Poster Session

Scientific Poster Abstracts

See the 36 poster abstracts below that were on display at the 2018 Scientific Symposium.

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2018 Poster Abstracts

1. Transplantation with GXHPC1 for Liver Cirrhosis: Phase 1 Trial

Presented by: Po-Cheng Lin, Gwo Xi Stem Cell Applied Technology
Authors: Huang, Ko-Chang, China Medical University Beigan Hospital; Chuang, Ming-Hsi, Chung Hua University; Lin, Yi-Chun, Gwo Xi Stem Cell Applied Technology; Chiou, Tzyy-Wen, National Dong Hwa University; Harn, Horng-Jyh, Buddhist Tzu Chi General hospital; Lin, Po-Cheng , Gwo Xi Stem Cell Applied Technology; Lin, Shinn-Zong, Buddhist Tzu Chi General hospital

Currently, the only effective therapy for cirrhosis of the liver is liver transplantation. However, finding a compatible liver is difficult due to the low supply of healthy livers and the ever-increasing demand. However, stem-cell therapy may offer a solution for liver cirrhosis. For example, GXHPC1 therapy preparation that contains adipose-derived mesenchymal stem cells (AD-MSCs) and was developed for treatment of liver cirrhosis. In our previous report, animal studies suggested that treatment of a diseased liver via GXHPC1 transplantation can abrogate liver fibrosis and facilitate recovery of liver function. In our current human trial, patients with liver cirrhosis were included. Their adipose tissue was harvested from the subcutaneous fat of the abdominal wall during surgery. AD-MSCs were cultured and suspended at a concentration of 100 million cells in one milliliter of physiological saline (i.e., GXHPC1). This human study passed the Taiwan Food and Drug Administration IND inspection and received the Phase I clinical trial permission. The trial was conducted with six subjects with liver cirrhosis to demonstrate the safety and efficacy of administering GXHPC1. Intrahepatic injection of GXHPC1 did not cause any safety issues in the analysis of adverse drug reactions and suspected unexpected serious adverse reactions and showed a tendency for improvement of liver function, METAVIR score, Child-Pugh score, MELD score, and quality of life for subjects with liver cirrhosis.

Key Words: stem-cell therapy, adipose-derived mesenchymal stem cells, Intrahepatic injection, liver cirrhosis

2. Multiparametric Signature of Glioblastoma Differentiation Revealed by Imaging of Cellular Epigenetic Landscapes

Presented by: Chen Farhy, Sanford Burnham Prebys Medical Discovery Institute
Authors: Farhy, Chen, Sanford Burnham Prebys Medical Discovery Institute; Hariharan, Santosh, Sunnybrook Research Institute; Ylanko, Jarkko, Sunnybrook Research Institute; Andrews, David W., Sunnybrook Research Institute; Terskikh, Alexey, Sanford Burnham Prebys Medical Discovery Institute

The resistance of glioblastoma multiform (GBM) to conventional cytotoxic drugs has prompted novel therapeutic strategies, including differentiating tumor propagating cells (TPCs) into less tumorigenic cells using small molecule inducers of TPC differentiation. However, high-throughput screening for such molecules is hampered by the lack of robust markers of GBM differentiation. To obtain a signature of differentiated TPCs, we developed “Microscopic Imaging of Epigenetic Landscapes” (MIEL), which captures patterns of nuclear staining for epigenetic marks to derive feature-fingerprints of individual cells. We confirmed MIEL’s ability to accurately distinguish multiple cell fates and identified a multiparametric epigenetic signature of differentiated TPCs. Critically, we validated epigenetic imaging-based signature using global gene expression thus providing the proof of principle for the MIEL’s ability to select and prioritize small molecules, which induce TPC differentiation.

Key Words: glioblastoma, cancer stem cells, epigenetics, high content analysis differentiation

3. Chondrocyte-derived Extracellular Matrix Facilitates Rapid Isolation and Expansion of Human Articular Chondrocytes with Minimal Loss of Phenotype

Presented by: Travis Block, StemBioSys
Authors: Block, Travis, StemBioSys; Mao, Yong, New Jersey Center for Biomaterials; Singh-Varma, Anya, New Jersey Center for Biomaterials; Sheldrake, Anne, StemBioSys; Leeth, Rachel, StemBioSys; Griffey, Sy, StemBioSys

A significant expansion of autologous chondrocytes in vitro is required for cell-based cartilage repair. However, the in vitro expansion of chondrocytes under standard culture conditions inevitably leads to the dedifferentiation of chondrocytes, and contributes to suboptimal clinical outcomes. To address this challenge, we focused our efforts on developing a better in vitro expansion condition, which shortens the expansion time with decreased dedifferentiation. It is well-known that the tissue microenvironment plays critical roles in regulating the cellular functions of resident cells and provides guidance in tissue-specific regeneration. We hypothesized that chondrocyte extracellular matrix (ECM) mimics a native microenvironment and serves as an ideal expansion condition in vitro. In this study, we prepared a decellularized ECM from allogeneic human articular chondrocytes (AC-ECM) and studied its effect on the in vitro expansion of primary human articular chondrocytes (HAC). As a comparison, an ECM derived from bone marrow stromal cells (BM-ECM) was also prepared. The differential composition and physical properties of these two ECM were analyzed by mass spectrometry and atomic force microscopy. Interestingly, very minor differences in composition manifested drastically different structure and mechanical properties as well as substantial differences in the phenotype of cells expanded on the substrates. Comparing with the standard tissue culture polystyrene (TCP), HAC cultured on AC-ECM and BM-ECM proliferated faster and reached a similar level of expansion in about 50% of time required for cells on TCP. The HAC showed dedifferentiation on all substrates. However, the ratio of COL2A1/COL1A1 remained the highest in cells cultured on AC-ECM. Furthermore, a pellet culture study demonstrated that cells expanded on AC-ECM produced a more cartilage-like ECM than cells expanded on BM-ECM or TCP. This is the first report on modulating chondrocyte expansion and dedifferentiation using cell type specific ECM. Ongoing work aims to optimize production of AC-ECM to support isolation and expansion of high quantity and quality HAC for cell-based therapies.

Key Words: autologous chondrocyte implantation, autologous chondrocyte transplantation, aci cartilage, chondrocyte, oa, osteoarthritis, tissue engineering, extracellular matrix, regenerative medicine, cell therapy niche

4. A Non-canonical BRD9-containing BAF Chromatin Remodeling Complex Regulates Naïve Pluripotency in Mouse Embryonic Stem Cells

Presented by: Jovylyn Gatchalian, Salk Institute for Biological Studies
Authors: Gatchalian, Jovylyn, Salk Institute; Malik, Shivani, Salk Institute; Ho, Josephine, Salk Institute; Lee, Dong-Sung, Salk Institute; Kelso, Timothy WR, Salk Institute; Shokhirev, Maxim, Salk Institute

The mammalian BRG1-associated factors (BAF) complex is a multi-subunit chromatin remodeling complex that is an important component of the embryonic stem cell (ESC) transcriptional regulatory network. However, the role of individual subunits in BAF complex targeting and function needs to be elucidated. Here, we find that the Bromodomain containing protein 9 (BRD9) and Glioma tumor suppressor candidate region gene 1 (GLTSCR1; also known as BICRA) or its paralog GLTSCR1-like (also known as BICRAL) define a smaller, non-canonical BAF complex (GBAF for GLTSCR1/1L-containing BAF complex) in mouse ESCs that is distinct from the canonical embryonic stem cell BAF (esBAF) and the polybromo-associated BAF (PBAF) complexes. GBAF lacks several esBAF subunits, including BAF47, BAF57 and ARID1A. We demonstrate that GBAF and esBAF complexes are targeted to different features of the genome and are co-bound with different sets of pluripotency transcription factors. Specifically, GBAF complexes co-localize with key regulators of naïve pluripotency, KLF4 and Sp5, on the genome. Consistent with this, we provide evidence that GBAF’s specific function is to regulate the transcription of genes involved in the maintenance of naïve pluripotency, including Nanog and Prdm14. Additionally, we show that BRD9 is displaced from chromatin by the selective BRD9 bromodomain inhibitor, I-BRD9, and that this leads to changes in target gene expression. The function of the GBAF complex in ESCs is highly correlated with the function of BRD4, consistent with an association between GBAF complexes and BRD4. We find that GBAF complexes are directly recruited to chromatin by BRD4 in a bromodomain-dependent fashion and we identify a set of I-BRD9- and JQ1-sensitive BRD9 binding sites that determine the functional similarity between these epigenetic regulators. Together, our results demonstrate functionally specific roles for BAF complex assemblies in maintaining the transcriptional network of pluripotency, highlighting the biological importance of complex heterogeneity.

Key Words: BAF complex, SWI/SNF, BRD9, naive pluripotency, chromatin remodeling, epigenetics

5. The Sanford Stem Cell Clinical Center: Accelerating Stem Cell Clinical Trials

Presented by: Jennifer Braswell, Sanford Stem Cell Clinical Center at UC San Diego
Authors: Braswell, Jennifer Briggs; Lawrence S.B. Goldstein, Catriona H.M. Jamieson, and David A. Brenner

In 2013, the Sanford Center saw that translating innovative stem cell research into patient diagnostics and therapy needed to accelerate. As the fifth year of the Sanford Center concludes, the Sanford Center reviews what it takes to accelerate complex stem cell clinical trials in an academic setting. Remarkable collaborations – across institutions, across disciplines, and with the private sector – are the key to accelerating the translation of scientific discoveries to patients. The commitment to develop trials from design-to-delivery requires operating through existing programs including UC San Diego Division of Regenerative Medicine, UC San Diego Stem Cell Program, CIRM Alpha Stem Cell Clinic at UC San Diego Health, Sanford Consortium for Regenerative Medicine, and the Sanford Surgical Training Center. The Sanford Gift cemented links between existing endeavors to catalyze design-to-delivery of stem cell clinical trials. Programs of the Sanford Center work through the departments of Medicine, Cancer Center, Bioengineering, Orthopedic Surgery, Animal Care, and other units at UC San Diego Health. Strong preexisting relationships enabled us to work quickly with known industry partners to mount UCSD’s first stem cell clinical trials. San Diego is a biotech hub for 1,100 life sciences companies and 80 independent and university-affiliated research institutes able to provide a critical ecosystem for operation of the Sanford Stem Cell Clinical Center.

