, and Emory University. Learn more: http://temenoff.gatech.edu.

Liisa Kuhn, Ph.D. University of Connecticut Health Center

Dr. Liisa Kuhn is a tenured Associate Professor in the Reconstructive Sciences Department at the University of Connecticut Health Center (UConn Health) and a faculty member of the UConn Biomedical Engineering Department. Prior to joining the faculty of UConn in 2002, Dr. Kuhn was Director of Orthopaedics Product Development for ETEX Corporation in Boston, MA, and Director of Development for NaturApatites Co., Inc., a bone graft substitute company which she co-founded. Her research in orthopedic biomaterials and drug delivery systems has been supported by federal (NIH), state and private

foundations (e.g. Coulter Foundation, Komen Foundation). For her distinguished service and exceptional contributions to medical product standards writing within the American Society of Testing and Materials (ATSM), Dr. Kuhn been recognized with ASTM's top three awards, including, in 2014, the Award of Merit with an accompanying honorary title of Fellow. Learn more: http://kuhnbiomaterials.uconn.edu

April 24th 2015 Symposium Program:

8:00 Registration

• Coffee & pastry

8:30 Opening remarks:

• Greeting & Introduction: William D. Hill, Ph.D, GRU • Welcome IRRM: Carlos M. Isales, M.D. Director of IRRM • Welcome GRU: Michael Diamond, M.D. GRU VP for Research and Director of Clinical

& Translational Research

8:45 Meeting Keynote Lecture: Dr. Arnold I. Caplan, PhD - Case Western Reserve University: "Adult MSCs: new logics for a new kind of Medicine"

9:45 - 10:40 - Engineered Stem Cell Microenvironments 1

• 9:45 Keynote Lecture: Eben Alsberg, Ph.D. - Case Western Reserve University: "Inductive high-density stem cell systems for tissue regeneration"

• 10:15 Edward A. Botchwey, Ph.D. - Georgia Institute of Technology: "Sphingosine-1-phosphate receptor-3 regulates hematopoietic stem cell retention in the bone marrow niche"

10:35 Coffee break

10:55 - 11:50 - Engineered Stem Cell Microenvironments 2

• 10:55 Keynote Lecture: Johnna Temenoff, Ph.D. - Georgia Institute of Technology: "Glycosaminoglycan-based Biomaterials for Protein Delivery in Orthopaedics"

• 11:25 Lohitash Karumbaiah, Ph.D. – University of Georgia: "Trophic Factor Enriching Chondroitin Sulfate Glycosaminoglycan-Based Hydrogels for TBI"

11:45 - 12:15 - Tissue Engineered Structure Standards

• 11:45 Keynote Lecture: Lissa T. Kuhn, Ph.D. - University of Connecticut Health Center: "Standardization of tissue engineered medical products from an ASTMperspective"

12:15 Lunch & Poster Session

1:30 - 3:00 - Pre-Clinical Trials in Large Animal Models

• 1:30 Keynote Lecture: Dr. Steve Stice, Ph.D. - Univ. of Georgia: "Dual purpose non rodent models for human and animal medicine"

• 2:00 John F. Peroni, DVM, MS, Dip ACUS - Univ. of Georgia: "A practical review of ovine fracture models for the purpose of bone repair."

• 2:20 Frank West, Ph.D. - Univ. of Georgia: "Regenerative Neural Stem Cell Therapy in a Pig Stroke Model."

• 2:40 Samuel P. Franklin, DVM/Ph.D. - Univ. of Georgia: "Comparison of PRP and Stromal Vascular Fraction Supplemented with a Novel Nanofiber Polymer for the Treatment of Cartilage Pathology in Dogs"

3:00 Coffee break

3:20 – 5:00 Clinical Trials

• 3:20 Keynote Lecture: David C. Hess, M.D. - Georgia Regents University: "Cell Therapy for Stroke: Results from early phase clinical trials"

• 3:50 Harold Solomon – Georgia Venture Lab: "Funding the Lean Approach to Commercializing Health Technologies"

• 4:10 Changwon Park, Ph.D. – Emory University: "Function of endothelial ETV2 in angiogenesis"

• 4:30 Adam Berman, M.D. - Georgia Regents University: "IxCell-DCM Clinical Trial and Cardiac Regenerative Medicine Research"

• 4:50 Ayman Al-Hendy, M.D. - Georgia Regents University: "Gene and Stem cell Therapy of Premature Ovarian Failure"

5:10 Closing Comments

5:15 – 6:00 Poster Session and hors d'oeuvres

Efficacy of Local Delivery of Mesenchymal Stem Cells for Large Animal Tendinopathy Alexandra Scharf1, Shannon Holmes1,2, Merrilee Thoresen1, Jennifer Mumaw1, and John Peroni1

1Department of Large Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia 30605 2Diagnostic Imaging, College of Veterinary Medicine, University of Georgia 30605

Tendinopathy is a career debilitating disease with a high rate of morbidity in both human and equine athletes. The overall goal of our research is to enhance flexor tendon repair by the local delivery of mesenchymal stem cell (MSC) therapy. Our hypothesis is that the therapeutic benefits of MSCs correlate to the efficacy of cell delivery and cell survival. The goal of this study was to a) evaluate the efficacy of MSC delivery following local delivery, with and without ultrasound guidance and b) determine the degree of migration and persistence of cells over time.

Images of equine and ovine tendon lesions were acquired using a 1.5 Tesla Magnetic Resonance Imaging (MRI) scanner and knee coil. These lesions were localized to the superficial and deep digital flexor tendons in equine and ovine subjects, respectively. Lesions were treated with 10*106 cells labeled with superparamagnetic iron oxide nanoparticles (SPIOs), which produce a magnetic field perturbation detectable by MRI. Previous studies have been performed to validate the biosafety and optimize imaging parameters for detection of labeled cells. Subjects were imaged at the time of injection, 2 weeks, 6 weeks, and/or 4 months post-injection to assess the location of cells and degree of healing.

The results of this study suggest that even under ultrasound guidance, localized cell injections do not ensure that the entire payload is delivered to the site of injury. Of notable concern is that the cells delivered to the paratenon and surrounding tissue demonstrate little migratory capability. Any cells that do not make it into the lesion upon injection are unlikely to migrate to the site of injury over time. Further studies need to be performed to determine if the location of cells influences their survival and degree of healing. Future work on this project will use histological and diagnostic imaging data to further evaluate the relationship between cell delivery and tendon healing that can be obtained at 6 weeks and 4 months post-injection.

Chondroitin Sulfate Glycosaminoglycan Hydrogel-Based Neural Stem Cell Carriers for Traumatic Brain Injury

Martha I. Betancur , Melissa Albarado , Meghan Logun , Ravi Bellamkonda , Lohitash Karumbaiah

Regenerative Bioscience Center, Department of Animal and Dairy Science at The University of Georgia, Athens GA 30602. Wallace H. Coulter Department of Biomedical Engineering at Georgia Institute of Technology, Atlanta, GA 30332 Chondroitin sulfate glycosaminoglycans (CS-GAGs) are integral components of the neural stem cell (NSC) niche extracellular matrix (ECM), where they play key roles in the maintenance of NSC self-renewal. We therefore hypothesized that the natural affinity of neurotrophins, stem cell self- renewal, and differentiation inducing factors to CS-GAGs will help increase NSCs survival, and help facilitate acute neuroprotection, and targeted cell differentiation when transplanted in a rodent moderate traumatic brain injury (TBI) model. To achieve this, we designed CS-GAG based hydrogels capable of carrying NSCs. We believe that CS-GAG based hydrogels have significant advantages over synthetic polymer – based hydrogels owing to their ability to provide biological recognition and function, and undergo natural degradation to facilitate tissue remodeling. To test our hypothesis, we developed a moderate to severe TBI model and a hydrogel intracortical injection model for histological and behavioral evaluation after treatment with NSC laden CS-GAG hydrogels. Using a pneumatically actuated controlled cortical impactor (CCI), we induced injuries of 3mm diameter by 2mm depth in a repeatable manner, using a velocity of 2m/s, and a dwell time of 250 milliseconds. Two days after receiving a TBI, rats were injected directly into the site of injury with CS-GAG hydrogels containing PKH26GL labeled NSCs, and the trophic factors bFGF and BDNF. Histological analysis at 4 weeks post-injury shows a significant difference in injury size between TBI control and TBI treated with CS-GAG containing NSCs. In addition, quantification of cell survival shows a significant increase in cell survival when NSCs were implanted in CS-GAG hydrogels when compared to cells implanted with no hydrogel. The evaluation of cell survival at 12 weeks and the assessment of functional recovery using standardized behavioral tests are currently ongoing. Figure 1.

Figure 1. CS-GAG hydrogel implants containing NSCs and trophic factors significantly reduce neural tissue loss. Cresyl violet stained coronal sections taken at the epicenter of the injury were imaged and quantified at the region of interest (ROI) indicated by the white boxes. The percent area occupied by neurons was quantified using Image J. A pair wise comparison to the TBI group using one-way ANOVA and a post hock Tukey HSD Pairwise Comparison revealed a significantly greater present of neuronal tissue area in the TBI group treated with CS-GAG hydrogel encapsulated NSCs+TF, as compare to the no-treatment TBI group and the TBI-NSCs only group.

Figure 2.

Figure 2. CS-GAG hydrogel implants containing NSCs and trophic factors demonstrate a significant increase in NSC presence within the lesion 4 weeks post injury. Sections stained for neurons (green) shows NeuN+ cells within injection site. Zoomed in box of an area of the injury reveals a large number of red PKH26GL+ NSCs. Quantification shows that the percent PKH26GL+ area is greatest for group implanted with NSCs in the CS-GAG-TF scaffold. A pairwise comparison to the TBI-NSCs group using one-way ANOVA and a posthock Tukey HSD Pairwise Comparison revealed a significant difference between NSCs-only control and the two GAG-hydrogel-NSC treatments.

GRU Regenerative Medicine and Cellular Therapy Research Symposium, 2015: Abstract

sHER3 Inhibits the Proliferation and Migration of Melanoma-derived Cells in a Tenascin-dependent Manner.

