Poster Session

Poster Presentations Announced!

Every year, the Cell & Gene Meeting on the Mesa invites the submission of abstracts that report new research developments in the areas of stem cell biology, gene therapy, genome editing, stem cell medicine, regenerative medicine or ethical and regulatory issues. Abstracts are welcome from physicians, scientists, researchers and trainees in all sectors, including academia, industry, government and education.

See the abstracts that will be on display for viewing at the 2017 Scientific Symposium below!

2017 Poster Abstracts

1. Assessment of In Vitro Transduction and Functionality of Channelrhodopsin-2 Protein When Delivered Via Adeno-Associated Viral Vector to Predict In Vivo Application

Presented by: Alana Rainbow-Robinson, Allergan Biologics
Authors: Rainbow-Robinson, Alana, Senior Scientist; Li, Yong-Xin, Experimental design; Guo, Yuan-Xing, Recording; Vu, Chau, Cell culture; Yu, Ying, Infection; Donello, John, Group leader; Rodrigues, Gerry, In vivo experimental design

Purpose: Adeno-associated virus (AAV) is one of the most promising vehicles for ocular human gene therapy in research. Diseases such as Retinitis Pigmentosa cause degradation of the photoreceptor cells of the retina, this degrading causes initial tunnel vision followed by progressive blindness. Channelrhodopsin-2 (ChR2) is a light-gated ion channel protein derived from green algae, Chlamydomonas reinhardtii, driving phototropism. It is hypothesised that implementation of the ChR2 gene within cells of the retina will enable the protein to be generated and produce a stimulus when exposed to light of a specific wavelength. To demonstrate this, ChR2 gene was packaged into an adeno-associated virus (AAV) vector of serotype 2 and delivered to a human cell line. Transduction efficiency as well as ChR2 protein functionality were then assessed. Fusion plasmids containing ChR2 and fluorescent genes were also assessed to review localisation of the protein within the cell. Methods: AAV2 material was produced carrying the ChR2 gene. This material was then inoculated onto a human cell line (HEK-293) and incubated. Patch-clamp recordings were performed three days after infection. ChR2-mediated currents were activated with a blue LED attached to the microscope. Plasmids containing ChR2 and GFP fusion gene were transfected onto the human cell line as well as GFP only plasmids; these were incubated and then observed via confocal microscopy. Results: Light activated currents were recorded from the cells infected with AAV-ChR2 material when exposed to blue light. No current was detected from the cells infected with the AAV-EGFP material. Cells transfected with ChR2 and GFP fusion gene plasmids had a lower fluorescence than the cells transfected with the GFP only plasmids. Conclusion: These results indicate that the AAV2 is an efficient vehicle to deliver the ChR2 gene to cells for the treatment of blindness caused by degradation of photoreceptor cells within the retina. The results also indicate that review of in vivo transduction efficiency of the ChR2 gene could be problematic when fused with a fluorescent protein due to protein localization within the cell.

Key Words: Adeno-Associated virus Optogenetics Retinitis Pigmentosa Channelrhodopsin-2

2. IND Enabling Preclinical Studies of Intravenous Cardiosphere Derived Cells for Duchenne Muscular Dystrophy

Presented by: Jennifer Johnson, Capricor Therapeutics
Authors: Johnson, Jennifer, Capricor Therapeutics, Inc.; Kanagavelu, Saravana, Capricor Therapeutics, Inc.; Vaturi, Sharon, Capricor Therapeutics, Inc.; Sakoda, Chris, Capricor Therapeutics, Inc.; Li, Liang, Capricor Therapeutics, Inc.; Fournier, Mario, Cedars-Sinai Medical Center; Dawkins, James, Cedars-Sinai Medical CenterCapricor Therapeutics, Inc.

Cardiosphere Derived Cells (CDCs) modulate inflammation, limit fibrosis and promote regeneration. It has been shown that intramyocardial administration of CDCs improves cardiac function and increases long-term survival in aged mdx mice, a preclinical model of Duchenne Muscular Dystrophy (DMD). These results motivated the HOPE-Duchenne clinical trial to treat DMD cardiomyopathy by intracoronary delivery of CDCs (CAP-1002). The positive results observed in HOPE on both cardiac and skeletal muscle function motivate additional studies exploring new delivery routes and regimens that can maximize systemic efficacy of CDCs, allow for multiple administrations and reduce patient administration discomfort. Here we studied efficacy, biodistribution and safety of CDCs administered intravenously. We first analyzed the therapeutic activity of escalating doses of CDCs (37, 75 and 150×10^3) delivered intravenously into mdx mice. A dose dependent improvement in isolated soleus muscle function was observed 6 weeks after CDC treatment. Mdx mice treated with 150×10^3 CDCs showed an increase in exercise capacity compared to placebo. Histological analysis showed reduced muscle inflammation and fibrosis in CDC-treated mice compared to placebo. CDC biodistribution was evaluated (by qPCR for human Alu sequences) 10 minutes and 24 hours after intravenous administration of cells. Most CDCs were found in the lungs with no significant presence in liver, heart, spleen, blood, diaphragm, or soleus muscle. Less than 25% of the CDCs present in the lungs at 10 minutes remained at 24 hours. CDC clearance was evaluated 1 week and 3 weeks after intravenous administration. CDCs transiently redistributed from the lungs to other organs (primarily the heart) between 24 hours and 1 week. Less than 5% of the CDCs present in the lungs at 24 hours remained at 1 week, and clearance was complete from all organs by 3 weeks. Finally, we evaluated the safety of intravenous administration of escalating doses of CDCs (50, 100, 200×10^6) in pigs. A few animals experienced a dose independent, mild, transient decreases in oxygen saturation. No significant histological alterations were observed in the lungs of pigs injected with 200×10^6 CDCs. Escalation up to 200×10^6 in pigs was reasonably safe. These data show a dose dependent improvement in mdx mice with intravenous CDC delivery. Although most of the administered cells accumulate acutely in the lungs and are gone by 3 weeks, a reduction in fibrosis and an improvement in function was observed in skeletal muscle at 6 weeks. This effect is probably mediated by the release of extracellular vesicles into the bloodstream. Our results also demonstrate that intravenous delivery of CDCs is safe. Intravenous delivery of CDCs allows for administration of higher and more frequent doses than intracoronary. These findings support clinical evaluation of systemic administration and repeat dosing of CAP-1002 in patients with DMD to potentially maximize efficacy.

Key Words: cell therapy, cardiosphere, Duchenne, muscular dystrophy, biodistribution, safety, efficacy, intravenous

3. Characterize and Correlate Early Attributes of Adult Human Stem/Progenitor Cells and iPS Cells with Future Biological Performance Using New Automated High Content Imaging, Analysis and Selection Technology – CellX and Colonyze

Presented by: Venkata Mantripragada, Cleveland Clinic
Authors: Mantripragada, Venkata, Cleveland Clinic; Luangphakdy, Viviane, Cleveland Clinic; Kwee, Edward, Cleveland Clinic; Handerhan, Brian, Parker Hannifin; Powell, Kimerly, Ohio State University; Muschler, George, Cleveland Clinic

Cell therapy protocols, whether based on autologous somatic cells or induced pluripotent stem (iPS) cells are based on common paradigm: i) define optimized sources of stem and progenitor cells, ii) define processing methods to optimize purity, concentration and biological potential of the founding cell and iii) define critical quality attributes (CQAs) that predict efficacy and avoid clinical or biological risk. However, repeatable and reproducible success in cell therapy is often confounded by i) variation introduced during processing steps, ii) clonal differences among stem/progenitor cells resident in native human tissues and iPS cells variations, and iii) lack of systematic means to measure and manage these variations, including documentation. Tools and techniques are necessary to understand, characterize and selectively choose from the heterogeneous population of stem and progenitor cells iPS cells. To define and quantify CQA of the stem and progenitor cells in systematic repeatable and reproducible manner, we have developed “ColonyzeTM”, a generalizable AM-LIA software platform based on Automated Multimodal Large field of view Image Analysis (AM-LIA). The most important quantitative metrics analyzed include proliferation rate (cells per colony), cell surface markers, and cell and colony morphology. The principles and nomenclature enabling this approach are outlined in an ASTM Standard F2944-12. This method will eliminate problems encountered by manual methods for cell and colony counting that are time-consuming, imprecise and inconsistent within and between observers. Additionally, CQAs can be determined that predict the likelihood of desired biological activity to early attributes. To automate manipulation of cells in rapid, precise, repeatable and rigorously documented manner, we have partnered with Parker Hannifin (Cleveland, OH) to physically integrate ColonyzeTM imaging and analysis with a robust robotic system Cell XTM for automated imaging, colony picking and fluid management. Cell XTM enables rapid early performance based selection for cell sourcing. Using CellXTM, automated image analysis followed by targeted “biopsy” or “picking” desired cells or colonies for further propagation is possible. Alternatively a user could selectively remove (“weed”) undesired cells and colonies. The combination of ColonyzeTM and Cell XTM is directly related to four thrust areas: Cell selection and sourcing, bioprocess automation, tissue/product finishing and testing. These provides unprecedented ability to make quantitative measurement in a complex heterogeneous cell environment and then to act on those measurements to define highly documented reproducible methods for cell and colony “management” based on application specific CQAs.

Key Words: Imaging, selection, automation, stem cell, iPSCs

4. Entering a New World: Ambient Storage of Cellular Therapies

Presented by: Sandeep Dhall, Osiris Therapeutics
Authors: Dhall, Sandeep, Osiris Therapeutics Inc.; Sathyamoorthy, Malathi, Osiris Therapeutics Inc.; Kuang, Jin-Qiang, Osiris Therapeutics Inc.; Hoffman, Tyler, Osiris Therapeutics Inc.; Moorman, Mathew, Osiris Therapeutics Inc.; Jacob, Vimal, Osiris Therapeutics Inc.; Lerch, Anne, Osiris Therapeutics Inc.

