Poster Session

Naiara Akizu,  Joseph Gleeson, UC San Diego

Ciliopathies are a heterogeneous group of disorders characterized by a wide spectrum of clinical features that result from the impaired structure or function of the primary cilia, leading to symptoms predominantly in brain, retina and kidney. The individually rare but collectively not uncommon conditions include Joubert syndrome (JS), nephronophthisis (NPHP), Meckel-Gruber syndrome (MKS), Bardet-Biedl syndrome (BBS) and Polycystic kidney disease (PKD). Despite over 50 known genetic causes, the current animal models completely fail to recapitulate the organ specificity and severity observed in humans. Therefore, alternative models are required to understand the molecular bases that underlie the specific clinical manifestation in each of the genetic conditions of human ciliopathies. For this purpose we have designed an strategy to generate an iPSC model for three of most important organs affected in ciliopathies (retina, kidney and cerebellum), the goal of which is to preserve the ‘humanness’ of the diseased cells as well as the genetic background to study the complexity of human ciliopathies.

Key words: Ciliopathies, iPSC, Primary cilium, Photoreceptor, Outer segment, 3D spheroid, Polarized epithelium

2. WNT/beta-catenin Independent Signaling in Proliferation and Differentiation of Human Pluripotent Stem Cells

Matthieu Bauer, UC San Diego; David Cappellen, Université Victor Ségalen-Bordeaux 2; Karl Willert, UC San Diego

WNT signaling is important in many developmental processes and diseases. Multiple signaling pathways are triggered by the soluble lipid-modified WNT glycoproteins binding their receptors. Previous studies on the role of WNT signaling in hPSC are so far inconclusive and contradictory. Some show that WNT signaling promotes undifferentiated cell proliferation while other studies suggest that WNT signaling promotes differentiation into definitive endoderm. These contradictory results can be explained by the distinct cellular context in which these results have been obtained as well as the distinct pathways that are activated. One possibility is that distinct isoforms of the WNT5A gene mediate the differential effects. Determining which pathways and ligand/receptor pairs are able to trigger hPSC self-renewal and differentiation will allow us to better design strategies for the directed differentiation of hPSCs into a specific cell types. To further explore this possibility we used N-terminal peptide sequencing of purified WNT5A and found that the WNT5A gene is translated as two distinct proteins yielding a short and a long isoform. Previous experiments with cancer cell lines showed that these two WNT5A isoforms have distinct and opposite functions. While the short isoform promotes cell proliferation and survival and is upregulated in tumors, the long isoform inhibits cell proliferation and is downregulated in tumors. We extended these studies to hPSCs, and our preliminary studies show that undifferentiated hPSCs express low levels of WNT5A-L. Upon embryoid body-mediated differentiation, its expression is upregulated. This finding suggests that expression of WNT5A-L plays a functionally important role in differentiation of hPSCs. We are currently conducting experiments to characterize to what extent WNT5A expression mediates differentiation, determine the differentiated cell lineage promoted by WNT5A expression, and identify downstream targets of WNT5A signaling in hPSCs.

Key words: WNT, alternative transcripts, differentiation, self-renewal, hPSC

3. Comparative Proteomic and Phosphoproteomic Profiling of Human Embryonic Stem Cells and Their Pure PAX6+ Neuroectodermal Derivatives

Laurence Brill, Ilyas Singec, Junjie Hou, Brian Tobe, Andrew Crain, Evan Snyder, Sanford-Burnham Medical Research Institute

Total proteome and phosphoproteome analyses were performed on pure populations of pluripotent hESCs (OCT4+/PAX6-) and multipotent neural stem cells (NSCs; ~97% PAX6+/Nestin+). We identified 12,904 proteins, 76.4% of which were phosphorylated on a total of 59,680 sites. This is the largest comparative (phospho)proteomic resource to date in any biological system. There was a 75.9% overlap of proteins identified between hESCs and NSCs, but only 25.7% of the localized phosphorylation sites overlapped between the cell populations, suggesting that more regulation of protein activities was at the level of phosphorylation than expression. A significantly lower percentage of the phosphoproteins vs. those lacking detectable phosphorylation were enzymes in both hESCs and NSCs, whereas in both cell populations a significantly higher percentage of the phosphoproteins vs. proteins lacking detectable phosphorylation were kinases, receptors and channels. In addition, hESCs contained a significantly higher percentage of phosphorylated transcription factors than transcription factors lacking detectable phosphorylation. Most of all the known TGF-beta and WNT pathway members were identified, most with phosphorylation, and some were identified in only one cell population or the other. We detected surprisingly early expression and phosphorylation of proteins associated with later-stage neuronal function (e.g. synaptic vesicle proteins) and age-dependent neurological diseases (e.g. beta-synuclein, amyloid precursor protein). These findings shed light on pluripotency and human neural fate choice, their molecular basis, and should facilitate the rapid and systematic production of large numbers of defined neural phenotypes for developmental studies, disease modeling, drug discovery and systems biology.

Key words: human embryonic stem cells; neural stem cells; systems biology; phosphoproteomics

4. Modeling Parkinson’s Disease Using Human Pluripotent Stem Cells

Ben Campbell, Chris Tse, Leah Boyer, Salk Institute for Biological Studies; Beate Winner, Zacharias Kohl, Juergen Winkler, University of Erlangen; Fred Gage, Salk Institute for Biological Studies

Parkinson’s Disease (PD) is the most common neurodegenerative movement disorder. Historically, PD has been considered a strictly neuronal disease; however, clinical observations and evidence from animal models suggest inflammation may contribute to disease progression. It remains controversial whether glial activation, and the resulting inflammatory cascade, is a result or a cause of neuronal death. Towards resolving this distinction, we have established cultures of human dopaminergic neurons, astrocytes, and microglia. These neural cell lines are used to investigate the inflammatory response to extracellular alpha-synuclein, the protein whose mis-folding is the pathological hallmark for all forms of PD. We have found this protein is sufficient to induce the secretion of pro-inflammatory cytokines in both astrocytes and microglia. Our human cell based model establishes the neurotoxic response to this glial derived increase pro-inflammatory cytokines, allowing us to study the inflammatory contribution to the pathological development of PD in a human system. Furthermore, we are reprogramming fibroblasts from patients with idiopathic PD to investigate the contribution of inflammation to the progression of non-genetic forms of this debilitating disease. We aim to understand the role inflammation in PD and identify key molecular events involved at early stages in PD to exploit as potential targets for therapeutic intervention.

Key words: Disease Modeling, Parkinson’s Disease, Neuro-Inflammation, Dopaminergic differentiation.

5. Analysis of Human Embryonic Stem Cell-Derived Pancreatic Progenitors Utilizing NanoString and QPCR Gene Expression Platforms

Rosemary Cesario, Janice Payne, Jonathan Kelly, Jean Alfonso, Holly Young, Kevin D’Amour, Olivia Kelly, ViaCyte, Inc.

ViaCyte is developing a cell therapy for diabetes.  The therapy is comprised of pancreatic progenitor cells transplanted within a retrievable macro-encapsulation delivery device.  Using a step-wise differentiation protocol modeled after pancreatic development, we have generated pancreatic progenitor cells from human embryonic stem (hES) cells.  We have chosen to monitor gene expression changes as cells proceed through the stages of differentiation using nanoString technology, which measures gene expression levels through direct binding of color coded probes to mRNA from the gene of interest.  This technology allows multiplexing of hundreds of genes in one sample, has internal positive and negative controls for normalization, and removes the potential for variability induced via enzymatic reactions (cDNA and PCR).  