Chunlin Cai, Mojun Zhu*, Surendra Rajpurohit and Nita Maihle

Cancer Center, Georgia Regents University, Augusta, Georgia and *Yale School of Medicine, New

Haven, CT

The HER3 receptor tyrosine kinase is a member of the EGFR/HER family of cellular oncogenes.

While this receptor family has generally received wide attention and clinical success as a target in the development of biologically-targeted cancer therapeutics, drugs targeting the HER receptor axis have to date not been successful in the treatment of melanoma. Nonetheless, recent studies have shown that HER3 receptor expression is common in both primary and metastatic melanoma, and that high levels of HER3 expression are correlated with poor patient survival. However, no one has demonstrated the mechanistic basis for HER3 as a poor prognostic determinant in melanoma patients, even though these tumors poorly express other members of the EGFR/HER receptor family. In this study we present evidence demonstrating that 13 of 14 cell lines tested express HER3, supporting previous reports regarding the frequency of expression of this gene product in melanoma samples. We further report that a soluble isoform of HER3 (sHER3) that we previously have shown to be a potent inhibitor of neuregulin mediated, Akt-dependent breast cancer cell growth strongly inhibits the growth of HER3 expressing melanoma cells. Interestingly, however, the phosphorylation of Akt, a known mediator of ligand-dependent HER3 signaling is not reduced in melanoma cells growth inhibited by sHER3. Since the HER3 receptor lacks endogenous kinase activity by itself, it is typically dependent on co-expression of other HER receptor family members for promotion of cancer cell growth. Together, these observations suggested to us that the mechanism(s) by which HER3 promotes melanoma cell growth and metastasis may be distinct. To explore alternative mechanisms of HER3 signaling in melanoma, we identified sHER3 interacting partners using a mass spectrometry proteomic approach. Only one prominent sHER3 binding partner was identified in melanoma cell conditioned media: tenascin. This was an unexpected finding since ligand-dependent HER3 signaling has not previously been linked to tenascin. Interestingly, tenascin expression has independently been identified as another negative prognostic indicator in melanoma. Our initial studies to test the functional relationship between HER3 and tenascin suggest that tenascin expression is correlated with both HER3 expression and migration in all of the melanoma lines tested. Moreover, sHER3 inhibition of melanoma migration was dependent on the level of tenascin present. Treatment of melanoma cells with purified sHER3 inhibits the migration only in the presence of high levels of tenascin. These results suggest the existence of an unanticipated tenascin- and HER3-dependent signaling pathway in melanoma cells, and also suggest that this pathway is functionally correlated with melanoma cell migratory potential and poor patient survival. Since tenascin expression has recently been shown to define a stem cell niche in other cancers, and also to provide a protective signal in a therapy-resistant population of melanoma cells, together these results identify a novel and targetable Achilles heel for prevention of melanoma cell growth and metastasis. (These studies were supported by the Harry J. Lloyd Charitable Trust).

Role of the Hematopoietic Stem Cell During Osteogenesis and Fracture Repair

RR Kelly 1,2,3, MA McCrackin1,4, KR Wilson1,2, M Mehrotra1,2,3, AC LaRue1,2,3

1Research Services, Ralph H. Johnson VAMC, 2Department of Pathology and Laboratory Medicine, 3Hollings Cancer Center and Department of Comparative Medicine4, Medical University of South

Carolina, Charleston, South Carolina

This work is supported in part by the Biomedical Laboratory Research and Development Program* of the Department of Veterans Affairs (ACL) and NIH/NCI RO1 CA148772 (ACL)

*The contents of this abstract do not represent the views of the Department of Veterans Affairs or the United States Government.

Mesenchymal stem cells (MSC) have been the “gold standard” for cell-based fracture treatment. However, multiple MSC-based trials have been hampered by low engraftment rates and an inability to effectively and consistently isolate this population. Our studies and others have suggested hematopoietic stem cells (HSCs) may also contribute to the osteogenic lineage. Using a single cell-based transplantation model, we showed that the HSC gives rise to osteoblasts, osteocytes, and hypertrophic chondrocytes during non-stabilized fracture repair. We hypothesize that these HSC-derived cells can be exploited to enhance fracture repair. In vitro assays were first conducted to identify factors to promote osteogenesis from HSC-derived progenitors. Mineralized colonies formed when non-adherent BM, the fraction enriched for HSCs, or adherent BM, the fraction enriched for MSCs, was cultured under osteogenic conditions. Exogenous BMP-2 and BMP-9 had a synergistic effect on this mineralization. We then sought to determine if HSC-derived osteogenic precursors (CD34+OCN+) could be mobilized from the bone marrow and found that delivery of AMD3100, a CXCR4 antagonist, resulted in a 2-fold increase in CD34+OCN+ cells in circulation. Our ultimate goal is to test the functional impact of HSC mobilization and/or delivery of pro-osteogenic factors on fracture healing in both non-stabilized and atrophic non-union fracture. Towards this, we have generated an in vivo murine model of surgical non-union. Preliminary X-ray and micro-CT analyses demonstrate delayed healing in a subset of animals. Further longitudinal and histomorphometric analyses are underway to confirm non-union. Animals with non-stabilized fracture or non-union will then be randomized to control (no intervention) or experimental (+/-BMP, +/-AMD3100, +BMP/AMD3100) groups and temporal healing assessed and correlated to HSC-derived osteogenesis. Given that the HSC is an earlier, more easily mobilized, and better defined stem cell than the MSC, it may prove a more efficacious therapy for treating fractures, particularly difficult to heal non-unions.

Hematopoietic Stem Cells Give Rise to Chondrocytes Through a Monocytic Precursor Lineage

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KR Wilson1,2,3, Y Xiong1,2,4, RR Kelly 1,2,3, AC LaRue1,2,4 1Research Services, Ralph H. Johnson VAMC, 2Department of Pathology and Laboratory

Medicine, 3College of Graduate Studies, MUSC and 4Hollings Cancer Center, MUSC

This work is supported in part by the Biomedical Laboratory Research and Development Program* of the Department of Veterans Affairs (ACL) and NIH/NCI RO1 CA148772 (ACL)

*The contents of this abstract do not represent the views of the Department of Veterans Affairs or the United States Government.

Bone marrow consists of two types of stem cell populations, the mesenchymal stromal cell (MSC) and the hematopoietic stem cell (HSC). While MSCs have been demonstrated to have the capacity of differentiating into osteoblasts, chondrocytes, and adipoctyes, recent studies are beginning to delve into the possibility of hematopoietic stem cells also having this differential capability. Our laboratory has demonstrated an HSC origin for cells such as osteoblasts, cancer-associated fibroblasts, and immature adipocytes, revealing the ability of HSCs to give rise to cell types not typically associated with these lineages. Of particular relevance to this study are our in vivo studies which demonstrated that HSCs can give rise to hypertrophic chondrogenic cells during non-stabilized fracture repair using a clonal cell transplantation model. This work has led to the hypothesis that chondrocytes differentiate through an HSC lineage. To initially address this hypothesis, in vitro studies of culture conditions for HSC-derived chondroprogenitors was first elucidated. Past research has indicated an HSC and myeloid lineage for other cells such as osteoblasts/cytes and adipocytes, respectively. For this reason, the chondrogenic potential of monocytic precursor-derived cells was examined in an effort to explore a new source of cells to be considered for cartilage regeneration. Alcian Blue staining was used as an initial indicator of chondrogenic potential in determining glycosaminoglycan production. In addition to this, nodule formation was assessed in cultures in serum containing chondrogenic media. More specific studies involved monocytic precursor cells cultured in serum-free conditions and directed towards and chondrogenic lineage using TGF-β1. immunofluroescent expression of cartilage specific markers Aggrecan and Collagen II after induction indicated differentiation along with the negative expression of F4/80, a monocytic marker. Determining the mechanisms behind HSC contribution to chondrogenic lineages and the associated process of differentiation/maturation in future work has the potential to enhance stem cell therapies for cartilage repair.

Development and Characterization of a Piglet Cortical Impact Traumatic Brain Injury Model

Emily L. Wyatta,b, Holly A. Kindera,b, Jessica M. Hutchesona,b, Elizabeth W. Howerthc, W.

Matthew Hendersond, Kylee J. Dubersteina,b, and Franklin D. Westa,b

aRegenerative Bioscience Center, bDepartment of Animal and Dairy Science, cDepartment of Veterinary Pathology, dU.S. Environmental Protection Agency

University of Georgia, Athens, GA 30602, USA

Traumatic brain injury (TBI) is a leading cause of death and long-term disability among persons in the United States with toddler-aged children being one of the most affected age groups. Despite the number of people who are affected by TBI, an FDA-approved treatment remains elusive. Stem cell therapies offer new therapeutic potential as a treatment for brain injury by producing regenerative and anti-inflammatory growth factors a well as functioning as a cell replacement therapy. One of the difficulties in developing effective treatments for TBI has been the poor translatability of therapies from the widely-used rodent model to human patients. Therefore, the establishment of a more human-like and thus more predictive animal model is crucial for the development of cell therapies. The piglet brain is more similar to humans in anatomy and physiology with respect to neurodevelopmental sequence, size, gyral pattern, and gray to white matter ratio making it an excellent candidate as a TBI model. The objective of this study was to determine changes in lesion size, brain metabolism, cellular composition, and motor function in piglets that experienced a controlled cortical impact (CCI) at increasing impact velocities and penetration depths. In this study we assessed 4 treatment groups: (1) 2 m/s and 6 mm, (2) 4 m/s and 6mm, (3) 4m/s and 12mm, and (4) 4 m/s and 15mm impact velocity:penetration depth. Study results demonstrated a direct correlation between increasing impact velocity and penetration depth with increased brain lesion size, changes in brain metabolism, gliosis, and motor function deficits. At the tissue level, gross histology showed a significant increase in brain lesion size as impact velocity and penetration depth was increased. Animals receiving a CCI at 4 m/s impact velocity with a 15mm penetration depth had the largest lesion volume of 1236 mm3 one week post-TBI. At the cellular level, metabolomic analyses showed that there were significant changes in brain metabolites after injury; and furthermore, gray and white matter in the piglet brain exhibited differential responses to injury. Gliosis was quantified through immunohistochemistical analysis of the GFAP astrocyte and Olig2 oligodendrocyte protein markers. One week post-TBI, the amount of GFAP-positive astrocytes was significantly increased on the ipsilateral hemisphere relative to the contralateral hemisphere in piglets receiving a 4 m/s CCI with 6mm and 12mm penetration depths, while global increases of Olig2-postive oligodendrocytes were noted in piglets receiving a 4 m/s CCI with 12mm and 15mm penetration depths. At the functional level, analysis of motor function kinematics in piglets before and after injury showed that TBI resulted in motor instability characterized by increased stance time and preference to remain in 3-limb support versus 2-limb support. This study is the first to demonstrate that increasing velocity and penetration depth in a CCI piglet TBI model results in progressive deficits at the cellular, tissue, and functional levels. These quantifiable and reproducible changes in the piglet TBI model make it an attractive platform for the testing and development of novel cell therapies.

