Poster Session

Scientific Poster Abstracts

Every year, the Cell & Gene Meeting on the Mesa invites the submission of abstracts that report new research developments in the areas of cell biology, stem cell medicine, regenerative medicine or ethical and regulatory issues. The posters below were selected to be on display throughout the 2016 Scientific Symposium.

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2016 Poster Abstracts

1. Stromal Cell-Derived Extracellular Matrix Stimulates Mesenchymal Stem Cell Proliferation In Vitro and Bone Formation in Rat Femur Segmental Defects

Presented by: Sy Griffey, StemBioSys
Authors: Zamilpa, Rogelio, StemBioSys, Inc.; Pearson, Joseph, The University of Texas at San Antonio; Jennings, Sarah, The University of Texas at San Antonio; Montelongo, Sergio, The University of Texas at San Antonio; Navarro, Mary, StemBioSys, Inc.; Lemus, Karina, StemBioSys, Inc.; Navara, Chris, The University of Texas at San Antonio; Griffey, Sy, StemBioSys, Inc.; Guda, Teja, The University of Texas at San Antonio

Bone marrow mesenchymal stem cells (BM-MSCs) are a common treatment of non-union fractures due to their ability to immunomodulate the microenvironment as well as enhancing osteogenesis. One of the issues associated with the use of BM-MSCs for therapeutic treatments is the inherent variability found in patients which most often results in decreased cell yields for clinically relevant doses. To improve the cell yield of BM-MSCs, a cell culture substratum referred to as High Performance Micro Environment (HPME) was generated using bone marrow stromal cells. Mass spectrometry analysis indicated that the HPME is primarily composed of extracellular matrix proteins such as collagen types I, VI, XII, fibronectin, tenascin and transforming growth factor beta inducible protein. When used as a cell expansion substrate, the HPME significantly increased the yield of BM-MSCs, the expression of IL-10, and the expression of stage specific embryonic antigen (SSEA)-4 when compared to tissue culture plastic (all p<0.05). When used as a supplement in cell culture medium, an HPME protein extract significantly increased the BM-MSC yield in a dose dependent manner with a concomitant increase in the expression of SSEA-4 (41±6% difference compared to non-supplemented cultures). Interestingly, in combination with hydroxyapatite minigranules and bone marrow, the HPME protein extract stimulated bone regeneration in rat femur segmental defects when compared to empty defects or minigranules combined with bone marrow. These results demonstrate that the HPME increases the proliferation of BM-MSCs in vitro and stimulates bone formation in vivo.

Key Words: MSC, extracellular matrix, proliferation, SSEA-4, qRT-PCR, HA-TCP, IL-10, bone healing

2. Use of Non-modified RNAs for the Derivation of Clinically Relevant iPS Cell Lines From Human Blood, Urine and Skin Cells Using GMP-compliant Reagents

Presented by: Trevor Perry, Stemgent, a ReproCell Group Company
Authors: Eminli-Meissner, Sarah, Stemgent a ReproCell Group Company; Yi, Kevin, Stemgent a ReproCell Group Company; Moon, Jung-Il, Stemgent a ReproCell Group Company; Poleganov, Marco, BioNTech RNA Pharmaceuticals; Beissert, Tim, TRON; Sahin, Ugur, BioNTech RNA Pharmaceuticals; Rana, Amer, University of Cambridge; Marjani, Mazy, Stemgent a ReproCell Group Company; Hamilton, Brad, Stemgent a ReproCell Group Company

Human fibroblasts can be reprogrammed with a cocktail of mRNAs into integration-free human induced pluripotent (iPS) cells. Human blood provides easy access to adult human cell types for reprogramming purposes. Notably, blood-outgrowth endothelial progenitor cells (EPCs) can be clonally isolated from only 10 mL of fresh or frozen mononuclear cell (MNC) preparations from both human peripheral and cord blood. The adherent nature and high proliferative capacity of EPCs makes them highly desirable for repeated transfection with RNA when compared to commonly isolated hematopoietic suspension cell types. In 2015 we published the unique application of non-modified RNA technology to the reprogramming of human blood-derived EPCs and adult fibroblasts. Since then we could extend this novel reprogramming technology to urine-derived epithelial cells (UDCs) which can be highly reproducibly isolated from only 30 mL of urine (the most non-invasive form of cell procurement). Here we present a flexible, yet powerful, RNA-based reprogramming method that combines a novel cocktail of synthetic, non-modified reprogramming [OCT4, SOX2, KLF4, cMYC, NANOG and LIN28 (OSKMNL)] and immune evasion mRNAs [E3, K3, B18-R] with reprogramming-enhancing mature, double-stranded microRNAs from the 302/367 cluster. This unique combination of different RNAs results in a highly efficient (up to 4%) and robust reprogramming protocol using only GMP-compliant substrates (iMatrix-511 and vitronectin), media compositions (xeno-free or human serum), and RNA to produce clinically relevant iPS cells from blood-derived EPCs, neonatal as well as adult fibroblasts, and for the first time from cells derived from human urine.

Key Words: RNA Reprogramming, clinical-grade human iPSC, pluripotency, GMP compatible and xeno-free reagents, urine, skin, blood, unique non-modified RNA technology

3. Implantation of an Induced Pluripotent Stem Cell Derived Cardiomyocyte Tissue Engineered Graft Improves Left Ventricular Function and Electro-Mechanical Coupling in Rats with Heart Failure

Presented by: Jordan Lancaster, University of Arizona
Authors: Lancaster, Jordan, University of Arizona; Koevary, Jennifer, University of Arizona; Goldman, Steven, University of Arizona

Chronic Heart Failure (CHF) is the leading cause of hospital readmissions and mortality in the US. Here we report the effects of surgically delivering a human bioengineered graft of human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) and neonatal dermal fibroblasts (hNDF) on left ventricular (LV) function in rats with CHF. We evaluate improvements in LV systolic and diastolic function, electromechanical coupling and gene expression after graft implantation. Methods: Adult male Sprague-Dawley rats underwent left coronary artery ligation and were randomized to Sham (N=8), CHF (N=8-21), and CHF+hiPSC-CM graft (N=20-24). Heterogeneous hiPSC-CMs were seeded and co-cultured onto a Vicryl matrix embedded with hNDF. Echocardiography was performed at 3 and 6 weeks post-randomization. Hemodynamic pressure measurements were performed at 6 weeks post-ligation with Millar solid state micromanometer pressure catheters. Open chest Electrophysiologic (EP) mapping was performed at 6 weeks post ligation. Gene expression was evaluated through qRT-PCR. Results: 48 hours into culture hiPSC-CMs grafts displayed synchronized and spontaneous contractions which developed in robustness over time. At maximal robustness, contractions were visualized across the full thickness of the construct. Contractions were recorded at 36+5 beats BPM. Three weeks after graft implantation (6 weeks post ligation) the hiPSC-CM graft decreased (P<0.05) LV EDP (45%), Tau (29%), E/e’ (23%) and increased (P<0.05), e’/a’ (36%) with trending improvements in EF (14%) and e’ (20%). EP studies show electro-mechanical coupling between the graft and the native myocardium with normal activation through the graft and increases (P<0.05) voltage amplitude in CHF versus hiPSC-CM graft treated rats (1±0.5 mV vs 6±1.5mV). Rats treated with the hiPSC-CM graft showed significant (P<0.05) fold expression of Cx43 (3.3), ANG-1 (13.63), VEGF (3.8), βMYH7 (6.4) and IGF-1 (22.9) versus control. Conclusion: Cardiac graft implantation with hiPSC-CMs is an effective and feasible method of treating CHF with improvements in systolic function, diastolic function, and electro-mechanical coupling in rats with CHF.