While a powerful technology, NanoString is not well-suited for rare event detection.  There is concern that cell products derived from human ES cells could contain residual undifferentiated ES cells, which could give rise to teratomas.  Because OCT4 (POU5F1) is well documented as being a marker of ES cells and germ cells, but not other somatic cell types, we are developing a QPCR assay to monitor our cell product for undifferentiated ES cells.  It is challenging to design primers that are unique to OCT4 message because OCT4 has multiple pseudogenes and is highly polymorphic.  With this in mind, we are optimizing primer design to increase assay sensitivity. 

Key words: human embryonic stem cells, pancreatic development, differentiation, NanoString, OCT4, POU5F1

6. Acetylation Within the Core Domain is Required for p53 Stabilization

Sun-Ku Chung, Yang Xu, UC San Diego

Tumor suppressor p53 is critical to suppress human cancers. To investigate the function and regulation of p53 directly in human cells, we employed human embryonic stem cells (hESCs) that can undergo unlimited self-renewal and differentiate into all cell types in the human body. Using knock-in technology, we introduced various missense mutations into the posttranslational modification sites of the endogenous p53 gene in hESCs. By analyzing the p53 stability and activity in the knock-in hESCs and their derivatives, we have identified the critical posttranslational modification events that are required for p53 activation. Some of these important posttranslational modifications are critical for p53 activation that are targeted for mutation in human cancers. In summary, we have developed an hESC-based approach to elucidate the function and regulation of human p53 in suppressing human cancer. 

Key words: p53, human embryonic stem cell, knock-in technology, posttranslational modification, stability

7. The Role of IGF2 mRNA Binding Protein 1 in Human Pluripotent Stem Cells

Anne Conway, Tony Essex, Kristen Brennand, Severine Landais, Leanne Jones, Salk Institute for Biological Studies

Igf2 mRNA binding protein 1 (IMP1/Igf2bp1) is an mRNA binding protein that is expressed in multiple tissues during development and in gonads post-natally.  Importantly, human IMP1 expression is highly up regulated in a number of cancers, including breast, testicular, and colorectal tumors and is used as a marker associated with poor patient prognosis. Previously characterized mRNA targets of human IMP1 include c-myc, Igf2, and b-TrCP1, a regulator of Wnt signaling.  Here we have used human pluripotent stem cells to probe the role for hIMP1 during development.  Western blotting analysis showed that hIMP1 is highly expressed in human embryonic stem cells (hESCs) and induced Pluripotent Stem cells (iPS), with little to no expression in fibroblasts, consistent with a possible role in early development.  Interestingly, expression of IMP1 peaks during differentiation of embryoid bodies (EBs), before declining to undetectable levels. Using immunoprecipiation followed by PCR, we have shown that hIMP1 can bind to key pluripotency mRNAs, including Oct4 and Nanog, and loss of hIMP1 results in upregulation of OCT4 and NANOG protein.  Therefore, we propose that hIMP1 acts as a switch from pluripotency to differentiation by regulating the stability and/or translation of key pluripotency factors.

Key words: stem cells, pluripotency

8. In Vitro Osteogenic Differentiation of Novel Human Embryonic Stem Cell Derivatives

Shreyasi Das, Jaqueline Wu, Sanford-Burnham Medical Research Institute; Sean Grogan, The Scripps Research Institute; David Larocca, Mandala Bioscience LLC; Darryl D’Lima, The Scripps Research Institute; Michael West, BioTime, Inc.; Evan Snyder, Sanford-Burnham Medical Research Institute

Repairing large bone defects after major trauma is difficult as the availability of using the patient’s own bone is often limited.  Based on the extensive investigation of various ways to regenerate bone, osteoblasts show great promise for application in human patients. Recently, we obtained derived human embryonic progenitor cells lines (ACTCelerate cell lines) that may generate a robust source of osteoblasts.  To determine the osteogenic potential of these cells, cell morphology, differentiation and mineralization were analyzed.  These cells exhibited a number of osteo-related markers including osteoblast-cadherin (CDH11), osteomodulin (OMD), secreted phosphoprotein 1 (SPP1), osteonectin (SPARC) as examined by microarray.  Additionally, these cells displayed mineralization as detected by Alzarian red staining. These results showed that ACTCelerate cell lines maybe useful as a surrogate model to study osteocyte differentiation and bone mineralization.

Key words: Osteogenesis, MSC, progenitor stem cells

9. High-resolution Mapping of Higher Order Chromatin Structure in Murine Embryonic Stem Cells

Jesse Dixon, Ludwig Institute for Cancer Research, UC San Diego; Ming Hu, Harvard University; Feng Yue, Siddarth Selvaraj, Jennifer Fang, Ludwig Institute for Cancer Research, UC San Diego; Ke Deng, Steve Qin, Emory University; Jun Liu, Harvard University; Bing Ren, Ludwig Institute for Cancer Research, UC San Diego

In eukaryotic cells, the linear DNA is packaged into a highly compact chromatin structure that allows for dynamic regulation of gene expression.  Despite intense efforts to study the nature of chromatin, higher order chromatin structure remains poorly understood.  Recently, techniques have been developed that allow for the genome wide identification of higher order interactions in the genome.  However, these studies have suffered either from being focused solely on a given transcription factor or from a lack of resolution. In order to better understand the higher-order chromatin structure associated with pluripotency and differentiation, we performed Hi-C in murine embryonic stem cells and obtained a genome-scale map of chromatin interactions at a resolution of 20 kilobases.  This high-resolution map allows us to build 3D models of chromosomes, identify target genes for distal enhancers, and examine the spatial relationships between different DNA binding proteins.  Interestingly, this map also reveals over 7,000 of interaction ‘hotspots’, which display unusually high frequency of interactions with others.  The ‘hotspots’ are enriched for promoters, enhancers, insulators, and transcription end sites. Surprisingly, these regions also include a large number of bivalent promoters. Future studies will focus on understanding the changes in genome structure that accompany differentiation of pluripotent cells into different lineages.

Key words: Chromatin, Stem Cells, Higher Order Structure

10. Regulation of Cell State and Fate by Adhesion Strength

Alexander Fuhrmann, Adam Engler, UC San Diego

Fibronectin (FN), an extracellular matrix protein, is known to form a fibrillar network, which surrounds cells and plays an important role in morphogenesis, e.g. FN null embryos cannot undergo gastrulation. Though FN is found in a fibrillar structure in vivo, simple ligand coatings of the protein have been found to induce initial embryonic stem cell (ESC) lineage commitment. Here we demonstrate that endoderm commitment occurs through an alpha-5-integrin dependent process and that adhesion strength corresponds to lineage specific differentiation. Preliminary results of investigating the attachment strength, and subsequently attachment mechanisms, of mESCs to the various substrates, challenge the current models of cell detachment under shear. 