Human placental amnion (AM) has a long history in the field of wound treatment. Advances in tissue preservation have helped to overcome the short shelf life of fresh AM and led to the commercialization of amnion products. Viable cryopreserved amniotic membrane (VCAM), which retains all components of fresh AM, has shown positive outcomes in wound management. However, cryopreservation requires specialized ultra-low temperature storage equipment, thus limiting widespread use of VCAM. Recently, a novel lyopreservation technology has been developed that allows for ambient storage of living cells and tissues. Fresh AM was lyopreserved using this method, and its tissue structure, cell viability, and wound-healing properties following rehydration were investigated in vitro and in vivo. Histological staining demonstrated that the structure of fresh AM was retained in viable lyopreserved AM (VLAM). Cell viability was assessed by calcein staining, which demonstrated that the number of viable cells was comparable in VLAM, fresh AM, and VCAM. Inhibition of tumor necrosis factor-α (TNF-α) secretion from activated immune cells, and a hypoxia-driven increase in vascular endothelial growth factor (VEGF) demonstrated VLAM’s anti-inflammatory and pro-angiogenic potential in wound microenvironment assays in vitro. Furthermore, VLAM was evaluated in two wound models in diabetic mice. Similar to fresh AM and VCAM, weekly applications of VLAM resulted in significantly faster rate of wound closure compared to the control group. Moreover, wound closure in the VLAM group correlated with a decrease in pro-inflammatory cytokines and an increase in antioxidant levels, vascularization, collagen deposition and dermal thickness in wound tissue samples. These data demonstrate that both VCAM and VLAM retain the structure, cell viability, and functional properties of fresh AM. However, VLAM is stored at ambient temperatures making VLAM more accessible for widespread use.

Key Words: Lyopreservation, Cold Chain, Cryopreservation, Inflammation, Angiogenesis, Wound Healing, Amnion

5. Releasable Hydrogel Microspheres Enable Multiparametric Cell Separation Independent of Magnetic and Fluorescent Tags for Improved Cell Therapy Bioprocessing Workflows

Presented by: Sean Kevlahan, Quad Technologies
Authors: Qin, Guokui, Quad Technologies; Jersuraj, Nithya, Quad Technologies; Kevlahan, Sean, Quad Technologies; Ball, Andrew, Quad Technologies

For cell therapy biomanufacturing, cell purification methods heavily influence process efficiency and clinical outcomes. Most T cell purification processes utilize magnetic beads which must be removed from the cell product prior to infusion. Magnetic de-beading is associated with reduced yields, complexity, and cost. We have developed magnetic bead-free hydrogel microspheres which capture specific cell populations from heterogeneous samples, rapidly release captured cells, and enable multiple marker-based cell separation. Methods An ionotropic copolymer hydrogel was formulated and used to fabricate hydrogel microspheres, which were conjugated to streptavidin. Various release buffers with chelating agents were evaluated for ability to rapidly dissolve microspheres. Several filter/column combinations were tested for optimal retention of microspheres and bound cells and elution of contents following application of release buffer. Biotinylated antibodies against CD34, CD3, CD4 and CD8 were used for separation of Kg1A cells, human CD3+ T cells, or human PBMCs. Multiple microsphere sizes were evaluated for efficiency of cell capture and release. Flow cytometry was used to assess biotin binding, cell count, viability and phenotype. Results Streptavidin-conjugated microspheres ranging in size from 20µm to 200µm diameter were fabricated. First, Kg1A cells were incubated with biotinylated anti-CD34 and streptavidin-conjugated hydrogel microspheres (30µm diameter), then loaded into a column/strainer containing a mesh filter (20µm pore). 2 mM EDTA was the optimal release buffer for microsphere phase-change/release (95%). 85% of the Kg1A population were captured and released using hydrogel microspheres. We applied this system to capture and release CD4+ or CD8+ T cells from a CD3+ cell population, with purities of 94% and 99% respectively, and viability >99%. We performed the same experiment using PBMCs, resulting in purities of 96% (CD4+) and 97% (CD8+). Finally, we performed sequential separations of CD4+ and CD8+ cells from PBMCs. Unbound cells from the first separation (CD4-) pass through the column/strainer device while bound cells (CD4+) are retained. Unbound cells were applied to a second column/strainer device for CD8+ separation. Upon release, highly purified populations of CD4+ (97% purity) cells and CD8+ (95% purity) cells were recovered (>99% viability). Conclusions We developed a technology platform which efficiently captures target cells from heterogeneous samples, rapidly releases high purity/viability bound cells, and enables multiparametric purification via sequential capture and release. The releasable hydrogel microspheres are versatile, scalable, automation-compatible, compliant with USP cell therapy manufacturing guidance, and amenable to cGMP manufacturing. This technology offers the potential to eliminate magnetic beads from cell therapy biomanufacturing.

Key Words: Cell therapy Bioprocessing Cell separation Adoptive T cell therapy CAR-T Biomanufacturing Hydrogel microspheres

6. The First Step to Conserving Endangered Species Through Stem Cells

Presented by: Marisa Korody, San Diego Zoo Institute for Conservation Research
Authors: Korody, Marisa, San Diego Zoo Institute for Conservation Research; Nguyen, Tom, San Diego Zoo Institute for Conservation Research; Pivaroff, Cullen, The Scripps Research Institute; Schell, J.P., The Scripps Research Institute; Tran, Ha, The Scripps Research Institute; Ryder, Oliver, San Diego Zoo Institute for Conservation Research; Peterson, Suzanne, The Scripps Research Institute

Since the development of induced pluripotent stem cells (iPSCs), stem cell research has expanded dramatically, but most of this research focuses on human and mouse models. The first reported generation of endangered species iPSCs was Since the development of induced pluripotent stem cells (iPSCs), stem cell research has expanded dramatically, but most of this research focuses on human and mouse models. The first reported generation of endangered species iPSCs was published in 2011. These iPSC lines, from the drill and the northern white rhinoceros (NWR), were reprogrammed using the integrating lentivirus. To make integration-free iPSCs, we have reprogrammed NWR fibroblast cell lines from several animals using Sendai virus. Fibroblast cell lines were obtained from the San Diego Zoo’s Frozen Zoo® repository of biomaterials, which contains cell lines from 12 different NWRs, and captures high genetic variability of this species. These cell lines were established from skin biopsies and banked over the last 38 years. Fibroblasts were grown in a medium optimized for the growth of rhino cells, which has now also been incorporated into the formulation of a rhino specific stem cell medium (KB medium). Compared to human iPSCs, NWR iPS cells and colonies have distinctive morphologies, which are consistent among lines from this species. Pluripotency and differentiation potential was confirmed with immunocytochemistry markers for pluripotency in the iPSCs and markers for all three germ layers in differentiated embryoid bodies (EBs). qRTPCR analyses using NWR specific primers confirmed expression of pluripotency-associated genes (CDH1, DNMT3b, LIN28a, NANOG, and SOX2) in the iPSCs. All fibroblast cell lines and corresponding iPSC lines were found to be karyotypically normal. The NWR is functionally extinct, with only three, non-reproductive, living individuals remaining. These NWR iPSCs are the first step in a plan to rescue this species through assisted reproduction.

Key Words: induced pluripotent stem cells, integration-free iPSCs, endangered species, northern white rhino, assisted reproduction

7. Expanding a “Youthful” Subpopulation of Mesenchymal Stem Cells From Elderly Donors on Bone Marrow – High Performance Micro Environment (BM-HPME)

Presented by: Travis Block, StemBioSys
Authors: Block, Travis, StemBioSys; Marinkovic, Milos, UT Health San Antonio; Tran, Olivia, UT Health San Antonio; Marshall, Amanda, UT Health San Antonio; Dean, David, UT Health San Antonio; Chen, Xiao-Dong, UT Health San Antonio

Due to increased life expectancy, age-related degenerative diseases are becoming an important public health concern. Although researchers have actively pursued stem cell-based therapies for retarding or reversing age-related degeneration, the results of clinical trials aimed at demonstrating stem cell efficacy have been inconsistent. Unfortunately, progress in developing autologous mesenchymal stem cell (MSC)-based therapies has been impeded by the fact that MSC quantity and quality decline with aging. Since elderly patients are the main target population for cell-based treatment of age-related diseases, it is essential that a reproducible strategy for providing sufficient quantities of high-quality autologous cells be developed and rigorously tested. In the present study, we propose that the ratio of young to old (aged) stem cells in the BM-MSC population reverses with aging and that old MSCs not only lose their self-renewal and differentiation capacity, but also release harmful factors that damage and/or suppress the younger “healthy” sub-population of MSCs. These changes result in an inexorable functional decline of the overall elderly MSC population. Here, we test the hypothesis that MSCs from elderly patients can be rejuvenated by: 1) isolating a sub-population of more “youthful” cells, using biomarkers (size and SSEA-4 expression), from the original population of elderly MSCs; and 2) preserving and expanding this “youthful” sub-population by culture on a “young microenvironment” using our established technology (BM-ECM produced by young cells). We demonstrate that it is possible to obtain large quantities of MSCs that are functionally and phenotypically similar to MSCs from young donors from elderly donors whose overall cell populations are highly compromised. The results are provocative and suggest that it may be feasible to bank large quantities of high-quality autologous MSCs for treatment of age-related diseases.

Key Words: Stem Cell, Regenerative Medicine, Aging, Tissue Engineering, Cell culture, Extracellular matrix, biomaterial, mesenchymal stem cell, bone marrow, degenerative

8. Using hiPSC-Derived Neuronal Cultures to Assess the Safety of Pre-Screened Potential Therapeutic Compounds Against Zika Virus

Presented by: Cassiano Carromeu, StemoniX
Authors: Slavin, Ileana, StemoniX; Dea, Steven, StemoniX; Montefusco, Sandro, UC San Diego; Siqueira-Neto, Jair, UC San Diego; Zanella, Fabian, StemoniX; Carromeu, Cassiano, StemoniX

The recent global threat of Zika Virus epidemic has highlighted the need for sophisticated screening systems capable of detecting unintended toxicity of candidate compounds against this serious infection. Toxicity to the Central Nervous System (CNS) is a key aspect in safety pharmacology evaluation of drugs under development. The characterization of the toxicological profiles of new chemicals to the CNS involves extensive investigation using in vitro and in vivo models. Currently, primary cultures and animal models are popular platforms for those studies. In spite of their importance, those platforms typically are not amenable to larger scale toxicity screens. Human induced pluripotent stem cell (hiPSC) technology has enabled the ready availability of large and consistent batches of neural cells and tissues for wider toxicity screens, having the potential to change the current paradigm in pharmacological research. Through hiPSCs and state-of-the-art differentiation protocols, researchers now have available unlimited source of neural cells, able to mimic early and late stage of human CNS development. These sophisticated cellular models hold great potential in reducing the time to assess toxicity of developing drugs. Here we investigate the toxicological profile of 29 compounds recently described in the literature as potential therapeutic compounds against Zika Virus infection. hiPSCs-derived neural cells at different developmental stages were challenged with this library of compounds in two-dimensional cultures as well as three-dimensional mini-brain organoids. We observed greater susceptibility of the neural tissues to compound toxicity at early stages of development, and decreasing toxicity as the neuronal cultures mature in vitro. Compounds with the safest profiles were further evaluated in high throughput calcium flux and multi-electrode array assays for assessment of potential functional side effects on the normal function of the CNS. In summary, our work highlights the power of a human CNS model in predicting toxicological profiles of proposed drugs against Zika Virus. Moreover, this system can be applied to investigate the safety profiles on new chemical entities, improving predictivity of clinical outcome and reducing overall drug development costs.

