Scientific Report for the Gladstone Institute of Cardiovascular Disease

Gladstone Institutes Findings 2018

MissionGladstone Institutes uses visionary science and technology to overcome major unsolved diseases.

VisionGladstone Institutes believes that life science research provides the most effective solutions to overcome major unsolved diseases and enables society to address health-related humanitarian issues worldwide.

Based in San Francisco’s Mission Bay neighborhood, the Gladstone Institutes is an independent state-of-the-art biomedical research institution that empowers its world-class scientists to find new pathways to cures. It has a close academic affiliation with the University of California, San Francisco.

Unified by a common vision, everyone at Gladstone believes that the best discoveries will come from bringing diverse thoughts, approaches, and people together to tackle scientific challenges in creative ways.

Gladstone Institutes

Gladstone LeadershipAndrew S. Garb Trustee

William S. Price III Trustee

Nicholas J. Simon Trustee

Deepak Srivastava, MD President

Scientific Report StaffMegan McDevitt Vice President of Communications

Thomas Becher Producer for Web

Gary Howard, PhD Editor

Julie Langelier Editor

Giovanni Maki Art Director

Sarah Gardner Graphic Designer

Teresa Roberts Design Assistant

ContributorsMartyna Ziemba-Martinez Graphic Designer

Diana Rothery Photographer

The reference period for this scientific report is from January 1, 2016, to December 31, 2017.

1650 Owens Street San Francisco, CA 94158 415.734.2000 gladstone.org

@gladstoneinst /gladstoneinstitutes

© 2018 Gladstone Institutes

Gladstone Institutes

ContentsGladstone by the Numbers 2

Message from the President 3

Institutes at Gladstone 4

Scientific Advisory Board 6

Major Research Achievements 6

Laboratory Reports

Thomas P. Bersot, MD, PhD 8

Benoit G. Bruneau, PhD 10

Bruce R. Conklin, MD 12

Sheng Ding, PhD 14

Saptarsi Haldar, MD 16

Todd C. McDevitt, PhD 18

Deepak Srivastava, MD 20

Shinya Yamanaka, MD, PhD 22

Programs and Initiatives 24

Research Infrastructure 25

Recent Discoveries 26

Findings 2018

2

$35.6M

Federal Grants

$4.7M

State Grants

$7.7M

Commercial Revenue

$12.5M

Gladstone’s Endowment

$20.5M

Private Support

People472

Trainee Careers (since 2012)

After their Gladstone training, postdoctoral scholars have moved on to:

Graduate Students

45

Administration/ Support Sta�

99

PostdoctoralScholars

103

Investigators(including a sta� research investigator and a visiting investigator)

26

Scientific Sta�

199

Other (government,non-science related,further studies)

31%

Science-related professions (including policy and business development)

8%

Industry

26%

Academia (tenured and non-tenured faculty positions)

35%

Gladstone by the Numbers

Finances$81M

Science292Publications in scientific journals from January 1, 2016, to December 31, 2017.

24Laboratories

9Core facilities and technology center

The financial information presented above is unaudited and has been prepared in accordance with accounting principles generally accepted in the United States.

Message from the PresidentAt the Gladstone Institutes, we believe that the key to making groundbreaking and paradigm-shifting discoveries lies in the power of interdisciplinary teams. When scientists from different fields come together to tackle a common problem, the combi-nation of their unique perspectives leads to more creative and comprehensive solutions. Ultimately, that’s how we can maximize the impact of biomedical research on improving human health.

This successful model has spread throughout our organization. In the past few years, it led to the creation of several new research centers aiming to foster collaborations around broad thematic areas and exceed the potential of any single laboratory.

Within the Gladstone Institute of Cardiovascular Disease (GICD), our team science approach has united basic biologists, chemists, computer scientists, and engineers, all working towards a shared goal of unraveling the most fundamental aspects of the cardio-vascular system. We also join forces with investigators through-out Gladstone and the San Francisco Bay Area, as well as the experts in our core facilities and technology centers.

This breadth of knowledge and variety of viewpoints provide a rich learning environment for our trainees. A central part of Gladstone’s mission, mentoring is a priority for all our investigators as we recognize the importance of preparing graduate students and postdoctoral scholars for successful scientific careers.

At GICD, our research focuses on understanding how the entire cardiovascular network develops and functions, both in health and disease. We explore the cellular and molecular mecha-nisms underlying pluripotency and cardiogenesis, as well as the basic concepts in gene regulation, particularly those disrupted in human disease. We combine stem cell biology, gene editing techniques, and chemical biology approaches, while also engi-neering 3D tissues and organoids, and developing novel tech-nologies. Our goal is to fill existing gaps in cardiac regeneration and genetics and to find better therapeutic strategies for patients with cardiovascular disease.

Building on our discoveries, Gladstone launched a new biophar-maceutical company, Tenaya Therapeutics Inc., in 2016. This spin-off company was formed from the BioFulcrum initiative, developed to enhance Gladstone’s translational efforts by merging our basic science expertise with the resources and translational know-how of the biotechnology industry. We are very proud of Tenaya and continue to be closely involved in their work, which strives to create new therapeutics for cardiac regen-erative medicine and drug discovery for heart failure.