Key words: adhesion, fibronectin, mouse embryonic stem cells

11. In Vitro Differentiation of Human Parthenogenetic Stem Cells into Neuronal Progenitor Cells

Ruslan Semechkin, Ibon Garitaonandia, Tatiana Abramihina, Tatiana Zogovic-Kapsalis, International Stem Cell Corporation; Richard West, West Michigan University; Andrey Semechkin, International Stem Cell Corporation

Human parthenogenetic stem cells (hpSC) are pluripotent stem cells derived from unfertilized oozytes. hpSC behave the same way as human embryonic stem cells (hESC) in their proliferation capacity and multilineage differentiation in vitro. Depending on the way they are derived, hpSC can be either heterozygous or homozygous. Homozygous hpSC have the potential to serve as a useful source of cells in clinical therapy because if the HLA type is common, their differentiated derivatives can match millions of individuals. In this study we derive NSC from hpSC using an approach based on an adherent model, in which rosettes of neuroepithelial cells are formed by culturing hpSC in feeder-free conditions. The generated rosettes express markers PAX6, SOX1, NES (Nestin), MSI1 (Musashi-1), but not OCT4 (POU5F1) as determined by qRT-PCR. The rosettes are then isolated, dissociated into single cell suspension, and propagated as an adherent cell culture giving rise to human parthenogenetic neural stem cells (hpNSC). The hpNSC can be propagated for up to 30 passages and express the neural markers Nestin, SOX2, and Musashi-1.  Expression of OCT4 was not detected at the RNA and protein levels and the hpNSC can be cryopreserved without losing expression of neural markers. hpNSC can be further differentiated into neurons and glia. The hpNSC derived neurons express TUBB3 (Tubulin beta III) and MAP2, elicit action potentials, and outward and inward currents. In conclusion these results indicate the enormous potential of human parthenogenetic stem cells for the treatment of neurodegenerative diseases.

Key words: Human parthenogenetic stem cells, Homozygous, Heterozygous, Human neural stem cells, Neurons

12. Development of Synthetic Microenvironments for Stem Cell Growth and Differentiation

Sarah George, Karl Willert, David Brafman, UC San Diego

Fully defined and scalable culture conditions are essential to the development of human pluripotent stem cell (hPSC) based therapies. Synthetic materials that replace the currently utilized, expensive, and variable matrix and media components, could provide an inexpensive and reliable alternative for hPSC expansion. To identify such alternative culture conditions, we developed a technology platform to screen the effect of thousands of combinations of synthetic polymers on cell attachment, proliferation, differentiation and gene expression. We used this cellular microarray technology to systematically screen a library of synthetic polymers (with various functional groups, charge density, molecular weight, and hydrophobicity) for their ability to support hPSC growth and pluripotency. We identified several polymers that can support self-renewal of hPSCs. While most of these polymers provide support for only a short period of time, we identified a synthetic polymer poly(methyl vinyl ether-alt-maleic anhydride) (PMVE-alt-MA) that supported the long-term attachment, proliferation and self-renewal of HUES1, HUES9, and iPSCs in defined media conditions. HPSCs cultured on PMVE-alt-MA maintained their characteristic morphology, expressed markers of pluripotency, and retained a normal karyotype. We are currently performing studies to apply this polymer in large-scale-two-dimensional (2D) and suspension culture systems of hPSCs. To this end, PMVE-alt-MA is either combined with polyacrylamide in a semi-interpenetrating polymer network (semi-IPN), or as a thin coating on polyacrylamide gel. In addition to screening polymers for hPSC expansion and maintenance, we are employing the high throughput array-based screening approach to identify polymers that can support self-renewal of hPSC derived neural progenitor cells (NPCs).

Key words: Human pluripotent stem cells, biomaterials, defined culture conditions, hydrogel

13. Characterizing the Hb9 Targetome Using ChIP-Sequencing Within Embryonic Stem Cell-Derived Motor Neurons

Wesley Gifford, Todd Macfarlan, Shawn Driscoll, Salk Institute for Biological Studies; Samuel Pfaff, Howard Hughes Medical Institute and Salk Institute for Biological Studies

Within the central nervous system, the transcription factor Hb9 is expressed exclusively in the motor neurons (MNs) of the spinal cord and brain stem. MNs in Hb9 knockout (KO) mice take on characteristics unique to a specific class of nearby spinal interneurons. Conversely, ectopic expression of Hb9 within the dorsal spinal cord generates ectopic MNs. Therefore, the Hb9 transcription factor is a critical regulator of MN cell fate. Despite its importance, there are only two known binding sites for Hb9. We set out to identify all the genomic Hb9 binding sites within embryonic stem cell-derived MNs (ESC-MNs) using chromatin immunoprecipitation followed by high throughput sequencing (ChIP-Seq) to obtain an unbiased look at the Hb9 ‘targetome.’ To functionally annotate binding sites, mice harboring an Hb9 null allele were crossed to generate WT and Hb9 KO ESC lines and were assayed using RNA-Sequencing. Candidate Hb9 binding sites and genes misexpressed upon deletion of Hb9 have been identified. We have uncovered several novel features of Hb9’s control of MN fate specification and are now in the process of determining whether Hb9 regulates gene expression in MNs by directly influencing the general transcriptional machinery or indirectly by first modifying chromatin domains.

Key words: ChIP-Seq, RNA-Seq, Epigenetics, Chromatin, Motor Neurons, Development, Cell Fate Specification

14. The pan-BCL2 Inhibitor, Sabutoclax, Sensitizes Chemotherapy-resistant Chronic Myeloid Leukemia (CML) Stem Cells to BCR-ABL Inhibition

Daniel Goff, Kristen Smith, Heather Leu, UC San Diego; Jun Wei, Dayong Zhai, Sanford-Burnham Medical Research Institute; Anil Sadarangani, UC San Diego; Maurizio Pellecchia, John Reed, Sanford-Burnham Medical Research Institute; Catriona Jamieson, UC San Diego

Leukemia stem cells (LSC) play a critical role in the development and progression of chronic myeloid leukemia (CML). Although BCR-ABL targeted tyrosine kinase inhibitors (TKI) eradicate the majority of CML cells, they fail to eliminate LSC that drive relapse. Here, we demonstrate that CML LSC (CD34+CD38+lin-) upregulate pro-survival BCL2 family members during CML disease progression and, when in the bone marrow niche, are resistant to dasatinib, a potent TKI. This resistance is cell-type and niche specific and commensurate with further elevation of BCL2 expression. These data led us to speculate that marrow LSC may be sensitive to BCL2 inhibition and that this treatment may sensitize LSC to TKI therapy. Treatment of LSC with a pan-BCL2 family inhibitor, sabutoclax, in vitro led to a dose-dependent increase in apoptotic cell death while normal human progenitor cells were less sensitive to sabutoclax treatment with IC50 approximately 5 times higher than that for LSC. Treatment of LSC transplanted mice with sabutoclax led to a significant reduction in engraftment of LSC in all hematopoietic organs analyzed and sabutoclax pre-treatment also sensitized bone marrow LSC to dasatinib ex vivo. Finally, combination treatment with both sabutoclax and dasatinib eliminated bone marrow LSC and significantly improved the survival of secondary recipient mice transplanted with treated marrow. These results demonstrate that marrow niche LSC are sensitive to sabutoclax both in vitro and in vivo.

Key words: BCL2, BCR-ABL, CML, leukemia stem cells

15. Bone Marrow Derived Mesenchymal Stem Cells Enhance Survival and Bacterial Clearance in Murine E. coli Pneumonia

Naveen Gupta, The Scripps Research Institute; Anna Krasnodembskaya, Michael Matthay, University of California, San Francisco

Mesenchymal stem cells (MSCs) have been demonstrated to provide protection against acute inflammatory lung injury, however, their potential therapeutic role in the setting of bacterial pneumonia has not been well studied.

This study was focused on testing the therapeutic and mechanistic effects of MSCs in a mouse model of gram-negative pneumonia.   Syngeneic MSCs from wild-type mice were isolated and administered via the intratracheal (i.t.) route to mice 4 hours after the mice were infected with E. coli.  3T3 fibroblasts and PBS were used as controls for all in vivo experiments.  Survival, lung injury, bacterial counts, and indices of inflammation were measured in each treatment group.  Treatment with wild-type MSCs improved 48 hour survival (MSC, 55%; 3T3, 8%; PBS, 0%; p < 0.05 for MSC vs 3T3 and PBS groups) and lung injury compared to control treated mice.  In addition, wild-type MSCs enhanced bacterial clearance from the alveolar space as early as 4 h after administration, an effect that was not observed with the other treatment groups. The antibacterial effect with MSCs was due, in part, to their upregulation of the antibacterial protein, lipocalin 2.  Treatment with MSCs enhanced survival and bacterial clearance in a mouse model of gram-negative pneumonia.  The bacterial clearance effect was due, in part, to the upregulation of lipocalin 2 production by mesenchymal stem cells.