Key Words: human induced pluripotent stem cells, neural progenitors, neurons, neurospheres, toxicity, neurotoxicity, Zika, viability, multi-electrode arrays, high throughput, screening, re-purposing, high throughput calcium flux

9. Characterization of hiPSC-derived Cardiomyocytes on a Micro-engineered High Throughput Screening Platform

Presented by: Riccardo Contu, StemoniX
Authors: Contu, Riccardo, StemoniX; Spangenber, Stephan, StemoniX; Si, Wonjong, StemoniX; Padilla, Robert, StemoniX; Fanton, Alison, StemoniX; Sampietro, Kristen, StemoniX; Vierra, Brittney, StemoniX

Current approaches on the study of acute cardiac safety and toxicity have been transformed with the availability of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Cardiac toxicity plays an important role in the failure of therapeutic agents in late stages of clinical trials, as well as for the removal of approved drugs from the market. The Comprehensive in vitro Proarrhythmia Assay (CiPA) constitutes a novel safety screening proposal intended to replace current regulatory strategies that have failed to predict the acute cardiotoxic effects of developing drugs. Through the CiPA initiative, researchers from diverse organizations such as the FDA, academic institutions, and pharmaceutical companies are evaluating hiPSC-CMs as an integral tool for the safety assessment of novel therapeutic compounds. Nonetheless, key challenges under consideration for the hiPSC-CM system are sub-ideal cardiomyocyte geometry, sub-cellular structural organization and overall electro physiological maturity. As an example, hiPSC-CMs frequently display, undefined or disarrayed sarcomeric organization when plated in standard cell cultureware. Here we describe and characterize a novel high-density screening platform employing hiPSC-CMs that is micro-engineered to emulate correct cardiac muscle fiber organization. This platform allows for passive self-alignment of hiPSC-CMs, leading to sarcomeric organization perpendicular to the cell body, with readily identifiable, correctly and constantly patterned myofibrils. Concomitantly, directionality of contraction of hiPSC-CM preparations were observed to be increased in our platform. Comprehensive analysis of calcium flux in hiPSC-CMs and observed that hiPSC-CM alignment in this platform influences cardiomyocyte physiology. Specifically, changes in beating rate, increased peak amplitude, decreased peak spacing standard deviation, decreased peak rise time, and decreased peak decay time were observed when compared to standard flat cell cultureware across two independent hiPSC-CM lines. Importantly, the observed changes can be of great importance for the evaluation of compound cardiotoxicity as they may provide greter resolution over drug-induced effects. Altogether we describe a novel hiPSC-derived cardiomyocyte platform with greater physiological and functional relevance that is pre-formatted to high throughput screening.

Key Words: induced pluripotent stem cells, cardiomyocytes, physiology, physiologically relevant, sarcomeres, alignment, high throughput, screening, cardiotoxicity, cardiac, geometry

10. Neural Stem Cell Therapy for Parkinson’s Disease: Magnetic Resonance Imaging-Guided Delivery

Presented by: Marcel Daadi, Texas Biomedical Research Institute
Authors: Daadi, Marcel, Texas Biomedical Research Institute

Parkinson’s disease is characterized by the degeneration of the dopamine-producing neurons in the substantia nigra. Neural stem cell transplantation to the basal ganglia has the potential to improve dopaminergic function in patients afflicted with Parkinson’s disease. Optimal neural stem cell delivery procedures are critical for the success of the cell therapy approach. Unlike small molecules, growth or neurotrophic factors, cells are sensitive to the microenvironment they are in direct contact with. Cell injection variables such as the rate of infusion and needle size affect the penetration and dispersion of the cells in the target tissue and thus may compromise the efficacy of the therapeutic approach. Magnetic resonance imaging (MRI) can be used as a minimally invasive tool to guide the cell transplantation. In these experiments, the cells were labeled with Super Paramagnetic Iron Oxide (SPIO) nanoparticles, which can be visualized as hypointense regions on MRI. The injection parameters such as infusion speed and cannula diameter were established using the agarose gel model by injecting SPIO-labeled cells into 0.6% agarose gel phantoms and using real-time MRI to monitor the cellular dispersion. These injection parameters were translated to MRI-guided injections into the baboon cadaver brain, using the ClearPointTM system (MRI Interventions, Irvine, CA) to identify the injection target and guide the cannula insertion. A whole-brain 3D MRI series was acquired, which the ClearPoint Software used to establish a 3D coordinate system. A cannula trajectory guide (SmartFrameTM) with four MRI-sensitive fiducial markers was attached to the skull. The fiducial markers allowed the software to segment the SmartFrameTM and determine the trajectory of the cannula. Iterative imaging of the fiducial markers and adjustment of the SmartFrameTM was used to align the cannula trajectory with the target until there was less than 1mm of error between the desired target and cannula trajectory. Once the cannula trajectory was finalized, a burrhole was drilled at this location. The cannula, loaded with SPIO-labeled cells, was inserted through the guide into the brain and the injection was initiated at 1μL/min and recorded in real-time with a TurboFlash series. A whole-brain 3D image series was acquired post-injection to visualize the graft. The phantom experiments showed that the injection speed of 1μL/min is appropriate to prevent clogging and backflow of the cells along the cannula track. The injection recording in both the phantom and MRI-guided baboon experiments showed pulsatile flow of the cells, which exited the cannula in distinct bursts throughout the injection. The post-injection MRI images confirmed that the cells were successfully delivered to the putamen. The use MRI guidance was determined to be an effective and minimally invasive method of cell delivery to the baboon brain.

Key Words: Parkinson’s disease, stem cell therapy, neural stem cells, dopaminergic neurons, Transplants, nonhuman primate model, Brain imaging, MRI-guided stem cell delivery, Preclinical Development

11. An Endogenous Metabolite Enhances the Maturation of Oligodendrocytes

Presented by: Brittney Beyer, The Scripps Research Institute
Authors: Beyer, Brittney, The Scripps Research Institute; Fang, Mingliang, The Scripps Research Institute; Sadrian, Benjamin, The California Institute for Biomedical Research; Montenegro-Burke, Rafael, The Scripps Research Institute; Plaisted, Warren, The California Institute for Biomedical Research; Kok, Bernard, The Scripps Research Institute; Kondo, Toru, Hokkaido University

A promising approach for the treatment of Multiple Sclerosis (MS) involves the identification of small molecules that enhance oligodendrocyte precursor cell (OPC) differentiation, a process which becomes limiting during progressive stages of demyelinating diseases. A complement to this approach is the use of metabolic profiling to further understand cellular programming and mechanisms associated with cell fate transitions. Previously, our lab identified a compound that directly stimulates OPC differentiation to induce remyelination in MS models [(Nature 502, 327 (2013)]. Extending upon this work, here, we demonstrate the use mass spectrometry-based metabolomics to identify metabolites that are significantly altered over the course of the OPC differentiation process. Levels of taurine, an amino sulfonic acid possessing pleotropic biological activities and broad tissue distribution properties, among other related metabolites, was found to be significantly elevated (~20-fold) over the course of oligodendrocyte differentiation and maturation. When added exogenously at physiologically relevant concentrations, taurine was found to dramatically enhance the processes of drug-induced OPC differentiation and in vitro myelination of co-cultured axons. Mechanism of action studies suggest that the OPC differentiation- and myelination-enhancing activities of taurine are driven primarily by its ability to directly increase available serine pools, which serves as the initial building block required for the synthesis of the glycosphingolipid components of myelin that define the functional oligodendrocyte cell state.

Key Words: somatic stem cells; drug discovery; metabolomics; multiple sclerosis

12. Characterization of Human Neural Precursor-Like Cells That Promote Neurologic Recovery in the JHMV Model of Multiple Sclerosis

Presented by: Aditi Shankar, The Scripps Research Institute
Authors: Shankar, Aditi, The Scripps Research Institute; Coleman, Ronald, The Scripps Research Institute; Wang, Yanling, The Scripps Research Institute; Mclntyre, Laura, University of California, Irvine; Lane, Thomas, University of Utah School of Medicine; Walsh, Craig, University of California, Irvine; Loring, Jeanne, The Scripps Research Institute

Multiple sclerosis (MS) is a chronic degenerative autoimmune disease of the central nervous system (CNS). It is the most common cause of non-traumatic neurological disability in the US. In MS an immune-mediated attack destroys myelin-producing oligodendrocytes in the CNS, disrupting communication between neurons. This leads to a wide variety of specific pathologies depending on the area of the CNS that is attacked. Previously, using the JHMV mouse model of multiple sclerosis (MS), we showed that intraspinal transplantation of human embryonic stem cell-derived neural precursor like cells (hNPLCs) resulted in a sustained clinical recovery. The mice showed continued clinical improvement for 3 months and sustained recovery for 6 months (longest duration of experiment) post-transplantation. Spinal cords of 21 day post-transplant mice showed increased numbers of CD4(+) FOXP3(+) T regulatory cells (Tregs) within the spinal cords and remyelination of spinal neurons. The human cells did not engraft into these immunocompetent mice, and were rejected by 8 days post-transplantation. We hypothesize that the transplanted cells secreted factors that induced changes in T-cells and oligodendroglial precursors that later led to the long-term recovery. The cells that elicited clinical improvement have a very specific gene expression profile that is unlike any cell type that has been characterized before. We are investigating the developmental potential of these cells. Our studies indicate that hNPLCs are primarily neural crest cells and their derivatives. We are also focusing on secreted proteins made by these cells that may be responsible for the dramatic transient effects of the transplants. Our long-term goal is to identify factors that may have clinical use for MS patients.

