6th annual conference stem cell research regenerative...

Host Institution

FEBRUARY 6–7, 2020

Stem Cell Research6th Annual Conference on

& Regenerative Medicine

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About RegenMed SA

RegenMed SA is a citywide organization in San Antonio designed to facilitate networking and to promote interactions among individuals, institutions, centers, installations, companies and foundations with interests in stem cell research, tissue engineering, regenerative medicine and/or biotechnology related to these areas.

RegenMed SA includes representation of individuals, facilities and resources within San Antonio related to stem cell research and regenerative medicine. Those organizations include the University of Texas at San Antonio, UT Health San Antonio, the Southwest Research Institute, the Texas Biomedical Research Institute and the Southwest National Primate Research Center, the U.S. Army Institute of Surgical Research, the U.S Air Force 59th Medical Wing, BioBridge Global and GenCure, StemBioSys, and many other biotechnology companies. RegenMedSA.org

Mission and Vision

The mission of RegenMed SA is to facilitate and promote communication, interaction and collaboration among the many people and facilities within San Antonio and neighboring regions that share interested and resources related to areas of stem cell research, tissue engineering and regenerative medicine. The San Antonio Conference on Stem Cell Research and Regenerative Medicine is designed to provide a forum for the exchange of information describing ongoing research, education, innovation, clinical application and product development in these areas.

RegenMed SA Steering Committee

Andre Cap, U.S. Army Institute of Surgical ResearchBecky Cap, GenCureSy Griffey, StemBioSysJian Ling, Southwest Research InstituteJohn McCarrey, University of Texas at San AntonioChristi Walter, UT Health San AntonioErik Weitzel, U.S Air Force 59th Medical Wing

Baboon iPS cells reprogrammed from adult peripheral blood mononuclear cells (PBMCs). A trio of highly conserved transcription factors (NANOG, POU5F1 and SOX2) serve as the master regulators of pluripotency. POU5F1 (green) is labeled in this image and is observed in the nucleus of each cell. DNA is labeled with DAPI (blue). Image courtesy of Dr. Christopher Navara, Ph.D., Director of the UTSA Stem Cell Core and Associate Professor of Research, Department of Biology, University of Texas at San Antonio.

On the cover:

Overview

Mission and VisionMission and Vision

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Welcome to the Sixth Annual Welcome to the Sixth Annual Conference on Stem Cell Research Conference on Stem Cell Research

& Regenerative Medicine & Regenerative Medicine organized by RegenMed SAorganized by RegenMed SA

Very special thanks to our conference sponsors

Platinum:

Silver:

Bronze:

Vendor:

Sponsors

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Thursday, February 6

8:25 a.m. Welcome – Becky Cap

SESSION 1 [Session Chair – John McCarrey, UTSA]

8:30 a.m. Xiaowu Wu (USA-ISR) Systemic Administration of Bone Marrow Derived Stromal Cells: Hemostasis and Acute Immune Response in Rats with Polytrauma and Hemorrhagic Shock

8:50 a.m. Dimitrios Miserlis (UT Health San Antonio) Vascular Surgery and Biomedical Engineering Research

9:15 a.m. Gabrila Uribe (UTSA) Bioelectronic Medicines to Control Neural Activity

9:40 a.m. Jian Ling (SwRI) Update of SwRI’s Novel Cell Manufacturing Bioreactor for Regenerative Medicine

10:00 a.m. Thomas Orzak – Norton Rose Fulbright

10:05 a.m. Coffee break

10:25 a.m. Brian Hermann (UTSA) Examining Spermatogonial Stem Cell Fate In Vitro

10:50 a.m. Barbara Christy (USA-ISR) Cell Therapy for Traumatic Injury: Promise and Challenges

11:10 a.m. Keynote Lecture Introduced by Christi Walter (UT Health San Antonio) Feng-Chun Yang (UT Health San Antonio)

ASXL Family Proteins in Stem Cell Biology and Disease

12:10 a.m. Lunch (provided)

Program

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SESSION 2 [Session Chair – Andre Cap, USA-ISR]

1:00 p.m. Travis Block (StemBioSys) Cell-Derived Extracellular Matrix Supports Rapid Maturation of iPSC-Derived Cardiomyoctyes in 2-D Culture

1:20 p.m. Tiffani Chance (USA-ISR) The Angiogenic Potential of hMSC-EVs

1:40 p.m. Peng Zhang (UT Health San Antonio) Loss of INTS11 Destabilizes the PRC2 Complex and induces p21-Dependent Cell Cycle Arrest in Hematopoietic Stem Cells

2:00 p.m. Andrew Sheen (USAF) Novel regenerative approaches to sports medicine injuries

2:15 p.m. Vanesa Nieto-Estevez (UTSA) Precision Models of ARX-Associated Genetic Epilepsies

2:40 p.m. Joe McDonough – Southwest Research Institute

2:45 p.m. Coffee break

3:00 p.m. Nicholas Clay (USA-ISR) A Plasma-alginate Composite Material Provides Improved Mechanical Support or Dual Stem Cell and Antimicrobial Delivery

3:20 p.m. Bruce Bunnell (Tulane Center for Stem Cell Research & Regenerative Medicine) Adipose Stem Cells Efficiently Inhibit Ongoing Inflammation In Vivo

3:45 p.m. Xiao-Dong Chen (UT Health San Antonio & StemBioSys) Co-Transplantation of Islets with Adipose-derived Stem Cells Synergistically Reduced Blood Glucose in STZ-induced Diabetic Rats

4:10 p.m. Lindsey Macpherson (UTSA) Rewiring Taste with Semaphorin Signals

4:35 p.m. Mingjiang Xu (UT Health San Antonio) Regulation of Hematopoietic Stem Cell Function by Long Non-Coding RNAs

5-7 p.m. Poster Session and Networking Reception

Program

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Friday, February 7

SESSION 3 [Session Chair – Erik Weitzel, USAF 59th Medical Wing]

8:30 a.m. Jae Choi (USA-ISR) Effects of Systemic Stem Cell Therapy on End Organ Injury Severity in ARDs

8:50 a.m. Anibal Diogenes (UT Health San Antonio) Oral Stem Cells and Neurogenesis