!FIRST! REPORT! OF! A! POTENTIALLY! NOVEL! ONCOGENIC! MECHANISM!AND!THERAPEUTIC!TARGET!IN!ANAPLASTIC!THYROID!CANCER!!Leslie! Peard! BS1,! DeHuang! Guo! PhD1,2,! Kamran! Mohammed! BS2,!Tiffany!Coleman!BS1,2,!and!Paul!Weinberger!MD1,2,3!1Department!of!Otolaryngology,!Medical!College!of!Georgia,!Georgia!Regents!University;! 2Center! for! Biotechnology! and! Genomic! Medicine,! Georgia!Regents! University,! and! 3GRU! Cancer! Center,! Georgia! Regents,! University,!Augusta,!GA!!BACKGROUND:"Anaplastic"thyroid"cancer"(ATC)"is"a"rare"form"of"thyroid"carcinoma"with"extremely"high"morbidity"and"fast"disease"progression."There" are" no" effective" treatments," and" 5@year" overall" survival" is" 4.3%.""We" and" others" have" noted" elevated" expression" of" keratin" 8" (CK8)" in"several"ATC"cell"lines,"most"notably"the"highly"aggressive"lines"with"fast"population" doubling" time" (Td)." Keratins" are" intermediate" filament"proteins"with"known"role"as"a"structural"member"of"the"cytoskeleton"in"epithelial"cells."METHODS:"Highly"characterized"thyroid"cancer"cell"lines"were" obtained."Western" blot" and" immunohistochemistry" was" used" to"determine" CK8" expression." Using" a" stable" lentiviral@CK8" shRNA"construct,"we"performed"a" knockdown"of"CK8" in"ATC1" cells." Following"puromycin" selection," culture" wells" were" imaged" daily" to" determine"effect."RESULTS:" Fast" growing" thyroid" cancer" cell" lines"ATC1," 29T," and"11T" had" elevated" expression" of" CK8." Slow" growing" cell" lines" 16T,"FTC133,"and"11T"had"undetectable"CK8"levels." In"ATC1"cells,"scrambled"lentivirus" controls" demonstrated" expected" recovery" and" proliferation"following" infection." Wells" with" no" virus" (negative" control)" underwent"apoptosis."The"CK8" lentivirus"wells" showed"near" total"growth"arrest"of"ATC" cells." CONCLUSION:" Rather" than" simply" being" a" biomarker" for"epithelial"cancer,"CK8"may"play"a"direct"role"in"anaplastic"thyroid"cancer"progression." If"true,"this"represents"a"novel"mechanism"of"action"and"a"potentially"druggable"target"for"a"disease"that"currently"has"no"cure."

Assessment of Learning and Memory in a Piglet Model

Holly A. Kinder1,2, Emily L. Wyatt1,2, Franklin D. West1,2

1Regenerative Bioscience Center, 2Department of Animal and Dairy Science, University of Georgia, Athens GA, 30622

The use of the piglet in studying neurodevelopment has become of increased interest due to similarities in brain structure and development to toddler-aged children. Several neural injury and disease models have recently been developed creating a need for further validation of behavioral tests to better assess learning and memory in piglet models. The present study evaluated the ability of 5 week-old piglets to perform 4 behavioral tests, a spatial T-maze test, an object recognition test, a social recognition test, and an open field test, to assess different aspects of cognition including learning and spatial memory, spontaneous trial-unique memory, social memory, and normal/abnormal behaviors, respectively. For spatial-T maze testing, piglets were trained to locate a food reward at a constant place in space by using extra-visual cues, despite starting the task at alternating locations. Piglets were significantly faster at making a reward arm choice by day 5 (p< .05), indicating acquisition of the task. Additionally, piglets reached a performance criterion of at least 80% correct reward arm choices by day 4 (p<0.05), indicating that piglets were able to create and use spatial memories to find a food reward. Correct choices decreased after reversal of the reward, but improved after several days of testing (p <0 .05). In object recognition testing, pigs were exposed to two similar objects in a sample trial, followed by a 10 minute inter-phase interval, and then exposed to one familiar and one novel object in a test trial. Piglets explored the novel object significantly more than the familiar object (p<0.05) in the test trial. Additionally, piglets explored the familiar object significantly less in the test trial than in the sample trial (p<0.05), indicating the piglet formed a spontaneous trial-unique memory of the familiar object. In the social recognition test, piglets were exposed to an unfamiliar pig and a novel object in a sociability test, followed by a 10 minute inter-phase interval, and then exposed to a familiar pig and a novel pig in a social recognition test. Piglets spent significantly more time with the unfamiliar pig in the sociability trial than the novel object (p<0.05) and tended to spend more time with the novel pig in the social recognition trial than the familiar pig (p=0.01) indicating that the piglet had retained a social memory of the familiar piglet. Finally, in the open field test, piglets were placed in an open arena for 10 minutes to monitor different aspects of behavior such as ambulation, exploratory interest, and anxiety. Piglets generally explored fewer zones and became less ambulatory over time, demonstrated significantly decreased exploratory interest over time (p<0.05), and generally became less anxious over time. This test overall suggests that piglets became habituated to the testing arena, a characteristic of normal piglet behavior. Taken, together these tests provide an ideal platform with which to continue behavioral testing in neural injury and disease models and to assess the effectiveness of different treatments on improving learning, memory, and behavior.

A novel TNF-derived peptide activating alveolar liquid clearance during acute lung injury I. Czikora1, A. Alli2, D. Kaftan3, S. Sridhar1, Z. Bagi1, T. Chakraborty4, D.C. Eaton2 and R. Lucas1. 1Georgia Regents University - Augusta, GA/US, 2Emory University School of Medicine - Atlanta, GA/US, 3University of South Bohemia- Ceske Budejovice/CR, 4Justus-Liebig University - Giessen/DE. (Supported by an AHA post doc award to IC)

RATIONALE. Alveolar liquid clearance (ALC) is regulated by vectorial Na+ transport through the apically expressed epithelial sodium channel (ENaC) and basolateral Na+-K+-ATPase in type II alveolar epithelial cells. ALC is critical to prevent life-threatening pulmonary edema formation in the alveoli, for which no standard treatment exists to date. Since the pneumococcal virulence factor pneumolysin impairs ENaC activity1, it is of the utmost clinical importance to identify novel mechanisms that increase the channel’s function, defined by both its expression level and its open probability time, during pneumonia. The lectin-like domain of the cytokine TNF, mimicked by the 17 residue TIP peptide, activates ENaC-dependent Na+ uptake2-4. This study was designed to determine the mechanism(s) by which the peptide increases ENaC activity in the absence and presence of the pneumococcal pore-forming toxin pneumolysin. RESULTS. The TIP peptide, upon caveolae-dependent internalization, binds to the intracellular carboxy-terminal domain of the alpha subunit of ENaC4, thus increasing the open probability of the channel. By inhibiting pneumolysin-mediated activation of the enzymes protein kinase C-alpha and ERK, which are known to blunt ENaC open probability and expression, respectively, the lectin-like domain of TNF restores the channel’s activity in H441 cells. As suggested by a molecular docking study using ASIC-1 as a template4, mutation of ENaC-alpha residues Val567, Glu568 and Glu571 to Ala significantly blunted its interaction with the TIP peptide, indicating that these amino acids are crucial for ENaC activation by TNF. The TIP peptide significantly increased ALC in patients with acute lung injury in a recent phase 2a clinical trial5.

References. 1Lucas et al., PNAS 2012; 2Lucas et al., Science 1994; 3Elia et al., AJRCCM 2003; 4Czikora et al., AJRCCM 2014.5Sieck and Wylann, editorial, AJRCCM 2014.

GILZ%Protects%TNF0α0induced%Bone%Loss%in%Mice%

Nianlan%Yang1,%Babak%Baban2,%William%D.%Hill3,%Mark%W.%Hamrick3,%Carlos%M.%Isales1,%4,%Xing0Ming%Shi1,%5%%

Departments%of%Neuroscience%&%Regenerative%Medicine1,%Pathology5,%Oral%Biology2,%Cellular%Biology%and%Anatomy3,%Orthopaedic%Surgery4,%Georgia%Regents%University,%Augusta,%GA%USA%%

Background:%Tumor%necrosis%factor0alpha%(TNF0α)%plays%a%key%role%in%the%pathogenesis%of%inflammatory%diseases%such%as%rheumatoid%arthritis%(RA).%It%is%known%that%chronic%inflammation%causes%bone%loss.%We%showed%previously%that%glucocorticoid%(GC)0induced%leucine%zipper%(GILZ),%a%GC%anti0inflammatory%effect%mediator,%can%enhance%osteogenic%differentiation%of%bone%marrow%mesenchymal%stem%cells%(MSCs),%and%antagonize%the%inhibitory%effect%of%TNF0a%on%MSC%differentiation.%However,%it%is%not%known%whether%GILZ%will%have%the%same%effects%in%vivo%and%thus%serve%as%a%potential%new%anti0arthritis%drug%candidate.%%

Methods:%We%created%TNF0α:GILZ%double%transgenic%mice%by%crossing%a%human%TNF0α0expressing%transgenic%mouse%with%a%GILZ%transgenic%mouse,%in%which%the%expression%of%GILZ%is%under%the%control%of%a%3.6kb%type%I%collagen%promoter%fragment%(Col3.6).%The%expression%of%TNF0α%and%GILZ%in%double%transgenic%mice%was%confirmed%by%RT0PCR,%Western%blot%and%ELISA,%and%the%bone%protective%effects%of%GILZ%were%assessed%with%standard%bone%biology%and%histology%techniques.%%