Key words: mesenchymal stem cells, innate immunity, lipocalin 2, pneumonia, lung injury

16. Neuronal Induction in Grafts of Neuronal Progenitors to Sites of Spinal Cord Injury

Verena Haringer, UC San Diego; Paul Lu, Mark Tuszynski, UC San Diego, VA Medical Center

Spinal cord injury (SCI) causes loss of neurons and axons at the lesion site, frequently resulting in permanent loss of function. One repair strategy is to use cell transplantation to replace injured neurons, providing a potential mechanism for forming polysynaptic relays across sites of injury. In the present study embryonic neural cells were cultured and pre-defferentiated in vitro before transplantation into the injured adult spinal cord.

Fetal NPCs were generated from neurospheres derived from E14 spinal cord. When grafted into the adult injured spinal cord, these cells differentiated into glia. To increase neuronal differentiation/maturation, the cells were pre-differentiated into neuronal progenitors as monolayer cultures for 3, 5, 7 days using retinoic acid and sonic hedgehog. The cells expressed the early neuronal progenitor markers, beta-III tubulin and Nestin. When grafting these pre-differentiated neuronal progenitors/early neurons into adult SCI lesion sites, almost no mature spinal cord neurons were detected 4 weeks after transplantation. Instead the graft consisted mainly of glial cells. Overall graft survival was good.

These findings confirm the pro-glial environment of the adult spinal cord, indicating that even pre-differentiation into neuronal phenotypes are insufficient to lead to a neuronal fate in vivo. Further differentiation steps are required to promote retention of the neuronal phenotype in grafted neural progenitors.

Key words: spinal cord injury, neurospheres, neural stem cells

17. Engineering Cell-material Interfaces for Long-term Expansion of Human Pluripotent Stem Cells

Yongsung Hwang, UC San Diego; Chien-Wen Chang, National Tsing Hua University; David Brafman, Thomas Hagan, Susan Lin, Shyni Varghese, UC San Diego

Developing cost-effective and scalable synthetic matrices for long-term expansion of human pluripotent stem cells (hPSCs) is an important intermediate step to meet the need for large cells required to realize their applications, ranging from drug screening platforms to regenerative medicine. Here, we report the development of a synthetic hydrogel comprising of heparin mimetic moieties, capable of supporting long-term expansion of hPSCs for over 20 passages in chemically defined Stempro           medium. HPSCs expanded on these hydrogels maintained their characteristic morphology, colony forming ability, karyotypic stability, and differentiation potential. HPSCs expanded on this synthetic matrix exhibited pluripotency markers comparable to those cultured on Matrigel. Additionally, we report the correlation between various material properties, such as functional group, hydrophobicity, charge density, and rigidity, on various cellular responses of hPSCs. The observed cellular responses are explained through matrix mediated binding of ECM proteins and growth factors, along with subsequent upregulation of specific adhesion molecules in adhered hPSCs. Such synthetic matrices comprising of off-the shelf components are easy to synthesize and do not require any sophisticated processing thus making them cost-effective and translational.  Beyond this promising translational application, synthetic matrices with defined bulk and interfacial properties are an ideal tool to probe the molecular mechanisms that control fate and commitment of hPSCs.

Key words: Self-renewal , Pluripotency, Human pluripotent stem cells (hPSCs), Synthetic matrices, Hydrogels

18. Elucidation of Novel RNA Editing Mechanisms Driving Human LSC Generation

Qingfei Jiang, Alice Shih, Daniel Goff, Angela Court-Recart, Heather Leu, Wenxue Ma, Kristen Smith, Anil Sadarangani, Ifat Geron, Catriona Jamieson, UC San Diego

Our research focuses on dissecting the role of RNA editing in both normal HSC development and the progression of human chronic myeloid leukemia (CML) from chronic phase (CP) to blast crisis (BC). Our qRT-PCR data suggest that blast crisis LSCs harbor higher levels of IFN responsive ADAR1 p150 isoform than chronic phase progenitors and normal cord blood progenitors (p=0.014). In vitro study of lentiviral ADAR1 p150 transduced progenitors from normal cord blood and chronic phase shows a significant change for preferred differentiation to GMP (Granulocyte-macrophage progenitor) population, which has been shown to be the leukemia stem cells in CML. A similar inclination was observed in lentiviral shRNA ADAR1 transducer progenitors from blast crisis phase and chronic phase. ADAR1 may also play a role in self-renewal, as a significant of decrease in self-renewal capacity was observed in shRNA transducer chronic phase progenitors. The preliminary data suggests a crucial role for ADAR1 in both cell differentiation and self-renewal of hematopoietic stem cells.

Key words: RNA editing, CML progression

19. Modulation of Synapse Number in Human iPS Cell-derived Neurons by Neuroligin-4

Ji-Eun Kim, Matthew O’Sullivan, Anirvan Ghosh, UC San Diego

We have previously described a protocol to direct the differentiation of human induced pluripotent stem cells (iPSCs) into forebrain neurons by exposing them to developmentally relevant signals.  Human iPSC-derived neurons (or ‘iPS-neurons’) mature into electrophysiologically functional neurons and display a range of characteristics typical of forebrain neurons.  Human iPS-neurons may be applied in studies investigating molecular mechanisms of human neurodevelopmental diseases such as autism.  We have used them in a synapse induction assay where iPS-derived neurons are co-cultured with HEK293T cells that have been transfected with synapse-inducing molecules such as the Neuroligins.  Human iPS-neurons are able to efficiently form Neuroligin-induced synapses onto HEK293T cells.  The synapse-inducing ability of autism-linked mutants of Neuroligin-4 (NLGN4) can be directly compared in the same assay.  NLGN4 overexpression in iPS-neurons resulted in enhanced density of presynaptic structures in three-week-old neurons. Conversely, NLGN4 knockdown in the iPS-neurons resulted in a decrease in density of synapsin-positive puncta and an increase in puncta area.  These experiments provide further evidence that iPS-neurons are a robust experimental system to investigate the effects of human disease-related mutations in neuronal synapse formation and function.

Key words: Neuroligin, autism, synapse, iPS

20. bFGF Induces an Unfolded Protein Response During Early Human ES Development

Heike Kroeger, Oleg Sten, Jonathan Lin, UC San Diego

Human embryonic stem cells (hESCs) dramatically expand the production of proteins and lipids as they differentiate into terminal mature cell types. The Endoplasmic Reticulum (ER) is responsible for performing these vital processes. The Unfolded Protein Response (UPR) encompasses a family of intracellular signalling pathways that enable the ER to perform its functions in response to physiologic or pathologic demands by activating broad transcriptional and translational programs. In metazoan cells, three distinct ER resident transmembrane proteins, IRE1, PERK, and ATF6, are the molecular ‘gatekeepers’ of the UPR, and activate distinct signal transduction cascades. Here, we investigated the role of UPR signalling during early human embryonic stem cell differentiation from embryoid bodies into all three germ layers. We found a striking induction in IRE1, PERK, and ATF6 expression concomitant with the loss of pluripotency markers and the expression of differentiation markers.  We found that this induction was triggered in part by bFGF, whereby bFGF supplementation enhanced UPR induction, while bFGF signalling inhibition by PD173074 impaired UPR induction.  Last, we found that induction of UPR resulted in dramatic expansion of ER in mature germ layers.  Intriguingly, only PERK and ATF6 signaling were activated during this process.  Our data reveal a novel link between bFGF signalling and UPR signalling during hESC development.  Our data suggest that UPR signalling plays a vital role in hESC development by controlling growth and function of the ER in hESCs.