9:15 a.m. John McCarrey (UTSA) Validation of Baboon Pluripotent Cells as a Model for Translational Stem Cell Research

9:40 a.m. Jaime Garza (UT Health San Antonio & Texas Center for Cellular Therapy & Research) Review of Cell Therapy Clinical Trials for OA in Texas

10:05 a.m. Asif Maroof (UTSA) Identifying Pathophysiological Phenotypes Associated with Neurodegenerative Disease Progression using Human Pluripotent Stem Cells

10:30 a.m. Jennifer Potter – UT Health San Antonio

10:35 a.m. Coffee break

11:10 a.m. Panel Discussion on… Stem Cell Therapy – The Wrong Way & The Right Way Introduced by Sy Griffey (StemBioSys)

Moderated by Jessica Raley (BioBridge Global)

Laura Beil (Texas freelance author) “Frightening Introduction to the Stem Cell Clinics” Bruce Bunnell (Tulane University) “What is a Stem Cell?” Jaime Garza (UT Health San Antonio) “How Do You Do a Clinical Trial the Right Way?” Adrienne Mendoza (BioBridge Global) “Manufacturing of Stem Cells – Quality and Regulatory Measures”

12:10 a.m. Lunch (provided)

Program

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SESSION 4 [Session Chair – Jian Ling, SwRI]

1:00 p.m. Bernard Arulanandam (UTSA)

1:05 p.m. Wensheng Zhang (USAF) Exosome Analysis of Neural Regeneration

1:20 p.m. Emily Boice (USA-ISR) Isolation and Characterization of Extracellular Vesicles from Human Induced Pluripotent Stem Cells for Neuroprotective Application Post Retinal Injury

1:40 p.m. Steve Harris (UT Heath SA) Interaction of the Estrogen Receptor, Wnt Signaling, and the Bmp Gene in Periodontal Stem/Progenitor Cell Specification and Differentiation

2:05 p.m. Guillermo Vela (NeuScience) Using Computer Vision and Deep Learning to Assess Treatment Efficacy at the Single Cell Level

2:30 p.m. Conference adjourns

Program

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Speakers

Feng-Chun Yang (UT Health San Antonio)ASXL Family Proteins in Stem Cell Biology and Disease

Feng-Chun Yang, M.D./PhD, the A.B. Alexander Distinguished Chair in Cancer Research, is a Professor of the Department of Cell Systems & Anatomy at the UT Health Science Center San Antonio. Dr. Yang received her bachelor degree in Medicine at the Hebei Medical University, Hebei, China in 1983. She continued to excel in Ph.D. at Shinshu University School of Medicine, Japan followed by a two years of postdoc training in Dr. Tatsutoshi Nakahata’s laboratory to study the hematopoietic stem cell biology in Tokyo University. In 1998, Dr. Yang joined Dr. David Williams’s lab at Indiana University as a postdoctoral fellow of the Howard Hughes Medical Institute, studying the hematopoietic stem cell biology. She became an Assistant Professor in 2005 at the Herman B Wells Center, Department of Pediatrics, Indiana University School of Medicine. Since then, her team made several important contributions in understanding the role of tumor

suppressor, NF1, and epigenetic dysregulation (ASXL1/2) in hematopoietic stem cell functions and these gene mutations in the pathogenesis of myeloid malignancies. These works provided the first evidence that targeting interactions between tumor microenvironment and cancer stem cells are capable of slowing down the progression of plexiform neurofibromas (Cell 2008), which led to a successful phase I/II clinical trial in NF1 (Lancet Oncology 2012). Recently, they also discovered that ASXL1/2 exerts their functions via the cohesin complex to maintain normal sister chromatid separation and gene expression (Science Advances 2017, Nature Communication 2017, Cell Discovery 2018,). They also discovered that ASXL1 truncation mutations result in a gain-offunction by gaining interactions with BRD4 and BAP1, leading to malignant transformation (Blood 2018, Leukemia 2019). Currently, the Yang Lab is focusing on understanding the molecular mechanisms underlying epigenetic alteration mediated myeloid malignancies, with a specific focus of ASXL1, ASXL2 and PHF6 gene mutations. The goal of her research is to develop novel therapeutics to target myeloid cancers.

Laura Beil (Texas freelance author)“Frightening Introduction to the Stem Cell Clinics”

Laura Beil is an independent journalist specializing in medicine, health policy and science. She was the recipient of the Victor Cohn Prize for Medical Science Reporting in 2018, one of the topawards in medical journalism. Her work has appeared in numerous publications, including The New York Times, Cosmopolitan, Reader’s Digest, Texas Monthly, Men’s Health, and Science News. She began freelancing in 2007 after working as medical writer for the Dallas Morning News from 1992 to 2006. In 2018, she reported and hosted the podcast Dr. Death, which has been downloaded more than 50 million times. Her latest podcast, Bad Batch, looks at the for-profit stem cell industry: https://podcasts.apple.com/us/podcast/bad-batch/id1482851200 https://ipscell.com/2019/10/bad-batch-podcast-is-must-listen-for-stem-cell-community/ https://ipscell.com/2019/11/bad-batch-podcasts-dark-picture-of-perinatal-stem-cellindustry/

Keynote SpeakerKeynote Speaker

Panel MembersPanel Members

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Speakers

Bruce Bunnell (Tulane University)“What is a Stem Cell?”

Bruce Bunnell, PhD is the Director of the Tulane Center for Stem Cell Research and Regenerative Medicine and Professor in the Department of Pharmacology in the Tulane University School of Medicine. In addition, he holds the Aron Family Regents Distinguished Chair in Gene Therapy. Dr. Bunnell obtained his PhD in Microbiology from the University of Alabama at Birmingham School of Medicine. He then pursued Postdoctoral Fellowship research at the Howard Hughes Medical Institute in the School of Medicine at the University of Michigan and the National Human Genome Research Institute at the National Institutes of Health in Bethesda, MD. Dr. Bunnell was an Assistant Professor at the Nationwide Children’s Hospital Research Institute, part of the Ohio State University School of Medicine prior to joining the faculty at Tulane University in 2002. Dr. Bunnell’s research program is focused on both the basic science and

translational applications of adult stem cells isolated from the bone marrow and adipose tissue.