Key Words: Multiple Sclerosis, JHMV, human embryonic stem cells, neural crest cells, neural differentiation

13. The Cost-utility of Investing in Curative Therapies for Orphan Indications: A Case Study of Choroideremia Gene Therapy

Presented by: Celine-Lea Halioua-Haubold, The University of Oxford
Authors: Halioua-Haubold, Celine-Lea, The University of Oxford; Jolly, Jasleen, The University of Oxford; Brindley, David, The University of Oxford; MacLaren, Robert, The University of Oxford

Background: Choroideremia (CHM) is an orphan degenerative retinal disease caused by an X-linked recessive mutation of the CHM gene. The mutation causes a loss-of-function of the Rab escort protein 1 (REP1), resulting in progressive vision loss and eventual blindness. Gene therapy (GT) offers a potential cure for CHM by delivering a functional copy of the REP1 gene, rescuing the degenerating photoreceptors. The aim of this study was to determine the long-term financial value, if any, of a national healthcare system, here the United States, investing in and ensuring wide adoption of a therapeutic indicated for a small population; a gene therapy for Choroideremia is used as an example. Methods: Two Markov models (A and B) were built to calculate the net expected Medicare costs and lifetime quality-adjusted life-years (QALYs) of a CHM patient treated either with a curative GT or the current standard-of-care (SOC) which is functionally equivalent to no treatment. Model A calculated the benefit of treating all current Choroideremia patients, which encapsulates a variety of degeneration levels and ages, with the GT versus SOC. Model B calculated the hypothetical benefit of treating all CHM patients at age 20 years. The model outputted the expected loss of retinal area (RA) of a patient from ages 20 to 65 years. RA percentage remaining was assigned a utility value (in QALYs) and annual expected Medicare costs upon patient eligibility, discounted 3%. Monte Carlo microsimulation (n = 1000) was used to calculate the outcome values. Results: Immediate GT treatment for all non-blind current CHM patients (Model A) would give a net benefit of approximately $20,000 in future, discounted Medicare costs and 8 QALYs; net GT value is approximately $420,000 per patient. For the average 20-year-old patient carrying the CHM mutation (Model B), preliminary results show immediate GT treatment saves approximately $74,000 in future, discounted Medicare costs and 18 lifetime QALYs over current standard of treatment. Using the common minimum accepted willing-to-pay per QALY of $50,000 in the US, this gives a net GT value of approximately $974,000 per patient. This analysis did not take into account healthcare costs accrued before 65 years of age due to national variances. Conclusions: Assuming the cost of a GT is determined on a healthcare value basis, these results give the hypothetical maximum justifiable cost of the GT; these numbers raise significantly when using a QALY value of $100,000-plus, as commonly accepted. The population-wide prevention of CHM-associated vision degeneration at an early age could justify significant immediate therapy costs due to its long-term financial benefits and significant increase in patient quality of life. However, the total financial benefit of the GT is accrued over each patient’s lifetime, while the GT cost burden is immediate. Therefore, the real-world implementation of these results may still present significant challenges.

Key Words: Gene therapy, Choroideremia, Retina, Vision loss, AAV, orphan

14. A Cell Therapy Systems (CTS) Sendai Virus Reprogramming Kit for Xeno-Free Generation of iPSC

Presented by: Chad MacArthur, Thermo Fisher Scientific
Authors: MacArthur, Chad, Thermo Fisher Scientific; Lakshmipathy, Uma, Thermo Fisher Scientific

The field of cellular reprogramming for the generation of induced pluripotent stem cells (iPSC) has rapidly matured in the past decade, from initial basic research to now moving towards clinical applications. As iPSC move towards the clinic, it becomes important to ensure that these cells are generated with safe, consistent methods and that the cells themselves are of high quality. The reprogramming workflow should be free of any animal origin components that can be potential sources of adventitious agents which may compromise safety. Many traditional reprogramming and cell culture protocols include the use of animal origin components such as fetal bovine serum or bovine serum albumin, and so protocols must be altered to remove these components while ensuring consistent reprogramming performance. We generated high quality iPSC with xeno-free workflows using a Sendai based reprogramming kit which is free of animal origin components, and is manufactured under a certified ISO 9001 Quality Management System. Using this kit, iPSC were generated from both blood-derived cells and skin-derived dermal fibroblasts. For both starting cell types, beginning from somatic cell isolation all the way through to iPSC generation and banking, completely xeno-free workflows were used. These iPSC were characterized and shown to have a normal karyotype, no detectable presence of Sendai reprogramming vectors, expression of typical PSC self-renewal markers, and functional pluripotency for the three germ layers. The ability to consistently create high quality iPSC in completely xeno-free workflows will allow researchers to more easily transition to clinical applications.

Key Words: iPSC, reprogramming, Sendai, xeno-free, stem cells

15. Investigation of Neurogenic Defects in Fragile X Syndrome

Presented by: Ai Zhang, The Scripps Research Institute
Authors: Zhang, Ai, The Scripps Research Institute; Szucs, Attila, UC San Diego; Wang, Yanling, The Scripps Research Institute; Boland, Michael, The Scripps Research Institute; Bratt-Leal, Andres, The Scripps Research Institute; Loring, Jeanne, The Scripps Research Institute

Fragile X Syndrome (FXS) is the leading monogenic cause of intellectual disability and autism spectrum disorder. It is caused by an expansion of a trinucleotide repeat in the 5’UTR of the Fragile X Mental Retardation-1 (FMR1) gene. We have previously reported that induced pluripotent stem cells (iPSCs) derived from FXS patients exhibit reduced neurogenesis and genome-wide differences in DNA methylation and gene expression compared to controls (Brain, 2017). In the current study, we have found that the impaired neurogenic competence in FXS cells is likely due to flawed establishment of dorsal telencephalic identity, which is manifest by low expression of the dorsal forebrain gene FOXG1 and abnormal neural rosette formation and neuroepithelial polarity. Our gene expression analysis suggests dysregulation in the FXS cells of Notch, WNT, and TGF? signaling pathways that are essential for dorsal fate specification in the telencephalon. We are dissecting the roles of signaling pathways underlying the neurogenic phenotype using genomic rescue experiments and assessing the phenotypic outcomes with gene expression, DNA methylation, and electrophysiological analyses. We are developing assays to screen small molecule candidates for their ability to rescue or reduce the impaired patterning and neurogenesis in the FXS cells. Our long-term goal is to develop a phenotypic assay that is amenable to high-throughput therapeutic drug screening.

Key Words: Disease modeling, Neural progenitors, Autism Spectrum Disorders

16. Role of Human iPSC-derived Macrophages in Rett Syndrome Pathogenesis

Presented by: Pinar Mesci, UC San Diego
Authors: Mesci, Pinar, UC San Diego; Muotri, Alysson, UC San Diego

Microglia are brain-resident macrophages that are involved in key functions in the developing brain such as synaptogenesis, apoptosis, phagocytosis, angiogenesis and astrogliogenesis. They are able to release different factors such as pro-inflammatory cytokines, neurotrophic factors, reactive oxygen species and glutamate. Yet, microglial cells have been mainly studied because of their implication in pathological conditions such as neurodegenerative diseases. Their role in Rett syndrome (RTT) has been the subject of controversy, likely due to inconsistencies in mouse models. Moreover, there is no indication, so far, that microglia are implicated in RTT in a human context. By generating human induced pluripotent stem cell (hiPSC)-derived macrophages from RTT patients, we are aiming to clarify the controversies surrounding the impact of these cells. Our preliminary data shows that hiPSC-derived macrophages possess the appropriate expression of cell surface markers such as CD14 and CD68. In addition, upon activation with lipopolysaccharides (LPS), hiPSC-derived macrophages released elevated levels of IL-6 and TNFa and were also able to perform phagocytosis. Altogether, we have strong preliminary data showing successful generation of functional hiPSC-derived macrophages. Given the current insufficient knowledge about the ontogeny and development of human microglia, and the absence of specific markers to differentiate microglial cells from macrophages, we have chosen to use hiPSC-derived macrophages as an alternative to study these immune cells in a context of a neurodevelopmental disorder. Therefore, we propose to test the hypothesis that RTT macrophages could be implicated in the neuronal defects observed in RTT, and that the immunomodulation of these immune cells could be a valuable therapeutic strategy.

Key Words: Rett, macrophage, microglia, hiPSC

17. Subtype-Specific Cardiomyocyte Drug Screening and Disease Modeling Platform

Presented by: Michael Yu, UC San Diego
Authors: Yu, Michael, UC San Diego Department of Bioengineering; Spiering, Sean, Sanford Burnham Prebys Medical Discovery Institute; Savtchenko, Alex, Sanford Burnham Prebys Medical Discovery Institute; Mercola, Mark, Stanford University Department of Medicine; Colas, Alexandre, Sanford Burnham Prebys Medical Discovery Institute

The lack of subtype-specific cardiomyocyte (CM) model that can recapitulate the unique target space of subtype-specific cardiac disease, ex. atrial fibrillation, hinders the understanding of the disease-causing mechanisms and the discovery of treatment. We first establish subtype-specific cardiomyocyte (CM) differentiation protocol by introducing differentiation cues on day 5 during hiPSC-derived CM differentiation. We are able to generate highly homogeneous ventricular-like cardiomyocytes (VCM) and atrial-like cardiomyocytes (ACM) expressing subtype-specific markers respectively. Furthermore, the phenotypical action potential recorded through electrophysiology from these subtype-CMs recapitulate the adult human data. We arrayed these subtype-CMs in 384 well microtiter plates using liquid handling robotics. By plating CMs sparsely, we limited the electrical coupling between CMs to achieve single-cell-like action potential analysis. CMs were then loaded with voltage-sensitive probes and recorded in 100Hz for 5 seconds through high-throughput/high-content automated microscopy. The individual action potential changes over time of each cell were then analyzed by batch processing software, and the physiological parameters such as beat rate, action potential duration at 90% (APD90), were also calculated. The establishment of the single-cell level subtype-specific CM functional readout platform not only enables high-throughput functional screenings between CM subtypes, but also increases the accuracy and samples size dramatically. We then demonstrate concepts of functional genomics and drug discovery screenings respectively on the platform. Since KCNA5 is responsible for atrial-specific ultra-rapid delayed-rectifier current (IKur). By inhibiting this potassium channels on CMs, we should observe the prolongation of action potential duration only in the ACM, not in the VCM. The result of the treatment of siKCNA5 showed that only APD90 of ACM was prolonged, but the APD90 of VCM remains unchanged. This result indicated that we are able to performed functional genomics by screening through siRNAs and deconvolute their effect on action potentials on VCM and ACM respectively. Moreover, 4-aminopyridine (4-AP) is a selective potassium ion channel blocker of the KCNA family. Our result showed that the treatment of 4-AP prolonged only the APD90 of ACM, but not VCM. Again, we demonstrated that we could apply drug screenings on the platform and exam the efficacy in a subtype-specific manner from compound libraries.