Results:%%Overexpression%of%GILZ%blocked%TNF0α0mediated%bone%loss%in%double%transgenic%mice%as%demonstrated%by%a%significantly%increased%bone%mineral%density%(BMD)%in%the%femur%(+13.5%,%p<0.01),%tibia%(+12.9%,%p<0.05)%and%vertebra%(+14.5%,%p<0.05)%compared%with%that%in%the%TNF0α%transgenic%mice.%GILZ%also%reduced%the%expression%levels%of%IL06%mRNA%and%serum%levels%of%RANKL,%as%well%as%the%levels%of%overexpressed%human%TNF0α.%%Furthermore,%GILZ%antagonized%the%inhibitory%effect%of%TNF0α%on%osteocalcin%expression%in%GILZ0TNF0α%double%transgenic%mice.%%As%predicted,%the%TNF0α%transgenic%mice%developed%poly%arthritic%symptoms%between%week%809%along%with%significant%body%weight%loss%and%bone%loss.%The%double%transgenic%mice,%somehow,%showed%little%effect%on%inflammatory%arthritic%symptoms%compared%with%the%TNF0α%transgenic%mice.%This%could%be%due%to%the%restricted%GILZ%transgene%expression%in%osteoblastic%lineage%cells%and%ubiquitous%and%high%level%of%TNF0α%transgene%expression%in%double%transgenic%mice%because%significant%reductions%of%TNF0α%was%only%observed%in%bone%marrow%mesenchymal%lineage%cells%(043.5%)%but%not%in%hematopoietic%(or%osteoclastic)%lineage%cells.%%

Conclusion:%Overexpression%of%GILZ%in%osteoprogenitor%cells%protects%bone%loss%in%mice%with%chronically%elevated%levels%of%proinflammatory%cytokines%such%as%TNF0α.%%

Repurposing Dimethylfumarate for Parkinson's disease- Preclinical evidence

Manuj Ahuja1, Navneet Ammal Kaidery1, Lichuan Yang1,7, Noel Calingasan3, Natalia Smirnova4, Arsen

Gaysin5, Irina Gaysina6, Takao Iwawaki8, John Morgan2, Rajiv Ratan4, Irina Gazaryan4, Anatoly

Starkov3, Flint Beal3, Bobby Thomas1,2,*

1Departments of Pharmacology & Toxicology and 2Neurology, Medical College of Georgia, Georgia Regents University, Augusta, Georgia 3Brain and Mind Research Institute, Weill Medical College of Cornell University, New York, New York 4Burke Medical Research Institute, White Plains, New York 5Department of --- Northwestern University, Chicago 6Department of Medicinal Chemistry, University of Illinois 7Kunmig Biomed, China 8Gunma University, Japan Targeting oxidative stress either by providing exogenous antioxidants or by enhancing the endogenous antioxidative capacity has been intensely investigated for PD therapies. The latter includes the activation of nuclear-factor-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway which regulates the expression of a battery of genes encoding anti-oxidative, anti-inflammatory, and cytopro-tective genes. Tecfidera is an oral formulation of dimethylfumarate (DMF) approved for Multiple sclerosis based on its promising beneficial effects. Fumaric acid esters such as dimethyl and mono-methyl fumarate have been found to exert neuroprotective effects by activating the Nrf2/ARE signaling pathway. We investigated in vivo pharmacokinetics, effects on Nrf2/ARE signaling both in vitro and in vivo and its ability to block 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity and associated oxidative damage, impaired mitochondrial function, and inflammation in mice. We found that dimethyl and mono-methylfumarate selectively activate Nrf2 pathway using Neh2-luciferase reporter, and OK48-luc reporter transgenic mice. Assessment of mRNA and protein levels showed upregulation of several cytoprotective and antioxidative genes in discrete mouse brain regions commensurate with DMF pharmacologic levels in vivo, and in wild type mouse embryonic fibroblasts but not in Nrf2 null fibroblasts in vitro. Oral adminis-tration of both dimethyl and mono-methylfumarate at (10, 25, 50, and 100mg/kg) dose dependently pro-tected against acute MPTP neurotoxicity assessed by stereological cell counts of total and tyrosine hydroxylase positive neurons of substantia nigra and striatal levels of catecholamines, employing HPLC electrochemistry, in wild type but not Nrf2 null mice. Dimethyl and mono-methylfumarate blocked against MPTP-induced oxidative damage assessed by 3-nitrotyrosine and inflammation determined CD68 immunoreactivity and expression of pro-inflammatory cytokines in the midbrains. Both dimethyl and monomethylfumarate enhanced mitochondrial bioenergetics assessed by oxygen consumption rate in vitro in mouse embryonic fibroblasts in an Nrf2 dependent fashion. Our results suggests that fumaric acid esters protect against nigrostriatal dopaminergic neurotoxicity and associated oxidative damage, inflam-mation, and mitochondrial dysfunction by virtue of its ability to activate neuroprotective Nrf2/ARE ge-netic program. The current results clearly put forth pre-clinical evidence favoring DMF to be an ideal therapeutic intervention that should be repurposed for PD.

Hdac3 regulates osteoblastic glucocorticoid and lipid metabolism during aging Meghan E. McGee-Lawrence, Lomeli R. Carpio, Ryan J. Schulze, Mark A. McNiven, Sundeep Khosla, Merry Jo Oursler, Jennifer J. Westendorf Histone deacetylase 3 (Hdac3) removes acetyl groups from lysine residues in histones and other proteins to epigenetically regulate gene expression. Hdac3 interacts with skeletal transcription factors and is essential for bone development and maintenance. We previously reported that conditional deletion of Hdac3 (Hdac3 CKO) in osteochondral progenitors, using Osx1-Cre, caused osteopenia and increased marrow adiposity, both hallmarks of an aging skeleton. Here we demonstrate that Hdac3 mRNA expression is reduced in bone cells from elderly women and primary bone marrow stromal cells (BMSC) and osteocytes from aged mice. More significantly, phosphorylation of S424-Hdac3, an event that stimulates deacetylase activity, is suppressed in osseous cells from old (22-26 months) mice as compared to young (1-2 months) animals. Lipid droplet formation was prevalent in the aged wildtype cultures as well as in osteogenic cultures of young Hdac3-depleted BMSCs. Gene expression analyses revealed insignificant changes in mRNA levels of Ppar gamma and fatty acid synthase, but Hdac3 CKO cultures demonstrated 10- to 20-fold increases in expression of genes related to lipid storage (Cidec, Perilipin1) and 2 to 3-fold changed in genes regulating glucocorticoid metabolism (11β-hydroxysteroid dehydrogenase type 1 (Hsd11b1)), suggesting that Hdac3 regulates local osteoblastic activation of glucocorticoids and lipid storage. In support of this hypothesis, lipid-containing cells in the Hdac3 CKO cultures expressed Runx2. In vitro lipid droplet formation was dependent on glucocorticoid signaling, as lipid storage and gene expression returned to near wildtype levels when dexamethosone (Dex) was excluded from the culture medium. Hdac3 attenuated Dex-induced activation of the glucocorticoid-responsive MMTV-luciferase reporter. Taken together, our data suggest that suppression of Hdac3 activity in bone cells contributes to reductions in bone density and increases in marrow adiposity that are associated with aging and long-term glucocorticoid treatment.

Early&Craniofacial&Defects&Occur&Following&Knockdown&of&the&Extracellular&Matrix&Protein,&TINAGL1&

H.&Neiswender1,&S.&Navarre2,&D.J.&Kozlowski1,2,&and&E.K.&LeMosy1&

1Dept.'of'Cellular'Biology'and'Anatomy,'and'2Institute'of'Molecular'Medicine'and'Genetics,'Georgia'Regents'University,'Augusta,'GA'30912'

&

Abstract&

Introduction:'The'TINAGL'family'of'secreted'basement'membrane'proteins'(TINAGL1'and'TINAG'in'mammals;'TINAGL1'in'lower'species)'is'highly'conserved'but'functionally'opaque.'Limited'data'from'humans'and'mice'has'suggested'functions'in'cell'adhesion,'renal'and'vascular'development,'cranial'suture'closure,'and'suppression'of'metastasis,'while'the'fly'TINAGL1'appears'to'act'as'a'positive'Wnt'(Wg)'cofactor.'''

Results/Methods:'We'are'using'morpholino'(MO)'knockdown'to'study'TINAGL1'function'in'zebrafish'development.'A'consistent'pattern'of'pharyngeal'arch'cartilage'defects'is'observed'with'three'independent'TINAGL1'MOs'but'not'their'misRmatch'controls,'and'is'observed'with'coRinjection'of'subRthreshold'doses'of'the'two'bestRcharacterized'MOs.'Substantial'rescue'(e.g.,'35%'vs'95%'larvae'affected)'is'observed'by'coRinjection'of'TINAGL1'mRNA.'In'situ'hybridization'demonstrates'endogenous'TINAGL1'expression'in'ventral'tissues'underlying'the'hindbrain'at'15R22'hpf.'In'24'hpf'morphant'zebrafish,'the'neural'crest'cell'marker'dlx2a'is'reduced'or'absent'in'posterior'pharyngeal'arch'domains'that'show'severe'defects.''

Conclusions:'These'results'suggest'that'TINAGL1'is'required'for'survival'or'migration'of'neural'crest'cells'in'some'domains'of'the'pharyngeal'apparatus.''We'postulate'roles'involving'cell'adhesion'in'tissues'through'which'the'neural'crest'cells'migrate,'or'involving'regulation'of'Wnt'localization'and'activity'within'these'tissues.'Future'experiments'will'address'neural'crest'cell'behavior'in'these'morphants,'and'whether'TINAGL1'genetically'and/or'physically'interacts'with'Wnts'important'during'early'craniofacial'development.'

Translational&Impact:'Craniofacial'skeleton'development'is'a'model'for'birth'defects'and'cartilage/bone/matrix'regulation.'In'future,'the'zebrafish'model'provides'a'platform'for'testing'drugs'and'other'interventions'rationally'identified'via'mechanistic'studies.'