Jaime Garza (UT Health SA)“How Do You Do a Clinical Trial the Right Way?”

Dr. Jaime Garza is currently Professor of Orthopedic Surgery, Professor of Otolaryngology, Professor in the Center for Stem Cell Research and Regenerative Medicine at Tulane University School of Medicine; Professor of Surgery and Professor of Otolaryngology at UT Health-SA. He was the Founder and Chairman of their Division of Plastic and Reconstructive Surgery. He served on the University’s Executive Committee as Associate Vice President for South Texas Health Affairs. Dr. Garza is President of the Texas Center for Cellular Therapy and Research at the Texas Center for Athletes and is a past president of the Texas Society of Plastic Surgeons. In 2011, then Texas Governor Rick Perry appointed Dr. Garza a Regent to the Texas State University System, where he served as a two-term Chairman of the Board, and is now serving his eighth year as chair of the Academic and Health Affairs Committee and as a member of the Governmental Relations

Committee. An accomplished clinical researcher, he is one of the first clinician/scientists in the nation to receive an Institutional Review Board approval for a research study involving the use of human regenerative cells for the treatment of osteoarthritis of the human knee joint. His work was published in The Journal of Regenerative Medicine. He is one of three principal investigators in a national FDA approved Phase IIB stem cell trial for the treatment of knee osteoarthritis that has been accepted for publication in The American Journal of Sports Medicine.

Adrienne Mendoza (BioBridge Global)“Manufacturing of Stem Cells – Quality and Regulatory Measures”

Adrienne Blevins Mendoza is BioBridge Global’s Vice President of Global Quality, reporting to Chief Compliance Officer (CCO) Richey Wyatt. Adrienne serves on the company’s senior leadership team and oversees all quality & regulatory compliance matters, including strategies to delight the customer, improve execution of all GxP relevant processes, control risks, and improve efficiencies through Quality Systems Management. Adrienne joined BioBridge Global in 2016 from a background in Pharmaceuticals and Biologics, including Cell and Gene Therapies and Transfusion Medicine spanning nearly 20 years. Prior to joining BioBridge Global, she previously worked with Bachem, StemCyte, the American Red Cross Blood & Tissue Services, and as a partner in Blevins CUBED Architects. Adrienne earned a bachelor’s degree in Business Administration from California Coast University and participated in Administrative and Executive

Leadership programs at the Kellogg School of Management at Northwestern, and the University of Oklahoma.

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Poster Presentations

Optimizing Conditions and Analysis for Cold Storage of Mesenchymal Stromal Cells

Isaac Abaasah, Tiffany Heard, Maryanne Herzig, Barbara Christy, Christopher Delavan, Andrew P. Cap, James A. Bynum

Blood and Coagulation Research Department, US Army Institute of Surgical Research, Fort Sam Houston, Texas

Background

Mesenchymal stromal/stem cells (MSCs) are adult stem-like cells that show promise as cell therapy agents for the treatment of acute traumatic injury. Their potential benefits include anti-inflammatory and immune-modulatory activities that may lessen or interrupt damage progression following injury and reduce overall morbidity and mortality. Because MSCs are relatively immune-privileged, off-the-shelf use is possible without the need for tissue matching. MSCs are routinely cryopreserved for shipping, allowing for long shelf life but necessitating washing and handling. There is generally reduced viability and function immediately after thawing compared to freshly harvested cells. These conditions limit the utility of live MSCs for clinical use, especially in combat trauma. Optimization of conditions to allow successful cold storage of MSCs in solution for a matter of days, while maintaining an acceptable level of viability and function may ultimately allow preparation of cells prior to shipping and storage in the cold, increasing the feasibility of utilizing live cell therapy with MSCs in combat hospitals or even in remote locations. The studies detailed here focus on metabolic analysis of MSCs after cold storage in a variety of media.

Methods

Commercial human MSCs, thawed and expanded under standard culture conditions, were harvested using TrypLE (Gibco), washed 3X with Dulbecco’s phosphate buffered saline, divided into aliquots and resuspended in the different storage media to be tested and subsequently stored at 4oC. Cell testing was performed on day 0 (day of storage set-up) and at multiple timepoints thereafter. Daily testing included cell count and viability determination by trypan blue exclusion and determination of ATP levels using Cell Titre-Glo (Promega). Metabolic function was evaluated using the Seahorse XFe24 (Agilent) on day 0 and several subsequent days.Cells were affixed to the plate using Cell Tak (Corning) for immediate analysis to avoid adaptation back to adherent culture conditions. Following the Seahorse assay, plates were frozen at -80oC; DNA content per well (for normalization) was determined using FluoReporter Blue Fluorometric dsDNA Quantitation Kit (Molecular Probes) or Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen/Thermo Fisher Scientific).

Results & Conclusions

In these preliminary studies MSCs can be maintained in solution at 4oC after cell harvest for at least 96h while maintaining an acceptable level of viability and metabolic function. Supplementation with basic saline solutions with energy sources as well as pH stabilization have the greatest effect on sustaining cell viability and energetics. Determining metabolic function can be achieved both with a rapid ATP assay and an in depth metabolic panel. Studies continue to determine the optimal conditions to maximize the retention of MSC function for ultimate clinical benefit.