Key Words: iPSC-Cardiomyocytes, Tissue Engineering, Graft, Heart Failure, LV Function

4. Evaluation of a Novel Human Platelet Lysate Formulation Compared to Fetal Bovine Serum for Human Cord Blood and Tissue-Derived CD34+ and Mesenchymal Stem Cell Culture

Presented by: Robert Tressler, San Diego Blood Bank
Authors: Tressler, Robert, San Diego Blood Bank; Monte, Nicholas, San Diego Blood Bank; Galel, David, San Diego Blood Bank; Barlow, James, San Diego Blood Bank; Davey, Will, San Diego Blood Bank

There has been rapid growth in the field of stem cell therapeutics for immune and regenerative medicine applications utilizing umbilical cord and bone marrow-derived mesenchymal (MSCs) and CD34+ stem cells. In vitro expansion of cells for therapies by the use of fetal bovine serum (FBS) has raised safety concerns related to immunogenicity and zoonotic risk. Xeno-free media preparations that give high cell yields, viability and retention of desired cell phenotypes for human cell therapies are needed to support the development of these new treatments for diseases. Various formulations of human platelet lysate (hPL) were evaluated as a media supplement for comparison with FBS and to develop an optimized hPL for cell culture. Cell-based assays utilizing the optimized hPL formulation were carried out to characterize the proliferation and viability of MSCs and CD34+ cells isolated from fresh or frozen cord blood and tissue. Parameters evaluated included doubling time, extended expansion time, morphology, CFU and FACs analysis. Comparisons were made with FBS at concentrations ranging from 1-10% in media. Growth assays were conducted for culture durations of up to 168 hours to assess effects on media refeeding of cultures as well. When MSCs and CD34+ cells were cultured in the hPL formulation and compared to cultures in FBS, the hPL supplement demonstrated superiority for proliferative capacity and culture maintenance over time with excellent viability compared to FBS (>200% proliferation at 96-144 hrs post cell seeding with cord blood MSCs). Dose-ranging potency comparisons to FBS with the hPL supplement using MSCs showed shorter doubling times compared to FBS at concentrations of 2%, 5% and 10%. Manufacturing lot to lot variability of the hPL supplement was also evaluated on various cell types and demonstrated excellent consistency in terms of cell growth potency. These data demonstrate that hPL is a viable alternative to FBS and can be produced in a consistent cGMP-compliant manner for in vitro expansion of cord-derived CD34+ cells and MSCs.

Key Words: Human platelet lysate Cell culture Mesenchymal stem cells CD34 stem cells Cord blood Cord tissue

5. Delivery Highways: Tunneling Nanotubes Facilitate Transfer of Therapeutic Molecules for Gene Therapy Treatment of Cystinosis

Presented by: Spencer Goodman, UC San Diego
Authors: Goodman, Spencer, UC San Diego; Chua, Emily, UC San Diego; Naphade, Swati, UC San Diego; Sharma, Jay, UC San Diego; Chevronnay, Héloïse, Université Catholique de Louvain; Courtoy, Pierre, Université Catholique de Louvain; Cherqui, Stephanie, UC San Diego

Cystinosis is a lysosomal storage disorder caused by mutations in the CTNS gene, encoding the lysosomal transmembrane transporter cystinosin. As a consequence, cystine builds up in all tissues and eventually causes multi-organ degeneration, especially affecting the kidney and eye. We previously showed in Ctns-/- mice that transplantation of ex vivo lentiviral-transduced hematopoietic stem and progenitor cells (HSPCs) resulted in abundant integration of bone marrow-derived cells into all tissues and long-term kidney and eye preservation. We observed that HSPCs differentiated into macrophages that extended intercellular bridges called tunneling nanotubes (TNTs). TNTs are long actin-rich structures that provide a dynamic cytosol-cytosol connection enabling cellular communication as well as transfer of protein and organelles. In vitro co-culture of DsRed Ctns-/- fibroblasts with macrophages expressing cystinosin-GFP allowed visualization of bidirectional lysosome transfer through these TNTs. In vivo, we visualized TNTs in multiple tissues in lentiviral-modified HSPC-transplanted Ctns-/- mice. We also observed the transfer of cystinosin-bearing lysosomes from macrophages into diseased host cells such as proximal tubular cells in the kidney and keratocytes in the eye. These findings represent the first observed instance of TNTs delivering a functional protein to facilitate tissue regeneration following gene therapy. Utilizing both genomic and proteomic approaches, putative novel constituents of TNTs were discovered and subsequently validated via RNAi. We also designed an application using ImagePro to automatically quantify and measure TNTs in vitro via morphological filters. With our ImagePro workflow and FACS-assisted immunoblotting, contributions of candidate proteins to TNT frequency, morphology and transfer efficiency were assessed. We further investigate the molecular nature of the TNTs and established new important protein candidates. In conclusion, HSPC transplantation can preserve near-normal kidney and eye architecture in cystinosis via TNT-based transfer of functional protein. Understanding the mechanisms underlying TNT repair could spur the development of novel stem cell-based gene therapies for numerous genetic disorders.

Key Words: tunneling nanotubes, cystinosis, regenerative medicine, gene therapy, hematopoietic stem cells, lysosomal transfer, macrophages, tissue repair

6. Towards a Phase I Clinical Trial for Autologous Hematopoietic Stem Cells Transplantation in Cystinosis

Presented by: Tatiana Lobry, UC San Diego
Authors: Lobry, Tatiana, UC San Diego; Sharma, Jay, UC San Diego; Ur, Sarah, UC San Diego; Lau, Athena, UC San Diego; Rocca, Celine, UC San Diego; Kohn, Donald B, University of California, Los Angeles; Carbonaro, Denise, University of California, Los Angeles; Cabrera, Betty, UC San Diego; Hernandez, Laura, UC San Diego; Cherqui, Stephanie, UC San Diego

Cystinosis is a metabolic hereditary disease characterized by intracellular accumulation of cystine in every organ, which leads to multi-organ dysfunction. Affected individuals typically present with proximal tubulopathy before one year of age and eventually progress to end-stage renal failure. The current treatment is the drug cysteamine which reduces the intracellular cystine content. However, cysteamine does not prevent the proximal tubulopathy nor the end-stage renal failure and only delay the progression of the disease. The long-term objective of this project is to develop a new treatment for cystinosis by autologous transplantation of Hematopoietic Stem and Progenitor Cells (HSPC) genetically modified ex vivo to express a functional CTNS gene. Using the mouse model for cystinosis, the Ctns-/- mice, we showed that transplantation of syngeneic Sca1+ HSPC expressing Ctns resulted in abundant bone marrow-derived cell engraftment and significant reductions of cystine content in all the tissues tested. This treatment also prevented the progression of kidney dysfunction. We obtained the same results with ex vivo transduced HSPC using our lentiviral vector construct, pCCL-CTNS. We submitted a pre-Investigator New Drug (IND) application in 2013 and we are now working on the pharmacology/toxicology studies required to obtain an IND for a phase I clinical trial for cystinosis. The in vitro studies first require optimization of the transduction of human CD34+ cells using GMPc pCCL-CTNS virus preparation to obtain a VCN in the range of 1-3. The FDA requires transduction of CD34+ cells from 3 healthy donors and 3 patients affected with cystinosis. So far, the Colony Forming Unit assays did not show aberrant differentiation of the transduced CD34+ cells from the healthy donors and cystinotic patients. Finally, no immortalized clones were observed with the IVIM assays suggesting a good safety profile of our vector. The in vivo studies consist in a serial transplantation in Ctns-/- mice. So far, eleven primary and eight secondary Ctns-/- mouse transplanted with pCCL-CTNS-transduced Ctns-/- HSPCs have reached the end of the study. The primary mice had a mean VCN of 1.714 and the secondary 2.04. Clinical evaluations, histopathology and Vector Integration Site (VIS) analyses revealed no adverse event so far suggesting a good safety profile of our product. Moreover, cystine content was significantly decreased in all tissues tested. Analysis of the remaining primary and secondary recipient mice is in progress.