As the new president of Gladstone, I am honored to represent my scientific colleagues and their outstanding research. I invite you to read this scientific report, which highlights the accomplish-ments of GICD’s eight investigators from 2016 and 2017.

4

Findings 2018

Institutes at GladstoneGladstone houses four major institutes, each representing different yet interconnected areas of focus. Interwoven are multiple research centers that bring together common approaches or themes throughout the organization. Driven by their inquisitive nature, investigators have the freedom to follow their research wherever it leads, and work closely with their colleagues in all institutes to deeply probe important questions in biomedicine. Above all, they champion highly interactive, creative, and mold-breaking approaches to science as they seek prevention, treatments, and cures for major diseases.

Supported by state-of-the-art core facilities and professionally trained staff, Gladstone scientists rely on the latest technologies to advance their work. And to deliver results to patients, as urgently as possible, they join forces with the Office of Corporate Ventures and Translation to develop fruitful collaborations with the San Francisco Bay Area biomedical industry.

Gladstone investigators also actively invest in the future of research. They remain strongly committed to mentoring graduate students and postdoctoral scholars, who will have to overcome tomorrow’s scientific and medical challenges.

5

Gladstone Institutes

Gladstone Institute of Cardiovascular DiseaseCardiovascular disease remains the world’s leading cause of death, with heart failure alone afflicting over 26 million people around the globe. Despite decades of work, patients and doctors still need scientific and medical breakthroughs to combat these devastating diseases, which include heart attacks, congenital heart defects, and other disorders.

To address this critical situation, the Gladstone Institute of Cardiovascular Disease (GICD) leverages important genetic, developmental, chemical, biological systems, computational, and engineering approaches to the study of heart disease and stem cell biology.

Recently, much of their work expands on two paradigm-shifting discoveries: induced pluripotent stem cells and CRISPSR-Cas9 gene editing. Gladstone scientists uncovered new and more efficient ways to use these technologies to study cardiovascular disease and transform their research into therapies that help repair damaged hearts.

Gladstone Institute of Virology and ImmunologySince its foundation 25 years ago, the Gladstone Institute of Virology and Immunology (GIVI) has made significant contri-butions to fighting HIV/AIDS, which ranks among the deadliest infectious epidemics ever recorded. Its investigators defined the life cycle of HIV, paved the way for many medications currently in use, and led a global and groundbreaking effort in HIV prevention.

Today, antiretroviral drugs can help prolong lifespan and improve the quality of life for people with HIV. However, patients require lifelong treatment of daily medications, because the virus persists in a latent form. Scientists in GIVI are uniquely positioned to explore the biological basis for HIV latency, which represents the greatest barrier to a cure.

The major successes in HIV research have provided Gladstone with an opportunity to shift this institute’s focus to a new field. Investigators are working to identify a new area of research that could be most impacted by capitalizing on the existing strengths of GIVI.

Gladstone Institute of Neurological DiseaseDiseases that affect the brain or other parts of the central nervous system raise fascinating neuroscientific questions and are among the most devastating and complex conditions plaguing humankind. As populations around the world are living longer, neurodegenerative disorders are rising in prevalence at an unprecedented pace. However, many of these diseases remain without effective treatment options.

Consequently, the Gladstone Institute of Neurological Disease (GIND) maintains a strong focus on neurodegenerative and neuroinflammatory diseases, including Alzheimer’s disease, fron-totemporal dementia, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and multiple sclerosis. Given the overlap between these conditions and other challenging brain diseases, they also study epilepsy and neuropsychiatric disorders, such as autism and depression.

GIND investigators believe that pathogenic convergence points among these conditions will allow them to identify therapeutic interventions that might benefit multiple disorders. They are using unconventional approaches to yield such multi-faceted solutions, while also expanding capabilities in regenerative and personalized medicine.

Gladstone Institute of Data Science and BiotechnologyIn recent years, the deployment of advanced technologies has become crucial in driving novel scientific discovery. Researchers increasingly depend on creative data analysis and integration to interrogate biological questions. To respond to this growing need, the Gladstone Institute of Data Science and Biotechnology was launched in 2018.

Building on the success of the Convergence Zone, this new insti-tute’s mission is to decode biomedical knowledge that is missed without rigorous statistical approaches. Their efforts will combine multiple intellectual and physical assets, as well as new machine learning and artificial intelligence approaches, that will impact numerous disease areas. They will also develop new research technologies and platforms to propel groundbreaking science throughout Gladstone and the San Francisco Bay Area.