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The Role of the Kynurenine Pathway on Neural Stem Cell function Morgan Bucknor, Erzsebet Kokovay University of Texas Health Science Center at San Antonio Abstract | Neurogenesis continues throughout life in the subventricular zone (SVZ) that lines the lateral ventricles of the mammalian brain. This region of the brain serves as a specialized microenvironment which harbors the pool of adult neural stem cells (NSCs) and supports the production of functional new neurons. Neurogenesis is a highly intricate process which can be mediated by both intrinsic and extrinsic factors. The Kynurenine Pathway (KP) is the main route of tryptophan metabolism and believed to be associated with the pathophysiology of a number of neurodegenerative diseases. Accumulating evidence has showed some KP metabolites have neuroactive properties which can exert either a beneficial or detrimental biological effect. Quinolinic Acid (QA) is an endogenous excitotoxin of the KP which is secreted by microglia during inflammatory conditions. In contrast, Kynurenic Acid (KA), possesses anti-inflammatory and antioxidant properties, and is secreted by astrocytes and endothelial cells. It remains unclear how NSC’s are affected by this pathway and how its dysregulation directly effects the process of neurogenesis. Here, we propose to examine the neuroprotective properties of KA and its precursor Kynurenine (KYN) on NSC function. Our data shows that NSCs possess the enzymes necessary to generate KA and neuroblasts express NMDA receptors of which KA acts as an antagonist on. Furthermore, we have found elevated levels of NSC proliferation in KMO -/- mice (n=7), which have elevated levels of KA and KYN. Thus, we hypothesize that KYN and/or KA increase neurogenesis. We propose to test KYN and KA’s effects directly in vitro; specifically, on NSC proliferation, self-renewal and differentiation and test if neurogenesis is increased in vivo. Novel insights into the mechanisms by which KA exerts protective effects will be instrumental for understanding NSC function and could lead to novel strategies to increase brain repair.

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Daniel Carlisle, Katherine Smith, Nicolas Morton-Gonzaba, Chimobi Emukah, Alvaro Moreira M.D. The University of Texas Health and Science Center at San Antonio A Systematic Review of Mesenchymal Stem Cells for Rotator Cuff Injury

Background: Rotator cuff injury (RCI) is a leading cause of shoulder pain. Current treatments for RCI have varied outcomes and high reinjury rates. Animal models of RCI suggest mesenchymal stromal cells (MSCs) are safe and promising novel agents, which have now translated to early phase human clinical trials. The purpose of our study was to collate and methodically evaluate current human clinical trials examining the efficacy and safety of MSCs for RCI. Methods: Literature search was performed in five databases. Two independent reviewers screened and examined the effects of MSCs on: (i) rotator cuff function, (ii) rotator cuff integrity, and (iii) safety and long-term outcomes. ROBINS-I was used to assess risk of bias and the study protocol was submitted to PROSPERO. Results: After reviewing 858 potential articles, six interventional studies were included (total n= 319 participants). Autologous administration of MSCs was the most used mode of transplantation, while the most common tissue source was bone marrow. All studies administered a single injection of 25,000 to 4.7 x 108 cells. MSC treatment improved activities of daily living, decreased pain scores, and reduced retear rates. Overall, MSCs were well-tolerated and did not have serious adverse events. Conclusion: In this systematic review of RCI, MSCs improved overall shoulder function and were safe. These results should be interpreted with caution as our review was limited by a small sample, large heterogeneity, and moderate risk of bias.

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Reducing Tissue Factor Expression in Mesenchymal Stromal Cells to Improve Safety Christopher Delavan, Barbara Christy, Patrick Ketter, Maryanne Herzig, Andrew Cap, James Bynum. Blood & Coagulation Research Department, U.S. Army Institute of Surgical Research

Background. Mesenchymal Stromal Cells (MSC) and their products show therapeutic potential for cellular therapy and regenerative medicine. Multiple clinical trials are ongoing for treatment of a wide variety of immune related disorders, wound healing and tissue regeneration. However safety should be a concern with these cells with intravenous (IV) injection. Previously we have shown that MSCs express Tissue Factor (TF) on their surface and potentiate the coagulation ofhuman blood or plasma; adipose-derived MSC express higher levels of TF and are more pro-coagulant than bone marrow-derived MSC. The hypothesis for our studies is that reducing TF expression will increase the safety profile of adipose MSC. Here we detail the 3 different approaches used to reduce TF expression: (1) introduction of TF-specific siRNAs; (2) introduction of a TF-specific shRNA plasmid; and (3) transduction with retroviral vectors expressing TF-specific shRNAs.Methods. For the TF siRNA transfection, human adipose MSC were seeded into a 24-well plate at 50,000 cells/well. The next day TF or negative control siRNAs (37.5, 75, 150ng) were added with HiPerfect transfection reagent (1.5, 3, 4.5µL). Transfection was allowed to proceed for 24 hours and relative TF gene expression was then measured with RT-PCR. With TF shRNA plasmid transfection, human adipose MSC were seeded into a 6-well plate at 200,000 cells/well. After 24h, plasmids that were either TF-specific or negative control shRNA plasmid and also containing the gene for green fluorescent protein (GFP) and puromycin resistance (Origene)were transfected into the MSCs using calcium phosphate and allowed to proceed for 24 hours. Successful transfections expressed green fluorescent protein (GFP) and were puromycin resistant. Lastly, for the TF shRNA retroviral packaging, the TF shRNA and pVSV-g plasmids were co-transfected into a HEK293 packaging cell line. The retroviral supernatant was collected 48 hours later and frozen at -80oC. Human adipose MSC were seeded into a 6-well plate at 200,000 cells/well; after overnight incubation fresh media containing 6µg/mL polybrene was added, along with retroviral supernatant. Plates were centrifuged at 1200 x g for 1 hour and then incubated for 24 hours. Successfully transduced MSC were resistant to puromycin.Results. After transiently transfecting MSC with TF siRNA, TF gene knockdown was observed with as little as 37.5ng of siRNA; better results were achieved with 75ng and 150ng of TFsiRNA. Varying the volumes of HiPerfect transfection reagent did not have any discernable effect. Because it is difficult to obtain enough TF knockdown cells for functional testing using this method, alternative methods were evaluated. Direct transfection with the TF-specific shRNA plasmids suggested that MSC are difficult to transfect. Less than 5% of cells transiently expressed GFP after transfection, and very few colonies of cells stably expressing puromycinresistance were generated. As primary cells, MSCs are only able to undergo a finite number of population doublings before becoming senescent, therefore we were unable to generate a large population of stably transfected cells for testing. The retroviral transduction protocol is still in the early stages but has already shown encouraging results. We have generated much higher numbers of puromycin-resistant MSC following transduction with retroviral particles expressing both the TF-specific shRNAs and puromycin resistance and evaluation of TF expression and function is currently underway. Conclusions. The TF siRNA method gave promising early results as we were able to show a quantifiable decrease in TF gene expression compared to the control using RT-PCR. Unfortunately, it is difficult to produce enough transiently transfected cells to perform functional testing using this method. MSCs appear quite resistant to direct plasmid transfection, rendering this method impractical for testing as well. Because the retroviral transduction method appears to yield many more puromycin-resistant colonies, this method seems the logical way to go moving forward.