Key words: Unfolded Protein Response, Endoplasmic Reticulum Stress, bFGF signaling, hESC, Differentiation

21. Efficient Correction of Hemoglobinopathy-causing Mutations by Homologous Recombination in Integration-free Patient iPSCs

Mo Li, Keiichiro Suzuki, Jing Qu, Jungmin Lee, Ilir Dubova, Preeti Saini, Fei Yi, Ignacio Sancho-Martinez, Guanghui Liu, Salk Institute for Biological Studies

Mutations of the HBB gene lead to the development of hemoglobinopathies, such as sickle cell disease and beta-thalassemia. In particular, sickle cell disease is one of the most prevalent devastating monogenic diseases in the world. Efforts to cure the disease by viral vector-based gene therapy have met serious obstacles. The advent of induced pluripotent stem cell (iPSC) technology offers an exciting possibility of developing cell-replacement therapies based on patient-specific iPSCs, in which the disease-causing mutations could be repaired. By using a novel gene-targeting approach based on the use of Helper-Dependent Adenoviral Vector (HDAdV) in combination with improved episomal-based reprogramming strategies, in here we describe for the first time the efficient (up to 100%) and safe correction of disease-specific mutation on the HBB gene in transgene- and integration-free patient derived iPSCs.

Key words: gene editing, induced pluripotent stem cells, Sickle cell disease, integration-free reprogramming

22. The Role of ‘Np63’ in Human Cytotrophoblast Stem Cell Proliferation and Lineage Specification

Yingchun Li, UC San Diego

The main functional component the placenta is the trophoblast, which is derived from extraembryonic ectoderm. Little is known about the trophectoderm in the human embryo, as human trophoblast stem cells have yet to be isolated and characterized. We have previously identified p63 as a marker of proliferative trophoblast in human placental tissues.  Specifically, based on immunohistochemical staining, p63 is expressed in all Ki67-positive trophoblast, including cytotrophoblast in the floating chorionic villi and cytotrophoblast cell columns in anchoring villi; however, it is absent in differentiated trophoblast, both syncytiotrophoblast of the chorionic villi and the invasive extravillous trophoblast in the placental bed. Here, we have further studied its role in trophoblast proliferation, using the human trophoblast cell lines, BeWo and HTR8, and primary cytotrophoblast (CTB) isolated from first trimester or term placental tissue. BeWo cells can be induced to differentiate into syncytiotrophoblast by treatment with forskolin, while primary CTB differentiate spontaneously to syncytiotrophoblast over a 7-day period in culture. We show that p63 expression in all these cells is limited to the delta-Np63-alpha isoform and is lost as the cells (both BeWo and primary CTB) differentiate into syncytiotrophoblast.  We generated lentivirus, expressing the delta-Np63-alpha isoform, and showed that overexpression of this protein leads to increased trophoblast proliferation, as measured by BrdU uptake. Finally, p63 expression is induced in human embryonic stem cells, following differentiation into trophoblast lineage by short-term treatment with BMP4.  We hypothesize that p63 plays an important role in both trophoblast proliferation and lineage specification.

23. Examination of Cell Signaling Pathways During Early Endoderm Specification from Human Embryonic Stem Cells Utilizing Fluorescence Cell Barcoding and Intracellular Flow Cytometry

Nil Emre, Jason Vidal, Rosanto Paramban, Khooshbu Shah, John Apgar, Christian Carson, BD Biosciences

The ability of human embryonic stem cells (hESCs) to differentiate into cell lineages allows for the study of developmental biology and has applications in regenerative medicine and cellular therapy. To optimize differentiation protocols and answer biological questions, it is important to understand signaling pathways that are involved in lineage specification. We applied a multiparametric flow cytometric screening approach to address this problem in early endoderm differentiation from hESCs.  We screened cells at different time points of endoderm differentiation by flow cytometry for the activation of different cell signaling pathways. We tested multiple monoclonal antibodies to activated signaling molecules and correlated their expression to cells expressing either pluripotency or endoderm markers to delineate differentiation status. Pathways investigated included MAPK/ERK, Jak/Stat, PI3/AKT, and Wnt/b-catenin. Our data suggest a complex signaling network for endoderm specification of hESCs in culture. This screening methodology can readily be applied to various stem cell populations and their derivatives to explore cell signalling events such as self-renewal, reprogramming, and lineage specification.  The identification of cellular pathways involved in these events will not only aid in the understanding of biology but also enable the use of small molecule inhibitors to replace or modify endogenous cell signaling to increase efficiency of self-renewal or differentiation.

Key words: Flow cytometry, Pluripotent stem cells, endoderm, cell signalling, fluorescence cell barcoding

24. Examining the Role of Cancer Stem Cells in Micro-metastasis Formation by Direct in Vivo Visualization

Frederick Park, Konstantin Stoletov, Richard Klemke, UC San Diego

Many cancers are thought to be initiated by a small subset of stem-like tumor cells termed cancer stem cells (CSCs).  It has been postulated that metastatic tumors are likewise initiated by CSCs that have gained the additional ability to migrate away from the primary tumor and seed distant sites. However, it remains unclear which tumor cells initiate metastases and little is known about the factors within the metastatic niche that permit metastasis-initiating cell survival and proliferation.

To address these questions, we have developed unique animal models using the chick embryo and zebrafish to directly visualize tumor cell extravasation and micro-metastasis formation in vivo with single-cell resolution.  In both the chick and zebrafish models, we find that Wnt activity, as indicated by the TOP-GFP reporter, is dynamically upregulated during extravasation and formation of micro-metastases.  Since Wnt activity correlates positively with tumor-initiating ability, this suggests that extravasation and metastasis initiation may involve a transition toward stem-like identity.  Next, since connexins are directly upregulated by the Wnt pathway and since stem cell survival in certain developmental contexts requires gap junctions, we hypothesized that CSCs require connexin-mediated interactions to survive in the metastatic niche.  Indeed, using our chick model, survival of dormant, non-proliferating metastatic cells required connexin-mediated interaction with the vasculature.  This has direct implications for cancer recurrence after chemotherapy, which has been attributed to a sub-population of dormant, treatment-refractory, stem-like cells.  We are now preparing to extend these studies using primary human cancer cells transduced with fluorescent stem cell pathway reporters.

Key words: cancer, stem cells, metastasis, extravasation, niche, wnt, top-gfp, connexins

25. The Histone Demethylase LSD1 is Required to Endocrine Cell Formation in the Developing Pancreas

Nisha Patel, UC San Diego; Kevin D’Amour, Allan Robins, ViaCyte Inc.; Michael Rosenfeld, Maike Sander, UC San Diego

Much effort is being expended on attempts to derive transplantable insulin-producing beta-cells from human embryonic stem cells (hESCs) for treating diabetes mellitus. However, as current protocols still fail to support the generation of functional beta-cells, a better understanding of the molecular mechanisms underlying beta-cell development will be necessary to achieve this goal. The transcriptional network regulating neogenesis of beta-cells and other endocrine cell types in the embryonic pancreas has been comprehensively dissected. However, chromatin remodeling events which regulate the pancreatic transcriptional program to orchestrate proper endocrine cell development remain largely unexplored. The chromatin modifying enzyme, LSD1, has been shown to be required for lineage specification in several tissues. To examine the role of LSD1 in the developing pancreas, we utilized both mouse genetic approaches and a parallel hESC-based in vitro system of pancreatic cell differentiation. Using the first model, we found that inactivation of LSD1 in mice abrogates pancreatic endocrine cell differentiation. Extending this finding, spatially-controlled inactivation of LSD1 in the developing pancreas revealed that endocrine cell development requires LSD1 activity in multipotent, but not in endocrine-committed, pancreatic progenitor cells. Furthermore, chromatin-immunoprecipitation experiments on hESC-derived multipotent pancreatic progenitors in our second model demonstrated that LSD1 occupies regulatory sequences of the key transcription factors for endocrine fate commitment and differentiation. Taken together, these findings suggest that LSD1 governs pancreatic endocrine cell formation by regulating endocrine lineage-specific gene expression programs in multipotent pancreatic progenitor cells.