Findings 2018

Scientific Advisory BoardMembers of the 2018 Scientific Advisory Board for the Gladstone Institute of Cardiovascular Disease

Brian Black, PhD Cardiovascular Research Institute University of California, San Francisco

George Q. Daley, MD, PhD Children’s Hospital Boston Harvard Medical School Howard Hughes Medical Institute

Joseph Goldstein, MD Departments of Molecular Genetics and Internal Medicine University of Texas Southwestern Medical Center at Dallas

Andrew R. Marks, MD The Clyde and Helen Wu Center for Molecular Cardiology Columbia University

Deborah Nickerson, PhD Department of Genome Sciences University of Washington School of Medicine

Eric N. Olson, PhD Department of Molecular Biology University of Texas Southwestern Medical Center at Dallas

Marlene Rabinovitch, MD Department of Pediatrics, Cardiology Stanford Cardiovascular Institute Stanford University School of Medicine

Janet Rossant, PhD Developmental and Stem Cell Biology Program The Hospital for Sick Children University of Toronto

Christine Seidman, PhD Department of Genetics and Medicine Harvard Medical School

Irving L. Weissman, MD Institute for Stem Cell Biology and Regenerative Medicine Stanford University School of Medicine

Major Research AchievementsOver the past 40 years, investigators at the Gladstone Institute of Cardiovascular Disease made important discoveries that have significantly impacted the scientific community. These achievements became the foundation for numerous subsequent research projects that continue to advance knowledge in the field of cardiovascular biology, cellular reprogramming, and regenerative medicine.

7

Gladstone Institutes

Discovered genetic causes and underlying mechanisms of heart diseaseSince 1979, Gladstone investigators have been at the forefront of characterizing heart disease. Early on, they made significant contributions to the scientific community’s understanding of how cholesterol and apolipoproteins are involved in coronary artery disease. Specifically, they showed how apolipoprotein E2 contributes to type III hyperlipoproteinemia and premature heart disease. More recently, the scientists shifted their focus to better understand early heart development and birth defects that affect the heart. They showed the mechanisms underlying human cardiac septal defects and valve disease.

Identified and defined apolipoprotein E and its role in cholesterol metabolism and heart diseaseGladstone scientists identified and described the characteristics of apolipoprotein E (apoE), one of the major lipoproteins involved in cholesterol metabolism, heart disease, and neurological disease. They determined the amino acid and gene sequences of the three isoforms of apoE, their three-dimensional structures and their effects on function, and their involvement in cholesterol metabolism and heart disease. This influential research laid the basis for showing apoE4’s involvement in Alzheimer’s and other neurological diseases.

Identified and defined the function of lipid metabolism enzymesLipids, especially triglycerides, are the major energy storage molecules for animal and human cells. Excessive accumulation of triglycerides, however, is associated with human diseases, such as obesity, diabetes, and steatohepatitis. Little was known about the enzymes and synthetic pathways that make these fats, until Gladstone scientists identified and characterized those enzymes, including monoacylglycerol acyltransferases and diacylglycerol acyltransferases. Their studies helped define the possible roles of these important enzymes in human health and diseases.

Defined the sequence of molecular events in normal and abnormal heart development and homeostasisFor regenerative medicine to be successful in repairing human hearts, it is critical to understand normal and abnormal heart development, as well as the morphogenetic and patterning processes that occur to assemble all of the heart’s components into a functional organ. Gladstone scientists achieved this by deciphering a basic blueprint for the development of the heart. To do so, they systematically investigated the function of tran-scriptional and epigenetic regulators across the entire genome, and gained a deeper understanding of how networks of genes are deployed for important patterning and morphogenetic decisions in heart development. Recent recognition that broad epigenetic dysregulation in heart failure underlies the reactiva-tion of fetal gene programming and activation of fibrotic path-ways provides novel targets for treating cardiac dysfunction.

Reprogrammed resident cardiac fibroblasts to cardiomyocytes in situ to regenerate damaged heartsWhen a heart attack occurs, blood flow is lost to a portion of the heart muscle. Without a steady supply of oxygen, the heart muscle dies and cardiac fibroblasts—which make up about 50 percent of the heart—move in to form non-beating scar tissue. In a seminal advance, Gladstone investigators reprogrammed the abundant fibroblasts in a mouse heart into beating heart muscle. As a result, instead of forming scar tissue, the fibroblasts became cardiomyocytes, incorporated themselves into the heart tissue, and began beating. The approach of harnessing resident cells to regenerate the heart is now being developed toward clinical application within the spin-out company, Tenaya Therapeutics.

Developed cellular reprogramming process for multiple cell types using chemicalsThe initial discovery of reprogramming adult cells into stem cells revolutionized biology and energized research into regenerative medicine. Gladstone scientists identified small molecules that can replace the genetic material that was traditionally used to reprogram cells. More recently, Gladstone scientists success-fully reprogrammed fibroblasts to pluripotent stem cells using CRISPR-Cas9 technology to activate enhancers and promoters of pluripotency factors. They also identified discrete combinations of small molecules that can reprogram fibroblasts directly into heart, neural, liver, and pancreatic cells and control specific types of T cells, simplifying the process of reprogramming to specific cell types.

Laboratory Reports

Since 1990, Thomas P. Bersot has directed the Gladstone Lipid Disorders Training Center, helping educate health care providers to better manage risk factors for cardiovascular disease (CVD) in their patients, including overweight and obesity. The center has trained over 4,200 health care pro-viders in the Community Health Network of the San Francisco Department of Public Health and at San Francisco General Hospital. In addition to physicians, the courses are offered to nurse practitioners, clinical pharmacists, dietitians, and ex-ercise physiologists, given their substantial responsibility for managing CVD risk factors in primary-care patients.