Key Words: Hematopoietic Stem and Progenitor Cells Autologous transplantation Cystinosis Lentivirus vector Toxicology studies

7. The Effects of Composition and Processing on the Immunomodulatory and Stem Cell Regulatory Properties of Amniotic Membranes

Presented by: Katie Mowry, NuTech Medical
Authors: Mcquilling, JP, NuTech Medical; Vines, Jeremy, NuTech Medical; Mowry, Katie, NuTech Medical

Currently, there are a variety of processing methodologies for amniotic derived products including: dehydration, cryopreservation and hypothermic storage. In general, these tissues are thought to be of value for their capacity for promoting regeneration, angiogenesis, and down-regulating chronic inflammation. The purpose of this study was to compare the effects of these different processing techniques on the immunomodulatory properties of the amniotic tissue and its effects on MSC responses. To evaluate the immunomodulatory properties of amniotic grafts dehydrated amnion chorion (dHACM), cryopreserved amniotic membrane (CHAM), and hypothermically stored amniotic membrane (HSAM) were cultured with and without inflammatory cytokines for 72 hours. Following this incubation, cytokine content including TGFβ-1, TGFβ-3, and interleukin 10 (IL-10), and Macrophage Stimulating Protein alpha (MSPα) were evaluated. Additionally, HSAM and dHACM were evaluated for their effects on MSC cell migration and proliferation. MSCs were incubated for 24 hours in the migration chamber inserts with conditioned media loaded in the receiving chamber. Following 24 hours, cells which had migrated thought the membrane were stained and counted. MSC proliferation assays were conducted by co-culturing MSCs with or without HSAM or dHACM conditioned media. At 14 days of culture, Alamar Blue assays were used to determine cell proliferation. We observed a significant 41±17% increase in TGFβ-1 in HSAM grafts when stimulated with inflammatory cytokines compared to non-stimulated HSAM grafts (n=6, p<0.01) however we did not observe a significant change in TGFβ-1 in either dHACM or cHAM grafts. TGFβ-3 was significantly higher for HSAM grafts when stimulated with inflammatory cytokines (996±314% increase over control, n=6, p<0.01), where no differences were observed with either dHACM or CHAM grafts. IL-10 was significantly increased in response to inflammatory cytokines in both HSAM and CHAM grafts (74±50% and 49±37 increase over controls respectively, n=6 p<0.01) and no changes were observed with dHACM. MSPα was increased under inflammatory conditions with HSAM (170±52% increase over control, n=6, p<0.01). MSC migration was significantly increased in the presence of HSAM conditioned media (18±0.08% increase, n=12, p<0.001) however no significant difference in migration was observed with dHACM conditioned media. MSC proliferation at 14 days was also significantly increased over control with dHACM conditioned media (42±8%,n=6, p>0.001) but not with HSAM conditioned media. The results of this study demonstrate the impact composition and processing techniques for these tissues have on the tissue’s ability to respond to inflammatory conditions and to promote proliferation and migration of important cell types (MSCs). We hypothesize differences observed may be in part due to the presence or absence of the chorion, viable cells, and integrity of the extracellular matrix (ECM).

Key Words: Amnion, Chorion, Regenerative Medicine, Wound Healing, Mesenchymal Cells, Immunemodulation, Anti-Inflammatory

8. Novel Mechanisms of Non-Coding Genomic Regulation Identified in Cardiac Disease-in-a-dish Models

Presented by: Kirsten Wong, UC San Diego
Authors: Wong, Kirsten, UC San Diego; Thomas, Stephanie, UC San Diego; Kumar, Aditya, UC San Diego; Tenerelli, Kevin, UC San Diego; Lo Sardo, Valentina, The Scripps Research Institute; Ferguson, William, The Scripps Research Institute; Topol, Eric, The Scripps Research Institute; Baldwin, Kristin, The Scripps Research Institute; Engler, Adam, UC San Diego

Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) at gene loci that affect cardiovascular function, and while mechanisms in protein-coding loci are obvious, those in non-coding loci are difficult to determine. 9p21 is a recently identified locus associated with increased risk of coronary artery disease (CAD) and myocardial infarction. Associations have implicated SNPs in altering smooth muscle and endothelial cell properties but have not identified adverse effects in cardiomyocytes (CMs) despite enhanced disease risk. Using induced pluripotent stem cell-derived CMs from patients that are homozygous risk/risk (R/R) and non-risk/non-risk (N/N) for 9p21 SNPs and either CAD positive or negative, we assessed CM function when cultured on hydrogels capable of mimicking the fibrotic stiffening associated with disease post-heart attack, i.e. “heart attack-in-a-dish” stiffening from 11 kiloPascals (kPa) to 50 kPa. While all CMs independent of genotype and disease beat synchronously on soft matrices, R/R CMs cultured on dynamically stiffened hydrogels exhibited asynchronous contractions and had significantly lower correlation coefficients versus N/N CMs in the same conditions. Dynamic stiffening reduced connexin 43 expression and gap junction assembly in R/R CMs but not N/N CMs. To eliminate patient-to-patient variability, we created an isogenic line by deleting the 9p21 gene locus from a R/R patient using TALEN-mediated gene editing, i.e. R/R KO. Deletion of the 9p21 locus restored synchronous contractility and organized connexin 43 junctions. As a non-coding locus, 9p21 appears to repress connexin transcription, leading to the phenotypes we observe, but only when the niche is stiffened as in disease. These data are the first to demonstrate that disease-specific niche remodeling, e.g. a “heart attack-in-a-dish” model, can differentially affect CM function depending on SNPs within a non-coding locus.

Key Words: “heart attack-in-a-dish”, disease modeling, 9p21, induced pluripotent stem cell-derived cardiomyocytes, dynamic stiffening

9. SENP1 and Polycythemia: iPS-derived Human Experiment in Nature at High Altitude

Presented by: Priti Azad, UC San Diego
Authors: Azad, Priti, UC San Diego; Haddad, Gabriel, UC San Diego

Since polycythemia is a predominant trait in some high altitude dwellers (Chronic Mountain Sickness, CMS, or Monge’s disease) but not others living at the same altitude in the Andes, we took advantage of this human “experiment in nature” and studied both populations (with CMS and without, non-CMS). Although polycythemia could be advantageous at high altitude because it increases O2-carrying capacity, this adaptive pattern to high altitude has deleterious effects since blood increases its viscosity and induces serious morbidities, such as myocardial infarction and stroke in young adults. Methods: In order to understand the molecular basis for polycythemia of high altitude, we generated a disease in-the dish-model by re-programming fibroblasts from CMS and non-CMS subjects. Results: As compared to sea level controls and non-CMS subjects who responded to hypoxia by increasing their RBCs modestly or not at all, CMS cells increased theirs remarkably (up to 60 fold) with a dose-dependent response to graded hypoxia (1.5, 5, 10% O2). We knocked down SENP1 (a desumoylase) in CMS iPS cells using lentiviral constructs and observed a striking reduction (>90%) of the CMS excessive erythropoietic response to low O2. And, by over-expressing SENP1 in non-CMS iPS cells, the hypoxic response in these subjects increased enormously. By further analyzing RBC differentiation in hypoxia, we found that VEGF, GATA1 and Bcl-xL increased their gene expression in the CMS erythroid cells, in contrast to their minimal expression in the other two populations. We demonstrate also that, by utilizing a SUMO-GATA1 fused construct, GATA1 desumoylation, a target of SENP1, is critical for the CMS phenotype. Unlike GATA1, the over-expression of the anti-apoptotic gene Bcl-xL (a GATA1 target), only partially rescued the blunted erythroid response to hypoxia in non-CMS cells. Conclusion: We conclude that the increased erythropoietic sensitivity to hypoxia in CMS subjects is genetic in nature and depends on an increase in SENP1 expression and its desumoylation mediated activation of GATA1 under hypoxia. By combining the iPS technology with this unique Andean population that has adapted (or mal-adaptated) to chronic hypoxia over thousands of years, we have built an important in-vitro model. We believe that utility of this model lies not only for studying hypoxia-induced polycythemia but also other hypoxia-driven diseases experienced at sea level.