Key Words:stem cells, clinical trials, academic cGMP, regenerative medicine, academic medicine

6. SWI/SNF Complex Mutations in Intellectual Disabilities

Presented by: Fangjian Gao, Salk Institute for Biological Studies
Authors: Gao, Fangjian, Salk Institute for Biological Studies; Elliott, Nicholas, Salk Institute for Biological Studies; Hargreaves, Diana, Salk Institute for Biological Studies

SWI/SNF chromatin remodeling complexes play pivotal roles in regulating embryonic development and lineage specific cell identity. Mammalian SWI/SNF complexes, also known as BAF complexes, are large protein complexes that incorporate up to 15 subunits around a core ATPase, either BRG1 or BRM. There are a total of 29 genes that encode those subunits. Some are homologs from gene families that are alternatively used in specific cell lineages during development. The expression of each subunit is tightly controlled at each stage of development. For instance, during neural differentiation from embryonic stem cells (ESCs) to neurons, BRG1 is downregulated while BRM is upregulated. BAF complexes are ATP-dependent chromatin remodelers which are thought to use energy from ATP hydrolysis to move or displace histone octamers and alter the open/closed state of chromatin. Interestingly, heterozygous mutations in the BRM ATPase domain cause Nicolaides-Baraitser Syndrome (NCBRS), an intellectual disability syndrome. This suggests that alteration of the BRM ATPase function may disrupt proper neural development. However, the mechanistic role of BRM in regulating neural differentiation is unknown. Here, we introduced two NCBRS-derived heterozygous BRM mutations K755R or R1159Q in human embryonic stem cells (hESCs) using CRISPR and used an in vitro neural differentiation system to study how the BRM mutations affect neural development. As expected, BRMK755R/+ and BRMR1159Q/+ hESCs have normal morphologies and maintain pluripotent marker expression. Strikingly, neural progenitor cells (NPCs) derived from BRMK755R/+ and BRMR1159Q/+ hESCs have morphological defects and proliferate significantly slower than WT cells. Further characterization found that BRMK755R/+ and BRMR1159Q/+ NPCs have significantly reduced levels of NPC specific markers such as PAX6 and SOX2. We assessed the global chromatin accessibility status in WT and BRM mutant NPCs and found thousands of changed accessible sites with strong overlap between the two mutants. Surprisingly, the sites that lost chromatin accessibility mainly overlapped with active enhancer histone modifications H3K4me and H3K27ac in WT NPCs suggesting that BRM-containing BAF complexes are critical for maintaining accessibility for neural progenitor active enhancers. Motif search analysis identified that SOX and RFX family binding motifs are highly enriched in sites that lost accessibility indicating binding of neural specific transcription factors is likely impacted. RNA-seq analysis of NPCs expressing WT or BRM mutants identified a decrease in the expression of genes involved with neurogenesis and cell cycle, and an increase in the expression of genes involved with extracellular matrix organization. These data suggest that BRM-containing BAF complexes are required for proper neural differentiation by facilitating the access of neural transcription factors to transcriptional enhancers that specify the neural cell fate.

Key Words: BRM, SWI/SNF, neural development, chromatin remodeling, epigenomics, enhancer

7. Assessing Biological Mechanisms of Action of Soluble Paracrine Factors and Extracellular Vesicles Secreted by Human Cardiosphere-Derived Cells

Presented by: Kristin Luther, Capricor Therapeutics
Authors: Luther, Kristin, Capricor Therapeutics; Quon, Tanner, Capricor Therapeutics; Peck, Kiel, Capricor Therapeutics; Walravens, Ann-Sophie, Capricor Therapeutics; Holewinski, Ronald, Cedars-Sinai Medical Center; Van Eyk, Jennifer, Cedars-Sinai Medical Center

Cardiosphere-derived cells (CDCs) are a regenerative cell product that are being tested clinically for numerous disease indications including Duchenne muscular dystrophy, heart failure with preserved ejection fraction, and pulmonary arterial hypertension. The mechanism of action is understood to be paracrine in nature, through reduction of inflammation and fibrosis, and enhancement of viability and angiogenesis. CDCs have a complex secretome that includes extracellular vesicles (EVs), several growth factors, and many other proteins. While the EVs are believed to mediate many of the CDCs’ effects, the soluble growth factors might have similar functions to the EVs. The goal of this work was to develop bioassays that identify effects mediated by growth factors secreted by CDCs and distinguish them from those mediated by CDC-EVs. We began by concentrating EVs through ultrafiltration by centrifugation (UFC) using filters with different molecular weight cutoffs, which yielded EVs and varying amounts of soluble proteins. We thoroughly characterize the content of the different UFC products in terms of EV and protein concentration, miRNA content, proteomics, flow cytometry for EV marker proteins, and bioactivity in two in vitro bioassays. We conclude that while rapid activation of ERK1/2 and Akt signaling cascades in endothelial cells is dependent on growth factors co-isolated with EVs, anti-inflammatory effects occurring after 6 hours in macrophages is the result of EV uptake.

Key Words: cell therapy, extracellular vesicles, exosomes, ultrafiltration, paracrine effects, heart failure, inflammation, macrophages, endothelial cells, Akt, ERK1/2, growth factors, miRNA

8. Dysregulation of NEUROG2 Plays a Key Role in Focal Cortical Dysplasia

Presented by: Simoni Avansini, UC San Diego
Authors: Avansini, Simoni, UC San Diego; Torres, Fabio, UNICAMP; Vieira, Andre, UNICAMP; Rogerio, Fabio, UNICAMP; Coan, Ana, UNICAMP; Secolin, Rodrigo, UNICAMP

Focal cortical dysplasias (FCDs) are a type of malformations of cortical development and a significant cause of drug‐resistant epilepsy. Although recent literature points to the involvement of the mTOR pathway in FCD type II, the exact molecular mechanism leading to this type of cortical malformation, especially to the presence of aberrant cells, remains undetermined. In this work, we aimed to investigate whether abnormal gene regulation, mediated by microRNA, could be involved in FCD type II. We used total RNA from the brain tissue of 16 patients with FCD type II and 28 controls. MicroRNA expression was initially assessed by microarray. Quantitative polymerase chain reaction, in situ hybridization, luciferase reporter assays, and deep sequencing of genes in the mTOR pathway were performed to validate and further explore our initial study. The hsa‐let‐7f (p = 0.039), hsa‐miR‐31 (p = 0.0078), and hsa‐miR34a (p = 0.021) were downregulated in FCD type II, whereas a transcription factor involved in neuronal and glial fate specification, NEUROG2 (p < 0.05), was upregulated. We also found that the RND2 gene, a NEUROG2‐target, is upregulated (p < 0.001). In vitro experiments showed that hsa‐miR‐34a downregulates NEUROG2 by binding to its 5′‐untranslated region. Moreover, we observed a robust nuclear expression of NEUROG2 in balloon cells and dysmorphic neurons and found that 28.5% of our patients presented brain somatic mutations in genes of the mTOR pathway. Our findings suggest a new molecular mechanism, in which NEUROG2 has a pivotal and central role in the pathogenesis of FCD type II. In this way, we found that the downregulation of hsa‐miR‐34a leads to upregulation of NEUROG2, and consequently to overexpression of the RND2 gene. These findings indicate that a weak coupling in neuronal differentiation and migration mechanisms may explain the presence of aberrant cells and complete dyslamination in FCD type II. In the next step, we intend to establish an in vitro human epilepsy-associated model of type II FCD using cortical organoid to recapitulate cortical development and to speed-up screening and discovery of new candidate drugs for the treatment of this severely epileptogenic cortical malformation.

Key Words: cortical development, epilepsy, malformations of cortical development, neuronal differentiation, neuronal migration

9. Genetic Dissection of m6A RNA Methylation Role in Stem Cell Differentiation and Early Mammalian Development

Presented by: Lior Lasman, Weizmann Institute of Science
Authors: Lasman, Lior, The Weizmann Institute of Science

The role of DNA epigenetic modifications in regulation of gene expression and cell differentiation has been studied extensively. However, the role of RNA modifications has only started to be unveiled. N6-methyladenosine (m6A) is the most common mRNA modification found in eukaryotes, and has been lately identified as a critical gene expression regulator. Different proteins were found to be “writers”, “erasers” and “readers” of this modification, further supporting its function in epigenetic dynamics. Our group has previously identified Mettl3, an m6A RNA writer, as a critical regulator for the transition between naïve pluripotency and cellular commitment to differentiation. We found that Mettl3 KO in ESCs is sufficient for complete removal of m6A modifications from mRNA molecules. Moreover, we show that while Naïve ESCs and pre-implantation epiblast are viable and present normal pluripotent markers in the absence of m6A, they fail to adequately terminate this state, and subsequently undergo aberrant priming and differentiation. Our analysis identified m6A as a critical determinant, which destabilizes naïve pluripotency genes. m6A predominantly restrains transcript stability, therefore safeguards rapid exit from naïve pluripotency. I will present unpublished results regarding m6A reader proteins – YTHDF1,2 and 3. Using genetic Knock Out models, in vitro and in vivo, we show that these readers affect ESCs’ differentiation, in vivo development and fertility. Collectively, our work further promotes the understanding of mRNA epi-transcriptome in stem cell differentiation and early mammalian development.