AccomplishmentsThe Gladstone Lipid Disorders Training Center offers two types of courses several times per year.

The basic 2-1/2-day course covers the physiology and patho-physiology of plasma lipid metabolism, hypertension, and diabe-tes mellitus. Bersot and his team review the evidence supporting risk assessment tools and the use of diagnostic procedures and therapies. They devote extensive time to diet, exercise, and weight management, which are the cornerstones of CVD preven-tion. They also review safe and appropriate use of medications. In addition, attendees participate in a 1-day demonstration clinic where they see actual patients, thus providing them with practi-cal experience in patient management.

The other course is a 1-day update for previous students and covers new diagnostic methods for assessing the risk of sustain-ing a clinical vascular disease event and the treatment implica-tions of recently completed clinical studies. Bersot reviews new drug therapies and discusses significant developments in life-style management.

Bersot and his team stress the value of a healthy lifestyle, which can reduce vascular disease risk by 50 percent or more and add to the benefits of drug therapy and invasive treatments (angioplasty, stenting, and bypass surgery). The courses’ focus

Highlights Conducted two courses to train 155 health care providers in cardiovascular risk factorsOversaw the training of 68 residents in managing cardiovascular disease risk factorsServed as a member of the institutional review board for human research at San Francisco General Hospital

Thomas P. Bersot MD, PhDASSOCIATE INVESTIGATOR

9

Gladstone Institutes

Top Five Overall PublicationsLing H et al. Genome-wide linkage and association analyses to identify genes influencing adiponectin levels: the GEMS study. Obesity (Silver Spring). 2009.

Mahley RW et al. Low levels of high density lipoproteins in Turks, a population with elevated hepatic lipase. High density lipoprotein characterization and gender-specific effects of apolipoprotein E genotype. Journal of Lipid Research. 2000.

Rall SC Jr et al. Type III hyperlipoproteinemia associated with apolipoprotein E phenotype E3/3. Structure and genetics of an apolipoprotein E3 variant. Journal of Clinical Investment. 1989.

Bersot TP et al. Interaction of swine lipoproteins with the low-density lipoprotein receptor in human fibroblasts. Journal of Biological Chemistry. 1976.

Mahley RW et al. Identity of very low density lipoprotein apo-proteins of plasma and liver Golgi apparatus. Science. 1970.

is expected to be reversed in the next 5 years. The good news is that a significant proportion of the risk of heart disease is completely within our own hands.

Future DirectionBersot will continue his work to train physicians and other health care providers using the latest methods of managing cardiovas-cular disease risk factors. He hopes that drawing attention to the value of a healthy lifestyle will contribute to improving the management of these risk factors and, ultimately, reduce the number of deaths associated with CVD.

on these issues is a uniformly popular feature, because encour-aging patients to comply with lifestyle change recommendations is difficult.

Research ImpactCoronary heart disease is still responsible for one in six deaths in the United States. If stroke and heart failure are included, CVD caused about one in three deaths (≈690,000 deaths) in 2008.

The number of annual deaths caused by CVD has significantly decreased since 1968, due to better treatments and improve-ments in the management of CVD risk factors. However, the steadily increasing prevalence of overweight, obesity, and diabe-tes has increased CVD death in adults by nearly 20 percent. As of 2008, 68 percent of adults in the United States were over-weight or obese, as were one in three children 12–19 years of age, and nearly one-fifth of children 2–11 years of age.

If this trend of increasing overweight and obesity in young chil-dren persists, the decline in CVD mortality that began in 1968

Gladstone Lipid Disorders TrainingThe Center’s training courses are endorsed by the American Heart Association. Since the founding of the center in 1990, these courses have educated over 4,200 health care providers on ways to help patients reduce their risk of cardiovascular disease.

Laboratory Reports

Benoit G. Bruneau and his team aim to understand how the human genome is coordinately regulated to make specific cell types, such as cardiomyocytes, and how this normally stable blueprint is misread in inherited and acquired heart disease.

AccomplishmentsBruneau’s laboratory demonstrated the interactions between three disease-related transcription factors—TBX5, NKX2-5, and GATA4—at a genome scale. They found that these proteins co-localize across the genome to regulate the cardiac gene expression program, and elucidated some of the rules by which they co-recruit one another to active cardiac enhancers. The scientists also identified a protein-protein interaction that facili-tates their shared binding, through the crystal structure of TBX5, NKX2-5, and their shared DNA binding site.

In addition, the team studied the roles played by another tran-scription factor, CTCF, in embryonic stem cells. Using a new system that allows rapid and reversible depletion of CTCF, they showed that the three-dimensional organization of chroma-tin into structures called “topologically associated domains” is highly dependent on CTCF. Through these studies, they discovered new rules about chromatin organization and how it impacts gene regulation.

They also examined the importance of a disease-related histone-modifying enzyme called KMT2D. The gene that encodes this protein is often mutated in congenital heart disease. They deleted KMT2D in mice and showed that it controls a set of genes essential for embryonic cardiac function by depositing at regulatory elements in the genome a specific type of histone modification that helps genes become activated.