Key words: LSD1, Pancreas, Development

26. Cell Autonomous Phenotypes of iPSC-Derived Neurons from Alzheimer’s Disease Patients

Sol Reyna, Grace Woodruff, Mason Israel, Shauna Yuan, Lawrence Goldstein, UC San Diego

A limiting factor for modeling familial (fAD) and sporadic Alzheimer’s Disease (sAD) with human induced pluripotent stem cells (hIPSC) is the efficient and rapid generation of multiple lines per patient. Because of the need for more hIPSC lines, we adapted the recently published multiplex technique of fluorescent cell barcoding (FCB) to efficiently screen and identify the ‘best’ hIPSC colonies (Krutzik & Nolan, 2006). Criteria for selection includes high expression of a combination of pluripotency markers (tra-1-81, tra1-60, Nanog), the absence of differentiation markers, retroviral silencing, a normal karyotype, and capacity to efficiently differentiate into all three germ layers. The generation of hIPSC lines from many patients would provide the numbers to address the causative contribution of human genetic variability in both fAD and sAD.

The predominant hypothesis in the AD field implicates toxic amyloid-beta (A-beta) peptides in driving neuronal phenotypes. This hypothesis largely stems from the knowledge that the genes altered in fAD patients are involved in the processing of amyloid precursor protein and the generation of its secreted peptide, A-beta. Studies in the field, however, suggest that neuronal autonomous events could also be taking part. Specifically, it has been repeatedly observed that AD neurons contain enlarged early endosomes and abnormal lysosomes. In our AD hIPSC-derived neurons, we see evidence for both of these phenotypic alterations.

Key words: disease modeling, iPS, Alzheimer’s Disease, genetics

27. Modeling Schizophrenia Using Human Induced Pluripotent Stem Cells

Anthony Simone, Ngoc Tran, Kristen Brennand, Jessica Jou, Chelsea Gelboin-Burkhart, Lizzy Hoffman, Kameron Black, Sarah Sangar, Yan Li, Yangling Mu, Fred Gage, Salk Institute for Biological Studies

Schizophrenia (SCZD) is a debilitating neurological disorder with a world-wide prevalence of 1%; there is a strong genetic component, with an estimated heritability of 80-85%. Though postmortem studies have revealed reduced brain volume, cell size, spine density and abnormal neural distribution in the prefrontal cortex and hippocampus of SCZD brain tissue and neuropharmacological studies have implicated dopaminergic, glutamatergic and GABAergic activity in SCZD, the cell types affected in SCZD and the molecular mechanisms underlying the disease state remain unclear.  To elucidate the cellular and molecular defects of SCZD, we directly reprogrammed fibroblasts from SCZD patients into human induced pluripotent stem cells (hiPSCs) and subsequently differentiated these disorder-specific hiPSCs into neurons. SCZD hiPSC neurons showed diminished neuronal connectivity. Gene expression profiles of SCZD hiPSC neurons identified altered expression of many components of the cAMP and WNT signaling pathways. Key cellular and molecular elements of the SCZD phenotype were ameliorated following treatment of SCZD hiPSC neurons with the antipsychotic Loxapine.

Key words: stem cells, schizophrenia, neurons

28. Identification of Cell Surface Marker Signatures of Distinct Cell Subpopulations Defined by Expression of Sox1, Sox2, Pax6 and Doublecortin in Neural Induction Cultures of Human Embryonic Stem Cells

Jason Vidal, Jody Martin, Nil Emre, Christian Carson, BD Biosciences

The differentiation of human pluripotent stem cells to neural ectoderm presents an opportunity to study human neurogenesis and neurodegenerative diseases.  Previous work has demonstrated that it is possible to identify distinct cell populations that represent developmental transition points in neural induction cultures.  However, little is known about the extent of all of the different cell populations and their significance in modeling neural development and disease and their relevance for therapeutic applications. We combined intracellular marker expression with cell surface immunophenotyping to identify prospective cell populations in neural induction cultures.  We analyzed cells for expression of neural stem cell markers Pax6, Sox1 and Sox2 and the neuronal marker doublecortin (DCX) by intracellular flow cytometry.  We were able to detect 4 distinct cell populations: Pax6+/Sox2-/Sox1-/DCX-, Pax6+/Sox2+/Sox1+/DCX-, Pax6-/Sox2+/Sox1+/DCX- and Pax6-/Sox2-/Sox1-/DCX+.  We performed a large unbiased screen of 242 antibodies to cell surface markers to identify cell surface signatures of each cell population and identified putative signatures for Pax6+/Sox2-/Sox1-/DCX-, Pax6+/Sox2+/Sox1+/DCX- and Pax6-/Sox2-/Sox1-/DCX+ cell populations. These cell surface markers will permit the isolation and subsequent analysis of these cell populations and add to the growing list of markers that can be used to identify neural cell types derived from pluripotent stem cells.

Key words: Pluripotent stem cells, neural stem cells, flow cytometry, Cell surface markers, Differentiation

29. A Novel TGFb Selective Inhibitor Drives Cardiogenesis Specifically in ESC

Erik Willems, Joaquim Teixeira, Sanford-Burnham Medical Research Institute; Dennis Schade, Marion Lanier, Human Biomolecular Research Institute; Paul Bushway, Sanford-Burnham Medical Research Institute; John Cashman, Human Biomolecular Research Institute; Mark Mercola, Sanford-Burnham Medical Research Institute

High throughput screens on mouse embryonic stem cells (mESC) to discover small molecules were run to probe different steps of cardiac differentiation.  Over 17000 molecules were screened using an image-based setup relying on a cardiac specific Myh6-GFP reporter to monitor cardiogenesis.  One of the identified molecules was a dihydropyridine (DHP), which are well-known L-type calcium channel inhibitors.  To tie the activity of DHP to a pathway, 40 well-known receptor tyrosine kinase inhibitors were screened and identified Activin A/TGFb as a key pathway that had to be inhibited to allow cardiogenesis.  Subsequent pathway analysis in combination with an SAR study of over 200 DHP analogs pinpointed the activity to the Activin A/TGFb pathway, with high inhibition efficiency for TGFb signals. Further functional characterization of the DHP in the TGFb pathway positioned the activity at the receptor level, where DHP downregulates the amount of TGFb receptors on the cell surface.  This TGFb specific inhibitor was then applied in the mESC assay to probe the effects of inhibiting TGFb specifically at several stages of differentiation.  When given early in differentiation, DHP blocked all mesoderm, indicating that TGFb is essential for mesoderm induction.  Secondly, when DHP was added during mesoderm patterning, cardiogenesis was enhanced specifically whereas other cardiovascular lineages such smooth muscle, blood and endothelial cells were not affected.