Key Words: ips-derived polycythemia, genetics, molecular mechanism(s)

10. Methods for Consistent and Efficient Generation of iPSC from Blood-Derived Cells

Presented by: Chad MacArthur, Thermo Fisher Scientific
Authors: Azad, Priti, UC San Diego; Haddad, Gabriel, UC San Diego

Induced pluripotent stem cells (iPSC) hold the potential to create patient-derived cells that may be used in a number of different applications including disease modeling, drug screening, and personalized medicine. iPSC can be derived from a wide variety of different cell types, including skin fibroblasts and blood-derived cells. While fibroblasts collected by skin punch biopsy are a common source of cells used to create iPSC, this collection process is more inconvenient to the patient than a standard blood draw. Peripheral blood contains a variety of different cells types which have been shown to be able to be reprogrammed, including CD34+ hematopoietic stem cells, erythroblasts, and T-cells. In order to develop optimized protocols that would achieve consistent, high efficiency reprogramming of different cell types isolated from blood, we tested different methodologies within the reprogramming workflow. Different isolation and culture conditions of blood-derived cells, as well as different media and matrices used with reprogrammed cells each influenced the overall efficiency of reprogramming. Results indicated that PBMC reprogramming was enhanced by using freshly isolated – as opposed to cryopreserved – cells. Additionally, use of Laminin-521 matrix also greatly enhanced the reprogramming efficiency of both PBMCs and T-cells. The relative convenience of collection and the high number of reprogrammable cells make blood a viable source of cells to generate patient-derived iPSC.

Key Words: iPSC, reprogramming

11. Factors that Enhance Sendai Virus Mediated Somatic Reprogramming

Presented by: Aryan Zarrabi, Thermo Fisher Scientific
Authors: Zarrabi, Aryan, Thermo Fisher Scientific; Avagyan, Samvel, Rensselaer Polytechnic Institute; MacArthur, Chad, Thermo Fisher Scientific; Lakshmipathy, Uma, Thermo Fisher Scientific

During the last ten years induced pluripotent stem cells (iPSC) have gone from a novel idea in the laboratory of Shinya Yamanaka to a wide-spread research tool used in numerous fields. Improvements in iPSC reprogramming technologies have led to the generation of patient-specific stem cells, which has fueled hope for patient specific treatments in the future. As clinical applications become closer to reality, there will be an increased demand for iPSC generated from diseased cells and from various somatic cell sources. One of the difficulties of patient-derived iPSC is that of potential intrinsic resistance to reprogramming. Reprogramming enhancers that could be used as supplementary factors in defined xeno-free media systems would be critical for consistent and efficient generation of clinical-grade iPSC. Sendai viruses are non-integrating RNA viruses that enable a safe, efficient, and consistent way to generate iPSC from a wide variety of cell types. Using sendai virus mediated reprogramming, we examined the effects of small molecule epigenetic modulators, pathway inhibitors, and hypoxia mimics for enhancement of human fibroblast reprogramming efficiency. Additionally, various matrices were evaluated to see if any one matrix provided an increase in reprogramming efficiencies. Our research showed that HDAC inhibitors had consistent positive effects on reprogramming efficiencies. We also found that cells replated on the matrix laminin 521 showed greater reprogramming rates than those grown on Geltrex or vitronectin. While hypoxic conditions also had a positive effect on reprogramming efficiencies, the results varied across different stem cell media and matrices. Using one or multiple enhancers in conjunction would be instrumental in enabling a clinical-grade workflow.

Key Words: Stem Cell, induced Pluripotent Stem Cells, Clinical grade stem cells, Enhancers, Reprogramming, Sendai Virus, Xeno-free

12. Design of Articular Cartilage Implant to Promote Mesenchymal Stem Cell Chondrogenesis Following Marrow Stimulation to Improve Articular Cartilage Repair Outcomes

Presented by: Michael Sinclair, Osiris Therapeutics
Authors: Geraghty, Sandra, Osiris Therapeutics, Inc.; Kuang, Jin-Qiang, Osiris Therapeutics, Inc.; Danilkovitch, Alla, Osiris Therapeutics, Inc.

Marrow stimulation is the most common procedure for treatment of articular cartilage injuries. Following marrow stimulation, mesenchymal stem cells (MSCs) are released from bone marrow into an articular cartilage lesion. In concept, these MSCs should differentiate into functional chondrocytes and synthesize type II collagen required for repair. However, instead of chondrocyte differentiation, the MSCs often become fibroblasts resulting in the production of type I collagen and the formation of fibrocartilage, the biomechanical properties of which lead to treatment failure over time. Better outcomes following marrow stimulation are achieved in small lesions where the MSCs are in closer proximity to surrounding healthy cartilage. This suggests that MSC chodrogenesis in the lesion is driven by signals secreted from surrounding cartilage tissue. We designed a human porated cartilage disc (hPCD) that can be introduced into the lesion following marrow stimulation. MSCs released from bone marrow will be trapped in the pores of the hPCD, residing in close proximity to cartilage tissue required to induce MSC chondrogenesis. To prove this hypothesis, we utilized in vitro and in vivo models. For an in vitro model, pores of the hPCD were filled with 4×106 MSCs suspended in 100 µl of fibrin glue and cultured for 4 weeks. After 4 weeks, MSC chondrogenesis was evaluated by staining the samples for type II collagen. This experiment was repeated using a devitalized version of the hPCD to evaluate the importance of viable chondrocytes within the hPCD. Results showed that 70% of MSCs underwent chondrogenesis and produced type II collagen within the pores of the hPCD. In comparison, only 41% of MSCs underwent chondrogenesis within the pores of the devitalized hPCD. In an in vivo model, 9 Spanish goats each received two critical size cartilage defects (6 mm diameter) in the medial femoral condyle of the right anterior knee joint. Defects were treated with marrow stimulation alone or combined with hPCD implantation. Goats with the hPCD implant exhibited improved gross morphology (quantified by normalized grade scores) and histology (quantified by Pineda scores) compared to goats treated with marrow stimulation alone at 3 and 12 months. Histological evaluation showed full integration of the hPCD implant with host cartilage and the restoration of hyaline cartilage across the lesion by 12 months. These experiments demonstrate that the hPCD supports MSC chondrogenesis and that viable chondrocytes within the hPCD have a significant contribution. These results led to the development of Cartiform, a viable commercial tissue allograft product that can be used in conjunction with marrow stimulation for treatment of articular cartilage injuries. Initial case studies demonstrate improved clinical outcomes and prospective clinical trials are underway.

Key Words: mesenchymal stem cells, chondrogenesis, cartilage injury, marrow stimulation, articular cartilage implant

13. Use Of Patient-Derived Long Qt Syndrome Type 3 iPSC Cardiomyocytes To Develop New Anti-Arrhythmic Therapeutics

Presented by: Wesley McKeithan, Sanford Burnham Prebys Medical Discovery Institute
Authors: McKeithan, Wesley, Sanford Burnham Prebys Medical Discovery Institute; Ryan, Daniel , Human BioMolecular Research Institute; Okolotowicz, Karl, Human BioMolecular Research Institute; Gomez-Galeno, Jorge, Human BioMolecular Research Institute; Johnson, Mark, Human BioMolecular Research Institute; Savtchenko, Alex, Stanford University; Kass, Robert, Columbia University; Mercola, Mark, Stanford University; Cashman, John, Human BioMolecular Research Institute

Long QT syndrome type 3 (LQTS3) is a congenital channelopathy resulting from a gain-of-function mutation in the cardiac specific sodium ion channel, Nav1.5, and is treated with the class 1b antiarrhythmic drug, Mexiletine. At a dose close to the therapeutic window of Mexiletine, the compound inhibits the rapid repolarizing potassium current (IKr) which is antagonizes the therapeutic antiarrhythmic effect of Mexiletine as well as causing drug induced long QT in some patents. Thus, a safer version of the drug is needed. An iPSC disease-in-a-dish model of congenital LQTS3 was developed and used to guide the chemical optimization of Mexiletine. Using a Kinetic Imaging Cytometer (Vala Sciences) and a novel small molecule fluorescent voltage sensitive probe, we acquired and analyzed iPSC-derived cardiomyocyte action potentials and calculated physiological parameters using Cyteseer software. In iterative cycles of chemical synthesis and testing, 123 Mexiletine analogs were evaluated in both normal donor and LQTS3 patient iPSC-cardiomyocytes for the therapeutic modulation of action potential duration (APD) and pro-arrhythmic induction of early after depolarizations (EADs). A structure-activity relationship (SAR) was developed for the desired APD shortening in LQT3 cardiomyocytes and the undesired APD prolongation and EADs in normal cells. Electrophysiology in both heterologous expression systems as well as the LQTS3 patient derived cardiomyocytes confirmed the increased potency and selectivity against InaL as compared with the parent molecule. The study has yielded 4 new potent and selective inhibitors of the late sodium current for in vivo testing as a therapeutic candidate for LQTS3 and other indications.