Lab Members* indicates current lab membersLaure BernardAaron Blotnick*Pervinder ChoksiSteven CincottaWalter Devine*Bayardo GarayMatthew George*Piyush Goyal*Swetansu Hota*Vasumathi Kameswaran*Irfan Kathiriya*

Alexis Krup*Alejandra Lopez DelgadoLuis Luna-ZuritaDario Miguel-PerezAbigail Nagle*Elphège-Pierre Nora*Diego QuinteroKavitha Rao*Tanya SukonnikAlec UebersohnSarah Wood*

HighlightsDefined the mechanism underlying 3D genome organizationDiscovered how cardiac transcription factors work together to form a heartDiscovered how chromatin-remodeling complexes change their identity to shape the genome of a heart cell

Benoit G. Bruneau PhDASSOCIATE DIRECTOR AND SENIOR INVESTIGATOR

11

Gladstone Institutes

Research ImpactBruneau’s research is important for understanding basic concepts in gene regulation, and how they are dysregulated in disease. The demonstrated interactions between cardiac transcription factors provided new insights into the tight regula-tion of gene cohorts, which has had immediate implications in understanding how diseases in these transcription factor genes cause similar diseases. These findings are broadly impactful as they apply to any set of transcription factors, in any cell type. In addition, his team’s work on CTCF and 3D genome organization resolved several long-standing questions in various fields, and raised new questions that now need answers.

Future DirectionThis laboratory focuses on understanding how the earliest deci-sion by an embryo or a stem cell to become a heart precursor is regulated, and how global gene regulation is coordinated in this process. The scientists are further exploring the cellular and molecular mechanisms by which discrete cell types contribute to specific cardiac structures, such as the interventricular septum. They are also investigating how chromatin-remodeling complexes establish a “go/no-go” switch in cardiac differentiation. Finally, they use human induced pluripotent stem cell models to under-stand, at a single-cell resolution, how disease-causing mutations in TBX5 affect gene expression and chromatin states.

MediatorComplex

mRNA

Pol II Pol IIGeneralTFs

ChromatinRemodeling

Complex

Histone ModificationReader/Writer

TranscriptionFactors

Co-Factor

RepressedChromatin

BindingSites

DNACardiomyocyteMolecular Players for Transcriptional RegulationCis-regulatory elements containing DNA binding sites are bound by transcription factors and modulate the assembly of the Pre-Initiation Complex at promoters through physical contacts driven by a three-dimensional arrangement of chromatin, thereby acting as a molecular platform between cellular signaling and gene activity.

Selected Recent PublicationsSun X et al. Cardiac-enriched BAF chromatin-remodeling complex subunit Baf60c regulates gene expression programs essential for heart development and function. Biology Open. 2017.

Nora E et al. Targeted degradation of CTCF decouples local insulation of chromosome domains from genomic compartmentalization. Cell. 2017.

Hota S et al. ATP-dependent chromatin remodeling during mammalian development. Development. 2016.

Ang S et al. KMT2D regulates specific programs in heart development via histone H3 lysine 4 di-methylation. Development. 2016.

Luna-Zurita L et al. Complex interdependence regulates heterotypic transcription factor distribution and coordinates cardiogenesis. Cell. 2016.

Top Five Overall PublicationsNora E et al. Targeted degradation of CTCF decouples local insulation of chromosome domains from genomic compartmentalization. Cell. 2017.

Luna-Zurita L et al. Complex interdependence regulates heterotypic transcription factor distribution and coordinates cardiogenesis. Cell. 2016.

Devine W et al. Early patterning and specification of cardiac progenitors in gastrulating mesoderm. eLife. 2014.

Wamstad J et al. Dynamic and coordinated epigenetic regulation of developmental transitions in the cardiac lineage. Cell. 2012.

Takeuchi J et al. Directed transdifferentiation of mouse mesoderm to heart tissue by defined factors. Nature. 2009.

Laboratory Reports

Decoding human genetic disease allows Bruce R. Conklin and his team to develop models of pathology that can be directly tested with gene correction or targeted drug therapy. Dominant negative mutations are particularly promising thera-peutic targets since they are resistant to traditional therapies, and yet, precise excision of a disease-causing allele could provide a cure. This laboratory uses patient-derived induced pluripotent stem cells (iPSCs) to model diseases in tissues that are particularly susceptible to dominant negative mutations: cardiomyocytes, motor neurons, and retinal pigment epithelial (RPE) cells. By developing CRISPR genome surgery in human cells, they hope to devise improved cellular models and human therapies.

AccomplishmentsConklin’s team has successfully created stem cell models of genome surgery. By focusing on allele-specific gene excision, they can select gene mutations that are highly penetrant, with clear phenotypes in cell types that can be readily derived from iPSCs. They use whole-genome sequencing to identify common genetic polymorphisms, which can be used to selec-tively inactivate the disease allele with CRISPR nucleases. The diseased cell types allow them to decode the cellular signatures of disease and determine if the excision of the disease allele restores cellular functioning.