Key Words: epigenetics, RNA methylation, m6A modification, stem cell differentiation, early mammalian development, germ cell biology

10. Extracellular Vesicles from Human Liver Stem Cell Indicate a Disease Modifying Potential in Murine Model of Hepato-Cellular Carcinoma by Restoring the Underlying Liver Fibrosis and Inhibiting Tumor Growth

Presented by: Daniel Gau, Unicyte
Authors: Gau, Daniel, Unicyte AG; Tetta, Cirro, Unicyte AG; Camussi, Giovanni, Unicyte AG

Extracellular vesicles and exosomes (EVs), and in particular when derived from stem cells are emerging as a new therapeutic modality in regenerative medicine. Stem cell derived EVs are of high abundance, are not self-replicating, are not immunogenic, own a tissue specific targeting capability and thus address hurdles of stem cells for translation into indications with large patient impact. With more than 10 years research in the field, Unicyte has developed a strong therapeutic pipeline in fibrotic disorders and oncology, using it’s proprietary Human Liver Stem Cell and derived nano-EVs (HLSC-nEVs). With their natural targeting capability to liver tissue and to tumor cells, HLSC-nEVs have been investigated in Hepatocellular carcinoma (HCC) the most common cause of death in people with cirrhosis. In pre-clinical studies, HLSC-nEVs decrease the growth and survival rates of tumor liver cells, while at the same time protecting normal hepatocytes. In addition, HLSC-nEVs have demonstrated strong liver regeneration capabilities in various models of acute and chronic liver injury, ex-vivo scaffold re-cellularization and more recently on normothermic transplantation models. These results suggest that HLSC-nEV address the underlying liver disease and inhibit tumor growth in hepato-cellular carcinoma via a multi-factorial mechanism of action. Unicyte is currently preparing clinical transition of its HLSC-nEVs programs in various indications including HC.

Key Words: extracellular vesicle, exosomes, liver, oncology, hepato-cellular carcinoma, nash, fibrosis, cirrhosis, stem cell, regeneration

11. Nanostraw Physical Delivery of Proteins and Nucleic Acids into Immune Cells for Cell and Gene Therapy

Presented by: Sergio Leal-Ortiz, Stanford University
Authors: Leal-Ortiz, Sergio, Stanford University; Hjort, Martin, Lund University; Vossfeldt, Hannes, Charite University; Swoboda, Ryan, NAVAN Technology; Cao, Yuhong, Stanford University; Melosh, Nicholas, Stanford University

The redirection of antigen specificity of T-cells via the transfer of desired antigen receptor genes and the adoptive transfer of these genetically designed T-cells into patients holds excellent promise for individually tailored treatment of cancerous diseases. At the same time, newly arisen technologies like CRISPR/Cas9, other editing nucleases and transposons could be part of the solution for many different types of disease. In all these novel therapeutic approaches, it is vital for the cells to be altered by the respective cargo delivered to the target cells, which is often an issue for hard-to-deliver cargo into hard-to-transfect cells, like immune cells. Also, delivery methods should feature some other properties, like non-perturbation to facilitate an effective and convenient means of cell transfection which is at the same time cost-efficient, easily implementable and scalable for the clinical application. Nanostraws, hollow alumina nanotubes, provide fluidic access into cells thereby allowing the insertion of exogenous cargos for cell engineering purposes. Cells in contact with the Nanostraws form a tight interface while showing normal cellular behaviour and virtually unchanged gene expression. To ensure delivery of charged cargo such as DNA plasmids, RNA strands, and proteins into the cells, an electric field is applied to locally destabilize the lipid bilayer in contact with the Nanostraws. Very low pulses are utilized in this process, which mobilizes the cargo, driving it into the cell in a dosage-controlled fashion. Due to its versatility and simplicity combined with high efficacy and non-perturbation, the Nanostraw technology is an ideal candidate for the cargo delivery to immune cells such as T-lymphocytes. We utilize Nanostraws to transfer different types of cargos and show successful delivery into T-cells, monocytes, macrophages and immune cells line. Our emphasis is to deliver different types of cargos in unstimulated CD4+ T-cells and macrophages. All cell types exhibited a very high viability of more than 90% after delivery (irrespective of cargo type). Nanostraws present a robust method for intracellular delivery of virtually any cargo even into hard-to-transfect cells and consequently can fulfill the need for a gentle, capable, scalable, universal, dosage-controlled, and cost-efficient platform in the development of current and next-generation cell and gene therapies.

Key Words: gene therapy, nano technology, transposons, nucleases, T-cells, macrophages, stem cells, DNA, RNA, RNP

12. Profiling Compounds with Known Cardiotoxic Mechanisms of Action Using Physiologically-Relevant, Anisotropic 384-Well High Throughput hiPSC-Cardiomyocyte Cultures

Presented by: Alec Witty, StemoniX
Authors: Contu, Riccardo, StemoniX; Padilla, Robert, StemoniX; Si, Won, StemoniX; Spangenberg, Stephan, StemoniX; Van Hese, Brittney, StemoniX; Fanton, Alison, StemoniX

Drug removal from the clinical market, as well as late-stage failures in clinical trials, are often linked to unforeseen cardiac toxicity. hiPSC-CMs are an integral component of a new paradigm, the Comprehensive in vitro Proarrhythmia Assay (CiPA) Initiative, through which panels of compounds with known mechanism of cardiotoxicity are being evaluated in hiPSC-CM platforms across independent test sites and through cutting-edge technologies. Key challenges under consideration for the hiPSC-CM system are sub-ideal cardiomyocyte geometry, sub-cellular structural organization, and electro-physiological maturity. Bioengineering approaches developed to enhance hiPSC-CM maturity have shown improvements in aspects of hiPSC-CM physiology, hhowever those approaches have limited scalability and thus are not amenable to high throughput screening. hiPSC-CMs cultures plated on a high throughput platform which passively promote cardiomyocyte alignment have been shown to display physiologically-relevant features, including more physiological cellular geometry, coherent unidirectional contraction, cardiac cell junction re-modeling, and improved calcium handling. To evaluate whether the changes induced by this platform translated into differential responses to cardio-active compounds, high throughput calcium flux assays were performed on hiPSC-CMs cultured in standard high throughput screening cell cultureware or anisotropic 384-well plates and subsequently interrogated with the 28 compounds included in the CiPA initiative. Interestingly, differential responses were observed in nearly 60% of the compounds tested. Specifically, compounds in the high risk category showed a dose-dependent progression in the severity of the pro-arrhythmic phenotypes in anisotropy. This was associated with a higher severity of early afterdepolarizations (EADs). Six out of eleven compounds in the intermediate risk category showed a more sensitive response in anisotropy. No EADs were observed in either control or anisotropic conditions treated with low risk compounds. Altogether, anisotropic high throughput hiPSC-CM cultures formatted in the platform employed in this study showed better resolution over the progression and severity of pro-arrhythmic events.

Key Words: hiPSCs, cardiomyocytes, safety, toxicology, pharmacology, CiPA, arrhythmia, human induced pluripotent stem cells, iPS

13. Preclinical Development and Clinical Scale Manufacturing of HIV Gag-Specific, Lentivirus-Modified CD4 T Cells for HIV Functional Cure

Presented by: Haishan Li, American Gene Technologies International
Authors: Li, Haishan, American Gene Technologies International; Lahusen, Tyler , American Gene Technologies International; Xiao, Lingzhi, American Gene Technologies International; Liou, Mei-Ling, American Gene Technologies International; Muvarak, Nidal, American Gene Technologies International; Pauza, David, American Gene Technologies International

Strong activation, infection and eventual depletion of HIV-specific CD4 T cells is the crucial disease mechanism in AIDS. The specific lack of functional, HIV-specific CD4 T cell help underlies chronic immune system dysfunction and viral persistence. Adoptive cell therapy with autologous, antigen-specific CD4 T cells is a promising strategy for rebuilding effective immunity against HIV, which might provide a pathway to disease cure. However, the unique characteristics of HIV infection and their impact on multiple immune cell subsets, create technical obstacles that limit ex vivo expansion of HIV-specific CD4 T cells. AGT developed a method for activating and expanding CD4 T cells reactive to epitopes in the Gag protein, to create an autologous cell product for HIV therapy. High numbers of HIV-specific CD4 T cells were obtained by stimulating with synthetic Gag peptides followed by quantitative depletion of CD8+, CD56+, and CD19+ cells. Following stimulation and depletion, the enriched CD4 T cells were transduced with lentivirus vector AGT103 that protects them from HIV infection or depletion. The steps of cell preparation, activation, isolation and transduction were performed in a cGMP-grade platform for integrated and functionally closed manufacturing of engineered T cells with minimal user interaction. Enriched and transduced cells were transferred to a static culture vessel for cell expansion in a process lasting 12 days from receipt of apheresis products to cryopreservation. On average, cell products are composed of > 80% of CD4 T cells, approximately 10% of CD4 T cells are specific for HIV Gag epitopes and regulatory T cells are low or undetectable. The major subset has effector memory phenotype and >10% were central memory T cells, an immune subset enabling potential long-term persistence of T cells. The autologous cell product AGT103-T is entering process qualification prior to IND submission with an expected clinical trial start in 2019.

Key Words: HIV, immunotherapy, antigen-specific CD4, GMP, manufacture

14. Non-Coding Genomic Regulation Identified in Human Cardiomyocytes

Presented by: Daniel Cheah, UC San Diego
Authors: Cheah, Daniel, UC San Diego; Kumar, Aditya, UC San Diego; Thomas, Stephanie, UC San Diego; Wong, Kirsten, UC San Diego; Lo Sardo, Valentina, The Scripps Research Institute; Hou, Yang-Hsun, UC San Diego UC San Diego

Based on genome-wide association studies, single nucleotide polymorphisms (SNPs) in the non-coding region of the 9p21 locus have been associated with increased risk of arrhythmic cardiac death, suggesting an altered cardiac remodeling response. To assess how 9p21 SNPs regulate cardiac phenotypes associated with fibrotic remodeling, induced pluripotent stem cell-derived cardiomyocytes (CMs) from patients that are homozygous risk/risk (R/R) and non-risk/non-risk (N/N) for 9p21 SNPs were cultured on a methacrylated hyaluronic acid hydrogel to mimic stresses induced by cardiac fibrotic stiffening. While CMs contracted synchronously in physiological niche independent of genotype, only R/R CMs exhibited asynchronous contractions due to a loss of connexin 43 expression after stiffening. This phenotype was mediated a c-Jun N-terminal kinase (JNK) phosphorylation-based mechanism to impair gap junctions after stiffening. However, locus deletion or treatment with a JNK antagonist after stiffening was sufficient to prevent asynchronous contraction by maintaining gap junctions, suggesting a mechanism that could contribute to the asynchronous phenotype. Thus, specific niche changes including non-coding loci can differentially affect cell function and identify unique regulation.