Key Words: human induced pluripotent stem cells, cardiomyocytes, long QT syndrome, drug rescue

14. Cell Therapy – TRAcking, Circulation and Safety (CT-TRACS): The Health and Environmental Sciences Institute (HESI)’s New Collaborative Effort to Address the Challenges of Cell Therapies Translation

Presented by: Charles O’Hanlon, The Health and Environmental Sciences Institute
Authors: O’Hanlon, Charles, Celsense Inc., Pittsburgh, PA; Helfer, Brooke, Celsense Inc., Pittsburgh, PA; Murray, Patricia, University of Liverpool, UK; Yamamoto, Keiji, Takeda Pharmaceutical Company Limited, Japan; van der Laan, Jan Willem, Medicines Evaluation Board, The Netherlands; Mullen, Gregory, King’s College London, UK; Shingleton, William, GE Healthcare, UK; Pereira Mouriès, Lucilia, Health & Environmental Sciences Institute (HESI)

Cell therapies show great therapeutic promise in the fields of regenerative medicine and immunotherapy. To realize their full clinical potential there is a need for greater understanding of their mode of action, how they migrate after administration to deliver their therapeutic benefits, their persistence at sites of action, and whether their localization or distribution may cause safety issues. Currently, there are several existing and many emerging tools available to develop pharmacokinetics data on these cell-derived therapies to improve our understanding of the mechanism of action and demonstrate on target delivery, but adoption by clinical investigators has been limited. Furthermore, the regulatory landscape is not clearly defined for these emerging therapies. The Health and Environmental Sciences Institute’s Emerging Issues Committee recently launched a new multi-sector collaborative sub-committee to identify key needs for assessing the safety of cell therapies and identify opportunities to meet these needs. This program, the Cell Therapy – TRAcking, Circulation, & Safety (CT-TRACS) sub-committee, provides a neutral platform for cell therapy developers, researchers, regulators, imaging specialists and other stakeholders to interact, discuss current challenges and identify best practices to ensure that these therapies are safe and effective for use. It brings together an international and multi-disciplinary team of experts with interest in sharing their knowledge, common challenges and seek consensus on finding harmonized solutions. In particular, the sub-committee aims to bring awareness on how the application of existing cell tracking technologies, methods, and best practices can benefit the clinical translation of these new therapies. Since its inception in December 2015, CT-TRACS gathered about 50 members from 25 organizations across the United States, Europe and Japan. The sub-committee has convened monthly and the focus of the group has been narrowed to Cell Fate, i.e., distribution, survival/engraftment and phenotype, post-administration, in vivo, as well as evaluating the tumorigenic potential of cell-based therapies. Over the coming year, the sub-committee’s focus will be to: 1/ evaluate current cell-based therapies safety assessment practice and tools; 2/ develop best practices for application of available tools for safety assessment of cell therapies and/or identify gaps in safety assessment; 3/ organize a workshop to present findings of the sub-teams and develop recommendations for next steps; and 4/ initiate a manuscript describing the needs and gaps identified, to build confidence in safety assessment approaches for clinical cell therapy applications. The CT-TRACS project is open to all current HESI members as well as new participants with relevant technical expertise. The program also seeks creative funding partners and encourages inquiries by those with interest in providing financial support for these innovative efforts.

Key Words: Collaborative program – cell tracking- in vivo cell imaging – stem cells – immunotherapy – tumorigenicity – cell therapy safety assessment – best practices

15. Wnt9a Drives Hematopoietic Stem Cell Emergence During Zebrafish Development

Presented by: Jenna Richter, UC San Diego
Authors: Richter, Jenna, UC San Diego; Grainger, Stephanie, UC San Diego; Willert, Karl, UC San Diego; Traver, David, UC San Diego

Hematopoietic stem cells (HSCs) give rise to all terminally differentiated blood cells. Bone marrow transplants, which contain HSCs, are used to cure and treat hematopoietic cancers and disorders. Deriving an HSC in vitro from pluripotent precursors has been attempted for decades, however, a true HSC suitable for human therapeutics has not yet been made. This is due in part to an incomplete understanding of molecular cues that direct the development of HSCs in vivo. The Wnt signaling pathway is involved in HSC development, but a role for (a) specific Wnt ligand(s) has not been described. Here we present data establishing a stage-specific requirement for Wnt signaling in early instruction of HSCs. Furthermore, by screening Wnt expression in hematopoietic tissues at this stage, we identified Wnt9a as the most likely candidate for regulating HSC development. Knockdown of wnt9a, but not the closely related Wnt gene wnt9b, decreased the number of emerging HSCs. However, wnt9a knockdown had no effect on the expression of early markers of commitment to HSC fate, suggesting that Wnt9a acts to promote HSC emergence, not HSC specification. The Wnt9a loss of function phenotype was only rescued by morpholino-resistant wnt9a cDNA, and not by cDNAs encoding either the closely related wnt9b or the prototypical wnt3a ligands. These results suggested that wnt9a is specifically required for proper HSC emergenceIdentification of specific signaling molecules, such as WNTs, that instruct HSC fate will be invaluable in improving existing differentiation protocols to derive HSCs suitable for therapeutic application in the treatment of hematopoietic diseases, disorders, and cancers.

Key Words: Hematopoiesis, Wnt signaling, developmental biology, zebrafish, hematopoietic stem cells

16. Wnt9a is Required for the Aortic Amplification of Nascent Hematopoietic Stem Cells

Presented by: Stephanie Grainger, UC San Diego
Authors: Grainger, Stephanie, UC San Diego; Richter, Jenna, UC San Diego; Traver, David, UC San Diego; Willert, Karl, UC San Diego

In other studies, we use quantitative time lapse imaging of H9 human embryonic stem cell colonies to assess the heterogeneity of colonies and differences in preparations. We tracked large numbers of colonies (~680 in 3 preparations) over long periods of time (5 d imaged at 45 min intervals). Large amounts of image data (~ 1 TB) were generated. In order to track many intact colonies at high resolution, >200 fields of view were stitched together for each time point, allowing us to apply image analysis algorithms to colonies that moved, changed shape and substantially increased their area over time. Each composite image at one time-point was ~1 GB in size, precluding easy viewing of the data on a desktop computer. Software based on multiresolution pyramids and the Deep Zoom Javascript library allowed us to examine the entire image, or zoom into individual colonies, in a dynamic mode. We examined expression of green fluorescent protein that reported on Oct-4 promoter activity as an indicator of plurpotency. We found temporal stability in Oct-4 expression in cells in all of the preparations, but substantial heterogeneity of cellular Oct-4 expression within individual colonies in one of the preparations. In two preparations, heterogeneously expressing colonies were 14% and 15% of all colonies, but in the third preparation, 54% of the colonies demonstrated heterogeneous expression of Oct4. Interestingly, the rate of growth of these colonies was equivalent to that of colonies from the other preparations and colonies that were homogeneously expressing Oct-4.