Dioleoylphosphatidylglycerol (DOPG) To Accelerate Corneal Wound Healing Lawrence Olala, Xiaowen Lu, Mitchell Watsky, and Wendy B. Bollag Charlie Norwood VA Medical Center, Augusta GA 30904 and Medical College of Georgia at Georgia Regents University, Augusta, GA 30912

Primary roles of the cornea include maintenance of normal vision by refracting light onto the lens and retina and provision of a barrier to the external environment. These functions are sustained, in part, by the ability of the corneal epithelium, like the skin, to undergo continuous renewal. Similarly to skin, corneal epithelial renewal is dependent on a highly integrated balance between the processes of proliferation, differentiation, and cell death. Our laboratory has previously described a novel cell signaling mechanism in skin epidermal keratinocytes composed of the glycerol transporter, aquaporin-3 (AQP3) and the lipid-metabolizing enzyme phospholipase D2 (PLD2), a member of the phospholipase D (PLD) family of enzymes. AQP3 and PLD2 function to produce phosphatidylglycerol (PG), which regulates keratinocyte proliferation and differentiation. In particular, we have shown that PG species containing monounsaturated fatty acids, e.g., dioleoylphosphatidylglycerol (DOPG), stimulate mouse keratinocyte proliferation in slowly dividing cells, while PG species containing polyunsaturated fatty acids, inhibit rapidly dividing keratinocytes. A mixture of PG species, such as PG derived from egg, can normalize mouse keratinocyte proliferation in rapidly or slowly dividing keratinocytes. We also have obtained in vivo data demonstrating that egg PG promotes healing of a full-thickness skin wound. Interestingly, another group has shown that mice lacking the AQP3 gene (i.e., AQP3 knockout mice) show not only delayed skin wound healing but also impaired corneal wound healing. Thus, we hypothesized that DOPG could regulate corneal epithelial function in a similar manner to mouse skin and promote wound healing. Using AQP3 knockout mice, we show here that 250µg/ml DOPG enhanced the rate at which corneal wounds healed. A corneal epithelial scrape wound model was used to assess wound healing. By 12 hours there was a significant reduction in wound size compared to vehicle (PBS)-treated controls, and the corneal wounds were almost completely healed in the DOPG treatment group by 28 hours. We also compared the rate of wound healing between genders, and observed no differences. Thus, our results suggest that DOPG liposomes can be used to accelerate corneal wound healing, particularly in individuals exhibiting wound healing defects.

Dioleoylphosphatidylglycerol (DOPG) to Accelerate

Corneal Wound Healing !Lawrence!Olala,!Xiowen!Lu,!Mitch!Watsky!and!!Wendy!B.!

Bollag!

SDF-1β / BMP-2 Co-Therapy Augments BMSC-Mediated Healing of Critical-Size Mouse Calvarial Defects

Samuel Herberg (1), Alexandra Aguilar-Perez (2,3), R. Nicole Howie (3), Galina Kondrikova (3), Sudharsan Periyasamy-Thandavan (3), Mohammed E. Elsalanty (3), Xingming Shi (3), William D. Hill (3) and James J. Cray (4) Case Western Reserve University, Cleveland, OH (1), Universidad Central del Caribe, Bayamón, Puerto Rico (2), Georgia Regents University, Augusta, GA (3), and Medical University of South Carolina, Charleston, SC (4) Bone is a dynamic and highly vascularized tissue that has the innate capacity for healing upon damage. This regenerative process, however, often fails in patients with significant co-morbidities, requiring surgical intervention [1]. Tissue engineering combining biomaterial scaffolds, regenerative cells, and soluble growth factors may provide viable alternatives to standard therapies (e.g., autografts, allografts, xenografts) [2-4]. We have recently demonstrated that acellular DermaMatrix (ADM) scaffolds have native binding affinities for relevant growth factors, facilitating bone healing [5]. Furthermore, we have shown that stromal cell-derived factor-1β (SDF-1β) works in concert with bone morphogenetic protein-2 (BMP-2) to potentiate osteogenic differentiation of bone marrow-derived mesenchymal stem/stromal cells (BMSCs) in vitro [6], and to augment bone formation in vivo using both mouse and rat models [5,7]. Here, we investigate the regenerative capacity of BMSC/ SDF-1β/BMP-2 combination therapies delivered on ADM relative to controls.

The Age-Associated Rise in miRNAs from Muscle Target SDF-1 and Musculoskeletal Regulatory Genes is Reversed

with Caloric Restriction and Leptin Sudharsan Periyasamy-Thandavan (1), Samuel Herberg (6), Phonepasong Arounleut (1), Sunil Upadhyay (1), Galina Kondrikova (1), Amy Dukes (1), Colleen Davis (1), Maribeth Johnson (5), Xing-Ming Shi (3,4), Carlos Isales (2,3,4), Mark W. Hamrick (1,2,3,4), and William D. Hill (1,2,3,4). 1Department of Cellular Biology & Anatomy (1), Department of Orthopaedic Surgery (2), Institute of Molecular Medicine and Genetics (3), Institute for Regenerative and Reparative Medicine (4), Department of Biostatistics (5), Georgia Regents University, Augusta, GA, USA, Case Western Reserve University, Cleveland, OH, USA (6). MicroRNAs (miRNAs) have the potential to regulate broad changes in connected systems and pathways altering homeostatic gene expression. We have previously identified age-related changes in the expression profiles of miRNAs isolated from human bone marrow mesenchymal stem cells (BMSCs). These miRNAs targeted numerous genes associated with musculoskeletal development, maintenance, aging and osteoporosis. As well as, the chemokine SDF-1 (CXCL12), and its receptor CXCR4. The SDF-1 axis is critical in the migration, survival, and engraftment of stem cells, including BMSCs and muscle satellite cells (SCs). We have noted leptin can alter systemic and muscle SDF-1 levels in an age-related manner suggesting nutrient signaling may effect systemic, or tissue SDF-1 expression. We hypothesized that changes in nutrient signaling pathways may modulate specific miRNA in an age specific manner. In turn these miRNAs may alter SDF-1 and key osteogenic, or myogenic pathway genes. We tested this hypothesis by examining changes in miRNAs, we had previously identified as linked to aging in BMSCs, in muscles of mice aged 12 months and 20 months fed ad-libitum (AL) and mice 20 months on caloric restriction (CR). We also treated other mice aged 20 months on caloric restriction with recombinant mouse leptin for 10 days at 10 mg/kg body weight. Age-associated patterns of expression of miR-29b-1-3p, miR-29b-1-5p, & miR-1244 in murine muscle (12 vs 20 months) mirrors that seen in human BMSCs between young (under 45 years of age) and older (over 65 years of age) subjects. In both cases the miRs 29b-1-5p, and 1244 increased, while miR-29b-1-p3 did not change. Of interest the miR-29 family is well known to target muscluloskeletal and extracellular matrix genes. CR reduced the miRs 29b-1-5p, 1244 and 141 in 20 month old mice to levels equal to that of 12 month mice while miR-29b-1-3p was not altered. Of interest CR with leptin treatment further reduced expression of miRs 29b-1-5p, 1244 & 141 well below that seen in the 12 month mice and surprisingly the amount of miR-29b-1-3p rose significantly. Therefore, it appears that food restriction is a potent regulator of miRNAs, and suppresses miRNAs that target muscluloskeletal gene systems in aged muscle. Leptin further drives this effect significantly below what is seen in younger muscle, which may induce an inbalance in gene expression that might itself impair the potential for muscle regeneration.

Alternative Splice Variants Of The Osteogenic Cytokine SDF-1 Differentially Mediate CXCR4 and CXCR7 Expression in Bone Marrow MSCs

Aguilar-Pérez, A.1,3, Herberg, S.3, Periyasamy-Thandavan, S.3 Volkman B.F. 5, Kondrikova, G. 3, Shi, X.6,7,8, Cubano, L. 1, Hamrick, M.W.3,6,7,8, Isales, C.M.3, 6,7,8, Hill, W.D.2,3, 6,7,8 1Universidad Central del Caribe, Bayamón, Puerto Rico, USA, 2Charlie Norwood VA Medical Center, Departments of 3Cellular Biology and Anatomy, 4Department of Biochemistry, 5Medical College of Wisconsin United States has one of the highest fracture rates associated with aging with up to 33% of the over 50 population being affected. Aging-related osteopenic and osteoporosic mechanisms remain poorly defined. It is a medical challenge to provide accurate and early treatment in order to decrease morbidity, improve muscluloskeletal function and increase independence among the elderly population. Our studies are focused on stromal cell-derived factor-1 (SDF-1/CXCL12). SDF-1 is part of the CXC chemokine family and can signal through CXCR4 and CXCR7. SDF-1 supports bone marrow-derived stem/stromal cell (BMSC) survival, proliferation, and osteogenic differentiation (1,2). There are six known alternative mRNA splice variants for SDF-1 (3). SDF-1α and SDF-1β are the main isoforms in bone marrow. SDF-1α has been implicated in BMSC mobilization and osteogenic differentiation. SDF-1β is more resistant to proteolytic cleavage due to the four additional C-terminal amino acids compare with SDF-1α and has also been shown to be important for BMSC survival and in regulating osteogenesis (1,2). It is unclear whether there is a difference in the activation of gene expression and signaling pathways between these two constitutively expressed isoforms in BMSCs. CXCR4 second messenger signaling has recently been shown to be able to switch between GPCR and β-Arrestin pathways in response to ligand concentration (4,5). We assessed the effects of SDF-1α & β on the expression of SDF-1 ligands and receptors using a dose range know to differentially affect SDF-1α mediated CXCR4 signaling pathway choice due to ligand biased GPCR activation.