Key Words: induced pluripotent stem cells, cardiomyocytes, extracellular matrix, stiffness, ANRIL

15. Insulin-like Growth Factor-1 (IGF-1) Remodels the Proteome Dynamics of Rett Syndrome (RTT) Patient-Derived Neurons

Presented by: Nam-Kyung Yu, The Scripps Research Institute
Authors: Yu, Nam-Kyung, The Scripps Research Institute; Yates, John, The Scripps Research Institute; Muotri, Alysson, UC San Diego; Romero, Sarah, UC San Diego; Miranda, Helen, UC San Diego

IGF-1 has been thought as a promising drug candidate for RTT. Multiple studies have shown that IGF-1 can ameliorate the cellular and behavioral phenotypes of RTT model animals or cells. However, detailed molecular mechanisms of how IGF-1 treatment restores the RTT cells have not been comprehensively described. Even RTT itself has been rarely studied at proteome level while there are a number of transcriptomic studies on RTT. Here we identified and quantified the nascent proteome of neurons differentiated from RTT patient-derived iPSCs and studied how the proteome is remodeled after IGF-1 treatment. We used metabolic amino acid labeling, click chemistry followed by enrichment of the labelled peptides, and mass spectrometry-based quantitative analyses. By comparing with CRISPR-rescued neurons, we could identify molecular changes caused by MeCP2 deficiency and how they are changed by IGF-1 treatment. We found the decreased synthesis of proteins involved in neuron projection morphogenesis and extracellular matrix organization, which was recovered by IGF-1 treatment, providing a novel insight into RTT pathogenesis and IGF-1 effects. Sorting out the RTT proteomic features that are affected by IGF-1 treatment from those that are not affected may provide a clue to developing a more specific and efficient way for treating RTT.

Key Words: insulin growth factor-1, Rett syndrome, proteomics, iPSC-derived neuron mass spectrometry

16. Hair Follicle Regeneration by Differentiation of Human Pluripotent Stem Cells Engineered into Microscaffolds

Presented by: Antonella Pinto, Sanford Burnham Prebys Medical Discovery Institute
Authors: Pinto, Antonella, Sanford Burnham Prebys Medical Discovery Institute; Hamidi, Jasmin, Sanford Burnham Prebys Medical Discovery Institute; Chermnykh, Elina, Koltzov Institute of Developmental Biology; Kalabusheva, Ekaterina, Koltzov Institute of Developmental Biology; Steiger, Wolfgang, Technische Universität Wien; Ovsianikov, Aleksandr, Technische Universität Wien

iPSCs have been directed to various cell fates. However, the derivation of Dermal Papilla (DP) cells had not yet been reported. The DP plays a dominant role during hair follicle morphogenesis and is critical in defining hair thickness, length, and life cycle. Our research attempted to explore, for the first time, the derivation of functional DP cells from iPSCs using a Neural Crest (NC) cell intermediate. We investigated the effect of growth factors such as Wnt, FGF, BMP, and R-spondin on the differentiation of iPSC-derived NC cells into DP cells. We observed that the activation of the Wnt pathway enhances the differentiation process, as demonstrated by the expression of classical DP cell markers (Versican and Alkaline Phosphatase) and the up-regulation of relevant DP genes (Nexin, Corin, LEF1, SDC1, HEY1, EGR3). To evaluate the identity of the derived DP cells in vitro, we compared, on a single cell level, the gene expression profile of iPSC-DPs with that of human adult DP cells. To generate a homogeneous DP population, we implemented robust cell purification and enrichment strategies such as FACs analysis, using Syndecan-1 and Integrin-α9β1 as surface markers. Prior evidence has suggested the importance of an appropriate 3D environment for DP cells to maintain their hair follicle-inducing properties and the fundamental role of crosstalk between mesenchymal and epithelial cells during the hair follicle formation. Thus, we co-cultured iPSC-DP cells with keratinocytes inside microscaffolds and transplanted them into the back skin of Athymic nu/nu mice. After two weeks, the formation of ectopic hair follicles was observed. In conclusion, the turning point of this work is the derivation of functional iPSC-DP cells in a well-defined environment, capable of stimulating robust hair growth, providing an unlimited source of cells for transplantation. The use of iPSC might allow us to induce the neogenesis of the human hair follicle from autologous adult cells in vitro, contributing to create a highly customized, patient-focused approach to stem cell treatment for hair loss disorder.

Key Words: iPSC, hair follicle, microscaffolds, regenerative medicine

17. A Novel Commercial Utility of iPS Cells: Fully Human Biologic Manufacturing

Presented by: Theresa Deisher, AVM Biotechnology
Authors: Deisher, Theresa, AVM Biotechnology; Zahid, Yumna, AVM Biotechnology; Poulin, Kendra, AVM Biotechnology; Jarzyna, Peter, AVM Biotechnology; Koyama, Kumiko, AVM Biotechnology

Chronic treatment using biologics leads to formation of resistance in up to 60% of patients by development of anti-drug antibodies or neutralizing antibodies. This not only compromises efficacy but may also cross-react with endogenous factors to cause serious toxicity. The fact that many biologics are manufactured in non-human mammalian cell lines, such as CHO (Chinese Hamster Ovary) and BHK (Baby Hamster Kidney) cells, is likely a key driver for this resistance. Post-translational modification processes, especially glycosylation, influence the biochemical and therapeutic properties of biologics and contribute to development of drug resistance. We have used Induced Pluripotent Stem cells (iPSCs) for manufacturing biologics which will be fully human, manufactured from the patient’s own cells, have similar post-translational modifications and reduced risks of viral contamination. We used lentivector transduction of iPSCs to express generic IgG4 protein using traditional methods for screening high-producing recombinant clones. Our results showed that iPSCs can produce up to 0.001 ng/cell of protein, prior to any optimization in adherent static culture conditions in 6-well plates. This is comparable to CHO production levels of 0.01 ng/cell of protein in 1986 before it was engineered to become a high producer cell line. For manufacturing purposes, iPSCs were also adapted into suspension cultures. Results showed a significant decrease in the expression level in suspension iPSCs compared to adherent iPSCs at same passage number. To further optimize our process, we will be using a single-use bioreactor system that allows expansion of iPSCs in adherent culture. This novel biologic manufacturing platform using iPSC provides opportunities for lineage-specific as well as patient-specific biologics for patients in lifelong therapy.

Key Words: iPSC, induced pluripotent stem cell, biologics, personalized medicine, precision medicine, manufacturing, therapeutics, patient-specific biologics, enzyme replacement therapy, fully human biologics, hemophilia, therapeutic antibodies, universal manufacturing

18. Inhibition of p16 to Increase the Efficiency of Direct Cardiac Reprogramming for Treatment of Ischemic Heart Disease

Presented by: Shruthi Perati, Albany Medical College
Authors: Perati, Shruthi, Albany Medical College; Li, Xiuchun, Albany Medical College; Zhang, Shuning, Albany Medical College; Devejian, Neil, Albany Medical College; Bennett, Edward, Albany Medical Center; Cai, Chuanxi, Albany Medical College

Ischemic heart disease (and subsequent heart failure) is the leading cause of death in the U.S, killing more people annually than all cancers combined. Heart transplantation remains the established therapy for severe heart failure, as an injured adult mammalian heart has little regenerative capacity post-injury. Direct cardiac reprogramming (DCR) has thus developed as a regenerative approach, directly converting fully-differentiated cardiac fibroblasts into induced cardiomyocytes. Current approaches to direct cardiac reprogramming have had very low efficiency rates, due to the major barrier of cell aging/senescence. This study therefore aims to improve DCR efficiency by implementing the most successful reprogramming approaches – use of small-molecule compounds and environmental cues (low oxygen and gel matrices) – in conjunction with inhibition of aging/senescence pathways, specifically p16. The study hypothesizes that inhibition of aging/senescence pathways, via lentiviral knockdown of p16, enhances direct cardiac reprogramming. We demonstrated that aging and senescent human cardiac fibroblasts (HCFs) exhibit upregulated expression of p16, as well as other markers of senescence and quiescence. Next, we demonstrated that lentiviral knockdown of p16 increases replication rate of HCFs and decreases expression levels of other quiescence and senescence-associated genes in HCFs. P16 knockdown also resulted in upregulation of anti-oxidant proteins and Erk signaling, suggesting an underlying mechanism for the rejuvenated phenotype seen in adult HCFs with p16 knockdown. qPCR results from the 7-day DCR timepoint demonstrate elevated expression levels of cardiac marker genes, with the cardiac marker RYR2 particularly elevated upon DCR with p16 knockdown. Results from the 21-day DCR timepoint are also presented. Insight gained from this study elucidates the role of specific senescence/aging pathways in cardiac reprogramming, ultimately bringing us closer to the clinical treatment of autologous cell therapy for patients with ischemic heart disease.

Key Words: ischemic heart disease, direct cardiac reprogramming (DCR), p16, cell senescence/aging, autologous cell therapy

19. LIS1 Takes the RISC to Modulate Non-Coding RNA, Alternative Splicing and Chromatin Dynamics in Priming

Presented by: Aditya Kshirsagar, Weizmann Institute of Science
Authors: Kshirsagar, Aditya, Weizmann Institute of Science, Israel; Olender, Tsviya, Weizmann Institute of Science, Israel; Karzbrun, Eyal, Weizmann Institute of Science, Israel; Kaibuchi, Kozo, Nagoya University Graduate School of Medicine, Japan; Hanna, Jacob, Weizmann Institute of Science, Israel; Reiner, Orly, Weizmann Institute of Science, Israel

LIS1 mutations and deletions have been associated with lissencephaly; a condition wherein the cerebral cortex of the patients assumes a smooth shape. The LIS1 protein is involved in several key functions including cell proliferation and neuronal migration, and is involved in regulation of the molecular motor cytoplasmic dynein and the cytoskeleton. Increase in the dosage of the LIS1 gene also causes mild brain malformations and developmental delay. During early development, following embryonic day 3.5, regulation of RNA at the transcriptional and post transcriptional levels plays a pivotal role in the regulation of pluripotency and differentiation. Lis1 knockout mice are early embryonic lethal and the role of this protein during early development together with its function in the nucleus still remains to be elusive. Immunostaining of mouse wild type blastocysts affirms that LIS1 co-localizes predominantly in inner cell mass cells. Here, in this study, LIS1 is detected in the nucleus of mouse embryonic stem cells in association with chromatin embedded proteins, and most notably, chromatin modifiers, the RISC complex and splicing factors. Genome wide studies and proteomics using Lis1 mutant mouse ES cells (mESCs) showed that LIS1 together with Argonaute factors physically associate with splicing and transcription factors. Further, LIS1 mutant human embryonic stem cell (hESCs) lines were generated using CRISPR/Cas9 genome editing. We developed a novel on-chip platform to grow 3D cortical organoids from mutant hESCs and modelled reduced folding. Extra cellular matrix (ECM) related genes were differentially expressed when wild-type and LIS1 +/- organoids were compared at different growth stages. Our study reveals novel molecular roles of LIS1 in ESCs and during early stages of brain development, and provide a model system to understand the mechanism associated with lissencephaly.