Genome surgery is a rapidly advancing field that uses state-of-the-art techniques to push the boundaries of cell and molecular biology. This laboratory uses advanced microscopy, tissue engi-neering, and single-cell genomics to optimize precise editing. They are also developing computational methods to select optimal CRISPR/Cas9 combinations in diverse populations. They aim to produce therapies that are safe and cost effective so they can benefit the maximal number of people. In collaboration with clinical scientists and the Innovative Genomics Institute, they are preparing large animal models and clinical-grade reagents for human clinical trials.

Lab Members* indicates current lab membersAmanda Jayne CarrCarissa FelicianoVanessa Herrera*Kristin HolmesNathaniel Huebsch*Olga IvanovaChristina JensenLuke Judge*Kathleen Keough*Angela Ziqi Liu*Mohammadali Mandegar

Steven MayerlMeghan McKenna*Michael Olvera*Meiliang Pan*Juan Pérez-Bermejo*Edward ShinKenneth Tan*An TruongHannah Watry*Kenneth Wu*Perry Wu

HighlightsEstablished precise genome-editing methods for disease modeling and therapyEstablished an efficient method to produce single-base changes in iPSCsPioneered the use of CRISPRi to epigenetically control gene expression in iPSCs

Bruce R. Conklin MDSENIOR INVESTIGATOR

13

Gladstone Institutes

Future DirectionGenome engineering and stem cell biology have been the most disruptive technologies of this millennia. Advances in iPSC differentiation and cell modeling will allow more cell types and sophisticated multicellular models of disease. These will provide molecular insights into many diseases, which are likely to lead to improved drug therapy without gene correction. Conklin’s team aims to further enhance these sophisticated methods to intervene in genetic disease with epigenetic modification or base editing that will expand the field of genome surgery. They continue to leverage these new advances to reach their goal of decoding and repairing genetic disease.

Research ImpactA major benefit of testing genome surgery in authentic cellular models is the mechanistic insights into the disease process and the potential for functional recovery. The reversion of a cellular phenotype is proof that a dominant negative allele was causative and that the disease process is reversible.

Detailed cellular analysis often provides new insights into the mechanism of the disease. Genomic deletions require detailed knowledge of the non-coding elements that are poorly under-stood, such as enhancers, LncRNAs, and microRNAs. Each cell type allows the researchers to probe the 3D architecture and epigenetic state of the gene region, since distant DNA can be in close proximity, allowing efficient excision of larger genomic segments. Finally, as Conklin’s team learns to orchestrate precise repair, they will better understand the DNA repair machinery of each cell type.

Genome surgery is an emerging field of medicine that will drive a new level of investigation into the molecular physiology of diverse cell types, including cardiomyocytes, motor neurons, and RPE cells. Only by understanding the basic cellular and molecular physiology of these cells can scientists meet the challenges that lie ahead.

Cleavage

Cas9 gRNA

Target

3%Healthy

100%Healthy

97%Diseased

Excision

DominantNegative

AlleleTarget A Target B

Resulting Protein Complexes

NormalProtein

DiseasedProtein

Genome Surgery for Dominant Negative DiseaseThe CRISPR/Cas9 system can be used to selectively silence the disease allele without altering the normal allele. In the lower panel, a dominant negative disease allele (orange) poisons the protein complex. CRISPR excision blocks the disease allele to ensure all proteins remain healthy.

Selected Recent PublicationsJudge LM et al. BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress. JCI Insight. 2017.

Liu SJ et al. CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells. Science. 2017.

Huebsch N et al. Miniaturized iPS-cell-derived cardiac muscles for physiologically relevant drug response analyses. Scientific Reports. 2016.

Mandegar MA et al. CRISPR interference efficiently induces gene knockdown and models disease in iPSCs. Cell Stem Cell. 2016.

Miyaoka Y et al. Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing. Scientific Reports. 2016.

Top Five Overall PublicationsConklin BR et al. Engineering GPCR signaling pathways with RASSLs. Nature Methods. 2008.

Dahlquist KD et al. GenMAPP, a new tool for viewing and analyzing microarray data on biological pathways. Nature Genetics. 2002.

Redfern CH et al. Conditional expression and signaling of a specifically designed Gi-coupled receptor in transgenic mice. Nature Biotechnology. 1999.

Conklin BR et al. Substitution of three amino acids switches receptor specificity of Gqα to that of Giα. Nature. 1993.

Federman AR et al. Hormonal stimulation of adenylyl cyclase through Gi-protein βγ subunits. Nature. 1992.

Laboratory Reports

The team led by Sheng Ding develops new chemical biology approaches to study stem cell biology and regeneration. Their current work focuses on identifying and characterizing novel small molecules that control cell fate and/or function in various systems, including maintenance of tissue-specific stem cells, directed differentiation of pluripotent stem cells toward new cell lineages, reprogramming of lineage-restricted somatic cells to alternative cell fate, and regulation of cancer stem cells. The identified small molecules or generated cells are further characterized in vitro and in vivo. Furthermore, mech-anistic studies of these small molecules provide new insights underlying fundamental processes in cell fate regulation.