Key Words: Cardiomyocyte subtypes, High-throughput screening, Disease modeling, Drug discovery, Stem cell differentiation, Functional screening, Single cell level

18. β3 Integrin is a Stress-inducible Gene Enabling Tumor Cell Adaptation to Stress

Presented by: Maricel Gozo, UC San Diego
Authors: Maricel, Gozo, UC San Diego; Minami, Toshiyuki, UC San Diego; Aguirre, Aitor, UC San Diego; Yebra, Mayra, UC San Diego; Weis, Sara, UC San Diego; Cheresh, David, UC San Diego

Aggressive epithelial cancers often hijack physiological pathways to enable tumor progression. Here, we show that expression of integrin αvβ3 is enriched on normal epithelial cells during remodeling events, as well as on tumor cells exposed to various forms of cellular stress. Mechanistically, stress induces a series of histone modifications that promote an open chromatin state on the β3 promoter, allowing binding of the transcription factor HNF4A. Accordingly, histone acetyltransferase or demethylase inhibitors block these stress-mediated histone modifications to prevent the induction of β3 and sensitize cells to the effects of stress. Using a genome-wide CRISPR/Cas9 forward genetics screen, we link this stress tolerance to activity of YAP/TAZ, transcriptional coactivators suppressed in normal cells by Hippo kinases to limit uncontrolled growth. Expression αvβ3 recruits Src/Yes to the β3 cytoplasmic domain to promote expression of proliferation/survival genes mediated by the YAP/TAZ transcriptional coactivators. Our findings suggest that the well-established role of αvβ3 in tumor progression and drug resistance may reflect the status of integrin β3 as a stress-inducible gene that enables tumor cell adaption to multiple forms of cellular stress.

Key Words: Integrin B3, reprogramming, cellular stress

19. Integrin αvβ3 Remodels the Endothelium

Presented by: Joseph Wawrzyniak, UC San Diego
Authors: Wawrzyniak, Joseph, University of California San Diego; Cheresh, David, UC San Diego

Integrin αvβ3 is robustly expressed on endothelial cells during vascular development and tissue remodeling, then is suppressed during vascular maturation. Once expressed, αvβ3 can bind to a number of provisional matrices that emerge during this process such as vitronectin, fibronectin, and fibrinogen. Provisional matrices are associated with tissue repair stimulating proliferation and survival, while in contrast; intact basement membrane promotes a stable, non-proliferative state. The role of αvβ3 in regulating this process remains unclear. To investigate the role of αvβ3 in regulating the plasticity of endothelial cells during tissue remodeling, we created a model in which endothelial cells can be shifted between more mature and less mature states. To drive vascular maturation in vitro, proliferating human umbilical vein endothelial cells (HUVECs) were grown on a layer of Matrigel™ to simulate a basement membrane. Under these conditions, HUVECs promptly differentiated and formed tube-like structures. However, when HUVECs are placed on provisional matrix proteins such as vitronectin, fibronectin or fibrinogen, we found an increase in cell density and proliferation and, to our surprise, the expression of several pluripotency genes such as OCT4, NANOG, and SOX2. To determine whether the expression of these stems genes was specifically αvβ3 dependent; we discovered that clustering of αvβ3 or its ectopic expression was sufficient to drive their expression. Interestingly, not only did ectopic αvβ3 induced stem genes, but drove HUVECs to de-differentiate as evidenced by the complete loss of several endothelial lineage markers (CD31, VWF, and VEGFR2). These results prompted us to consider the novel concept of αvβ3 as a reprogramming factor capable of converting mature endothelial cells to dedifferentiated stem-like cells. Supporting our hypothesis, we found that ectopic αvβ3 in HUVEC was sufficient to promote their dedifferentiation to a stem-like state in which under specific conditions, could be then differentiated to neurons, astrocytes, and cardiomyocytes. Overall, our findings show that αvβ3 signaling converts mature endothelial cells to multipotent stem cells. The high degree of cellular plasticity conferred upon angiogenic endothelial cells by αvβ3 might play a significant yet unappreciated biological function in tissue regeneration during wound healing, organismal development, and cancer progression.

Key Words: Vascular remodeling, Stem cells, Endothelium, Extracellular Matrix Proteins

20. Mechanoresponsive Stem Cells to Target Cancer Metastases Through Biophysical Cues

Presented by: Linan Liu, UC Irvine
Authors: Liu, Linan, University of California-Irvine; Zhang, Shirley, University of California-Irvine; Liao, Wenbin, University of California-Irvine; Zhao, Weian, University of California-Irvine

Cancer metastases are responsible for over 90% of cancer deaths, however no current effective treatments directly and selectively target them. The unique mechanical properties of metastatic niche offer an interesting target for the development of therapeutics specifically targeting metastases. Systemically infused mesenchymal stem cells (MSC) preferentially home to tumors. Also, it has been established that tissue mechanical properties regulate MSC fate by driving expression of genes involved in differentiation. We hypothesize that increased matrix stiffness is a fundamental property of the metastatic niche that can be targeted with MSC-based, mechanoresponsive therapies. Here we presented a new approach for the treatment of cancer metastases by targeting the mechano-environment of the metastatic niche using promoter-driven, MSC-based vectors, referred to as mechanoresponsive cell system (MRCS). Our data suggest a strong correlation between collagen cross-linking and increased tissue stiffness at the metastatic sites, where our MRCS is specifically activated by the specific cancer–associated mechano-cues. MRCS has significantly reduced deleterious effects compared to MSC constitutively expressing cytosine deaminase which converts a non-toxic prodrug 5-fluorocytosine to an active chemotherapy drug 5-fluorouracil. MRCS indicates that biophysical cues, particularly matrix stiffness, are intriguing targets for cancer therapy due to their long persistence in the body, making them refractory to the development of resistance to treatment. Our MRCS can serve as a platform for future diagnostics and therapies targeting abnormal tissue stiffness in conditions including cancer and fibrotic diseases.

Key Words: mesenchymal stem cell, cancer metastasis, mechanoresponsive, diagnosis, therapeutics, cell engineering, mechano-sensing, biophysical cues, YAP/TAZ, stiffness, atomic force microscopy, second harmonic generation imaging

21. Modeling Preeclampsia Using Induced Human Pluripotent Stem Cells

Presented by: Mariko Horii, UC San Diego
Authors: Horii, Mariko, UC San Diego; Touboul, Thomas, UC San Diego; Sonsin, Francesca, UC San Diego; Chang, Ching-Wen, UC San Diego; Laurent, Louise C, UC San Diego; Parast, Mana M, UC San Diego

Preeclampsia (PE) is a hypertensive disorder which affects 2-8% of pregnancies, and is associated with adverse outcomes for both mother and baby during the pregnancy and later in life. PE has two clinical subtypes, based on the gestational age at onset of disease: early-onset (EO-PE) and late-onset preeclampsia (LO-PE) refer to disease either before or after 34 weeks gestational age, respectively. The primary etiology of this disease is due to abnormal development and function of placental cells, including poor invasion of maternal tissues and excess secretion of anti-angiogenic molecules such as soluble Flt (sFlt). This leads to shallow implantation, reducing blood flow and oxygen delivery to the fetoplacental unit, a phenotype that is significantly more severe in EO-PE. Studying human trophoblast differentiation is difficult due to lack of proper disease models. To overcome this problem, our group has established an in vitro model of trophoblast differentiation, using human pluripotent stem cells (hPSCs) treated with bone morphogenetic protein 4 (BMP4). Upon 10 days of BMP4 treatment, hPSCs differentiate first into cytotrophoblast (CTB), multipotent trophoblast stem cells, and then into terminally differentiated trophoblast, including extravillous trophoblast (EVT) and syncytiotrophoblast (STB). We have shown that this model can recapitulate abnormal trophoblast differentiation using Trisomy 21 cells. Using this model, we hereby report for the first time that preeclamptic iPSC show the expected defects in trophoblast differentiation and function. Methods: Human iPSC (from both EO-PE and LO-PE, and gestational age matched controls) were cultured with 10ng/ml BMP4 for 10 days under either 21% or 5% oxygen, the latter using an XVIVO hypoxia chamber. Samples were collected on days 4, 6, 8 and 10 and assessed by expression of various markers by flow cytometry, qRT-PCR, or ELISA. Results: Following differentiation, the EO-PE iPSC line produced no EVT cells, based on surface expression of HLA-G by flow cytometry, and similarly, showed reduced expression of EVT markers ITGA5 and ITGA1 by qRT-PCR, compared to its gestational age matched control iPSC. Interestingly, this phenotype was specific to EO-PE iPSC, as differentiated iPSC from a LO-PE case did not show a significant reduction in EVT markers compared to term non-PE iPSC. In addition, both EO- and LO-PE iPSC lines showed a 5.7 fold increase in sFlt secretion by ELISA, compared to their gestational age-matched control iPSC, but only when exposed to 5% oxygen. Conclusions: Our results suggest that EO- and LO-PE have distinct phenotypes in terms of EVT differentiation and that these phenotypes can be readily studied using iPSC in vitro. Thus, we propose that iPSC hold enormous potential for studying the underlying mechanisms of PE and potentially other placenta-based pregnancy disorders.

Key Words: Preeclampsia, Bone morphogenetic protein 4 (BMP4) , induced pluripotent stem cells (iPSC), disease-in-a-dish model, placenta, extravillous trophoblast

22. A Cancer Stem Cell Subpopulation Secretes a Bioactive Lipid Inducing Stem-Like Properties on Neighboring Cells

Presented by: Taha Rakhshandehroo, UC San Diego
Authors: Rakhshandehroo, Taha, UC San Diego; Cheresh, David, UC San Diego

Cancer stem cells (CSCs) represent a small but highly aggressive, resistant, and metastatic subpopulation. We considered whether CSCs impact the phenotype of non-CSCs within the tumor mass resulting in their reprogramming to a stem cell fate. We recently showed that integrin αvβ3 is necessary and sufficient to promote the reprogramming of epithelial cancers to a CSC and drug resistant state. Here, we report that tumor cells expressing αvβ3 secrete factors that reprogram non-CSCs to acquire stem cell properties such as anchorage independent growth and expression of pluripotent genes. Further investigation revealed that tumor cells expressing αvβ3 secrete bioactive lipid signaling molecules, specifically lysophosphatidic acid (LPA) and prostaglandins. Integrin αvβ3 was both necessary and sufficient for the expression of enzymes involved in producing LPA and Prostaglandins, Phospholipase A2 and Cyclooxygenase 1 respectively. Interestingly, LPA was detected at markedly higher levels in conditioned media from αvβ3 expressing cells. Furthermore, pharmacological inhibition of the LPA receptors diminished the activity of conditioned media from αvβ3 expressing cells, indicating that LPA is a functional secreted factor. These findings indicate that αvβ3 expressing CSCs can release LPA and, potentially, other lipid signaling molecules, which reprogram non-CSCs within their tumor environment to a stem-like fate. Targeting the LPA receptor pathway may offer a therapeutic approach to reverse this paracrine effect and thereby prevent the conversion of tumor cells to a stem-like drug resistant fate.