Key words: embryonic stem cells, small molecules, Tgfb inhibitor, cardiogenesis, high content screening

30. To Combine Episomal and Chemical Approaches to Reprogram Somatic Cells into Integration-free iPSCs

Zhen-Ning Zhang, Tongbiao Zhao, Zhe Li, Saiyong Zhu, Sheng Ding, Kun Zhang, Yang Xu, UC San Diego

Induced pluripotent stem cells (iPSCs) are generated from the somatic mammalian cells by expressing defined transcription factors. IPSCs derivatives have promising prospect for disease models and cell therapies. However, the major obstacles for iPSC applications are the persistence of genetic and epigenetic abnormalities during reprogramming, which increase genome instability and tumorigenic potential. Significant progress has been achieved recently in generating integration-free iPSCs with episomal approach and chemical biology approach. Here we show that combining episomal approach and a small chemical cocktail can greatly increase the reprogramming efficiency of mouse embryonic fibroblasts and human fibroblasts into integration-free iPSCs. By exome sequencing, we found that our approach induces fewer genomic mutations when compared to previous methods. Thus, the cooperation of episomal and chemical biology approaches represents useful strategy to effectively reprogram somatic cells into integration-free iPSCs with minimized somatic gene mutations.

Key words: iPSCs, episomal approach, chemical biology, genetic mutation

31. Immunogenicity of Induced Pluripotent Stem Cells

Tongbiao Zhao, Zhenning Zhang, Zhili Rong, Yang Xu, UC San Diego

Induced pluripotent stem cells (iPSCs), reprogrammed from somatic cells with defined factors, hold great promise for regenerative medicine as the renewable source of autologous cells. While it has been generally assumed that these autologous cells should be immune tolerated by the recipient from whom the iPSCs are derived, the immunogenicity of the cells derived from iPSCs has not been vigorously examined. We show here that while embryonic stem cells (ESCs) derived from inbred C57BL/6 (B6) mouse can efficiently form teratomas in B6 mice without any evident immune rejection, the allogeneic ESCs from 129/SvJ mice fail to form teratomas in B6 mice due to rapid rejection by recipients. B6 mouse embryonic fibroblasts (MEFs) were reprogrammed into iPSCs by either retroviral approach (ViPSCs) or a novel episomal approach (EiPSCs) that causes no genomic integration. In contrast to B6 ESCs, teratomas formed by B6 ViPSCs were mostly immune rejected by B6 recipients. In addition, the majority of teratomas formed by B6 EiPSCs were immunogenic in B6 mice with T cell infiltration while apparent tissue damage and regression was observed in a small fraction of teratomas. Global gene expression analysis of teratomas formed by B6 ESCs and EiPSCs revealed a number of genes commonly overexpressed in teratomas derived from EiPSCs, and several such gene products are shown to directly contribute to the immunogenicity of the B6 EiPSC-derived cells in B6 mice. These findings indicate that, in contrast to derivatives of ESCs, abnormal gene expression in some cells differentiated from iPSCs can induce T-cell-dependent immune response in syngeneic recipients. Therefore, the immunogenicity of therapeutically valuable cells derived from patient specific iPSCs should be evaluated before any clinical application of these autologous cells into the patients.

Key words: Immunogenicity; Induced pluripotent stem cells; T cell-dependent immune response; Episomal vector

32. Use of a Cell Based Screening Device to Decipher Complex Cell Microenvironment Interactions

Naira Serobyan, Justin Bingham, Marie Zhang, MicroStem, Inc.

Stem cells exist in a highly complex extracellular environment in vivo composed of growth factors, cytokines, small molecules and extracellular matrix (ECM) proteins which have been shown to play an important role in controlling cell behavior, such as self renewal and differentiation. When stem cells are cultured in vitro, it is necessary to recapitulate such microenvironment in order to provide physiologically relevant conditions. Current in vitro methods require a large number of cells as well as significant time and cost commitment.

MicroStem, Inc. has developed a cell screening technology that deciphers and leverages optimal ECM combinations as a method for immobilizing live cells in a physiologically relevant state for high content immuno-fluorescent study of cellular function and behavior. The technology is especially powerful for studying rare cell populations, such as stem cells isolated from human tissue. We have utilized this technology to study lung cancer stem (CD133+) cells isolated from patients and human mesenchymal stem cells isolated from bone marrow of healthy donors. Use of the technology led to determination of ECM proteins individually and in combinations that promote cell growth (cancer stem cells) and differentiation (mesenchymal stem cells) respectively. This unique technology allows researchers to screen hundreds of ECM components simultaneously to study stem cell behavior in a cost and time effective manner.

Key words: extracellular matrix, cell microenvironment, mesenchymal stem cell, cancer stem cell, high content screening

33. Stem Cell Modeling to Define the Molecular Basis of Spinocerebellar Ataxia Type 7

Jacqueline Ward, Alysson Muotri, Albert La Spada, UC San Diego

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disorder caused by a CAG/polyglutamine (polyQ) repeat expansion. It is characterized by cerebellar and retinal degeneration, which begins with a loss of color vision and central visual acuity, and ultimately progresses to complete blindness. Studying the biology of SCA7 and other neurological diseases is particularly difficult, as isolating the affected human neural cell types for in vitro studies is prohibitive. Although SCA7 mouse models have provided key insights into mechanisms of disease pathogenesis, marked differences between mouse and human at the molecular, phenotypic, and therapeutic levels have revealed the need for a more representative model system to study this disease. I hypothesize that using patient-derived induced pluripotent stem cells (iPSCs) to derive affected cell types will allow me to develop powerful new models, and thereby gain novel insights into the molecular basis of SCA7 neurodegeneration. My initial goal is to generate a stem cell model system for SCA7 by reprogramming patient-derived fibroblasts into iPSCs. I will differentiate these pluripotent stem cells into neural precursor cells (NPCs), and from the NPCs, derive retinal photoreceptors. I will fully characterize the molecular and cellular phenotypes for SCA7-derived retinal photoreceptors cells to assure that they recapitulate SCA7-associated features. As ataxin-7 is a core component of the STAGA transcription co-activator complex, I will study the transcription dysregulation that is believed to be a key factor in SCA7 disease pathogenesis. This will be accomplished by performing an expression comparison between control and SCA7-derived photoreceptors using RNA-Seq, and by completing genome-wide location analysis by ChIP-Seq to define alterations in ataxin-7 occupancy. Bioinformatics analysis of these results will allow me to identify novel targets and pathways involved in SCA7 retinal degeneration. Taken together, these studies should advance our understanding of the molecular basis of SCA7 retinal degeneration.

Key words: spinocerebellar ataxia 7; trinucleotide repeat; induced pluripotent stem cells

34. Modeling Lissencephaly in Patient-Derived iPS Cells

Ashleigh Schaffer, Brian Cattaneo, Stephanie Bielas, UC San Diego; Joseph GleesonUC San Diego and Howard Hughes Medical Institute

One of the most severe neurological disorders, Classical Lissencephaly (LIS), arises when developing neural progenitor cells (NPCs) fail to migrate to stratify the cerebral cortex lamina properly. The process of neural migration in humans begins early, at gestational age 7 weeks. Hence, patients with LIS are not typically diagnosed with the disorder until after the cortex has incorrectly developed, severely limiting treatment. Potential future therapies for LIS, such as genetic prenatal diagnosis or manipulation of defective gene regulatory networks during development, require extensive understanding of the genetic, cellular, and molecular mechanisms controlling neural migration. Traditionally, this knowledge was obtained by analyzing gene-deficient mice; however, phenotype comparisons between LIS1-deficient LIS patients and Lis1 mutant mice have shown that the genetic pathways underlying neural migration have redundancy in mice compared to humans. In addition, defects in neural migration may be more readily detectable in humans versus mice due to the extended distance neurons must travel in a human cortex to reach their target locations. Therefore, in order to understand the underlying cause of LIS and improve patient therapies, I have generated a human cell based, in vitro model for this disorder.