AccomplishmentsThe scientists recently developed a new paradigm in cellular transdifferentiation using the cell-activation and signaling-directed (CASD) lineage-conversion strategy. This method employs tran-sient exposure of somatic cells with reprogramming molecules (cell activation, CA) in conjunction with lineage-specific soluble signals (signal-directed, SD) to reprogram somatic cells into diverse lineage-specific cell types without entering the pluripotent state. The strategy was demonstrated by directly converting fibro-blasts into cardiac, neural, or definitive endoderm precursor cells in mouse and human systems.

Importantly, Ding’s laboratory identified specific chemically defined conditions that enable robust expansion of the repro-grammed lineage-specific precursor cells, which could then be further differentiated into mature functional cells in vitro. Transplanting those CASD-reprogrammed cells rescued disease phenotypes in corresponding mouse models, demonstrating potential utility in cell-based therapy. More significantly, they identified chemical cocktails that enable CASD-based repro-gramming without genetic manipulations to generate cardiac and neural precursor cells from fibroblasts.

Lab Members* indicates current lab membersNan CaoShengping HouKe LiChangsheng Lin*Kai LiuPeng LiuTianhua Ma

Shibing TangHaixia WangShaohua XuTao XuChen Yu*Mingliang Zhang

HighlightsReprogram human fibroblasts into cardiomyocytes with a cocktail of small moleculesReprogram mouse fibroblasts into neural progenitor cells with a cocktail of small moleculesReprogram human Th17 cells into regulatory T cells

Sheng Ding PhDSENIOR INVESTIGATOR

15

Gladstone Institutes

Future DirectionDing will continue to pursue the research paradigm of identifying and further characterizing novel small molecules that control cell fate and/or function in vitro and in vivo, especially in the context of disease and injury.

Additional discoveries by the team include reprogramming pro-inflammatory Th17 cells into immune suppressive regulatory T cells by a small molecule using a novel immunometabolism mechanism, reprogramming white adipogenic cells into brown/beige adipogenic cells, and showing in vivo reprogramming of those cell types by the small molecules and disease rescue in relevant mouse models.

Research ImpactDing and his team hope their continued studies will ultimately facilitate therapeutic applications of stem cells and the devel-opment of small-molecule drugs. These could be used to stim-ulate the body’s own regenerative capabilities by promoting survival, migration/homing, proliferation, differentiation, and reprogramming of endogenous stem/progenitor cells or more differentiated cells.

NeuralInductionMedium

Fibroblast

Epigenetically“Activated”

Cell Population

TransientTreatment with

Molecules

Small Molecule Inhibitorof Pluripotency

iPSC Mediumand ExtendedExpression of

iPSC TFs

CardiacInductionMedium

DefinitiveEndodermInductionMedium

iPSC

CardiomyocyteCardiacPrecursor

Pancreatic andHepatocyte

EndodermPrecursor

Neuron and GlialNeural

Precursor

A Novel Path to TransdifferentiationTemporally restricted ectopic overexpression of reprogram-ming factors in fibroblasts leads to the rapid generation of epigenetically “activated” cells, which can then be coaxed to

“relax” back into various differentiated states, ultimately giving rise to somatic cells entirely distinct from the starting population. TF, transcription factor. iPSC, induced pluripotent stem cell.

Selected Recent PublicationsXu T et al. Metabolic control of TH17 and induced Treg cell balance by an epigenetic mechanism. Nature. 2017.

Nie B et al. Brown adipogenic reprogramming induced by a small molecule. Cell Reports. 2017.

Cao N et al. Conversion of human fibroblasts into functional cardiomyocytes by small molecules. Science. 2016.

Zhang M et al. Pharmacological reprogramming of fibroblasts into neural stem cells by signaling-directed transcriptional activation. Cell Stem Cell. 2016.

Zhang Y et al. Expandable cardiovascular progenitor cells reprogrammed from fibroblasts. Cell Stem Cell. 2016.

Top Five Overall PublicationsLi H et al. Versatile pathway-centric approach based on high-throughput sequencing to anticancer drug discovery. Proceedings of the National Academy of Sciences. 2012.

Li W et al. Rapid induction and long-term self-renewal of primitive neural precursors from human embryonic stem cells by small molecule inhibitors. Proceedings of the National Academy of Sciences. 2011.

Efe JA et al. Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. Nature Cell Biology. 2011.

Shi Y et al. A combined chemical and genetic approach for the generation of induced pluripotent stem cells. Cell Stem Cell. 2008.

Chen S et al. Dedifferentiation of lineage-committed cells by a small molecule. Journal of the American Chemical Society. 2004.

Laboratory Reports

The goal of Saptarsi Haldar’s laboratory is to dissect the molecular mechanisms used by cells to control gene expres-sion during cardiovascular and metabolic homeostasis. Furthermore, his team seeks to understand how these gene regulatory mechanisms are dysregulated in disease and find novel approaches to manipulate the epigenetic signaling pathways for therapeutic gain.

AccomplishmentsThe research team discovered several novel epigenetic signaling mechanisms that govern cardiovascular and metabolic homeo-stasis. For example, they found that BRD4, a member of the BET bromodomain family epigenetic reader proteins, is a critical regu-lator of cardiovascular stress responses and is important in heart failure pathogenesis and vascular remodeling. They showed proof-of-concept that small-molecule inhibition of BRD4 has ther-apeutic potential in cardiovascular disease.