Key Words: Cancer stem cells, Paracrine signaling, Secreted factors, Lysophosphatidic Acid, Prostaglandins, Phospholipase A2, Cyclooxygenase Stemness, Anchorage Independence, Lipid Signaling, Integrin, αvβ3, Pluripotency genes

23. EPCR and TM Guide Hematopoietic Stem Cell Homing to the Bone Marrow Independent of Niche Clearance

Presented by: Francesca Avemaria, Weizmann Institute of Science
Authors: Avemaria, Francesca, Weizmann Institute of Science; Gur-Cohen, Shiri, Rockefeller University; Kollet, Orit, Weizmann Institute of Science; Avci, Seymen, Weizmann Institute of Science; Lin, Wei-Ling, National Cheng Kung University; Wu, Hua-Lin, National Cheng Kung University; Conway, Edward, University of British Columbia Weizmann Institute of Science

Bone marrow (BM) homing, recognition and lodgment of long term repopulating hematopoietic stem cells (LT-HSCs) are essential steps during embryogenesis and in clinical stem cell transplantation. BMLT-HSCsexpress endothelial protein C receptor (EPCR), which control their retention, survival and chemotherapy resistance by restricting nitric oxide (NO) production(Gur Cohen S et al, Nat Med 2015). We report that transplanted EPCR-expressing LT-HSC preferentially home to the BM and not to other hematopoietic organ such as the spleen.Homed EPCR+ LT-HSC localized adjacent to thrombomodulin (TM)- enriched arterioles in the BM, which are absent in the spleen.Moreover, LT-HSC also express surface TM, and its lectin domain mediatemolecular recognition between LT-HSC and BM arterial endothelial glycocalyx, cooperatively providing preferential homing of LT-HSC to the BM. Utilizing a duel color stem cell transplantation we show that EPCR engagement by its major ligand, aPC, provide significant advantage in competitive BM homing and repopulation. Homing enhancement by aPCreached a plateau, suggesting that BM niches availability is limited for newly arrived stem cells. Interestingly, EPCR+ LT-HSCs home with higher efficiency and can engraft the BM of non-irradiated recipients. These quiescent homed cells can later be awakened by treating engrafted hosts with NO donor or with low dose chemotherapy, revealing that preconditioning and clearance of occupied HSC niches are not necessary. Herein we define EPCR and TM as guidance molecules, navigating LT-HSC specifically to the BM, withoutniches preconditioning and clearance. The BM displays limited numbers of available niches, and EPCR/aPC increase LT-HSC BM homing and repopulation. Our study provides mechanistic insights concerning LT-HSC homing, which may lead to improved BM transplantation and be applied to prevent chemotherapy resistance of EPCR-expressing cancer stem cell.

Key Words: Long term hematopoietic stem cells, bone marrow homing, EPCR, Thrombomodulin, aPC

24. From Human Naive Pluripotent Stem Cells to Studying Early Human Development

Presented by: Jonathan Bayerl, Weizmann Institute of Science
Authors: Bayerl, Jonathan, Weizmann Institute of Science; Ayyash, Muneef, Weizmann Institute of Science; Gafni, Ohad, Weizmann Institute of Science; Viukov, Sergey, Weizmann Institute of Science; Zerbib, Mirie, Weizmann Institute of Science; Massarwa, Rada, Weizmann Institute of Science; Hanna, Jacob, Weizmann Institute of Science

The establishment of human pluripotent stem cells (hPSCs) have paved the way for an in-depth study of early development, disease modeling and regenerative medicine. Nevertheless, experimental approaches are still limited by 2-D culture models and protocols for hPSC differentiation are far behind relative to their mouse equivalents, which can be attributed to the failure of capturing hPSCs in their naïve state that resembles the ICM of the pre-implanted human embryo in-vivo. Thus, we are able to unravel and devise a novel naïve-like hPSC state, which shares properties with mouse naive pluripotent stem cells and overrides obstacles observed with conventional hPSCs in their primed state, including propensity for limited and variable differentiation potential during human primordial germ cell formation and putative oogenesis in-vitro, as well as lack of chimerism competence after injection of hPSCs into mouse blastocysts. My Ph.D. study focuses to address these challenges in-order to provide a system to study basic regulation of Human oogenesis „in-a-dish“ and to comprehensively study the integration and differentiation of naïve hPSCs in a cross-species humanized animal setting in-vivo. The latter may be a valuable tool for investigating hPSC differentiation in an in-vivo physiological environment, which may be potentially used for future regenerative purposes.

Key Words: human stem cells, naive/primed pluripotency; cross-species chimaerism, early embryonic development, primordial germ cells, oogenesis

25. Regenerative and Clonogenic Potential of Fetal Lung Progenitors

Presented by: Chava Rosen, Weizmann Institute of Science
Authors: Rosen, Chava, Immunology Department Weizmann Institute of Scienc; Shezen, Elizs, Immunology Department Weizmann Institute of Scienc; Orgad, Ran, Immunology Department Weizmann Institute of Scienc; Reisner, Yair, Immunology Department Weizmann Institute of Science

Recently, we showed that in a procedure akin to bone marrow transplantation (BMT), a single cell suspension of mouse or human fetal lung cells harvested at the canalicular phase of gestation (20–22 weeks of human and E15–E16 for mouse gestation) and infused I.V. following conditioning of recipient mice with naphthalene and 6GY TBI, led to marked lung chimerism within alveolar and bronchiolar lineages. Furthermore, using a 1:1 mixture of GFP and ‘tomato’-expressing donor lung cells, we found that at 6-8 weeks after transplantation, single red or green lung progenitors are capable of expanding within the recipient lung to form a discrete ‘patch’. Notably, these donors’ derived ‘patches’ contained epithelial, mesenchymal and endothelial cells, strongly indicating that the patch forming lung progenitor exhibits marked plasticity. However, it could be argued that our single cells fetal lung cell preparation used for transplantation, might include some doublets which could potentially represent different committed progenitors that can give rise to different linages. To address this possibility, we have now carried out a series of transplantation experiments using as donors Rosa26-Confetti mice, bearing a multicolor Cre reporter system in which each cell expresses stochastically just one color thereby markedly reducing the probability that doublets will comprise cells of the same color. Initially, we verified in control experiments that upon bone marrow transplantation into lethally irradiated mice, the spleen colonies formed within 2 weeks after transplantation are as expected of clonal origin displaying discrete monochromatic colors. Next, we transplanted fetal lung cells from Rosa26-Confetti donors into lung-injured recipients. Notably, immunohistochemistry and single-molecule fluorescence in situ hybridization, clearly revealed similar discrete donor derived monochromatic lung patches. Taken together, our results strongly suggest that these observed lung patches likely originate from clonal expansion of a single lung progenitor, offering for the first time a robust in-vivo assay for this highly regenerative stem cell.

Key Words: lung regeneration, confetti mice, spleen colonies

26. Using Human Induced Pluripotent Stem Cells and CRISPR/Cas9 Genome-Editing to Model Autism Spectrum Disorder

Presented by: Allison Songstad, UC San Diego
Authors: Songstad, Allison, UC San Diego; Roberts, Elizabeth, UC San Diego; Markmiller, Sebastian, UC San Diego; Aigner, Stefan, UC San Diego; Yeo, Gene, UC San Diego; Goldstein, Lawrence, UC San Diego

Autism spectrum disorder (ASD) affects 1 in 68 children in the US and involves brain development with symptoms that are unable to be treated with current medicine. Although individual genomic variations strongly influence ASD, they are difficult to model in vitro because patients show varying phenotypes. However, recent technological progress in genome-editing offers an opportunity to investigate the genetic causes of ASD. The overall goal of this project aims to reveal upstream pathological mechanisms that cause synaptic and network dysregulation observed in ASD. In this study, we partnered a well-characterized neurotypical human induced pluripotent stem cell (hiPSC) line with CRISPR-Cas9 genome editing to generate monogenic ASD pluripotent stem cell disease models. We used a modified CRISPR/Cas9 vector containing a green fluorescent protein (GFP) marker with sgRNAs targeting the following genes associated with monogenic forms of ASD: FMR1, NHE6, and TSC2. After transfecting the WT hiPSCs with the CRISPR/Cas9 vector, we used fluorescently activated cell sorting (FACS) to select hiPSCs that were transfected with the CRISPR/Cas9 vector. The selected hiPSCs were plated, the isogenic colonies were subsequently picked and expanded, and each clone was genotyped via TOPO-TA cloning. Overall, we successfully generated homozygous knockout hiPSC lines for FMR1, NHE6, and TSC2. Although the targeted genes were knocked out, the resulting hiPSCs lines maintained normal karyotypes. In conclusion, we established three monogenic ASD hiPSC model lines that will serve as valuable tools to investigate the underlying causes of various forms of ASD. These three knockout ASD lines are now being differentiated into cortical neurons, astrocytes, and microglia that will be analyzed using RNA-seq to identify cell type-specific RNA signatures that will shed light on the molecular pathways that cause ASD. Ultimately, this work will provide new avenues for future ASD studies to discover prospective drug targets and novel diagnostic approaches for this increasingly common disorder.

Key Words: CRISPR, human induced pluripotent stem cells, autism, genome editing, disease modeling

27. Single Cell RNA-Sequencing Reveals Distinct Skeletal Muscle Progenitor Populations Across Human Development

Presented by: Haibin Xi, UC Los Angeles
Authors: Xi, Haibin, University of California Los Angeles; Langerman, Justin, University of California Los Angeles; Gonzalez, Karen, University of California Los Angeles; Sabri, Shan, University of California Los Angeles; Van Handel, Ben, CarthroniX Inc.; Liebscher, Simone, Eberhard Karls University Tübingen; Evseenko, Denis, University of Southern CaliforniaUniversity of California Los Angeles