Caloric Restriction and the Adipokine Leptin alter the SDF-1 signaling axis and autophagy in Bone and MSCs

Sudharsan Periyasamy-Thandavan (1), Samuel Herberg (6), Phonepasong Arounleut (1), Sunil Upadhyay (1), Amy Dukes (1), Colleen Davis (1), Maribeth Johnson (5), Mark W. Hamrick (1,2,3,4), Carlos Isales (2,3,4), and William D. Hill (1,2,3,4). 1Department of Cellular Biology & Anatomy (1), Department of Orthopaedic Surgery (2), Institute of Molecular Medicine and Genetics (3), Institute for Regenerative and Reparative Medicine (4), Department of Biostatistics (5), Georgia Regents University, Augusta, GA, USA, Case Western Reserve University, Cleveland, OH, USA (6). Within the bone marrow (BM), both osteogenic and adipogenic lineage cells originate from multipotent mesenchymal stromal/stem cells (MSCs). MSCs are a major source of the secreted chemokine stromal cell-derived factor-1 (SDF-1), which is critical in MSC differentiation and BM residence, largely via its receptor CXCR4. A major factor in age-associated osteoporosis is the fate of MSCs. Growing evidence suggests that SDF-1 is critical in regulating MSC differentiation resulting in either a pro-osteogenic fate, or an adipogenic one that leads to reduced bone mass and mineral density, as well as increased BM adipocytes. Leptin, a cytokine-like hormone is secreted in large part by adipocytes, exhibits anti-osteogenic effects via hypothalamic receptors. However, peripheral administration of leptin can demonstrate bone protective effects. Previous studies in mice suggest that dietary restriction decreases circulating leptin while increasing serum SDF-1 levels similar to the effect of aging. In contrast, the opposite occurs with diet-induced obesity. This study investigates the effects of caloric restriction (CR) and leptin on the SDF-1/CXCR4 axis in bone and BM tissues. For in vivo studies, we collected bone, BM cells and BM interstitial fluid from 12 and 20 month-old C57Bl6 mice fed ad-libitum (AL), and 20 month-old mice on CR with, or without, leptin for 10 days (10mg/kg body weight). To mimic conditions of CR in vitro, murine BM derived MSCs (BMSCs) were treated with 1) control (Ctrl): normal proliferation medium, 2) CR: low glucose, low serum medium, or 3) CR+leptin: low glucose, low serum medium + 100 ng/ml rmLeptin for 6-72 h. Both SDF-1 and CXCR4 protein and mRNA expression in MSCs were increased by CR and CR + leptin. In contrast, the alternate SDF-1 receptor CXCR7 was decreased, this supports a change in SDF-1 signaling due to a shift in receptor availability. Additionally, autophagic flux was increased with CR and attenuated with the addition of leptin. However, mRNA isolated from bone shows SDF-1 and CXCR4 & 7 increase with age and this is reversed with CR, but addition of leptin returns this to the “aged” level. This suggests that in bone CR and leptin alter the nutrient signaling pathways in different ways to affect autophagy and the osteogenic cytokine SDF-1’s local action. Studies focusing on the molecular interaction between nutrient signaling and autophagy mediated by CR, leptin and SDF-1 axis may help to address age-related musculoskeletal changes.

Poster 19

Mediation of SDF-1/CXCR4 signaling in aged skeletal muscle by the adipokine leptin.

* Samuel Herberg, Case Western Reserve University, USA, Sudharsan Periyasamy-Thandavan, Georgia Regents University & Charlie Norwood VAMC, USA, Phonepasong Arounleut, Georgia Regents University (formally Georgia Health Sciences University), USA, Sunil Upadhyay, Georgia Regents University, USA, Amy Dukes, Georgia Regents University, USA, Colleen Davis, Georgia Regents University, USA, Galina Kondrikova, Georgia Regents University, USA, Maribeth Johnson, Georgia Regents University, USA, Carlos Isales, Georgia Regents University, USA, William Hill, Georgia Regents University & Charlie Norwood VAMC, USA, Mark Hamrick, Georgia Health Sciences University, USA

Aging is associated with a decline in both muscle and bone mass, which is thought to involve dysfunction in bone- and muscle-derived stem cells. The chemokine SDF-1 (CXCL12) and its receptor CXCR4 play important roles in the migration, survival, and engraftment of stem cells, including bone marrow stromal cells (BMSCs) and muscle satellite cells (SCs). Adult muscle constitutively expresses SDF-1, and CXCR4 expression in muscle is elevated during muscle regeneration. Previous work in mice has shown that, with food restriction, leptin levels are decreased while SDF-1 levels are increased, and the reverse is observed with diet-induced obesity. We therefore speculated that leptin and the SDF-1/CXCR4 axis may interact throughout adulthood to mediate musculoskeletal tissue repair and regeneration. We tested this hypothesis by examining SDF-1 and CXCR4 expression in muscles of mice aged 12 months and 20 months fed ad-libitum (AL) and mice 20 months on caloric restriction (CR). We also treated mice aged 20 months on caloric restriction with recombinant mouse leptin for 10 days at 10 mg/kg body weight. Serum SDF-1 was increased in the aged mice, but leptin treatment reduced serum SDF-1 to levels seen in the younger (12 month) animals. SDF-1alpha expression in muscle decreased with age, but was increased with caloric restriction, and leptin treatment reversed this increase. CXCR4 expression in muscle decreased slightly with age, but was increased almost four-fold with caloric restriction, and this increase was attenuated by leptin treatment. Together these data suggest that food restriction is a potent stimulus for SDF-1 and CXCR4 activation in aged muscle, and that leptin can antagonize this effect. Thus, while we have previously demonstrated that leptin can increase muscle mass and IGF-1 in leptin-deficient rodents, leptin may also impair the potential for muscle regeneration and satellite cell migration via its effects on SDF-1/CXCR4 interactions.

TITLE: Therapeutic and Imaging Potential of Umbilical Cord Blood Derived Endothelial

Progenitor Cells in Stroke and Glioma

AUTHORS: 1,2B.R Achyut, 2Branislava Janic, 2Nadimpalli Ravi S Varma, 1,2ASM Iskander,

1,2Adarsh Shankar 1,2Ali S Arbab

AFFILIATIONS: 1Biochemistry and Molecular Biology, Cancer Center, Georgia Regents

University, Augusta, GA. 2Cellular and Molecular Imaging Laboratory, Henry Ford Health

System, Detroit, MI

ABSTRACT

Nervous system has limited regenerative potential in disease conditions such as cancer,

neurodegeneration, stroke, and several neural injuries. Umbilical cord blood (UCB) derived

hematopoietic stem cells (HSCs) are capable of giving rise hematopoietic, epithelial, endothelial

and neural progenitor cells. Thus, suggested to significantly improve graft-versus-host disease

and represent the distinctive therapeutic option for brain diseases. Recent advances in strategies

to isolate, expand and shorten the timing of UCB stem cells engraftment have tremendously

improved the efficacy of transplantations. A subpopulation of CD34+ human HSCs identified by

the cell-surface molecule AC133+ (CD133+), have been shown to be more specific for

endothelial differentiation and vascular repair. In addition, emerging applications of UCB

derived AC133+ endothelial progenitor cells (EPCs) as imaging probe, regenerative agent, and

gene delivery vehicle have been studied by our lab and others. We have been exploited AC133+

EPCs for in vivo imaging modalities, importantly; magnetic resonance imaging (MRI) to monitor

the migration and engraftment efficacy of administered cells in stroke and glioma preclinical

animal models.

!

Establishment of Stro1/CD44 as new markers for human myometrial/ fibroid stem cell Mas A1, Nair S1, Laknaur A1, Diamond MP1, Simon C2, 3, Al- Hendy A1. 1Department of Obstetrics and Gynecology, Georgia Regents University (GRU), Augusta, Georgia, USA.2Fundación Instituto Valenciano de Infertilidad, Instituto Universitario IVI- University of Valencia, INCLIVA, Valencia, Spain;3Department of Obstetrics and Gynecology, School of Medicine, Stanford University, California, USA. Keywords: Uterine fibroids, stem cells, Stro1/CD44. Background: Uterine fibroids are benign monoclonal tumors, each arising from a single dysregulated myometrial smooth muscle cell, likely a stem cell. To date, putative stem cells have been isolated from several female reproductive organs, especially myometrium, using the side population (SP) technique. However, given the limitations of this approach, further studies are required to establish a more refined method of isolating stem cells based on the presence of specific and unique surface markers. Objective: To identify and characterize new specific myometrial/fibroid stem cell markers in human myometrium to better understand their implication in the development of uterine fibroids. Methods: Fibroids (F) and adjacent myometrium (MyoF) tissues were processed after signed informed consent from patients undergoing hysterectomy. After specific tissue digestion with collagenase, single cell suspensions were treated with CD44 and Stro1 antibodies-coated biotinylated Dynabeads. CD44-/Stro1- and CD44+/Stro1+ cells were isolated and cultured under hypoxic conditions (1-2% O2). Molecular analysis for stem cell markers, ABC transporters and hormonal receptors were performed in isolated cells. Cell characterization was also achieved by an immunophenotypic analysis using typical mesenchymal/ hematopoietic markers as well as in vitro differentiation assays.Additionally, isolated cells were transfected with iron-RhoB nanoparticles and injected under the renal capsule of female NOG mice with E2/P4 sex steroids supplement for 8 weeks. During this period, MRI and fluorescent imaging were performed on live animals and IHC assays were performed on the xenografts to assess the formation of myometrial/fibroid-like tissues. Results: Using Stro1/CD44 markers, we were able to isolate stem cells from MyoF as well as from F tissues. At mRNA level, these cells expressed ABCG2 transporter as well as other specific stemness markers such as Oct4, Nanog and GDB3. However, they showed a low expression of steroid receptors: ER-alpha and PR-A/PR-B, suggesting that they are not yet hormonally committed.Presence of typical mesenchymal markers (CD90, CD105, CD73) and absence of hematopoietic stem cell markers (CD34, CD45) supported their mesodermal origin. Moreover, we demonstrated the ability of these cells to differentiate in vitro into adipocytes, osteocytes and chondrocytes, further supporting mesodermal lineage derivation. Finally, their functional capability to form fibroid-like lesions, was established in an animal model by MRI findings and further confirmed by fluorescence imaging and histology staining, demonstrating the regenerative potential of putative fibroid stem cells in vivo. Conclusions: We have identified and characterized new specific fibroid stem cell markers in human myometrium, demonstrating the functional capacity of these Stro1+/CD44+ cells to induce fibroid like tissue by in vitro and in vivo approaches. These findings offer a useful tool to better understand the origin and initiation of uterine fibroids which can improve the development of more effective therapeutic options.