Key Words: Wnt, hematopoietic stem cell, proliferation, zebrafish

17. An Enteroid Model of Congenital Tufting Enteropathy Shows Alterations in Epithelial Architecture and Actin Disorganization

Presented by: Kevin Okamoto, UC San Diego
Authors: Okamoto, Kevin, UC San Diego; Mcgeough, Matt, UC San Diego; Marchelletta, Ron, UC San Diego; Sivagnanam, Mamata, UC San Diego

Congenital Tufting Enteropathy is a severe diarrheal disease of infancy. We previously identified mutations in Epithelial Cell Adhesion Molecule as the cause of CTE. We developed an inducible in vivo mouse model of CTE based on EpCAM mutations found in patients (Δ4). In order to establish enteroids, intestinal crypts were isolated from ind.EpcamWT/WT and ind.EpcamΔ4/Δ4 mice using cold chelation buffer (5mM EDTA/ HEPES buffer) and cultured in matrigel with DMEM/F12 supplemented with N-acetylcysteine, vitamin B12, and Glutamax. After incubation in specialized culture media supplemented with the required growth factors (priority reagents: Noggin, EGF and R-spondin) for 7 days, the organoids were isolated from matrigel and placed in standard culture media. Enteroids have now been successfully established in our lab. Like mice, enteroids are able to be induced by tamoxifen exposure. Post induction we recognized striking alterations in the structure of ind. EpcamΔ4/Δ4/TAM enteroids vs. ind.EpcamWT/WT/TAM enteroids, with disorganization, lack of clear luminal compartment and central heaping of cells. The intracellular domain of EpCAM has 2 binding sites for alpha actinin. Additionally, a recent Xenopus model showed PKC inhibition was shown to be caused by a short segment of the EpCAM cytoplasmic tail. Thus actin and PKC-epsilon staining were undertaken using Alexaflor 647 phalloidin and PKC. Murine enteroids with EpCAM mutation show actin disorganization and increases in PKC-e as compared with ind.EpcamWT/WT/TAM. These findings suggest EpCAM mutations may alter cytoskeleton components in the intestinal epithelium, which may serve as a mechanism of enterocyte disorganization and “tufting” seen in congenital tufting enteropathy. We also establish the first enteroid model of congenital tufting enteropathy which will allow for enhanced understanding of the pathophysiology of this disease and testing of therapeutic options.

Key Words: Enteroid Intestinal Failure EpCAM Disease Modeling Tufting Enteropathy Actin

18. Role of Human iPSC-derived (hiPSC-) 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 of the central nervous system. They play key functions in the developing brain by being implicated in 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 has been subjected to controversies, likely due to inconsistency in mouse models of RTT. Moreover, there is no indication so far that microglia are important to RTT in a human context. By generating hiPSC-derived macrophages from RTT patients, we are aiming to clarify the controversies about the impact of these cells. Our preliminary data shows that these cells possess the appropriate expression of cell surface markers such as CD14 and CD68. In addition, upon activation with lipopolysaccharides (LPS), hiPSC-derived macrophages released increased 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 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 Syndrome, Macrophage, iPS, Neuroinflammation

19. Modeling Multigenic Familial Dilated Cardiomyopathy with Patient-specific and Engineered Pluripotent Stem Cells

Presented by: Cassandra Happe, UC San Diego
Authors: Happe, Cassandra, UC San Diego; Erbe, Rossin, UC San Diego; Tenerelli, Kevin, UC San Diego; Tran, Vivien, UC San Diego; Deacon, Dekker, UC San Diego; Adler, Eric, UC San Diego; Chi, Neil, UC San Diego; Engler, Adam, UC San Diego

Dilated cardiomyopathy (DCM) is a multivariate disease with poorly understood mechanisms, but recently 30+ different mutations have been suggested to contribute to disease pathology. We have identified a family with high DCM incidence where heterozygous mutations in two dissimilar cytoskeletal proteins, i.e. thin filament-associated –tropomyosin (TPM1; +/c.G97A) and intercalated disc-associated vinculin (VCL; +/c.659dupA), co-segregate with DCM. To better understand disease mechanism, we generated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from the family cohort carrying the variants (proband vs. non-carrier mother) and modeled these variants in human embryonic stem cell-derived CMs (hESC-CMs) via RNAi and CRISPR to discern the functional consequences that could induce DCM. Proband CMs had half of the normal protein expressed for VCL with morphological and cytoskeletal abnormalities and a lower aspect ratio relative to non-carrier CMs. They also contracted with decreased energy and more irregular timing compared to non-carrier control CMs. Decreased sodium and potassium channel expression accompanied slowed action potential kinetics in affected vs. non-carrier-derived CMs, which together suggests that VCL and/or TPM1 mutations may cause unique downstream transcriptome regulation that leads to the dysfunction we observed in vitro. To assess the combinatorial regulation by mutations in these dissimilar genes, hESC-CMs were altered to mirror decreased protein expression, and we found that hESC-CMs lost expression of gap junction protein connexin43 after 50% loss of VCL; these cells also exhibited prolonged calcium transients and contractions with decreased energy and irregular timing, suggesting that reduced VCL creates CMs with dysfunctional mechanical properties, resulting in part from prolonged Ca2+ handling, which would likely adversely affect TPM1 mutant phenotypes leading to more severe disease and DCM observed when the variants co-segregated. Given the lack of disease in single variant carriers, these data provide a unique set of analyses that result in identification of how dissimilar but co-segregating variants can result in disease.

Key Words: vinculin, tropomyosin, multigenic, dilated cardiomyopathy, induced pluripotent stem cells

20. Neurotransmitter Profiling Reveals Upregulation of Catecholamines in Human Schizophrenia iPSC Neurons, with Development of Global Mass Spectrometry Neurotransmitter Analyses

Presented by: Christopher Lietz, UC San Diego
Authors: Lietz, Christopher, UC San Diego; Hook, Vivian, UC San Diego; Toneff, Thomas, UC San Diego; Brennand, Kristen, Mt. Sinai University; Kind, Tobias, University of California, Davis; Palazoglu, Mine, University of California, Davis; Fiehn, Oliver, University of California, Davis; Gage, Fred, The Salk Institute

Human patient derived induced pluripotent stem cell (hiPSC) neurons provide the opportunity to gain insight into the neurotransmitter mechanisms of the brain in normal health and in brain disorders. The objective of this study was to (1) assess the ability of human-induced pluripotent stem cell (hiPSC) neurons are capable of neurotransmitter production and regulated secretion that are fundamental for brain functions, and (2) evaluate the hypothesis that neurotransmitters are regulated in schizophrenia (SZ) modeled by hiPSC neurons, and (3) develop global neurotransmitter profiling by mass spectrometry technology. The methodology of this study uitlized hiPSC neurons derived from SZ patients and normal controls with evaluation of depolarization-stimulation of neurotransmitter secretion for the small molecule catecholamine neurotransmitters – dopamine, norepinephrine, and epinephrine – and the peptide neurotransmitters dynorphin and enkephalin measured by targeted assays. Further, global mass spectrometry analyses of classical small molecule neurotransmitters and the peptide neurotransmitters are being developed to address the question of what neurotransmitters are secreted and regulated by an unbiased, global strategy. Results demonstrated: (1) hiPSC neurons possess the fundamental neuronal property of activity-dependent secretion of neurotransmitters, composed of both classical catecholamine type neurotransmitters and peptide neurotransmitters, along with the expression of biosynthetic enzymes for such neurotransmitters, and (2) SZ hiPSC neurons displayed upregulation of the levels of secreted dopamine, norepinephrine, and epinephrine catecholamine neurotransmitters, providing new data for regulation of norepinephrine and epinephrine as well as dopamine in SZ, and (3) unbiased global mass spectrometry profiling of peptide neurotransmitters and small molecule neurotransmitters for model neuronal chromaffin cells in culture, combined with ongoing hiPSC neuronal studies. In conclusion, results support the use of hiPSC neurons as human neurobiological models for elucidation of neurotransmitter mechanisms in schizophrenia and other brain disorders. The human iPSC neuronal strategy for brain neurotransmitter mechanisms can lead to future drug targeting and biomarkers in drug discovery and development.