Skeletal muscle progenitor cells (SMPCs) derived from human pluripotent stem cells (hPSCs) are promising sources for regenerative medicine in treating muscle wasting disorders including muscular dystrophies and sarcopenia. Current protocols of generating hPSC-SMPCs result in highly heterogeneous cell populations with immature progenitors that are unsuitable for clinical implementation. These drawbacks are reflective of insufficient skeletal muscle specification in vitro, due to lack of knowledge of human skeletal myogenesis in vivo. Using the recently published Drop-seq approach, we performed single cell RNA-sequencing on developing human embryos (week 5.5-7.5 of gestation/Carnegie stage (CS) 15-21; whole hindlimb (HL)) as well as juvenile tissues (year 7; surgically removed skeletal muscles). This enabled us to unbiasedly identify distinct myogenic populations from the various stage human tissues. Interestingly, we found that in early embryonic HL (w eek 5.5/CS15), the myogenic progenitors express PAX3 but not PAX7, reminiscent of the migratory myogenic progenitors in mice. On the other hand, in more developed HL (week 7.5/CS 21) the progenitors mainly express PAX7 with low levels of PAX3. As development further progresses, the stem cells from the juvenile muscles express only PAX7 and no PAX3. We confirmed this dynamic expression pattern during embryonic development by immunofluorescent (IF) staining, showing the exclusive PAX3+ population in week 5-6/CS14-16 HL and their gradual transition to PAX7+ at a later stage (week 7/CS 18). To facilitate isolation and further characterization of the distinct myogenic populations, we examined the surface markers on these cells. Our Drop-seq data showed enrichment of CMET (MET) and NCAD (CDH2) in the early PAX3+ migratory cells. Thus, we performed IF co-staining of these candidate markers with PAX3 on week 5-6/CS14-16 human HL sections. Indeed, CMET along largely enriches for the PAX3+ po pulation, and NCAD further improves the purity. When FACS sorted, the CMET+NCAD+ cells enrich for the expression of migratory progenitor markers PAX3 and LBX1, and demonstrate in vitro myogenesis capacity. In summary, we used Drop-seq to unbiasedly identify myogenic populations from different developmental stage human samples. Our profiling and IF data revealed dynamic expression of PAX3 and PAX7, two of the critical myogenic specifiers during human embryonic myogenesis. Furthermore, we discovered candidate surface markers that could potentially enable the isolation of distinct myogenic populations, and employed IF and FACS to validate the use of CMET+NCAD+ to purify the early migratory myogenic progenitors. In the future, we will continue to profile and examine myogenic cells across human development, including fetal as well as adult stages. Our studies will not only help build a roadmap of human myogenesis but also generate useful information on efficiently deriving optimal hPSC-S MPC populations for cell-based therapies.

Key Words: skeletal muscle, myogenic progenitors, pluripotent stem cells, human development, single cell RNA-sequencing, Drop-seq, PAX3, PAX7

28. Large-scale Generation of iPSC-derived Cardiomyocytes for Functional Genomic Applications

Presented by: Margaret Donovan, UC San Diego
Authors: Donovan, Margaret K.R., Bioinformatics and Systems Biology; D’Antonio-Chronowska, Margaret K.R., Institute for Genomic Medicine; Benaglio, Paola, Department of Pediatrics; Matteo, D’Antonio; Megan, Cook, Institute for Genomic Medicine; Farnam, KathyJean, Institute for Genomic Medicine; Smith, Erin N., Department of Pediatrics; Frazer, Kelly A, Institute for Genomic Medicine

Regulatory genetic variation associated with molecular and physiological traits often shows effects restricted to a subset of cell types. Experimentally elucidating the functional molecular underpinnings of regulatory variants is challenging because of the lack of genetically diverse human cell models. Human induced pluripotent stem cells (iPSCs) can be derived into cell types that are relevant for disease, such as cardiomyocytes, and may be an effective system for the comprehensive functional characterization of genetic variants in relevant cell types. Here we used iPSCs from the iPSCORE (iPSC Collection for Omic Research) study to derive cardiomyocytes (iPSC-CMs) from 130 different individuals, all of whom had whole-genome sequence data. This collection included 87 individuals belonging to one of 33 families (which include 6 trios and 7 MZ twin pairs) and genetically related to at least one other person, as well as 43 singletons. To achieve large-sc ale and robust derivations of iPSC-CM, we developed a highly-standardized protocol, which yielded up to 5.7×108 iPSC-CM with an average purity of 87.5% and up to 99.5% of cTnT-positive cells measured by flow cytometry. To study how genetic variation influences molecular phenotypes in iPSC-CM lines derived from both the same and different individuals, we analyzed the transcriptome and epigenome (RNA-seq, ATAC-seq, ChIP-seq for H3K27ac) of a subset of 24 iPSC lines derived from 5 monozygotic (MZ) twin pairs. Comparison of these data to reference data revealed cell type-specific transcriptomic and epigenomic profiles that recapitulated those of fetal heart. We then examined genetic and non-genetic sources of variation in the molecular phenotypes by comparing iPSC-CM molecular phenotypes within and across the 24 iPSC lines derived from the 5 MZ twin pairs. We observed that non-genetic factors including the % of cTnT-positive cells and harvest density were associated with variability bet ween clones from the same subject. However, genetic background accounted for most molecular variability in iPSC-CM and these genetic effects were independent of variation associated with non-genetic factors, suggesting that inherited genetic variants associated with molecular traits could be identified. Our study demonstrates that large collections of iPSC-CMs enabling molecular phenotype genetic studies can be created and supports the use of iPSC-derived cells as a model to functionally characterize regulatory genetic variants.

Key Words: Stem cells, differentiation, cardiovascular system, genomics

29. The iPSCORE Resource: 222 iPSC Lines Enabling Functional Characterization of Genetic Variation Across a Variety of Cell Types

Presented by: Agnieszka D’Antonio-Chronowska, UC San Diego
Authors: D’Antonio-Chronowska, Agnieszka, UC San Diego, USA; Panopoulos, Athanasia D., Salk Institute for Biological Studies, USA; D’Antonio, Matteo, UC San Diego, USA; Benaglio, Paola, UC San Diego, USA; Williams, Roy, UC San Diego, USA; Hashem, Sherin, UC San Diego, USA; Garcia, Melvin, UC San Diego

Large-scale collections of induced pluripotent stem cells (iPSCs) could serve as powerful model systems for examining how genetic variation affects biology and disease. Here we describe the iPSCORE resource: a collection of systematically derived and characterized iPSC lines from 222 ethnically diverse individuals that allows for both familial and association-based genetic studies. Participants were recruited to include families, twins, and individuals of diverse ethnicity to enable genetic studies investigating the segregation of traits. Using high-throughput RNA-sequencing and genotyping arrays, we show that the iPSCORE lines are pluripotent with high genomic integrity (no or low numbers of somatic copy-number variants). We differentiated a subset of iPSCs from a family to cardiomyocytes and examined how the donor’s genetic background was associated with gene expression variation in derived cell lines. We show that iPSC-derived cardiomyocytes demonstrate gene expression patterns that cluster by genetic background, and can be used to examine variants associated with physiological and disease phenotypes. Finally, we examined and annotated how individuals in the iPSCORE resource carry SNPs associated with diverse genome-wide association studies (GWAS) phenotypes. The iPSCORE collection contains representative individuals for risk and non-risk alleles for 95% of SNPs associated with human phenotypes through genome-wide association studies. Our study demonstrates the utility of iPSCORE for examining how genetic variants influence molecular and physiological traits in iPSCs and derived cell lines.

Key Words: Stem cell(s), Cardiovascular system, Quantitative trait, Genomics, Genotype-phenotype correlations, Complex traits, Regulation of transcription, Copy number/structural variation, Differentiation, Statistical genetics

30. Subclonal Somatic Point Mutations in iPSCs are Enriched in Active Chromatin and Associated with Altered Gene Expression

Presented by: Matteo D’Antonio, UC San Diego
Authors: D’Antonio, Matteo, UC San Diego; Jakubosky, David, UC San Diego; Greenwald, William, UC San Diego; Donovan, Margaret, UC San Diego; DeBoever, Christopher, UC San Diego; Benaglio, Paola, UC San Diego; Matsui, Hiroko, UC San Diego

To examine the functional effects of somatic mutations in iPSCs we conducted whole genome sequencing of 18 skin fibroblast-derived iPSCs and their matched blood DNA. We detected 46,612 somatic single nucleotide variants (SNVs), 2,776 small indels and 563 copy number variants, ranging from 1 kb to 48 Mb. The number of SNVs and indels per sample is highly heterogeneous, ranging from 913 to 7,570 and is likely due to the presence of different numbers of somatic mutations in the cell of origin. Indeed, the eight samples with the highest number of mutations all harbor many CC>TT substitutions, which are known to be caused by UV damage. To investigate if CC>TT substitutions were under selection during reprogramming, we analyzed additional 255 blood and 19 skin fibroblast WGSs. These variants are strongly enriched in fibroblasts and iPSCs derived from fibroblasts, compared to blood, suggesting that the CC>TT substitution signature originates in skin fibroblasts. The fact that fibroblasts harbor more mutations than the iPSCs reprogrammed from fibroblasts indicates that fibroblasts with many CC>TT substitutions may be less likely to be successfully reprogrammed into iPSCs. To address functional differences between clonal (present in all cells in an iPSC line) and subclonal (present only in a fraction of the cells) SNVs, we investigated their associations with chromatin organization. By permuting the position of SNVs across the genome, we found that clonal SNVs are strongly enriched in repressive chromatin states and depleted in active chromatin. Compared with clonal SNVs, subclonal SNVs are enriched in active promoter regions (p = 0.0012, paired t-test) and are depleted in quiescent chromatin regions associated with ESCs, iPSCs and ES-derived cells (p = 0.0067). Using RNA-seq data, we also found that subclonal SNVs are more likely to be associated with aberrant expression of their neighboring genes than clonal SNVs (p = 2.2 x 10-17, Wald test). These results show that clonal variants, which are likely derived from the cell of origin, tend to occur outside of functional regions, while subclonal mutations, which occur during passaging after reprogramming, are more likely to be present in active chromatin regions and are more likely to have phenotypic effects. In summary, we demonstrated that using deep whole-genome sequencing, we can identify and classify somatic mutations based on their origins. We showed that subclonal mutations that occurred in vitro are more likely to be in active chromatin regions and result in stronger impacts on gene expression than somatic mutations that were already present in the tissue of origin of iPSCs. In general, our study suggests that, if both germline and iPSC genomes are sequenced, we can identify and predict the potential functional consequences of somatic mutations, which otherwise may confound the effects of inherited variants when using iPSCs as model systems for studying human genetic variation and disease.