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Mechanisms of Stem Cell-Induced Analgesia

Ganatra Shilpa BDS, Brock Robert BS, Bayat Saeed Mohaved DDS, Li Qun MS, Austah, Obadah, MS, DDS, Espitia Claudia MS, Bendele Michelle BS, and Ruparel Nikita Bharat MS, DDS, PhD

Management of post-endodontic pain with NSAIDs and opioids are associated with serious side effects. Stem cells participate in wound healing; however their role in regulation of nociception presents a gap in knowledge. Objective: To evaluate mechanisms of Stem Cells of Apical Papilla (SCAP)-mediated reversal of apical periodontitis (AP)-induced mechanical allodynia (MA). Methods: 1.) Using a mouse model of AP, we first established a model of orofacial MA using von Frey filaments, 2.) Post-baseline thresholds, pre-labeled SCAP were injected intravenously in one-half of AP animals once/week for 3 weeks; Sham animals served as controls; MA was measured 24 hours post injections, 3) Immunohistochemistry of periapical lesions and medullary dorsal horn (MDH) was performed for expression of SCAP and nociceptive marker, c-fos, 4.) Teeth with AP were extracted at 3 weeks and co-cultured with or without SCAP. Conditioned Media (CM) was collected and applied to mouse cultured trigeminal neurons (TGs) followed by application of capsaicin (15nM; CAP) to evaluate TG nociceptor activity using calcium-imaging. Statistics: 2-way ANOVA with Tukey’s post-hoc test. Results: MA peaked at day 21 in AP mice. This effect was fully reversed by SCAP injections. Immunohistochemistry demonstrated SCAP homing into periapical lesions with no expression in the MDH. AP mice showed robust expression of c-fos in the MDH, which was drastically reduced after SCAP injections. Lastly, CM from Teeth alone sensitized CAP-evoked calcium accumulation. However, CM from Teeth co-cultured with SCAP significantly reduced CAP sensitization. Conclusions: Collectively, this study for the first time establishes a model of MA in mice with AP, which is reversed by SCAP. This appears to be a peripheral effect as SCAP homed to periapical lesions and data from calcium-imaging suggest that SCAP release soluble factors in the periphery that inhibit TG activity. These findings provide significant insight into mechanisms of stem cell-mediated analgesia.

Funds: Start-up funds (PID 156778).

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Jake D. Lehle, Keren Cheng, and John R. McCarrey. The University of Texas at San Antonio Epigenetic Reprogramming in a Dish – An In Vitro Model of Transgenerational Epigenetic Inheritance.

It is now well established that either maternal or paternal behaviors, environmental conditions, or exposures can alter the epigenome to cause epimutations which manifest as changes in DNA methylation or histone modification patterns in human or animal models. Epimutations can predispose disease states that can be transmitted to descendants in subsequent generations through inter- or transgenerational epigenetic inheritance. This is surprising because epimutations would normally be expected to be corrected by the epigenetic reprogramming events that occur in the preimplantation embryo and developing fetal germ line. Previous studies have shown that exposure to environmental agents known as endocrine disruptor chemicals (EDCs) can alter the epigenome causing epimutations. Based on preliminary bulk RNA-sequencing of publicly available data sets and immunocytochemistry (ICC) staining, we hypothesize that somatic cells (Sertoli cells, granulosa cells) will be more vulnerable to epimutations induced by exposure to EDCs due to presence of endocrine receptors known to interact with the selected EDCs (vinclozolin, bisphenol F or tributyltin) while germ cells (primordial germ cells [PGCs], spermatogonial stem cells [SSCs]) and pluripotent cells (embryonic and induced pluripotent stem cells [ESCs and iPSCs]) will be less vulnerable due to their lack of endocrine receptors. To assess the relative susceptibility of somatic, pluripotent, and germ cell types to disruption of DNA methylation patterns (epimutations) following exposure to our selected EDCs, we will establish a standardized dose at which each EDC generates a disruption of DNA methylation patterns in iPSCs, and we will then determine the extent to which the same dose disrupts DNA methylation patterns in each of the other mouse cell types (Sertoli, granulosa, PGCs, SSCs, and ESCs). To accomplish this analysis, we will utilize the Access Array and the BioMark instrumentation from Fluidigm, which will allow us to examine DNA methylation levels within a set of genic and intergenic regions associated with 27 imprinted genes as well as regions within the promoter of multiple non-imprinted genes including three housekeeping genes, seven tissue specific genes, and seven genes known to resist the normal demethylation process in PGCs during germline reprogramming. This assay will be paired with analysis of changes to global DNA methylation levels using the Methylamp DNA Modification Kit from Epigentek. Once we have established the Lowest Observed Effect Level (LOEL) of each EDC in iPSCs, we will expose each of the other cell types (ESCs, PCGs, SSCs, Sertoli cells, granulosa cells) to the same dose of each EDC for 24 hours in culture and determine changes to DNA methylation and the relative susceptibility in each cell type. In past efforts, the study of epimutation transmission during embryonic or germline development has been difficult due to the extensive cost, time, labor, and animal use required to investigate this phenomenon in vivo over multiple generations. To overcome this, in the second part of this project we established a quintuple transgenic induced pluripotent stem cell (iPSC) system for the purpose of recapitulating in vitro the epigenetic reprogramming that normally occurs during the life cycle in vivo. This novel system is designed to allow us to induce epimutations in a controlled manner by exposure to known doses of specific EDCs, and then follow these epimutations through recapitulation of normal embryonic, germline, and somatic epigenetic programming and reprogramming over the equivalent of multiple generations in vitro. The establishment of this novel in vitro system will allow our lab to pursue a reductionist approach to gain a more complete understanding of inter- or transgenerational inheritance of epimutations induced by EDC exposure in a much more rapid and cost-efficient manner that will minimize our use of animal models and the challenges that accompany that approach.