Key Words: lissencephaly, human embryonic stem cells, 3D brain organoids, GWAS, mass spectrometry

20. NSCLC Upregulates BCL-XL in Resistance to TRAIL-Induced Apoptosis

Presented by: David Deng, UC Berkeley
Authors: Deng, David, UC Berkeley; Moleirinho, Susana, Brigham and Women’s Hospital; Kitamura, Yohei, Brigham and Women’s Hospital; Shah, Khalid, Harvard Medical School

NSCLC constitutes 80% of all lung cancers, the leading cause of cancer death worldwide, and highly resistant to traditional chemotherapy, such as Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL)-induced apoptosis. Several strategies have been developed to enhance TRAIL treatment efficacy, including engineering Mesenchymal Stem Cells (MSCs) to produce bi-functional fusion protein Enb-TRAIL and simultaneously target TRAIL-induced apoptotic pathway and EGFR extracellular domain. However the effects on many NSCLC are minimal. Here, we characterized several TRAIL-resistant and Enb-TRAIL-resistant NSCLC cell lines before and after treatment regimens to dissect their mechanism of resistance. We first investigated the surface expression of TRAIL-binding Death Receptor 4 and 5 (DR4/5). Then, we looked for changes in the downstream extrinsic and intrinsic apoptotic pathways under the exposure of TRAIL and Enb-TRAIL. Finally, we checked if cytoprotective macroautophagy induced by TRAIL has engulfed surface expression of DR4/5 to prevent further apoptosis for resistant NSCLCs. Our preliminary results show that although the highly resistance cell lines show high expression of DR4/5 and EGFR on their surface, they do not show reduced cell viability despite long exposures with high concentrations of TRAIL/Enb-TRAIL. We then did a time-course FACS analysis and showed that no surface expression was changed under cytoprotective macroautophagy. Finally, by doing western blotting against downstream pro-apoptotic and anti-apoptotic proteins, we found that resistant NSCLCs elevate expression of anti-apoptotic protein BCL-XL (B-cell lymphoma-extra large), possibly as a survival mechanism under treatments. Our research suggests that TRAIL-resistant cell lines could depend on BCL-XL for survival and combined inhibition of BCL-XL could enhance treatment efficacy.

Key Words: NSCLC, TRAIL-induced apoptosis, BCL-XL, death receptor 4/5

21. Role of the Transcription Co-factor VGLL1 in the Specification and Maintenance of the Trophoblast Lineage Using Multiple Human Stem Cell-based Models

Presented by: Francesca Soncin, UC San Diego
Authors: Sonci, Francesca, UC San Diego; Farah, Omar, UC San Diego; Horii, Mariko, UC San Diego; Pizzo, Donald, UC San Diego; Meads, Morgan, UC San Diego; Niakan, Kathy, Francis Crick Institute

INTRODUCTION: Understanding early mechanisms of trophoblast specification during human peri/early post-implantation stages is crucial to understanding the etiology of many placenta-associated diseases, ranging from miscarriages in the first trimester to preeclampsia and intra-uterine growth restriction in the third trimester. In a recent mouse-human comparative study of placentation across gestation, we identified vestigial-like protein 1 (VGLL1) as a human-specific transcription co-factor highly expressed in placental villous cytotrophoblast (CTB) cells. We have observed that VGLL1 is expressed as early as the pre-implantation blastocyst stage, specifically in the trophectoderm layer, which will give rise to the trophoblast cells of the placenta. We have now started to investigate the role of VGLL1 in trophoblast cells using multiple in vitro stem cell-based models. METHODS: We differentiated human pluripotent stem cells (hPSC) into CTB-like cells using an improved BMP4-based protocol to study trophoblast lineage specification. We isolated trophoblast stem cells (hTSC) from first trimester placentae, based on a recently-published protocol, to investigate the role of VGLL1 in trophoblast maintenance. RESULTS: VGLL1 was highly up-regulated during the conversion of hPSCs into CTB-like cells and it was highly expressed in hTSC. Expression of VGLL1-targeting shRNA caused impaired expression of TP63, a crucial marker of mature CTB, suggesting that VGLL1 acts upstream of this trophoblast marker. VGLL1 contains a highly conserved region binding the TEA domain family of transcription factors. In primary CTB and hPSC-derived CTB-like cells, VGLL1 co-localized with TEAD4 in the cell nuclei and we observed co-immunoprecipitation of these two factors in both models. CONCLUSIONS: We hypothize that, in human, VGLL1 acts in combination with TEAD4 to drive trophoblast lineage specification and trophoblast stem cell maintenance. Future studies include identification of direct down-stream targets of the VGLL1/TEAD4 complex by ChIP-seq as well as establishment of the transcription factor cascade necessary for trophoblast specification and maintenance using both hPSC and hTSC models.

Key Words: placenta trophoblast, stem cells development, pluripotent stem cells, trophoblast lineage, preeclampsia

22. Modeling Preeclampsia Using Human Induced Pluripotent Stem Cells

Presented by: Mariko Horii, UC San Diego
Authors: Horii, Mariko, UC San Diego; Bui, Tony, UC San Diego; Soncin, Francesca, UC San Diego; Farah, Omar, UC San Diego; Meads, Morgan, UC San Diego; Laurent, Louise, UC San Diego

Introduction: Preeclampsia (PE) is a hypertensive disorder, which affects 2-8% of pregnancies, and is associated with adverse outcomes for both mother and baby during the pregnancy and later in life. PE has two clinical subtypes, based on the gestational age at onset of disease: early-onset (EO-PE) and late-onset preeclampsia (LO-PE) refer to disease either before or after 34 weeks gestational age, respectively. The primary etiology of this disease is abnormal differentiation and function of placental cells, called trophoblasts. PE is difficult to study, due to the onset of the disease originating in early stages of placental development, to which access is very limited. Our group has established a simple two-step trophoblast differentiation protocol, which recapitulates both normal and abnormal trophoblast differentiation during early placental development, by using human pluripotent stem cells. Here, we have applied this two-step protocol to induced pluripotent stem cells (iPSCs), derived from placentas of EO-PE patients, to evaluate the trophoblast differentiation defects associated with this disease. Methods: EO-PE and gestational age- and fetal sex-matched control iPSCs were cultured in Essential-8 based media with 2uM IWP2 (Wnt inhibitor) and 10ng/ml Bone Morphogenetic Protein-4 (BMP4) for 4-5 days (step 1) to differentiate into cytotrophoblast (CTB) stem cells. Subsequently, cells were replated in feeder conditioned media + 10ng/ml BMP4 under either 2% or 21% oxygen to terminally differentiate these cells (step 2) into extravillous trophoblast (EVT) and syncytiotrophobalst (STB). Samples were collected on days 0, 4 or 5 for step 1, and on days +2, +4, and +6 after replating for step 2, and assessed by flow cytometry, qRT-PCR, and ELISA. Results: At the conclusion of step 1, there were no differences in CTB marker expression by flow cytometry (EGFR) or qPCR (EGFR, CDX2, p63); however, GCM1, a transcription factor and master regulator of both EVT and STB differentiation, was significantly down-regulated (4-fold, p<0.05) in EO-sPE-iPSC. At the end of step 2, EO-sPE-iPSC-derived trophoblast showed significant reduction in STB-associated genes (ERVW1, PSG4, CGB) (2-83-fold, p<0.05); a 4-fold reduction(p<0.05) in hCG secretion was also confirmed by ELISA. Finally, EVT markers (HLAG, HTRA4) were also significantly reduced (2-11-fold, p<0.05). Conclusions: Our results showed significant compromise of both STB and EVT differentiation in EO-PE-derived iPSCs. We hypothesize that a reduction in GCM1, which is involved in regulation of both trophoblast sub-lineages, is the root cause of this phenotype. Our study shows that, while additional studies are necessary, iPSCs hold enormous potential for studying underlying mechanisms of placenta-based pregnancy complications such as PE.

Key Words: preeclampsia, trophoblast, BMP4, GCM1, disease-in-a-dish-model

23. Qualification of iPSC-derived Using Cell Therapy Compliant Reagents and Workflows

Presented by: Chad MacArthur, Thermo Fisher Scientific
Authors: MacArthur, Chad, Thermo Fisher Scientific, Cell Biology; Pradhan, Suman, Thermo Fisher Scientific, Cell Biology; Lakshmipathy, Uma, Thermo Fisher Scientific, Cell Biology

Induced pluripotent stem cell (iPSC) research is rapidly moving towards translational and clinical applications. These applications require robust and consistent workflows that utilize high quality reagents that are xeno-free. When commonly used animal-origin components are replaced with xeno-free alternatives, performance often suffers thereby necessitating thorough qualification of reagents and the process. Methods that enhance consistency will minimize extra effort and costs associated with clones that fail to expand, or do not meet quality standards for downstream use. Previously, we reported the first off-the-shelf reprogramming kit specifically designed for clinical and translational research. To further streamline iPSC generation and ensure the consistent creation of high quality iPSCs, xeno-free workflows for blood-derived cells such T cells were optimized to minimize the effect of donor to donor variability. Optimization of a combination of conditions including hypoxia, matrix, and seeding density was shown to offer consistent iPSC generation from difficult to reprogram donor cells, albeit with varying efficiencies. The resulting iPSC liens were confirmed to be foot-print free, authenticated to be derived from the corresponding donor cells, and were subjected to comprehensive characterization methods to assess its quality and safety profile. Clones qualified to be pluripotent and possess trilineage differentiation potential were thoroughly investigated for genomic stability. All of the clones tested showed a normal karyotype with traditional G-banding and lower resolution arrays (2 Mb). However, higher resolution arrays that can detect aberrations as small as 25-50 kb, indicated sub chromosomal loss and gains in a fraction of the clones. Such clones did not have a consistent pattern in either chromosomal location or size and a few of the affected regions did not contain any genes in the affected region. Further assessment of oncogenic hotspot mutations that are known to occur at a higher frequency in ESC and iPSC, including TP53, indicated the absence of oncogenic structural variants in all of the clones tested. The combination of qualified reagent and defined xeno-free workflows, in combination with comprehensive and predictive characterization assays aids in easy transition of early investigation work towards translational and clinical research.