Myometrial Tumor-Forming Stem Cells in a Murine Model of Uterine Fibroids Reside in Hypoxic Niches Mas A1, Laknaur A1, O’Connor P.M 2, Walker C.L3, Diamond MP1, Simon C4, 5, Al- Hendy A1. 1 Department of Obstetrics and Gynecology, Georgia Regents University (GRU), Augusta, Georgia, USA. 2Department of Physiology, Georgia Regents University (GRU), Augusta, Georgia, USA. 3Center for Translational Cancer Research, Houston, Texas, USA. 4Fundación Instituto Valenciano de Infertilidad, Instituto Universitario IVI- University of Valencia, INCLIVA, Valencia, Spain; 5Department of Obstetrics and Gynecology, School of Medicine, Stanford University, California, USA. Keywords: Eker rat, stem cells, hypoxia. Background: The Eker rat is a murine model for uterine fibroid formation. They carry a germ line mutation in the Tsc2 tumor suppressor gene, producing uterine tumors with similar anatomic, histologic, and biologic features to human uterine fibroids. To date, the potential role of stem cells in the formation of uterine fibroids in Eker rats has not been investigated. Furthermore, prior studies revealed strong regulatory links between O2 availability and function of stem cells. Objective: In this study, we aim to identify and localize the myometrial stem cell population in the Eker rat's uterus using specific surface markers, explore their role in fibroid tumor formation, as well as to establish the role of hypoxia in the proliferation of myometrial stem cells. Methods: Uterine horns, cervix and fibroid tissues from Eker rat uterus were firstly examined via double immunohistochemistry (IHC) to co-localize the putative myometrial stem cell markers Stro1/CD44 with the established markers of undifferentiation such as c-KIT, OCT-4 and NANOG. Subsequently, the Eker rat uterus were digested at different points (cervix, lower, middle and upper horn) with collagenase to obtain single cell suspensions in order to compute by flow cytometry the percentage of Stro1/CD44 stem cells. To determine in vivo tissue oxygen tension (pO2) in different anatomical locations of Eker rat uterus, Clark type oxygen micronsensors (Unisense) were used and changes in hypoxia were also examined by injection of hypoxia-detecting dye pimonidazole followed 24 hours later by IHC as well as specific staining for the well-known hypoxic markers: CAIX and HIF-α. Results: In the Eker rat model, uterine fibroid lesions occur predominantly in the cervix in 85% of cases versus 15% in the uterine horns. We demonstrate the co-localization of Stro-1/ CD44 with other specific stem cell markers (Oct-4, c-kit, nanog) validating their undifferentiated status and their distribution in Eker fibroids, uterus horns and cervix. Interestingly, IHC quantification demonstrated that significantly higher number of stem cells are present in the cervical region compared to uterine horns. Flow cytometric analysis corroborated that the percentage of stem cells in cervix is significantly higher than in uterine horns (P<0.05). The quantification of in vivo oxygenation demonstrates that the pO2 levels in cervix (13.24 mmHg) were significantly lower than in uterine horns (29.47mmHg; pANOVA=0.0014). Moreover, pimonidazole, CAIX and HIF-α staining demonstrating stronger expression in cervix than in uterine horns confirm the hypoxic status of cervix region when compared to other regions of the uterus. Conclusions: Eker rats serve as a preclinical model to screen for and evaluate the origin of uterine fibroids. For the first time, we have identified the myometrial stem cell niche by stem cell markers and we have establish an association of hypoxic niches in uterine cervix which is the primary location of fibroid lesions in this model. These observations could provide a putative mechanism by which myometrial stem cells regulate transformations into tumor initiating cells.

Contact information for Symposium Participants

Last Name First Name Degree Institution Name Institution Department/Program City State Phone Number Email

Achyut Bhagelu PhD Georgia Regents University Biochemistry and Molecular Biology, Cancer Center Augusta GA 706-721-4375 bachyut@gru.eduAguilar-Perez Alexandra BA/BS Georgia Regents University Cellular Biology and Anatomy Augusta GA 706-721-4797 AAGUILARPEREZ@gru.eduAhuja Manuj PhD Georgia Regents University Pharmacology & Toxicology Augusta GA 706-721-0905 manuj.ahuja@gmail.com

Al-Hendy Ayman MD/Ph.D Georgia Regents University

Dean for Global Translational Research, Professor and Director Division of Translational Research, Obstetrics and Gynecology Augusta GA 706-721-3833 aalhendy@GRU.edu

Alsberg Eben PhD Case Western Reserve University

Director of the Stem Cell and Engineered Novel Therapeutics Laboratory, Dept. Biomedical Engineering and Dept. Orthopaedic Surgery Cleveland OH 216-368-6425 exa46@case.edu

Alsulami Meshal MD Georgia Regents University Cancer Research Center Augusta GA malsulami@gru.eduAndrews Seth University of Georgia Bioengineering Athens GA sha72433@uga.eduAngara Kartik BA/BS Georgia Regents University Biochemistry and Molecular Biology Augusta GA 706-721-4375 kangara@gru.eduAoued Hadj M.S. Georgia Regents University Neuroscience & Regenerative Medicine Augusta GA haoued@gru.eduArbab Ali MD/Ph.D Georgia Regents University Cancer Research Center Augusta GA 706-721-8909 aarbab@gru.edu

Averett Rodney PhD Georgia Institute of TechnologyGeorge W. Woodruff School of Mechanical Engineering/IBB Atlanta GA rdaverett@gatech.edu

Badr Marwa MD Georgia Regents University OB/GYN Augusta GA 228-343-3914 mbadr@gru.eduBalotin Jeanette MPA MA Georgia Regents University Medical College of Georgia Augusta Ga 706-721-3391 jbalotin@gru.eduBenson Krista BA/BS Georgia Regents University Biological Sciences Martinez GA 706-284-6941 kristab93@comcast.net

Berman Adam MD Georgia Regents University

Director, Cardiac Arrhythmia Ablation Services, Dept. Medicine, Institute for Regenerative and Reparative Medicine Augusta GA 706-721-7815 ABERMAN@gru.edu

Betancur Martha BA/BS University of GeorgiaAnimal and Dairy Science, Regenerative Bioscience Center Athens GA 678-896-0248 marthbet@uga.edu

Bollag Wendy PhD Georgia Regents UniversityPhysiology, Institute for Regenerative and Reparative Medicine Augusta GA 706-721-0698 wbollag@gru.edu

Botchwey Edward PhD Georgia Institute of Technology Parker H. Petit Institute for Bioengineering and Biosciences, Biomedical Engineering Atlanta GA 434-249-6040 edward.botchwey@bme.gatech.edu

Brakta Soumia MD Georgia Regents University OB/GYN Augusta GA 504-578-8804 sbrakta@gru.eduCaldwell R. William PhD Georgia Regents University Pharmacology and Toxicology Augusta GA 706-721-3383 wcaldwel@gru.edu

Caplin Arnold I. Case Western Reserve UniversityProfessor of Biology and Director of the Skeletal Research Center Cleveland OH 216-368-3562 aic@cwru.edu

Clark Carl PhD Georgia Regents University Office of Innovation Commercialization Augusta GA 706-721-4055 caclark@gru.eduCzikora Istvan PhD Georgia Regents University Vascular Biology Center Augusta GA 706-721-6759 iczikora@gru.edu

Diamond Michael P. MD Georgia Regents University

Sr. VP for Research, Assoc. Dean, the Brooks Chair, and Chair of OB/GYN, Director - Clinical & Translational Research Augusta GA 706-721-3591 michael.diamond@gru.edu

Edwards Connie RN Georgia Regents University OB/GYN Augusta GA 706-721-9680 coedwards@gru.eduElhusseini Heba MD Georgia Regents University OB/GYN Augusta GA 706-589-4276 HELHUSSEINI@gru.edu

El-Remessy Azza PhD University of Georgia & GRUAssociate Professor, Director, Clinical and Experimental Therapeutics Graduate Program 706-721-6760 AELREMESSY@gru.edu

Erion Joanna BA/BS Georgia Regents University Department of Neuroscience & Regenerative Medicine Augusta GA jerion@gru.edu

Eroglu Ali PhD Georgia Regents UniversityDepartment of Neuroscience & Regenerative Medicine, Institute for Regenerative and Reparative Medicine Augusta GA aeroglu@gru.edu

Contact information for Symposium Participants

Last Name First Name Degree Institution Name Institution Department/Program City State Phone Number Email

Franklin Sam DVM/PhD University of Georgia

Department of Small Animal Med & Surg., College of Veterinary Medicine, Regen Biosci Ctr

Athens GA 706-206-7752 spfrank@uga.edu

Fulzele Sadanand PhD Georgia Regents UniversityOrthopaedic Surgery, Institute for Regenerative and Reparative Medicine CB2509 GA 706-721-4850 sfulzele@gru.edu

Galipeau Jacques MD Emory University EPIC Atlanta GA 404-831-1209 jgalipe@emory.eduGavalas Charlotte BA/BS Georgia Regents University Clinical Trials Office/Orthopaedic Surgery Augusta GA 706-721-2199 cgavalas@gru.eduGhoshal-Gupta Sampa PhD Georgia Regents University Pathology Augusta GA samgupta@gru.eduGomillion Cheryl PhD University of Georgia College of Engineering/Biological Engineering Athens GA 706-542-0918 ctgomillion@uga.eduGuo De-Huang PhD Georgia Regents University Augusta GA 706-721-1470 dguo@gru.eduHalder Sunil PhD Georgia Regents University Department of Obstetrics and Gynecology Augusta GA 706-721-8907 shalder@gru.eduHan Qimei MD Georgia Regents University College of Graduate Studies Augusta GA 205-240-9206 qhan@gru.eduHanda Hitesh PhD University of Georgia Athens GA 706-542-8109 hhanda@uga.eduHardigan Trevor BA/BS Georgia Regents University Augusta GA 706-721-0925 thardigan@gru.eduHayman Joy M.S. Georgia Regents University RDS Research support Augusta GA 706-721-7436 jhayman@gru.eduHelwa Inas PhD Georgia Regents University Cellular Biology and Anatomy Augusta GA 706-721-4843 ihelwa@gru.edu