Key Words: hiPSC neurons, schizophrenia, neurotransmitters, catecholamines, neuropeptides, secretion, mass spectrometry, peptidomics, metabolomics, brain disorders

21. C-kit+ Cells Resident in Adult Heart are Susceptible to Aging

Presented by: Alessandra Castaldi, UC San Diego
Authors: Castaldi, Alessandra, UC San Diego; Dodia, Ram, UC San Diego; Orogo, Amabel, UC San Diego; Najor, Rita, UC San Diego; Gustafsson, Asa, UC San Diego; Purcell, Nicole, UC San Diego; Brown, Joan Heller, UC San Diego

Although c-kit+ cardiac progenitor cells (CPCs) are currently used in clinical trials there remain considerable gaps in our understanding of the molecular mechanisms underlying their proliferation and differentiation. One of the biggest concerns in the field is that despite the fact that cardiac injury affects mainly the older human population, studies aimed to understand CPC biology and their ability to induce cardiac regeneration have been performed on animal models at young age. In the present study we investigated whether CPCs isolated from young (3 months old) and old (24 months old) mice present different characteristics that could account for different outcomes in future CPC-based therapeutic treatments for cardiac injury. We observed that old CPCs proliferated at a slower rate than young CPCs and that expression of the stemness marker LIN28 was also lower. We subsequently treated the cells with Dexamethasone (Dex), routinely used to induce cardiac commitment in CPCs, for 7 days and analyzed the expression of cardiac lineage marker genes. While MEF2C, GATA4, GATA6 and PECAM mRNA were significantly upregulated in response to Dex treatment in the young CPCs (2.5, 4.3, 6.5 and 6.5 fold change respectively), their expression was not increased in old CPCs compared to cells not treated. These defects did not appear to reflect impaired transcriptional machinery, as both young and old CPCs were able to upregulate ER stress marker in response to tunicamycin. Interestingly, Dex treatment of young, but not old, CPCs led to increased expression of the mitochondrial protein Complex IV, consistent with a defect in mitochondria complex assembly in the old CPCs. Lastly we observed higher expression of the senescence marker p16 in old compared to young CPCs. We are currently investigating whether CPCs from old mice show metabolic impairment that could explain the slower proliferation and decreased ability to increase cardiac genes and mitochondrial complex expression in response to Dex treatment. This study will have an impact on the design of future CPC-based therapeutic approaches for the treatment of old patients suffering from cardiac injury.

Key Words: CPC Aging Cardiac commitment Stem Cells Senescence

22. Enhanced Electronic Consent (EEC) and Electronic Educational Materials (EEM) for Stem Cell Studies May Increase Protocol Adherence

Presented by: Cory Kozlovich, UC San Diego
Authors: Kozlovich, Cory, UC San Diego; Jamieson, Catriona, UC San Diego

Clinical trial participants retain 30-48% of informed consent information, including procedures and major risks. Given the complexity of first in human stem cell interventions and limited information on the long term effects, investigators may wish to exceed the minimum elements of consent in order to ensure patient self-determination and to increase their safety. Recent FDA guidance recommends facilitating consent electronically to enhance such understanding of the trial risks and to increase retention and comprehension of information which may lead to an increase in protocol adherence. Enhanced electronic consent may include many types of media, and including multimedia has been shown to significantly increase understanding by 31%. Likewise, enhanced consent has been shown to significantly increase comprehension by 41%. To achieve these goals in such novel studies, the CIRM Alpha Stem Cell Clinic at UC San Diego Health is piloting this implementation. We present our process. Our site designed enhanced consent that is delivered via iPad tablets and uses free software. Validating electronic participant signatures is complicated by stringent federal regulations, so our method supplements the paper form (participants continue to sign paper). We include visual aids such as a calendar of events, videos and interactive graphics; navigation tools such as a table of contents and internal hyperlinks; distinct protocol sections; formatted text to highlight areas of frequent concern or confusion; annotation features and a glossary. Beyond an enhanced consent document, we offer interactive educational e-books covering basic clinical trial information and a stem cell primer, including content from ISSCR. We expect rapid deployment and positive response by sponsors, participants and investigators. A collection of 2 enhanced consents and 3 enhanced educational reference books were created in-house in 2 months. The enhancements were received by our IRB with encouragement and enthusiasm during a preliminary consultation. The first consent (CLL) was approved by our IRB within 7 business days of expedited submission. Other sites may wish to institute enhanced documents from our free model.

Key Words: ethics and public policy, consent, clinical trial, human, leukemia/lymphoma

23. Metabolic Impact of Culture Conditions on hPSC Maintenance and Cardiac Differentiation

Presented by: Hui Zhang, UC San Diego
Authors: Zhang, Hui, UC San Diego Bioengineering; Spiering, Sean, Sanford Burnham Prebys Medical Discovery Institute; Badur, Mehmet, UC San Diego Bioengineering; Ajit Divakaruni, Ajit, UC San Diego Pharmacology; Murphy, Anne, UC San Diego Pharmacology; Mark Mercola, Mark, Sanford Burnham Prebys Medical Discovery Institute; Metallo, Christian, UC San Diego Bioengineering

Our study aims to identify metabolic impact of culture conditions on human puripotent stem cells (hPSCs) and hPSC-derived cardiac progenitors. Metabolism plays a central role in supporting nearly all cell functions, also including pluripotency. The maturation of mitochondrial oxidative respiratory machinery is the metabolic signature of the health cardiomyocytes. In contrast to proliferating hPSCs that rely on aerobic glycolysis for growth, cardiomyocyte can utilize multiple nutrients to generate energy through oxidative respiratory pathway. However, current hPSC and cardiac progenitor culture conditions rely on serum-free, chemically defined media that would limit the nutrient availability of cultured cells. Therefore, assessment of the potential metabolic impact of the culture media will bring novel insights for optimizing in vitro culture conditions for cardiac lineage-directed hPSC differentiation and maturation. To achieve our research goal, we adopted an interdisciplinary approach using quantitative methods drawn from different research fields, including stem cell biology, human physiology, and metabolic engineering. We comprehensively characterized the metabolic alterations in hPSCs cultured in different media and differentiated into cardiomyocyte. We also tested the ability of hPSCs and hPSC-derived cardiomyocytes to oxidize mitochondrial substrates for evaluating the activity of mitochondrial energy generation. This approach involved cultivating cells in custom media containing stable isotope-labeled tracers, using specific tracers (e.g. [13C]glucose, [13C]palmitate) and gas chromatography/mass spectrometry to quantify labeling in downstream intermediates, and interpreting experiment data in the context of a biochemical network to perform metabolic flux analysis. In this study, we demonstrated that chemically-defined media strongly influenced the state of mitochondrial respiration and fatty acid metabolism in hPSCs. Lipid deficiency in chemically defined media dramatically altered central carbon metabolism and mitochondrial respiratory. Lipid supplementation can mitigate the media-induced cellular metabolic reprograming and increase oxidative metabolism. During cardiac differentiation, we identified the consumption change of glucose, lactate, glutamine and branch amino acids in differentiating cells. Lipid deficiency in hPSC culture subsequently affected hPSC-derived cardiomyocytes and promoted metabolic immature phenotype. Lipid supplementation improved mitochondrial function and sarcomere structure in hPSC-derived cardiomyocytes. In summary, we demonstrated culture condition is the effective factor to determine biological outcomes of hPSCs. Our study indicated that metabolism of human puripotent stem cells (hPSCs) is a promising field to make fundamental discoveries that drive further understanding of the pluripotency phenotype and improve hPSC bio-applications.