Key Words: reprogramming iPSC stem cells, translational characterization

24. The E3 Ligase Fbxw7 is a Novel Regulator of Muscle Stem Cell Commitment and Skeletal Muscle Regeneration

Presented by: Michael Stec, Sanford Burnham Prebys Medical Discovery Institute
Authors: Stec, Michael, Sanford Burnham Prebys Medical Discovery Institute; Gromova, Anastasia, UC San Diego; Richards, Alicia, Sanford Burnham Prebys Medical Discovery Institute; Krogan, Nevan, UC, San Francisco; Sacco, Alessandra, Sanford Burnham Prebys Medical Discovery Institute

Skeletal muscle has a remarkable regenerative capacity and is able to fully restore its structure and function after injury. Muscle stem cells (MuSCs) are indispensable for proper muscle regeneration; however, the function of MuSCs is impaired during aging and chronic disease, resulting in a drastically reduced regenerative capacity. The ability of MuSCs to self-renew and regulate muscle repair following injury makes them an attractive therapeutic target for these conditions; however, a greater knowledge of the mechanisms regulating MuSC function is required before they can successfully be implemented in regenerative medicine. Here, we demonstrate that Fbxw7, an E3 ligase shown to control the activation and differentiation of several stem cell compartments, is a novel regulator of MuSC commitment and muscle regeneration. Using a transgenic mouse model in which Fbxw7 is deleted in MuSCs (Pax7Cre-ER/TdT/Fbxw7-flox), we show that Fbxw7 does not play a major role in maintaining MuSC quiescence; however, Fbxw7 is required for efficient skeletal muscle regeneration by promoting the commitment and differentiation of MuSCs. To identify the relevant Fbxw7 protein substrates that regulate MuSC commitment, we performed global proteomics analysis on FACS-isolated MuSCs from wildtype and Fbxw7-null mice following activation in vitro. Of the ~3,600 proteins detected, we identified 18 upregulated proteins as putative targets of Fbxw7. Several of these targets have been shown to play key roles in cell cycle progression by regulating ribosome biogenesis, mitotic spindle assembly, and DNA replication, highlighting the importance of Fbxw7-mediated degradation in controlling MuSC cell cycle exit and commitment. Overall, these findings demonstrate that Fbxw7 is a major regulator of MuSC commitment and muscle regeneration, and ongoing studies identifying the function of its substrates in MuSCs may provide us with potential therapeutic targets for enhancing muscle regenerative capacity during aging and muscle degenerative conditions.

Key Words: skeletal muscle, muscle stem cells, injury, regeneration, proteomics

25. The Stat3-Fam3a Axis Promotes Muscle Stem Cell Myogenic Lineage Progression by Inducing Mitochondrial Respiration

Presented by: David Sala, Sanford Burnham Prebys Medical Discovery Institute
Authors: Sala, David, Sanford Burnham Prebys Medical Discovery Institute; Cunningham, Thomas J, Sanford Burnham Prebys Medical Discovery Institute; Etxaniz, Usue, Sanford Burnham Prebys Medical Discovery Institute; Nicoletti, Chiara, Sanford Burnham Prebys Medical Discovery Institute; Puri, Pier Lorenzo, Sanford Burnham Prebys Medical Discovery Institute; Duester, Gregg, Sanford Burnham Prebys Medical Discovery Institute

Metabolic reprogramming is an active regulator of stem cell fate choices, and coordinated changes in metabolism are required for successful muscle stem cell (MuSC) myogenic lineage progression. Quiescent MuSCs display a low metabolic rate that mainly relies on fatty acid oxidation. Upon activation, MuSCs initially induce their glycolytic pathway in order to sustain cell proliferation, and next a switch to oxidative metabolism is required to allow further MuSC commitment to the myogenic lineage and differentiation of myogenic progenitors. However, the mechanisms that promote mitochondrial respiration during MuSC commitment and differentiation are still poorly understood. Our data show that Stat3, which is required for MuSC transition through the myogenic lineage, promotes mitochondrial respiration during MuSC differentiation via the cytokine-like protein Fam3a. Acute loss-of-function studies in vitro and Fam3a gene deletion in vivo demonstrated that Fam3a is required for proper MuSC myogenic lineage progression and skeletal muscle development. We also provide evidence that Fam3a is secreted by myogenic cells, and that exposure of Stat3-ablated MuSCs to recombinant Fam3a in vitro is sufficient to rescue their defects in both mitochondrial respiration and myogenic lineage progression. Finally, in vivo delivery of recombinant Fam3a normalized the number Stat3-ablated MuSCs during adult skeletal muscle regeneration. Together, these findings indicate that Fam3a is a novel Stat3-regulated secreted factor that promotes oxidative metabolism and differentiation of MuSCs, and suggests that Fam3a is potentially a powerful tool for regenerative medicine to modulate stem cell fate choices.

Key Words: skeletal muscle, muscle stem cells, Stat3, Fam3a, mitochondria, oxidative metabolism, metabolic reprogramming

26. The Extracellular Matrix Protein Tenascin-C Promotes Muscle Stem Cell Expansion and Regenerative Potential

Presented by: Mafalda Loreti, Sanford Burnham Prebys Medical Discovery Institute
Authors: Loreti, Mafalda, Sanford Burnham Prebys Medical Discovery Institute; Sacco, Alessandra, Sanford Burnham Prebys Medical Discovery Institute

Skeletal muscle disease or excessive exercise can induce muscle damage. Muscle stem cells (MuSC) are necessary for proper skeletal muscle development and maintenance and play a fundamental role during muscle repair. In the recent years, several studies have identified a critical role of the microenvironment in instructing MuSC behavior and temporally coordinating tissue repair and maintenance. However, the impact of these microenvironmental cues on MuSC function is poorly understood. We have recently shown that the extracellular matrix (ECM) Tenascin-C (TnC) protein promotes MuSC expansion and enhances skeletal muscle tissue repair. TnC is involved in modifying the interaction of cells with their microenvironment, regulating cell adhesion, migration and differentiation in several contexts. TnC is an hexameric ECM with multiple alternative splicing and protein modifications, such as glycosylation. However, how these post-translation modifications impact TnC function is not well characterized. Our goal is to investigate how TnC is involved in MuSC expansion and evaluate it as a potential tool to promote tissue repair in adult muscles. By Mass Spectrometry analysis, I identified a unique N-glycosylation site of TnC during embryonic development and skeletal muscle regeneration, which might be involved in TnC-protein interactions. Indeed, by co-immunoprecipitation, I showed that endogenous TnC interacts with Fibronectin, an ECM protein essential for maintenance and function of MuSC. I am currently generating a point mutation of the identified N-glycosylated site in order to assess whether the glycosylation is required for TnC function and is involved in the TnC-Fibronectin interaction. In parallel, I am characterizing the skeletal muscle of the TnC-knockout mice. These findings will identify TnC as a novel regulator of MuSC expansion and tissue repair, will improve our understanding of the complex interplay between MuSC and the extracellular microenvironment and will contribute to the development of novel strategies for muscle repair for regenerative medicine applications.

Key Words: skeletal muscle, regeneration, extracellular microenvironment, tenascin-c, glycosylation, muscle stem cells expansion

27. Phosphorylation of Critical Residues in Sox2 Regulatory C-Terminal Domain Blocks its Ability to Interact with Novel RNA-Binding Proteins

Presented by: Sergey Shiryaev, Sanford Burnham Prebys Medical Discovery Institute
Authors: Shiryaev, Sergey A., SBP Medical Discovery Institute; Toda, Tomohisa, Salk Institute for Biological Studies; Leyn, Simon A., SBP Medical Discovery Institute; Huang, Chun-Teng, SBP Medical Discovery Institute; Farhy, Chen, SBP Medical Discovery Institute; Pinto, Antonella, SBP Medical Discovery Institute SBP Medical Discovery Institute

Sox2 protein (sex-determining region Y (SRY)-related HMG box 2) is a member of the SOXB1 family of transcription factors. Sox2 plays important role in early stages of embryogenesis, organogenesis, and it is a key factor for stem cell regulation by maintaining stem cell properties, including their ability to self-renewal, pluripotency and proliferation. Sox2 C-terminal portion, including Group B homology region and TAD domain (transcriptional activation domain) is a subject for multiple posttranslational modifications. These modifications regulate its ability to interact with different regulatory proteins and form transcriptional complex. Unfortunately the structure of this Sox2 part is still unknown as well as the exact composition of transcriptional complex. Using the technology for incorporation of UV-reactive unnatural amino acids into the protein sequence on predetermined positions, we did the mapping of Sox2 C-terminal part binding interface via direct UV photocrosslinking in the live cells. That allowed us to identify new set of Sox2 interactors. Some interactors from this listt are directly involved in pre-mRNA splicing, processing, maturation and transport from nucleus to cytoplasm throughout the nuclear pore. We were able to confirm the interaction between Sox2 and one of the major RNA-binding protein in the nucleus as well as their ability to form together tertiary complex with Nup153 nucleopore protein. The area on Sox2 protein C-terminal part responsible for interaction with that RNA-binding protein has two phosphorylation sites. Mutation of these regulatory phosphorylated residues on Ala or Gly residues, as well as their substitution on negatively charged Glu and Asp residues mimicking phosphorylation block the interaction between Sox2 and RNA-binding protein. We believe that the formation of tertiary complex Sox2/RNA-binding protein/Nup153 is very important step in formation of fully active transcriptional complex and also is a regulatory mechanism linking heterochromatin network formation with pre-mRNA production, processing and transport.