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Yu-Huey Lin and John R. McCarrey The University of Texas at San Antonio Analysis of Differential Epigenetic Reprogramming Potential among Pluripotent, Germ and Somatic Cell Types Male infertility is frequently linked to defective male gamete development, which can be caused by genetic mutations or adverse environmental effects. Male gametes are the product of spermatogenesis, which is a process sustained by spermatogonial stem cells (SSCs), and epigenetic reprogramming is an important part of this process. In vitro spermatogenesis, derived from either SSCs or induced pluripotent stem cells (iPSCs), represents a potential solution for male infertility, and has been a long-sought objective in the field of Reproductive Medicine. The establishment of an accurate, efficient, and reproducible in vitro cell system that recapitulates normal germ cell development in vivo will provide a very significant new approach relevant to both basic and preclinical research into the mechanisms by which epigenetic programming impacts normal development. Moreover, the stable derivation of functional gametes in vitro has the potential to advance human fertility, contraception, and health. Nevertheless, although there has been significant progress, in vitro spermatogenesis remains an unfulfilled challenge. Epigenetic abnormalities observed in germ cell-like cells derived from iPSCs in vitro may result from retention of epigenetic memory, or defects in epigenetic programming, of the initial cell type(s) from which the iPSCs are derived. Thus, we propose to establish an in vitro system in which transitions among pluripotent, germ and somatic cell states can be induced and monitored, so that we can optimize conditions to recapitulate epigenetic programming/reprogramming in vitro in a way that is comparable to that which normally accompanies similar transitions during germ cell development in vivo. As a first step in this process, we derived iPSC lines from either somatic (mouse embryonic fibroblasts [MEFs] or adult tail-tip fibroblasts [TTFs]) or germ (primordial germ cells [PGCs] or SSCs) cell types carrying the DOX-inducible 4F2A reprogramming cassette transgene to determine if iPSCs derived from distinct cell types retain distinct source cell type-specific remnants of epigenetic memory. Here we show that the timing of first iPSC colony appearance differs among iPSC lines derived from different source cell-types as follows (from fastest to slowest): PGCs > SSCs > MEFs > TTFs, which likely reflects the extent to which epigenetic programming within each of these initial cell types differs from that in the resulting iPSCs. Each of these iPSC lines is now being characterized for expression of pluripotency markers as well as for karyotype normalcy and teratoma differentiation potential. Confirmed iPSC lines will then be further assessed for global patterns of DNA methylation and gene expression. Once optimized, this germ/somatic cell to iPSC transition system will be useful as an in vitro model of epigenetic reprogramming associated with changes in cell fate during normal development in vivo.

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John Martinez, Sarah Zoretic, Alvaro Moreira UT Health Science Center-San Antonio Safety and efficacy of stem cell therapies in congenital heart disease; a systematic review and meta-analysis of pre-clinical and clinical studies Purpose: Adult clinical trials have reported safety and therapeutic potential of stem cells for cardiac disease. These observations have translated to the use of regenerative therapies in pediatric heart disease. We conducted a systematic review and meta-analysis to assess: (i) safety and (ii) effects of cell-based therapies (CBT) for critical congenital heart disease. Methods: Literature search was performed in PubMed, Scopus, Web of Science, and Science Direct. Two independent reviewers screened studies that examined the effects of CBT on: (i) safety and (ii) cardiac function. Data is reported as odds ratios (ORs) or mean difference (MD). Results: Fifteen animal and thirteen human studies were included. Pre-clinical: Right ventricular dysfunction was the most common model. Cardiac-derived cells were a common tissue source, with intramyocardial delivery as the most frequent route. Dose ranged from 1.25 x 105 to 5 x 107 cells/kg. No difference was observed between CB vs. control groups with respect to adverse events (OR 0.7, 95%CI 0.29, 1.58, p=0.37) or mortality (OR 0.42. 95%CI 0.16, 1.08, p=0.07). CBT improved ejection fraction (MD 6.5, 95%CI 3.63, 9.36, p<0.01) and fractional shortening (MD 4.09, 95%CI 1.28, 6.91, p<0.01). Subgroup analysis favored intramyocardial infusion of umbilical cord cells. Clinical: Nine studies utilized cells in patients with hypoplastic left heart syndrome. Bone marrow and cardiac tissue were the most common tissue source, with most studies using the intracoronary route. Dose ranged from 3.0x 105 to 24 x 106 cells/kg. A decrease in adverse events was observed in the CB cohort (OR 0.17, 95%CI 0.10, 0.30, p<0.01). Mortality rates were similar between groups (OR 0.48, 95%CI 0.14, 1.64, p=0.24). Cell administration improved ejection fraction (MD 4.84, 95%CI 1.62, 8.07, p=0.003). Conclusion: In this meta-analysis of preclinical and clinical models of critical congenital heart disease, CBT were safe and improved specific measures of cardiac function. Implications from this review may provide methodologic recommendations for current and future clinical trials.

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Nikolas Merlock, Md Musaddaqul Hasib University of Texas at San Antonio Using machine learning to define cortical network features in epileptic cerebral organoids

Changes in neuronal network signatures due to gene mutation can be studied with three-dimensional brain organoids. These models open a new window into disease mechanisms and discovery. Machine learning, especially deep learning models can be applied to characterize context-specific organoid behaviors from local field potential (LFP) signals. However, interpreting those models to gain an understanding of the mechanisms by which organoids respond to the mutation is still a challenging task. We present in this poster deep learning solutions to identify the LFP signatures unique to CHD2-mutant and patient derived induced pluripotent stem cells (iPSCs) organoids. We show that our deep learning model interpretation methods are better capable to interpret organoids variability than traditional SVM features. We first propose an end-to-end convolutional neural network (CNN) to differentiate CHD2-mutant organoids from control and obtained an area under the curve (AUC) performance of 98.2%. In contrast, a support vector machine (SVM) approach selected five frequency band-power features for 4-month-old organoids and achieved AUC 71.4%. To identify features learned by CNN, this poster proposes two knowledge distillation-based methods. First, we trained a decision tree (DT) regression model to identify the signatures unique to the CHD2 mutant organoids. Second, a frequency band activation method that provides sensitivity to frequency band in identifying CHD2 mutant organoids or controlled organoids. Both interpretation methods identified Alpha frequency band (7.5– 12.5 Hz) as the signature character to identify CHD2 mutant organoids.