Key Words: human pluripotent stem cell, cardiomyocyte, metabolism, culture, chemically defined media, differentiation, maturation, mass spec, metabolic flux analysis, stable isotope, mitochondria, respiration, lipid deficiency, lipid supplementation

24. Reducing Uncertainty in the Evaluation of Stem Cell Colonies

Presented by: Anne Plant, National Insitute of Standards and Technology
Authors: Halter, Michael, NIST; Lund, Steven, NIST; Bajcsy, Peter, NIST; Peskin, Adele, NIST; Li-Baboud, Ya-Shian, NIST; Amelot, Julian, NIST; Chalfoun, Joe, NIST; Bhadriraju, Kiran, NIST; Plant, Anne, NIST

Stem cell colonies are passaged based on their visible microscopic characteristics that presumably indicate desirable properties of pluripotency, viability, etc. In an effort to improve consistency and control in expansion, we used machine learning, based on training sets from experts, to automate the process of rating colonies for their desirability. Systematic testing of models and algorithms indicates that differences in experts’ opinions is a major source of uncertainty. Agreement between two experts is imperfect with a mean squared error of 0.6. A model was developed that predicted expert ratings better than the agreement between two experts, resulting in a mean squared error of 0.4. However, covariance analysis suggests that 20 % of the variance could be accounted for by features that both experts use, but are not accounted for in our models. In other studies, we use quantitative time lapse imaging of H9 human embryonic stem cell colonies to assess the heterogeneity of colonies and differences in preparations. We tracked large numbers of colonies (~680 in 3 preparations) over long periods of time (5 d imaged at 45 min intervals). Large amounts of image data (~ 1 TB) were generated. In order to track many intact colonies at high resolution, >200 fields of view were stitched together for each time point, allowing us to apply image analysis algorithms to colonies that moved, changed shape and substantially increased their area over time. Each composite image at one time-point was ~1 GB in size, precluding easy viewing of the data on a desktop computer. Software based on multiresolution pyramids and the Deep Zoom Javascript library allowed us to examine the entire image, or zoom into individual colonies, in a dynamic mode. We examined expression of green fluorescent protein that reported on Oct-4 promoter activity as an indicator of plurpotency. We found temporal stability in Oct-4 expression in cells in all of the preparations, but substantial heterogeneity of cellular Oct-4 expression within individual colonies in one of the preparations. In two preparations, heterogeneously expressing colonies were 14% and 15% of all colonies, but in the third preparation, 54% of the colonies demonstrated heterogeneous expression of Oct4. Interestingly, the rate of growth of these colonies was equivalent to that of colonies from the other preparations and colonies that were homogeneously expressing Oct-4. These approaches will allow validation that putative markers of pluripotency and differentiation that are present at early times in culture result in the fate expected. Time-lapse imaging also allowed us to identify a small number of rare events where colonies abruptly changed Oct-4 expression levels, or split into 2 colonies, one which was Oct-4 expressing and another which was not.

Key Words: stem cells, quantitative imaging, machine learning, image analysis, time-lapse, pluripotency, automate, biomarkers

25. A Physiologically-relevant and Screening-ready, Human iPSC-derived Cardiomyocyte Platform

Presented by: Fabian Zanella, StemoniX
Authors: Zanella, Fabian, StemoniX; Contu, Riccardo, StemoniX; Spangenberg, Stephan, StemoniX; Si, Wonjong, StemoniX; Selke, Janet, StemoniX; Peris, Matthew, StemoniX; Gordon, Ryan, StemoniX; Yeh, Ping, StemoniX; Petcavich, Robert, StemoniX

Human induced pluripotent stem cells (hiPSCs) have been perceived as a powerful tool to study organ and system-specific diseases and toxicity. hiPSC-derived cardiomyocytes have been increasingly adopted in cardiac disease modeling and cardiotoxicity research. Despite their inarguable value in current research pipelines, challenges pertaining to correct cell geometry, as well as sarcomeric and cardiac cell junction assembly and organization still hinder a wider adoption of this powerful and refined model. Here we describe a novel platform formatted into high density (384 and 1536-well) screening plates that contains hiPSC-derived cardiomyocytes pre-plated in a microstructure that emulates correct cardiac muscle fiber organization. Our platform leads to improved sarcomeric organization, as seen by readily identifiable, correctly patterned myofibrils along the cell body which tend to be absent, undefined or disarrayed when cardiomyocytes are plated in standard cell cultureware. Plakogoblin, a key component of cardiac cell junctions, shows correct targeting to distal intercalated discs in our platform, as opposed to an unrestricted, peripheric localization to the cell membrane in standard cell cultureware. In our platform we also observe increased gene expression of ryr2, atp2a2, and pln, key components of cardiomyocyte calcium handling pathways, which are crucial for cardiac physiology. The expression levels of cardiac ion channel genes such as cacnac1c, scn5a, kcne1, kcnq1 as well as cardiac cell junction components gja1, gja5 and dsp are also increased.

Key Words: Human iPS cardiomyocytes physiological accuracy

26. Guided Lymphangiogenesis for the Treatment of Lymphedema: a Pilot Study

Presented by: Michael Paukshto, Fibralign Corporation
Authors: Hadamitzky, Catarina, Helios Clinic, Hildesheim, Germany.; Zaitseva, Tatiana, Fibralign Corporation, USA; Katz, Nathan, JoinTechLab, Illinois, USA.; Escarraman, Manuel, Instituto Oncológico, Dominican Republic; Paukshto, Michael, Fibralign Corporation

To address the limitations of current treatments for secondary lymphedema, our study group developed an experimental surgical procedure based on Autologous Lymph Node Fragment (ALNF) transfer supplemented by nanofibrillar collagen scaffold with and without autologous Adipose Derived Stromal Cells (ADSCs). The efficacy of this scaffold was demonstrated before by histological, functional and imaging analyses in a porcine model of secondary lymphedema. The use of ADSCs has major potential demonstrated in preclinical and clinical studies, but their use is often hampered due to difficulties in harvesting and delivery. The cell injections require large dosages and still have limited effect due to poor survival and migration from the target site. We address these challenges by using ADSC-seeded scaffolds to deliver the cells, support cell survival, maintenance and function precisely at the targeted site. The ongoing pilot study has 8 patients currently enrolled. The ALNF transfer was performed in all patients and additionally 6 patients received the collagen scaffolds alone and 2 patients received the ADSC-seeded scaffolds. Lymphoscintigraphy was conducted before the treatment and will be conducted at the end of the study, 12 months after the initial surgery. Volume and bioimpedance measurements (L-Dex U400) were scheduled for 0, 3, 5, 8 and 12 months after surgery. A portable closed-loop Mini-Stem system was used for extraction of ADSCs from patient lipoaspirate. Also, a 1.7 ml trocar was used for cell seeding on the scaffolds (~2 hours in incubator) and for subcutaneous delivery to bridge the area of impaired lymphatics. While this is a small ongoing study, safety has been demonstrated as there has been no complications reported over the first nine months. For the first set of patients (n = 3), the affected/unaffected limb volume ratio was reduced on average from 1.49 to 1.33 at 8 months after surgery. For two patients in the second set (n = 5), the affected/unaffected limb volume ratio was reduced from 1.23 to 1.1 at 3 months after surgery. More preliminary data will be presented at the time of the conference. While vascularized lymph node transfer is considered to be a more advanced technique than ALNF transfer, there is a great interest in developing countries to have a simpler surgery that could help patients with lymphedema. On the other hand, if the current treatment improves lymphedema, the concept of guiding lymphangiogenesis with collagen scaffolds could potentially improve the efficiency of established vascularized lymph node procedures.

Key Words: Lymphedema, Adipose Derived Stromal Cells, nanofibrillar scaffold, clinical study

27. ERBB3 and NGFR Mark Distinct Skeletal Muscle Progenitor Cells in Human Development Enabling Enrichment and Maturation of hPSC Muscle

Presented by: Michael Hicks, UCLA
Authors: Michael Hicks, Julia Hiserodt, Katrina Paras, Majib Jan, Haibin Xi, Courtney Young, Denis Evseenko, Melissa Spencer, Ben Van Handel, April Pyle (UCLA)

Human pluripotent stem cells (hPSCs) can be directed to differentiate into skeletal muscle progenitor cells (SMPCs). However, the myogenic potential of hPSC-SMPCs compared to human fetal or adult satellite cells (SCs) remains unclear. This study demonstrates hPSC-SMPCs derived by commonly used protocols are functionally less mature than freshly-isolated human fetal or adult SCs. We utilized RNA-SEQ of human fetal SCs to identify differentially expressed genes including NGFR, ERBB3, which enriched for MYOD+ or PAX7+ cells. Furthermore, inhibition of TGFβ (TGFβi) improved hPSC-muscle maturation in three independent lines as measured by TEM and expression of myosins. Engraftment of CRISPR/Cas9-corrected Duchenne Muscular Dystrophy (DMD) hiPSCSMPC subpopulations treated with TGFβi in vivo, increased dystrophin-positive fibers to levels of engrafted cultured fetal SCs in mdx mouse models of DMD. This work provides the first characterization of the developmental status of hPSC-SMPCs and identifies candidates to enable robust myogenic activity in vitro and in vivo.