Key Words: Sox2, transcriptional factor, phosphorylation, protein-protein complex, UV-crosslinking, RNA-binding protein, RNA processing, RNA transport, super resolution imaging

28. Adaptation of a Nitroreductase-based System for Selective Ablation of Retinal Ganglion Cells and Characterization of Injury Response in 3D Retinal Organoids Derived from hPSCs

Presented by: Alanna Koehler, UC San Diego
Authors: Koehler, Alanna, UC San Diego; Diaz, Emily, UC San Diego; Anderson, Ryan, UC San Diego; Martin, Heather, UC San Diego; Jones, Melissa, UC San Diego; Xu, Guorong, UC San Diego; Fisch, Kathleen, F. Hoffmann-La Roche; Enke, Ray, James Madison University; Wahlin, Karl, UC San Diego

Retinal degenerative diseases constitute a leading cause of hereditary blindness worldwide and, because humans lack the capacity for retinal regeneration following injury, these diseases remain largely untreatable. Retinal ganglion cells (RGCs), which project axons to the brain via the optic nerve, are a retinal cell type that is particularly susceptible to degeneration and often dies in patients with glaucoma. Recently-developed protocols to culture complex retinal tissue from human pluripotent stem cells (hPSCs) provide a platform to investigate neural retina development and degeneration in vitro, but, to date, we lack a sophisticated method for the selective ablation of specific retinal cell types in these models. In this study, we adapted a genetic ablation system often used in in vivo studies of development and regeneration. This system is designed to selectively ablate RGCs in order to induce an injury response and establish a model for detecting successful induction of newly-regenerated RGCs in future studies. Methods. This approach uses hPSCs genetically engineered to express a bacterial nitroreductase (NTR) enzyme and a fluorescent mNeonGreen reporter protein under the control of the endogenous BRN3B promoter, which is highly active in developing and mature RGCs. In the presence of NTR, prodrug metronidazole is reduced to a toxic byproduct that kills the cell. The cell type specificity afforded by BRN3B-driven expression of NTR allows for titration of inducible apoptosis of BRN3B+ RGCs. In order to find the optimal dose that depletes RGCs yet is not toxic to the rest of the retina, dose response treatments with metronidazole are carried out in retinal monolayers and 3-dimensional retinal organoids. Selective ablation of RGCs with spatiotemporal resolution in vitro —and particularly in 3-dimensional organoids — allows us to better characterize the cellular response to injury in this system and is a first crucial step in an attempt to stimulate regeneration of human RGCs. Results. We have engineered human pluripotent stem cells with a nitroreductase gene and retinal reporter. These cells have been differentiated into 3-dimensional retinal organoids which show a cell type specific distribution of fluorescently tagged genes consistent with neural retina formation. This lays the framework for a titratable, drug-inducible cell ablation system suitable for studies of human retinal regeneration.

Key Words: stem cells, organoids, retina, ganglion cells, regeneration, nitroreductase, genetic engineering, degeneration, blindness

29. Temporal Transcriptomic Analysis of Human Pluripotent Stem Cell Derived Retinal Organoids

Presented by: Melissa Jones, UC San Diego
Authors: Jones, Melissa, UC San Diego; Ogata, Anna, UC San Diego; Chen, Bolin, UC San Diego; Seid, Justin, UC San Diego; Su, Feit, UC San Diego; Birmingham, Amanda, UC San Diego; Xu, Guorong UC San Diego

Current advances with pluripotent stem cells (PSCs), gene editing techniques, and sequencing technologies have allowed for futher understanding of human tissue development. Differentiation of human PSCs (hPSCs) into 3D organoids provides new insight into aspects of eye formation, specifically retinogenesis, in vitro in real time. Cell fate specification and maturation follows a sequential time course and some known genetic markers enable characterization of different stages of retinal development, yet many of the molecular changes that occur during human eye development are unidentified. Gene editing technologies using fluorescent markers allow for the tracking of cells, and in conjunction with known developmental genes allows for the identification and detection of retinal cells. Additionally, transcriptomic studies of PSC-derived retinal cells during differentiation and maturation will further elucidate the mechanisms underlying human eye development. The aim of this study was to identify early gene expression changes during hPSC derived 3D retinal organoid differentiation. The CRISPR-Cas9 gene editing system was used to create a SIX6:GFP and VSX2:tdTomato dual fluorescent reporter hPSC line. Briefly, a TET-inducible eSpCas9 iPSC line was created and cells were selected by sequential enrichment. Doxycycline treated cells were transfected with a SIX6:GFP DNA donor and genotyped. SIX6 homozygotes were subsequently transfected with a VSX2:tdTomato donor and similarly genotyped for homozygosity. The SIX6:GFP/VSX2:tdTomato PSCs were differentiated into 3D organoids and selected for SIX6:GFP and VSX2:tdTomato positivity using an automated high-content 3D imaging microscope. Organoids were collected at specific time points during retinogenesis, and transcriptomic studies via RNA-seq were performed. Organoids expressed GFP+ and tdTomato+ signals, indicating expression of SIX6 and VSX2, respectively. Transcriptomic analysis of a developmental time course of retinal development identified gene expression changes occurring during the transition from early SIX6:GFP+ eye field progenitors to VSX2:tdTomato+ retinas. Bioinformatic analysis identified significant upregulation of neural and retinal developmental processes during differentiation from PSCs to SIX6:GFP+ eye field organoids and upregulation of specific retinal developmental processes during transition to SIX6:GFP+/VSX2:tdTomato+ retinal organoids. Further analyses of these mechanisms will lead to better understanding of gene regulation during retinal development as well as cell fate specification. Further knowledge of gene expression changes that occur during retinal development may allow for more efficient and faithful in vitro retinal induction and cell fate commitment.

Key Words: stem cells, retina, organoids, CRISPR, gene editing, transcriptomics, bioinformatics, development, differentiation, RNA-seq

30. Development of an Automated Platform for Predicting the Teratogenic Potential of Drugs Using Human Induced Pluripotent Stem Cells

Presented by: Cecile Terrenoire, The New York Stem Cell Foundation
Authors: Terrenoire, Cecile, The New York Stem Cell Foundation; McCarthy, Barry, The New York Stem Cell Foundation; Paull, Daniel, The New York Stem Cell Foundation; Bauer, Lauren, The New York Stem Cell Foundation; Otto, Reid, The New York Stem Cell Foundation; Jaklin, Manuela, F. Hoffmann-La Roche; Schaefer, Nicole, F. Hoffmann-La Roche Ltd; Kustermann, Stefan, F. Hoffman-La Roche; Noggle, Scott, The New York Stem Cell Foundation; McGinnis, Claudia, F. Hoffman-La Roche

The mouse embryonic stem cell test (mEST) is an in vitro assay widely used by the pharmaceutical industry to predict the teratogenic potential of new compounds. It combines a cytotoxicity study with a differentiation test to assess teratogenic potential using a statistical prediction model. Because it uses results obtained from a murine model to infer the teratogenic potential of compounds in humans, translatability could be limited. To improve translatability, reproducibility and throughput, we have developed an automated human iPSC-based teratogenicity test using the NYSCF Global Stem Cell ArrayTM. This test is composed of a cytotoxicity study using 3 cell lines (human fibroblasts, undifferentiated hiPSCs and embryoid bodies derived from hiPSCs), combined with a gene expression-based teratogenic study carried out with embryoid bodies derived from hiPSCs. Preliminary results for 8 compounds (4 teratogens and 4 non-teratogens) showed that teratogens caused substantial variations in the expression of genes associated with the 3 germ layers at doses below the cytotoxicity threshold, whereas non-teratogens produced little to no effects on global gene expression. This automated platform is being developed to facilitate rapid and efficient screening of a panel of compounds on human iPS cells and evaluate their teratogenic potential based on novel cytotoxicity and differentiation studies. Once validated, this test will provide a new preclinical, high throughput, automated approach for assessing the teratogenic potential of drug candidates using a human in vitro model.

Key Words: teratogens, embryotoxicity, cytotoxicity, human induced pluripotent stem cells, embryoid bodies, automation, gene expression

31. Macrophage Polarization Results in Differential Formation and Efficacy of Tunneling Nanotube-Mediated Delivery of Cystinosin to Cystinotic Cells

Presented by: Spencer Goodman, UC San Diego
Authors: Goodman, Spencer, UC San Diego; Nephade, Swati, UC San Diego; Chua, Emily, UC San Diego; Cherqui, Stephanie, UC San Diego

Tunneling nanotubes (TNTs) are a recently discovered method of long range intercellular communication. We have observed TNT formation from macrophages following hematopoietic stem and progenitor cell (HSPC) transplantation therapy for cystinosis, a genetic lysosomal storage disorder wherein cystine accumulates in the lysosome and causes tissue degeneration and multi-organ failure. We previously showed in Ctns-/- mice that a single HSPC treatment causes life-long preservation of tissue morphology and function. This occurs via transport of cystinosin-bearing lysosomes from HSPC-derived macrophages to diseased cells through TNTs. Macrophages have broad functions ranging from phagocytic and pro-inflammatory to homeostatic and anti-inflammatory. We polarized bone marrow derived macrophages (BMDMs) expressing cystinosin-eGFP to either a pro- or anti-inflammatory phenotype and then developed novel imaging analysis programs to quantify TNT formation and intercellular trafficking to diseased fibroblasts following co-culture. We discovered that fewer TNTs formed with less transfer of cystinosin-eGFP in the pro-inflammatory condition vs. anti-inflammatory or control. To extend these findings in vivo, BMDMs were isolated from two transgenic mice strains deficient in anti-inflammatory macrophage polarization (MAFIA and Rac2-/-). To our surprise, Ctns-/- mice responded equally well to transplantation of mutant and wildtype HSPCs. Furthermore, there was no difference in TNT formation or organelle trafficking in vitro. Our data highlight how in vivo models are essential to understand physiological macrophage behavior.