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Louis Perez, Clyde F. Phelix, Lyndsay Millican, Lois Randolph, Richard G. LeBaron University of Texas at San Antonio Potential Gender Differences in Lubricin Expression in Estrogen-Treated Temporomandibular (TMJ) Cells: Implications in TMJ Disorders. Characterized cell lines provide a valuable tool for research into a disorder or disease. Here we aim to establish a model to help investigate the temporomandibular joint (TMJ) comprising, in part, an articular disc located between the mandibular condyle and fossa. The TMJ is a bilateral joint that exhibits hinge and sliding movements. The articular disc experiences both compressive and tensile forces as the jaw opens, closes, and relaxes. The disc must be able to handle these stresses in order to function properly, thus, the composition of its microenvironment is important as changes may lead to loss of integrity. Defects of the joint may lead to temporomandibular joint disorder (TMD) characterized, mostly in females, by symptoms of facial pain, jaw displacement (lock-jaw), and limited range of motion. The exact cause of TMD is unknown, although evidence suggests a gender component and a combination of factors including trauma, hormonal stasis, or changes in the ECM microenvironment play roles in the development of TMD. We recently discovered that estrogen suppressed expression of the articular joint lubricating molecule called lubricin. In order to aid further investigation to a cause of TMD we obtained cells from the TMJ of a male and female olive baboon to perform comparative studies of the effects of estrogen, and document in vitro cell characteristics. Our early studies indicate estrogen suppresses lubricin gene expression in hTERT-immortalized female TMJ cells. Recent studies focus on early-passage, non-immortalized, female and male baboon disc cells. We aim to characterize TMJ disc, synovial, and chondrocyte early-passage discontinuous cells for comparison to immortalized cells.

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Lorena Roa-de la Cruz, Shinnosuke Suzuki, Victoria D. Diaz, John R. McCarrey and Brian P. Hermann. University of Texas at San Antonio Developmental Origin Of Foundational Spermatogonial Stem Cells In The Mouse Testis. Spermatogenesis, the process by which sperm are produced in the testis, is maintained throughout adulthood by Spermatogonial Stem Cells (SSCs). One theory describing the mechanism of SSC specification during testis development holds that SSCs arise from a distinct subpopulation of prospermatogonia (fetal and early postnatal precursors of spermatogonia, including SSCs) that is determined. Published single-cell RNA-seq of E16.5 fetal prospermatogonia demonstrated that a subpopulation exhibit an SSC-like signature, which is consistent with the determination theory, but fails to prove that this subpopulation becomes the foundational SSCs. In subsequent analyses, we found that the subpopulation of fetal prospermatogonia bearing SSC-like transcriptomes was also proliferative based on RNA signatures at E15.5 (our data) and E16.5 (published data) and therefore hypothesized that this subpopulation could be lineage-traced by S-phase labeling to establish whether they become putative SSCs. To test this hypothesis, we injected pregnant Id4-Egfp transgenic mouse dams at E14.5 (at the end of the dividing M-prospermatogonia phase), or at E15.5, E16.5 or E17.5 (during the quiescent T1-prospermatogonia phase) with 5–ethynyl–2′–deoxyuridine (EdU) and collected testes from P0 male pups. P0 testis sections were co-stained for prospermatogonia (DDX4), EdU incorporation, and Id4-EGFP to distinguish the nascent SSCs. After birth, male germ cells that are DDX4+/Id4-EGFP+ are nascent SSCs, while DDX4+/Id4-EGFP- are not SSCs. When labeled at E14.5 during the M-prospermatogonia proliferation phase, we found that some P0 prospermatogonia in both populations were EdU+ (20.98% of DDX4+/GFP+ and 3.58% of DDX4+/GFP-). Likewise, when labeled at E15.5 when prospermatogonia are largely quiescent and the supposed SSC-determined subpopulation is proliferative, we also found that some P0 prospermatogonia in both populations were EdU+ (4.95% of DDX4+/GFP+ and 4.86% of DDX4+/GFP-). No P0 prospermatogonia were EdU-labeled when dams were treated at either E16.5 or E17.5, matching out dogmatic expectations that all male germ cells become quiescent late in fetal testis development. These results support an alternative mechanism of foundational SSC specification which is based on stochastic selection, rather than determination from a specified germ cell subset.

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DEVELOPMENT OF NITRIC OXIDE RELEASING BIO-ACTIVE WOUND DRESSING FOR COMBAT BURN WOUND INFECTION TREATMENT

Jahnabi Roy1, Jitendra Pant2, Lori Estes2, Robert Christy1, Hitesh Handa2,

Shanmugasundaram Natesan1

1 US Army Institute of Surgical Research, JBSA Fort Sam Houston, TX; 2 School of Chemical, Materials and Biomedical Engineering, University of Georgia, Athens

Infection has become the second most common cause of death in combat wounded. Providing adequate infection control will remain a capability gap in future delayed evacuation scenarios. Additionally, a delay in care results in infection-related complications which increase mortality and morbidity. The vast majority of antimicrobial agents only target wound infection while other important host cell responses such as angiogenesis, fibroblast proliferation, and migration are left to their natural fates. Nitric oxide (NO) has the potential to assist these natural wound healing processes while providing infection control. Notably, NO acts against a variety of microorganisms, including gram-positive and -negative bacteria, fungi, yeast, and viruses. Our overall goal is to develop NO-releasing bioactive wound dressings that can kill >99% bacteria (gram-positive and negative) while also improving natural wound healing processes.

In this study we have developed a novel wound dressing integrated with a NO donor (S-nitroso-glutathione, or GSNO) in a hybrid formulation of alginate and poly(vinyl alcohol) (PVA) to prevent and treat burn wound infection. The dressings developed in this work showed sustained NO release over a 72 hour period. Furthermore, they were tested for their efficacy against S. aureus and P. aeruginosa in in vitro studies. Finally, these dressings have no effect on the viability and proliferation of human dermal fibroblasts. Next, we applied the dressings to a porcine model of infection. A 3-cm wound infected by either S. aureus or P. aeruginosa was treated by vehicle control, standard of care, or treatments at two different NO concentrations. At pre-determined times, biopsies were taken from the wound and colony forming units were assessed as a primary end-point. Additionally, a biopsy strip was taken from each wound to histologically assess the effect of NO on burn progression.