Key Words: iPSCs, somatic mutations, whole genome sequencing, subclonal mutations

31. Fibro-Adipogenic Progenitors (FAPs) are Composed of Discrete Subpopulations with Distinct Temporal Dynamics, Specific Transcriptional Profiles and Biological Activities

Presented by: Usue Etxaniz, Sanford Burnham Prebys Medical Discovery Institute
Authors: Etxaniz, Usue, Sanford Burnham Prebys Medical Discovery Institute; Malecova, Barbora, Sanford Burnham Prebys Medical Discovery Institute; Gatto, Sole, Sanford Burnham Prebys Medical Discovery Institute; Passafaro, Magda, IRCCS Fondazione Santa Lucia; Giordani, Lorenzo, Institut Cochin, Université Paris-Descartes, CNRS; De Bardi, Marco, IRCCS Fondazione Santa Lucia; De Santa, Francesca, Institute of Cell Biology and Neurobiology, Sanford Burnham Prebys Medical Discovery Institute

Fibro-Adipogenic Progenitors (FAPs), multipotent mesenchymal cells located in the interstitium of muscle fibers, are currently defined by their anatomical position, expression of non-specific membrane-associated proteins, ability to adopt multiple lineages and to perform different biological activities in response to physiological or pathological stimuli. Gene expression analysis at the single cell level revealed the intrinsic heterogeneity of FAPs and uncovered discrete subpopulations (subFAPs), which can be prospectively isolated by FACS, based on the expression levels of Tie2 and Vcam1. Tie2-expressing (Tie2+) and Tie2/Vcam1 double negative (DN) subFAPs were present in unperturbed muscles and promptly expanded upon acute injury. By contrast, Vcam1-expressing subFAPs (Vcam1+) were undetectable in unperturbed muscles, but appeared “de novo” upon acute injury and expanded with a different kinetic compared to Tie2+ and DN FAPs. While Vcam1+ subFAPs were completely cleared upon resolution of muscle regeneration following acute injury, they persisted in chronically injured muscles from the mdx mouse model of Duchenne Muscular Dystrophy (DMD). “Nested” RNA-seq analysis of subFAPs revealed subpopulation-specific gene expression profiles that were dynamically regulated throughout the regeneration process, predicted specific biological activities and functional interactions with other cell types, and exhibited DMD-specific signatures. In particular, Vcam1+ subFAPs showed enrichment in fibrotic gene expression and myofibroblast markers, and their altered clearance in acutely injured muscles upon macrophage depletion was associated with fibrosis. These data reveal the individual contributions of distinct subFAPs to the physiological and pathological repair of skeletal muscles, thereby providing new insights into the pathogenesis of muscular dystrophies and new targets for selective therapeutic interventions.

Key Words: Fibro-Adipogenic Progenitors (FAPs), Single cell analysis, muscle regeneration, Duchenne Muscular Dystrophy

32. Identification of Genetically Associated Changes in 3D-chromatin Architecture by Leveraging Haplotype Information Across a Three-Generation Family

Presented by: William Greenwald, UC San Diego
Authors: Greenwald, William, UC San Diego; Li, He, UC San Diego; Benaglio, Paola, UC San Diego; Schmitt, Anthony, UC San Diego; Qiu, Yunjin, UC San Diego; Ren, Bing, UC San Diego; D’Antonio, Matteo, UC San Diego

Genetic variation that affects gene expression can occur in regulatory regions that are far from the gene’s locus, yet colocalize spatially through 3D-chromatin loops. However, it is unclear if this genetic variation acts by modulating regulatory region activity, or by inducing changes in the 3D-architechture of the genome itself. To study this question, we obtained paired iPSC and iPSC-derived cardiomyocyte (iPSC-CM) lines from 7 individuals in a 3-generation family with whole genome sequence data. We performed in-situ Hi-C and obtained a total of ~6 billion read pairs which we analyzed using Fit-Hi-C and HICCUPS to identify 17,567 and 19,003 significant loops in iPSCs and iPSC-CMs, respectively, 9,888 of which were common to both cell types. To identify loops associated with genetic variation, we performed allelic imbalance analyses with the HiC reads. To assign reads to either the maternal or paternal haplotype, we initially obtained long-range haplotypes from the HiC data; however, when we compared the resulting haplotypes to expected inheritance patterns, we observed a 1.8% point error rate. We thus devised a method which leveraged family structure, enabling us to resolve 93% of these point errors and resulting in 4.7M phased SNVs across the genome. To estimate allelic imbalance, we assigned reads within 25kb of a loop’s anchor that carried an allele of a phased heterozygous SNV to the corresponding maternal or paternal haplotype of the individual, performed a binomial test, and combined the individuals’ associations at each loop using Fisher’s combined p-value. In total, we identified 35 genetically associated chromatin loops with FDR < 0.05 across both cell types. Across all loops, 8 overlapped 2 somatic CNVs identified in these iPSCs, and for both CNVs we observed genetic effects with either a corresponding gain or loss, supporting that our method accurately detects genetically associated changes in chromatin loop formation. These data suggest the 3D-chromatin architecture of the genome may be largely invariant and only modestly affected by genetic variation; we are currently validating this observation by testing whether the genetic variants that are located in the distal end of a chromatin loop and are associated with allele-specific RNA expression at the genic end of the loop are also associated with corresponding differences in chromatin loop formation.

Key Words: Genetics, Genomics, Epigenetics, Chromatin Conformation, Hi-C, Gene Expression, Bioinformatics

33. Nanoparticle Delivery of Cas9 Ribonucleoprotein and Donor DNA In Vivo Induces Homologous Directed DNA Repair

Presented by: Kunwoo Lee, GenEdit
Authors: Lee, Kunwoo, GenEdit; Conbody, Mike, UC Berkeley; Park, Hyo Min, GenEdit; Murthy, Niren, UC Berkeley

CRISPR/Cas9-based therapeutics, especially those that can correct gene mutations via homology directed repair (HDR), have the potential to revolutionize the treatment of genetic diseases. However, HDR-based therapeutics are challenging to develop because they require simultaneous in vivo delivery of Cas9 protein, guide RNA and donor DNA. Here, we demonstrate that a delivery vehicle composed of gold nanoparticles conjugated to DNA and complexed with cationic endosomal disruptive polymers can deliver Cas9 ribonucleoprotein and donor DNA into a wide variety of cell types, and efficiently correct the DNA mutation that causes Duchenne muscular dystrophy in mice via local injection, with minimal off-target DNA damage.

Key Words: CRISPR, Cas9, Duchenne muscular dystrophy, HDR, Gene editing

34. Characterization of Bone Marrow Aspirate Concentrate (BMAC) Using Single-Cell RNA Sequencing

Presented by: Conner Paez, UC San Diego
Authors: Paez, Conner, UC San Diego School of Medicine; Walker, Todd, UC San Diego Department of Orthopaedic Surgery

Bone marrow aspirate concentrate (BMAC) has gained increasing popularity in recent years due to its promising clinical potential. As of 2016, there are at least 570 clinics in the United States offering “stem cell” treatments, with BMAC making up a significant portion of the services offered by orthopaedic surgeons. The therapeutic potential of BMAC is believed to be largely due to the potential presence of mesenchymal stem cells (MSCs). However, there is no accurate nor universal method of MSC identification or characterization. This is because most studies do not describe the process of obtaining and identifying MSCs, nor do they follow the minimal criteria set forth by the International Society for Cellular Therapy to identify MSCs. Both the actual concentration of MSCs in BMAC and an accurate description of BMAC itself are unknown. Single cell RNA sequencing has allowed the possibility to evaluate and identify specific cell populations based on gene expression profiles at the single cell level. This study will utilize single cell RNA sequencing to identify cells in patient BMAC expressing MSC genes. Bone marrow aspirate was obtained from the anterior superior iliac crest of patients receiving scheduled orthopaedic procedures. The aspirate was then concentrated using an industry device to yield approximately 2-5 ml of BMAC. The BMAC underwent single-cell suspension followed by single-cell RNA sequencing. A gene expression profile was generated of the individual cells in each patient’s BMAC sample. By comparing MSC gene expression profiles in the literature and choosing the most commonly present genes, a list of the top 12 MSC genes was composed. This list consisted of the genes COL1A1, COL1A2, SPARC, TGFβ, EEF1A1, TUBA1B, FTH1, VIM, FN1, CTGF, ACTG1, and TAGLN. Cells closely resembling MSCs were identified based on expression of these genes. Patient sample 1 had a cell count of 670. One cell expressed all 12/12 of the top MSC genes. 3 cells expressed 11/12 MSC genes. 3 cells expressed 10/12. One cell expressed 9/12 and one cell expressed 8/12. There were two other cells that expressed 9/12 genes, however, the log twofold change of the maximum gene expression was very low. In total, 9 of 670 (1.34%) cells analyzed from patient BMAC expressed MSC genes at a high level. Patient sample 2 had a cell count of 1,178. 1 cell expressed 10/12 MSC top genes. 1 cell expressed 9/10. And one cell expressed 8/10. In total, 3 out of 1,178 (0.25%) cells analyzed expressed MSC genes at a high level. This data suggests that a small subset of cells in bone marrow aspirate concentrate present with genes similar in composition to MSC when compared to the gene expression profile of MSC in the literature. This is the first time that single-cell RNA sequencing has been used for this purpose.

Key Words: Mesenchymal stem cells, orthopaedics, bone marrow aspirate concentrate, single-cell RNA sequencing

35. Profound TET Loss-of-function Results in Genome Instability and Oncogenesis

Presented by: Isaac Lopez, La Jolla Institute for Allergy and Immunology
Authors: Lopez, Isaac, La Jolla Institute for Allergy and Immunology; Tsagaratou, Ageliki, La Jolla Institute for Allergy and Immunology; Delatte, Benjamin, La Jolla Institute for Allergy and Immunology; Georges, Romain, La Jolla Institute for Allergy and Immunology; González-Avalos, Edahí, La Jolla Institute for Allergy and Immunology; Heinz, Sven, UC San Diego; Benner, Christopher, UC San DiegoLa Jolla Institute for Allergy and Immunology

TET (Ten-eleven-translocation) enzymes are a family of 2-oxoglutarate- and Fe (II)-dependent dioxygenases that oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidation products (oxi-mCs) in DNA. They act as epigenetic regulators, involved in DNA demethylation and in the creation of new epigenetic marks. TET loss-of-function is strongly associated with cancer. TET2 mutations are frequently observed in myelodysplastic syndromes and myeloid leukaemias, as well as T cell lymphomas. Additionally, low 5hmC levels attributable to profound TET loss-of-function have been observed in numerous cancers, leading us to examine the consequences of profound TET loss-of-function by deleting both Tet2 and Tet3. Mice with conditional disruption of both Tet2 and Tet3 genes in the T cell lineage during thymic development (here termed Tet2/3 DKO) showed a rapid, cell-autonomous and antigen-driven expansion of invariant NKT (iNKT) cells, a minor subpopulation of T cells that recognize lipid antigens presented on the nonclassical major histocompatibility protein CD1d. The resulting iNKT cell leukaemia was transferable to fully immunocompetent mice; moreover as the expansion progressed, the abundance of a particular clone increased, such that Tet2/3 DKO iNKT cells expanded in recipient mice were practically monoclonal. Whole-genome sequencing revealed structural variations, copy number aberrations, and particularly recurrent aneuploidies in expanded Tet2/3 DKO iNKT cells, concomitantly with increased accumulation of DNA double-strand breaks. Our results outline a role for TET proteins in safeguarding genome integrity, maintaining normal ploidy and preventing oncogenesis.