Hess David C. MD Georgia Regents UniversityPresidential Distinguished Chair, Chair Dept of Neurology Augusta GA (706) 721-1691 DHESS@gru.edu

Hill William D. PhD Georgia Regents University

Professor Depts. Cellular Biology & Anatomy, Orthopaedic Surgery, Neurology and Institute for Regenerative and Reparative Medicine Augusta GA 706-721-2019 whill@gru.edu

Holloway Joan BA/BS Georgia Regents University Clinical and Translational Sciences Augusta GA 706-721-9942 jholloway@gru.eduHunter Monte MD Georgia Regents University Chair, Dept Orthopaedic Surgery Augusta GA 706-721-6172 mohunter@gru.edu

Isales Carlos MD Georgia Regents University

Regents' Professor, Vice Chair for Clinical Affairs, DNRM, Vice Chair of Clinical and Translational Research Orthopaedic Surgery, Director of Institute for Regenerative and Reparative Medicine Augusta GA (706) 721-0692 CISALES@gru.edu

Jain Meenu PhD Georgia Regents University Cancer Research Center Martinez GA 301-204-7490 jainm25@hotmail.com

Karumbaiah Lohitash PhD The University of GeorgiaAssistant Professor, Regenerative Bioscience Center, ADS Athens GA 706-542-2017 lohitash@uga.edu

Kelly Ryan BA/BS Medical University of South CarolinaVAMC Research Services/Department of Pathology and Laboratory Medicine Charleston SC 410-409-2558 kellyrr@musc.edu

Khan Mohammad B PhD Georgia Regents University Neurology Augusta GA 706-721-1671 mbkhan@gru.eduKim Ha Won PhD Georgia Regents University Vascular Biology Center Augusta GA hkim3@gru.eduKinder Holly BA/BS University of Georgia Regenerative Bioscience Center Athens GA 7702861810 HollyK117@gmail.comKondrikova Galina BA/BS Georgia Regents University Cell Biology and Anatomy Augusta GA gkondrikova@gru.edu

Kuhn Liisa T. PhD University of Connecticut Health Ctr Reconstructive Sciences Department, Biomedical Engineering Department Farmington CT 860-679-3922 lkuhn@uchc.edu

LaRue Amanda PhD Medical University of South CarolinaPathology and Laboratory Medicine Charleston SC 843-789-6713 laruerc@musc.eduLaserna Candelario BA/BS Georgia Regents University Clinical Trials Office Augusta GA calaserna@gru.eduLeMosy Ellen MD/Ph.D Georgia Regents University Cellular Biology and Anatomy Augusta GA 706-721-0876 elemosy@gru.edu

Lin Sen PhD Georgia Regents University Department of Neuroscience & Regenerative Medicine Augusta GA 706-721-5780 selin@gru.eduLiu Yutao PhD Georgia Regents University Cellular Biology and Anatomy Augusta GA 706-721-2015 yutliu@gru.eduLocklin Jason PhD University of Georgia Chemistry/Engineering Athens GA 706-542-2359 jlocklin@uga.eduLucas Rudolf PhD Georgia Regents University Vascular Biology Center Augusta GA 706-721-9470 rlucas@gru.edu

Contact information for Symposium Participants

Last Name First Name Degree Institution Name Institution Department/Program City State Phone Number Email

Luo Tong PhD Georgia Regents University Department of Neuroscience & Regenerative Medicine Augusta GA 706-721-8910 tt342400@gmail.comMas Aymara PhD Georgia Regents University OB/GYN Augusta GA amasperucho@gru.edu

McGee-Lawrence Meghan PhD Georgia Regents UniversityCellular Biology and Anatomy, Institute for Regenerative and Reparative Medicine Augusta GA 706-446-0128 mmcgeelawrence@gru.edu

McKenzie Marie M.S. University of Georgia Environmental Health Science Athens GA 678-642-8547 mdopson@uga.eduMoreno Ricardo PhD Medical University of South CarolinaRegenerative Medicine Charleston SC 843-647-0996 morenor@musc.eduMortensen Luke PhD University of Georgia Regenerative Bioscience Center Athens GA luke.mortensen@uga.eduNeiswender Hannah BA/BS Georgia Regents University Cell Biology and Anatomy Augusta GA 706-721-0893 hneiswender@gru.eduOh Se-Yeong PhD Emory University Pediatrics Atlanta GA 404-727-9961 se-yeong.oh@emory.eduOlala Lawrence PhD Georgia Regents University Physiology Augusta GA 706-721-0704 lolala@gru.edu

Pan Jinxiu MD Georgia Regents University Department of Neuroscience & Regenerative Medicine Augusta GA 706-721-5780 JPAN@GRU.EDU

Park Changwon PhD Emory UniversityAssistant Professor, Department of Pediatrics, Center for Cardiovascular Biology Atlanta GA 404-727-7143 changwon.park@emory.edu

Park Mary Anne M.S. Georgia Regents UniversityManager Clinical Trials Office, Clinical & Translational Sciences/CTO Augusta GA 706-721-0193 mpark@gru.edu

Peard Leslie BA/BS Georgia Regents University Medical College of GA Augusta GA 404-403-0505 lpeard@gru.edu

Periyasamy-ThandavanSudharsan PhD Georgia Regents UniversityCellular Biology and Anatomy, Institute for Regenerative and Reparative Medicine Augusta GA 706-721-4797 spthandavan@gru.edu

Peroni John DVM, MS, Dip ACUSUniversity of GeorgiaDept. Large Animal Medicine, College of Veterinary Medicine, Reg. Biosci. Ctr. Athens GA jperoni@uga.edu

Pierce Jessica BA/BS Georgia Regents University Cellular Biology & Anatomy Augusta GA 678-467-8244 jepierce@gru.edu

Platt Simon

BVM&S MRCVS Dipl. ACVIM, Dipl. ECVN University of Georgia

Neurology & Neurosurgery Svc, Dept. of Small Animal Med. & Surg./ College of Vet Med Athens GA 706-206-6692 srplatt@uga.edu

Rajpurohit Surendra PhD Georgia Regents University Cancer Center Augusta GA 706-267-4590 srajpurohit@gru.eduRosson Brenda BSN Georgia Regents University Clinical Trials Office Augusta GA 706-721-9680 brosson@gru.eduScharf Alex University of Georgia College of Veterinary Medicine Athens GA ascharf@uga.eduSen Nilkantha PhD Georgia Regents University Neuroscience & Regnerative Medicine Augusta GA 706-721-8185 nsen@gru.eduSeremwe Mutsa PhD Georgia Regents University Joint Physiology & Cell Biology and Anatomy Augusta GA mseremwe@gru.eduShaaban Sherif MD Georgia Regents University Cancer CenterBiochemistry and Molecular Biology Augusta GA 216-632-2986 SSHAABAN@gru.eduShalaby Shahinaz MD Georgia Regents University OB/GYN Augusta GA 615-419-5718 sshalaby@gru.eduShankar Adarsh BA/BS Georgia Regents University Biochemistry and Molecular Biology Augusta GA 706-721-4375 ashankar@gru.eduShin Eric MD Emory University Cardiology Atlanta GA 765-532-6707 e.y.shin@emory.eduSolomon Harold BA/BS Georgia Institute of Technology VentureLab Atlanta GA 404-939-6271 harold.solomon@venturelab.gatech.edu

Stevens Mark DDS Georgia Regents UniversityCollege of Dental Medicine/Oral and Maxillofacial Surgery Augsta GA 706-721-2411 MASTEVENS@GRU.EDU

Stice Steve University of Georgia

Georgia Research Alliance Eminent Scholar Chair, Professor and Director of the Regenerative Bioscience Center, UGA Director of the Regenerative Engineering and Medicine partnership Athens GA 706-583-0071 sstice@uga.edu

Su Yun PhD Georgia Regents University Institute for Regenerative and Reparative Medicine Augusta GA 706-721-7920 YUSU@gru.edu Tang Yaoliang MD/Ph.D Georgia Regents University Vascular Biology Center Augusta GA 909-643-7388 yaotang@gru.edu

Contact information for Symposium Participants

Last Name First Name Degree Institution Name Institution Department/Program City State Phone Number Email

Temenoff Johnna Georgia Institute of Technology

Associate Professor, Coulter Department of Biomedical Engineering at Georgia Tech/Emory, GIT Director of the Regenerative Engineering and Medicine partnership Atlanta GA 404-385-5026 johnna.temenoff@bme.gatech.edu

Thomas Bobby PhD Georgia Regents University Pharmacology, Toxicology and Neurology Augusta GA 706-721-6356 bthomas1@gru.eduWest Franklin PhD University of Georgia Assistant Professor, Regenerative Bioscience Center Athens GA (706) 542-0988 westf@uga.edu

Wikesjö Ulf DDS Georgia Regents UniversityOral Biology, Institute for Regenerative and Reparative Medicine Augusta GA 706288-8649 uwikesjo@gru.edu

Wilson Katie M.S. Medical University of South CarolinaPathology and Laboratory Medicine Charleston SC krw34@musc.eduWilson Kyle BA/BS Georgia Regents University Department of Medicine, Hematology/Oncology Augusta GA 706-871-6591 kylewilson778@gmail.comWu Dona MD/Ph.D Emory University Microbiology & Immunology / Vaccine Center Atlanta GA 404-712-0563 dona.wu@emory.eduWyatt Emily BA/BS University of Georgia Regenerative Bioscience Center Athens GA elwyatt@uga.eduYang Nianlan MD/Ph.D Georgia Regents University DNRM Augusta GA 706-721-0519 nyang@gru.eduYoon Young-Sup Emory University Stem Cell Research Group Cardiology Atlanta GA 404-712-1733 yyoon5@emory.eduZhang Maoxiang PhD Georgia Regents University PHAMARCOLOGY Augusta GA 706-306-6423 mazhang@gru.eduZhang Mingzhen PhD University of Georgia Athens GA zhangmz@hotmail.comZhao Qun PhD University of Georgia Regenerative Bioscience Center Athens GA qunzhao@uga.eduZimmerman Arthur M.S. Georgia Regents University Department of Cellular Biology and Anatomy Augusta GA azimmerman1@gru.edu