35. Using High-Throughput Screening Methods to Identify Optimal Conditions for Stepwise Differentiation of Human Embryonic Stem Cells to Insulin-Producing Beta Cells

Nathan Kumar, David Brafman, Karl Willert, UC San Diego

The directed differentiation of human pluripotent stem cells (hPSCs) into insulin-producing beta-cells provides a potential source of cells for Type I diabetes cell replacement therapy. We and others have developed a five stage protocol in which hPSCs are sequentially differentiated to definitive endoderm (DE; STAGE1), primitive gut tube (PGT; STAGE2), posterior foregut (PFG; STAGE3), pancreatic progenitor (PP; STAGE4), and finally hormone producing endocrine cells (EC; STAGE5). However, the efficiency of differentiation is highly variable among distinct hPSC lines and to date functionally mature beta cells have not been produced in vitro. This suggests additional factors are required for the efficient differentiation of these cells. We first investigated the role of several signaling pathways and found that treatment with Wnt3a increased differentiation in a concentration dependent manner. Furthermore, screening of small molecules revealed that CHIR98014, a GSK-3β inhibitor, was able to effectively replace Wnt3a. In another line of investigation, we are employing a novel arrayed cellular microenvironment platform to screen conditions that promote DE expansion. To that end we have obtained an hPSC line in which enhanced green fluorescent protein (eGFP) has been introduced into the SOX17 locus, so that expansion of a DE cell population can be monitored in real time. Lastly, using a combination of three cell surface markers we have developed a FACS-based assay to isolate a population of cells with characteristics of PGT/Stage2. We are currently establishing assays to perform a cellular microarray screen that promotes the differentiation of these cells to later stages, in particular PFG/Stage3. In sum, these studies set the stage for identifying the optimal conditions for the stepwise generation of pancreatic progenitors from hPSCs.

36. Sourcing Human Blood-Derived Raw Material: Optimization, Qualification and Control

Pete van der Wal, Anna Stock, HemaCare Corporation; Scott Burger, Advanced Cell & Gene Therapy LLC

Human cells and tissue are critical raw material for cell therapy, tissue-engineered, and ex vivo gene therapy products. Quality of this cellular raw material is a major determinant of final product characteristics, but is complicated by the inherent heterogeneity and inter-individual variability of living biologics. Inconsistent collection procedures amplify this variability, adversely affecting the manufacturing process from the outset. Controlling and qualifying the cell collection step is essential to minimizing operational variability, and greatly increases the likelihood of success in manufacturing. HemaCare’s core competency is apheresis collection, and, building on 34 years of experience, has addressed this need for optimized cell collection with a program for controlling and qualifying its apheresis procedures and collection sites, to supply human-derived blood components for development and qualification of novel cell and gene therapies, assays, and medical devices. HemaCare’s apheresis program includes comprehensive staff qualification and training, documentation that supports its cGMP environment and programs to monitor effectiveness of equipment and procedures in accordance with an established quality system. Donor recruitment, screening and IRB-approved consents follow the requirements of Good Tissue Practices (GTPs). From 2006-2010, 55,262 apheresis procedures were performed, including collection of patient and normal-donor peripheral blood mononuclear cells, mobilized peripheral blood progenitor cells, and plateletpheresis products, to support clinical studies spanning Phase I-Phase III, preclinical research, and, for the first time, commercial cell therapy applications.

Key words: apheresis collection, cellular therapy, immunotherapy, cell collection, human-derived blood components, phase I-III, preclinical research, cell therapy manufacturin

37. Functional Consequences of Somatic Mutation in Human Induced Pluripotent Stem Cells

Athurva Gore, UC San Diego; Sergio Ruiz, Salk Institute for Biological Studies; Zhe Li, Ho-Lim Fung , Juan Carlos Izpisua Belmonte, Salk Institute for Biological Studies; Kun Zhang, UC San Diego

Defined transcription factors can induce epigenetic reprogramming of adult mammalian cells into induced pluripotent stem cells (iPSCs). Recent studies revealed that human iPSC lines acquire chromosomal rearrangements after long-term culture and single nucleotide point mutations enriched in cancer-related genes after short-term culture. However, the exact source of reprogramming-associated mutations remains unclear. It is also unknown if mutations are functional “drivers” for the reprogramming process or if they are “passengers” acquired due to clonal selection. Here we show that iPSC lines acquire point mutations regardless of donor age, progenitor cell type, or amount of passaging, implying that multiple mechanisms are responsible for reprogramming-associated mutations. We also demonstrate that clonal selection alone is not responsible for mutations.  We functionally characterized the effects of several mutations in key cancer and pluripotency genes, and determined that most appear to be nonfunctional during the process of reprogramming. Our results emphasize the need for further rigorous work on mutational load during iPSC generation. Extensive genetic screening should become a standard procedure to ensure stem cell safety before clinical use.

Key words: Induced Pluripotent Stem Cells, Somatic Mutation, Genomic Integrity

38. A Genome-wide Epigenetic Map Unveils Chromatin States of Pancreatic Cell Differentiation from Human Embryonic Stem Cells

Ruiyu Xie, UC San Diego; Logan Everett, Hee-Woong Lim, University of Pennsylvania; Nisha Patel, Chia-Yao Lee, Allen Wang, UC San Diego; Jonathan Schug, University of Pennsylvania; Kevin D’Amour, Evert Kroon, Olivia Kelly, Allan Robins, ViaCyte, Inc.; Kyoung-Jae Won, Klaus Kaestner, University of Pennsylvania; Maike Sander, UC San Diego

Epigenetic events control the transcriptional program of each cell by regulating chromatin structure. In undifferentiated human embryonic stem cells (hESCs), histones at gene loci of key developmental regulators are ‘bivalent’, carrying both the activating H3K4me3 and repressive H3K27me3 modification. This bivalent mark (H3K27me3 and H3K4me3) poises genes for rapid activation of transcription during cell differentiation by removal of the repressive H3K27me3 mark. However, what is still unclear is when bivalent states are resolved during the progression from the pluripotent to the terminally differentiated state, or whether this mechanism is relevant for restricting the plasticity of progenitors during their developmental progression.

To investigate these important questions during pancreatic endocrine differentiation, we utilized a stepwise differentiation protocol of hESCs into pancreatic endoderm (PE), which was subsequently transplanted for further differentiation into mature insulin-producing beta-cells. By performing RNA-seq and ChIP-seq analysis on eight distinct cell populations during the differentiation path, we have unveiled the global changes in chromatin modifications that are associated with lineage progression towards the beta-cell. We uncover that polycomb group (PcG) proteins, which mediate H3K27 trimethylation, govern each developmental step during the progression of hESCs towards the terminally differentiated state. PcG derepression underlies activation of definitive endoderm (DE), PE, and beta-cell genes, respectively, while endodermal genes and genes involved in the development of non-pancreatic lineages are PcG-repressed during lineage progression towards pancreas. This suggests that PcG proteins play on important role in restricting cell plasticity during embryonic development. Stage-specific manipulation of PcG complexes could prove an effective strategy to induce robust pancreatic and (beta-cell differentiation from a variety of hESC and human induced pluripotent stem cell (iPSC) lines.

Key words: Pancreas differentiation, hESCs, histone modification, PcG repression

39. Generating Patient Specific Hepatocytes by Reprogramming Human Fibroblasts from Patients with Alpha-1 Antitrypsin (AAT) Deficiency to Probe Mechanisms of Liver Failure and Liver Disease