Key Words: tunneling nanotudes, macrophage polarization, cystinosis, regenerative medicine, organelle transfer, hematopoietic stem cells

32. Correction of Hyperglycemia Through Adenoviral Mediated Compartmentalized Liver Transduction of the Human Insulin Gene in a Type 1 Diabetes Mellitus Pig Model

Presented by: Gustavo Cabrera, Global BioTherapeutics
Authors: Cabrera, Gustavo, Global BioTherapeutics

Type 1 diabetes mellitus (T1DM) is a complex metabolic disease characterized by elevated blood glucose levels caused by the autoimmune destruction of insulin producing pancreatic ß-cells. Lifelong daily monitoring of blood glucose levels and administration of precise doses of exogenous insulin, with the goal of controlling glycemic fluctuations and therefore prevention of life-threatening complications remains the cornerstone of T1DM management and a colossal burden for patients and physicians. Gene therapy strategies aim at ameliorating glycemic fluctuations by achieving the secretion of physiologic levels of insulin from non-pancreatic-ß-cells. Using streptozotocin to induce T1DM on pigs, our group assessed the glycemic effect exerted by the secretion of human insulin dictated by an expression cassette driven by a glucose response element (GRE) carried by E1-E3 deleted tape 5 adenovirus and delivered through compartmentalized liver transduction (CLT). Compartmentalized liver transduction is a novel mode of hepatic vector administration achieved by the intra-parenchymal injection of the vector into a blood flow isolated portion of the liver. At a dose of 2x10e10 IFUs/kg (2x10e12 VPs/kg), correction of fasting and postprandial hyperglycemia was achieved and followed for two years in Vietnamese male pigs (n=2). Our results further demonstrate that CLT is a safe and effective mode of vector delivery. Additionally, our results demonstrate that glycemic fluctuations are manageable through gene therapy strategies and should be further explored for the treatment of T1DM.

Key Words: type 1 diabetes, adenovirus, liver transduction, insulin, hyperglycemia

33. Health Policies about Gene and Cell Therapies in European Countries

Presented by: Guvenc Kockaya, CarthaGenetics
Authors: Kockaya, Guvenc, CarthaGenetics; Cavus, Filiz, CarthaGenetics; Sharaf, Amir, CarthaGenetics; Amasya, Burcu, CarthaGenetics; Ben Mansour, Samira, CarthaGenetics; Bidani, Aroussi, CarthaGenetics; Campana, Stephan, CarthaGenetics; Kaya, Erhan, CarthaGenetics; Carteron De Balmont, Philippe, CarthaGenetics

Objectives: The concept of gene and cell therapies is to cure rather than to stabilize the diseases like progression free survival. Depending on that approach, there is an increasing attention on gene therapies by health industry, physicians, patients and health authorities as well. The aim of the study is to understand the current situation of gene and cell therapies in European Countries. Method: France, Germany, Portugal, Spain, Turkey and United Kingdom (UK) have been selected as representatives of European Countries. A computer based search has been conducted to find the defined regulatory process, health technology assessment reports, reimbursement and pricing decisions. The search conducted from June 2008 to June 2018 on Google Scholar search engines. Results: France, Germany, Portugal, Spain and United Kingdom (UK) follow European Medicine Agency decisions for marketing authorization approval process. However, Turkey, as a European Union candidate country, is not following EMA decisions and has not any defined legislation for gene and cell therapies. France, Germany and UK have been publishing HTA reports in recent years. However, there is not any published HTA reports from Portugal, Spain and Turkey. Conclusions: There is no common approach for gene&cell therapies across European Countries yet depending on search. However, it is very well known that new gene&cell therapies are coming to market soon. Health authorities need to establish a good working process and improve the infrastructures of health systems for gene and cell therapies as soon as possible before gene&cell therapy tsunami hits to Europe.

Key Words: European Union, gene and cell therapies, reimbursement, market access, regulatory

34. Stem Cell Research Aboard the International Space Station U.S. National Laboratory

Presented by: Rachel Clemens, Center for the Advancement of Science in Space
Authors: Mulligan, Molly, Center for the Advancement of Science in Space; Giulianotti, Marc, Center for the Advancement of Science in Space

The demand for stem cells for the use of biological therapies is growing rapidly, yet the development of new, innovative technologies to address this demand is not keeping pace. Since 2010, the microgravity environment of the International Space Station US National Laboratory ISS-NL has been used as a unique platform to study stem cell expansion, stem cell mediated recellularization, stem cell differentiation, and stem cell regeneration. We propose that the ISS-NL offers unparalleled opportunity for innovation in technology development to satisfy the needs of the tissue engineering and regenerative medicine sector. Life science research in microgravity has a number of fascinating ground-based applications. Most importantly, studying cells in the microgravity environment of space elucidates a large number of processes not possible on Earth. For example, cells form 3D structures without the use of scaffoldings and results show that those 3D structures behave more like those in the human body. Cellular gene expression has been shown to change in published data from stem cell research on the ISS-NL. Of note, studies done on station have demonstrated that stem cell expansion is accelerated in microgravity, making it a compelling environment to scale-up production of cells for cell-based therapies or tissue engineering. It has been demonstrated that cardiovascular progenitor cells have increased expression of DNA repair genes and paracrine factors, as well as enhanced migration. Taken together, projects highlighted here present a window into this one-of-a-kind testing facility meant to help develop innovative technologies for regenerative medicine and tissue engineering. Studies conducted at the ISS-NL also provide the foundation for potentially manufacturing high-quality tissues and organs for transplant. We will present current efforts to push innovation in regenerative medicine being conducted at the ISS-NL, how that work is supported, and what the future might hold for technology development leveraging microgravity.

Key Words: stem cells, regenerative medicine, microgravity, technology development

35. The Pipeline: Future Products, Indications and Treated Patients for Curative Therapies in the U.S.

Presented by: Colin Young, Massachusetts Institute of Technology
Authors: Young, Colin, Massachusetts Institute of Technology; Quinn, Casey, Massachusetts Institute of Technology

OBJECTIVE: Estimate curative therapy new product launches over the next 5 years and 10 years, including indications likely to launch and the number of patients likely to be treated. METHODS: Pipeline data for curative therapies (treatment effect beyond 18 months) were obtained from Informa/Citeline Pharmaprojects and extracted. Industry probabilities of success and cycle times were used to estimate future launches by indication and year. Estimates for treatment eligibility, including rates and timing of uptake for new patients and of clearance for patients with the disease were by our research team from publicly available data. Access to new therapies was combined with existing epidemiological data on disease incidence and prevalence to generate estimated numbers of treated patients. RESULTS: 663 products were identified that met our criteria. These products are being developed in 212 different indications, which we divided into 63 orphans (<10,000 patients in the US), 95 larger indications, and 54 from oncology. Based on standard industry success rates and cycle times, this industry pipeline would be expected to lead to 40-50 launches, with 20-30 launching within the next five years. The preponderance of the latter launches is expected to be in B-cell (CD-19) lymphomas and leukemias. In total, around 2 million patients will have been treated with up to 50-60 products by 2027. In 2027, 400,000 patients per year may be treated with cell and gene therapies. Estimates though are dependent on incidence in exactly which diseases have new products, and assumes launches in relapsed/refractory disease. Cancer and hematology account for the bulk of this activity; however, treated patient population sizes – and costs – are driven by the number of prevalent cases. The next decade will likely see phenomenal changes in the healthcare system with major improvements in the treatment paradigms for numerous diseases that previously had high morbidity and mortality. These will lead to great needs for the overall system to adapt, including in how treatments are reimbursed and financed as we move from chronic palliative therapies to acute curative ones. Tools for precision financing need to be developed and in place so as to address these challenges when they arise. LIMITATIONS: Informa Citeline/Pharmaprojects is based on published clinical trial information and may misrepresent actual pipelines. In particular, there may be a lag between the effective failure of a project and its public termination, and early projects may be underrepresented due to lack of disclosure. Success rates and timelines for curative products are likely to differ substantially from industry standards, but there is insufficient historical information to determine the direction and magnitude of these differences.

Key Words: cell therapies, gene therapies, pipeline, precision, finance

36. Formulation Development: Enabling Robust Vein to Vein Delivery of Autologous Cell and Gene Therapy Products

Presented by: Rita Majithiya, GlaxoSmithKline
Authors: Majithiya, Rita, GSK; Pianella, Monica, GSK; Esmaeili, Farnaz, GSK; Neagle, Sarah, GSK; Apte, Anjali, GSK; Gatliff, Olivia, GSK

Commercialization of cell and gene therapies to transform clinical successes into medicinal products will require meeting technical and logistical challenges include ensuring sterility through end-to-end closed processing systems, storage and distribution of cell based drug products between manufacturing and clinical sites, and drug product logistics at clinical sites. A rationally designed formulation development program incorporating a risk based approach can assure delivery of a robust product to the patient. Formulations which improve the shelf life of drug products mean the development of commercial ex-vivo autologous cell therapies and shifting from fresh drug products (shelf life of hours) to cryopreserved drug products (shelf life of months/years). To develop a robust cryopreserved drug product for T-cells, various DMSO containing formulations which included in-house and commercially available formulations were screened. Cryoformulations were prepared using a controlled rate freezing process and post-thaw cell viability and % recovery were used as methods for preliminary selection of the formulations. Post-thaw cell viability ranged from 23.15% to 98.75% and the % recovery ranged from 22.90% to 144.62% during preliminary formulation screening. In addition to the requirement for a robust formulation, robust freezing and thawing processes are critical to ensure the quality of a cryo-preserved product. To evaluate the robustness of the controlled rate freezing process, the impact of different container closure systems and fill volumes on product quality were studied. Studies conducted using TF-1 cells suggested that there was no impact of container closure and fill volumes on drug product in terms of cell viability and % recovery. Post-thaw viability ranged from 92.07% to 93.45% and the % recovery ranged from 114.43% to 140.37% for cells in either cryovials or cryobags (mimicking lowest and highest fill volumes) as container closure systems.

Key Words: formulation development, robust process development, autologous cell and gene therapy