In conclusion, we have developed an anti-microbial dressing that results in sustained release of antimicrobial NO. Furthermore, the dressings show antimicrobial activity in in vitro studies. Finally, the dressings are being tested in pre-clinical porcine models of infected burn wounds to determine their efficacy.

Acknowledgements: This research was supported in part by an appointment to the Postgraduate Research Participation Program at the U.S. Army Institute of Surgical Research administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and USAISR. This research was funded by the U.S. Army MRDC and Combat Causality Care Research Program (K_25_2018_CCCRP).

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Olivia N. Tran, Hanzhou Wang, Andrey D. Malakhov, Aaron O. Gonzalez, David D. Dean, Chih-Ko Yeh, Xiao-Dong Chen University of Texas Health Science Center at San Antonio Trans-differentiation of Bone Marrow-derived Stem cells to Epithelial lineage by Salivary Gland Extracellular Matrix Proteins Salivary gland (SG) hypofunction is associated with xerostomic drugs, radiation for head and neck cancer, or autoimmune diseases (e.g. Sjogren’s syndrome), resulting in severe effects on oral and systemic health. At present, therapy is primarily symptomatic, consisting of saliva substitutes or stimulants, with no definitive cure. Researchers have investigated a number of potential treatments using SG stem cells to improve or restore SG function. However, SGs contain very low numbers of adult stem cells of high quality, and it is difficult to control the differentiation of multi-potent stem cells to the desired cell. Here, we propose a new approach using SG extracellular matrix (ECM) to induce the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) to the SG epithelial cell lineage in vitro. Submandibular glands (SMG) from Lewis rats were harvested and decellularized to obtain cell-free SG-ECM. The ECM was next processed by homogenization into small fragments (i.e. 300-500um diameter). To induce differentiation, primary Lewis rat BM cells (passage 1) were incubated with SG-ECM for one hour at 37°C, and then transferred to a flask or 6-well plate containing α-MEM and 20% FBS. At day 7 and day 14, morphology, gene and protein expression of SG epithelial cell-related markers were analyzed between the SG-ECM treated group and the untreated group. The results revealed that a small percentage of the BM-MSCs attached to the SG-ECM and formed sphere-like aggregates, while the remaining BM-MSCs formed a monolayer on the culture surface. The cells within the aggregates, but not the monolayer or untreated BM-MSCs, expressed SG epithelial cell markers including Mucin 10, krt14, claudin 3, claudin 10 and Mist 1. Subsequently, flow cytometry showed a remarkable increase in a subpopulation of cells expressing CD133+CD45- after treatment with SG-ECM. Finally, cells within the aggregates showed positive PAS staining and the presence of secretory granules in transmission electron microscopy images. Our findings suggest that treatment of BMSCs with SG-ECM promotes attachment and directs their trans-differentiation to the SG epithelial cell lineage.

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HoxBlinc LncRNA Regulates Hematopoietic Stem Cell Function via Cis- and Trans-acting Mechanisms

Ganqian Zhu1, Huacheng Luo2, Shi Chen1, Ying Guo1, Feng-Chun Yang1, Stephen D. Nimer3, Suming Huang2 and Mingjiang Xu1

1Department of Molecular Medicine, University of Texas Health San Antonio, San Antonio, TX; 2Department of Pediatrics, Pennsylvania State University Hershey College of Medicine, Hershey, PA; 3Department of Medicine & Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL.

Long non-coding RNAs (lncRNAs) have emerged as important regulators of gene expression and cell fate decisions although their roles in hematopoietic stem cell (HSC) function remain elusive. HoxBlinc is a HoxB gene associated lncRNA locates between HoxB4 and HoxB5. Giving that HoxBlinc regulates anterior HoxB gene expression, and HoxB genes are critical for HSC biology, we investigated the role of HoxBlinc in HSC behavior and explored the underlying mechanism. We found that HoxBlinc is highly expressed in HSCs and hematopoietic progenitor cells (HPCs), and loss of HoxBlinc in myeloid precursor cells impaired their proliferation and expression of genes critical for HSC/HPCs such as anterior HoxB genes, Runx1, and HoxA9.

Since HOXBLINC is overexpressed in patients with AML, we generated a transgenic mouse model within which HoxBlinc is driven by a hematopoietic-specific Vav1 promoter and characterized their hematopoietic phenotype. We found that HoxBlinc overexpression (HoxBlincTg) enhanced the self-renewal and proliferation but impaired the differentiation of HSC/HPCs. HoxBlincTg mice had strikingly increased Lin-Sca1+cKit+ (LSK) and myeloid progenitor (GMP) pools and all of the mice exhibited abnormal hematopoiesis such as anemia, splenomegaly, and myeloid malignancies. To determine the intrinsic role of HoxBlinc in HSC/HPCs, competitive transplantation was performed which showed that HoxBlincTg HSC/HPCs have increased repopulation capacity as compared to WT HSC/HPCs.

Analysis of datasets of RNA-seq, ATAC-seq, ChIP-seq showed a unique gene expression pattern and epigenetic signature of HoxBlincTg LSK cells as compared to WT LSK cells. HoxBlinc overexpression up-regulated the transcription of not only anterior HoxB genes but also other genes critical for HSC/HPC biology such as Wnt5a, HoxA9, and Runx1, likely by recruiting the MLL1 complex as MLL1 ChIP-seq and HoxBlinc ChIRP-seq showed that both MLL1 and HoxBlinc co-localized at the promoter regions of these genes, thus increasing the H3K4me3 occupancy and accessibility at their promoters.

In addition, 4C-seq and Hi-C data indicated that HoxBlinc lncRNA regulates the formation of the topologically associated domain (TAD) encompassed anterior HoxB genes. These data support that HoxBlinc may increase the expression of the anterior HoxB genes in the HSC/HPCs by changing the chromatin structure.

The study provides compelling evidence that HoxBlinc is a potent epigenetic regulator of HSC function.

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7th Annual Conference on Stem Cell7th Annual Conference on Stem CellResearch & Regenerative MedicineResearch & Regenerative Medicine

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