Haldar’s team also found that another epigenetic protein, CDK7 (the core kinase in the TFIIH complex), is a novel druggable target in heart failure pathogenesis. In addition, they identified Salt-inducible kinases (SIKs) as novel effectors of cardiomyocyte transcription control and pathological cardiac remodeling. They also used deep epigenomic interrogation of smooth muscle cells to discover novel core regulatory transcriptional circuitry that drives smooth muscle phenotypic switching, findings which have major implications for vascular diseases.

In the field of metabolism, they discovered that the transcription factor KLF15 is a master regulator of hepatic cortisol binding globulin production and is an essential regulator of systemic glucocorticoid bioactivity during physiology and disease. Using CRISPR-Cas9–based genome editing, they epitope tagged the KLF15 allele in mice, and are now discovering the first endoge-nous cistromes and interaction partners for this nodal metabolic transcription factor.

Lab Members* indicates current lab membersMichael Alexanian*Priti Anand*Rohan BhardwajAnna ChenQiming Duan*Previn Ganesan

Austin Hsu*Zhen Jiang*Sarah McMahon*Arun Padmanabhan*Sarah Wood*

HighlightsDiscovered several novel epigenetic regulators of cardiovascular homeostasis that may be druggable targets in heart failureLeveraged deep epigenomic interrogation to uncover novel core regulatory circuitry in smooth muscle cell phenotypic plasticityDiscovered novel molecular pathway in the liver that is essential for systemic glucocorticoid hormone homeostasis

Saptarsi Haldar MDASSOCIATE INVESTIGATOR

17

Gladstone Institutes

Future DirectionThe team engineered a suite of genetically modified cells and mice to deeply probe the molecular function of several epigen-etic regulators, including BET proteins, CDK7, SIKs, and KLF15, both in physiological homeostasis and disease. In a new discov-ery effort, they are leveraging deep epigenomic interrogation to decipher the core transcription factor regulatory circuitry that controls postnatal cardiomyocyte maturation, which represents a major knowledge gap in the cardiac regeneration field. Finally, they are developing genetic screens to discover novel regulators of cardiomyocyte homeostasis and plasticity.

Research ImpactThe Haldar laboratory discovered new epigenetic signaling mechanisms used by cardiovascular and metabolic tissues to control gene expression. For a number of these gene regulatory pathways, they showed how mechanisms go awry in disease and provided proof-of-concept that specific epigenetic signaling effectors can be pharmacologically targeted in conditions, such as heart failure, vascular dysfunction, and muscular dystrophy. Through a detailed mechanistic understanding of cardiovascular and metabolic gene regulation, they ultimately hope to find new therapies for human disease.

CellularLevel

OrganLevel

Diseased andDysfunctional

Cardiomyocyte

Diseased Heart

MatureCardiomyocyte

Mature Heart

NewbornHeart

CommittedCardiomyocyte

TF/Chromatin

TF/Chromatin

Haldar’s team aims to understand how gene expression is controlled in the postnatal heart during physiology and disease at both the cellular and whole-organ levels. This includes studying how DNA binding transcription factors (TFs) signal in the context of chromatin to fashion the mature cardiomyocyte and adult heart during postnatal growth and development, and how these mechanisms go awry in the diseased heart. The same conceptual framework is applied to understanding gene control mechanisms underlying cell state changes in blood vessels and key metabolic organs, such as liver and skeletal muscle.

Selected Recent PublicationsNewman J et al. Ketogenic diet reduces midlife mortality and improves memory and aging in mice. Cell Metabolism. 2017.

Duan Q et al. BET bromodomain inhibition suppresses innate inflammatory and profibrotic transcriptional programs in heart failure. Science Translational Medicine. 2017.

Stratton M et al. Signal-dependent recruitment of BRD4 to cardiomyocyte super-enhancers is suppressed by a microRNA. Cell Reports. 2016.

Top Five Overall PublicationsDuan Q et al. BET bromodomain inhibition suppresses innate inflammatory and profibrotic transcriptional programs in heart failure. Science Translational Medicine. 2017.

Morrison-Nozik A et al. Glucocorticoids enhance muscle endurance and ameliorate Duchenne muscular dystrophy through a defined metabolic program. Proceedings of the National Academy of Sciences. 2015.

Anand P et al. BET bromodomains mediate transcriptional pause release in heart failure. Cell. 2013.

Jeyaraj D et al. Circadian rhythms govern cardiac repolarization and arrythmogenesis. Nature. 2012.

Haldar S et al. Klf15 deficiency is a molecular link between heart failure and aortic aneurysm formation. Science Translational Medicine. 2010.

Laboratory Reports

The overall goal of Todd C. McDevitt’s laboratory is to develop novel technologies to enable the translation of stem cells for therapeutic and diagnostic applications. Much of their work focuses on engineering 3D microscale tissue constructs from human stem cells that recapitulate the phenotypic and func-tional properties of native tissues. They employ scaffoldless tissue-engineering approaches to study the morphogenesis of pluripotent stem cells using a combination of biomateri-als-based approaches and cell-engineering techniques. They are also interested in characterizing and exploiting morpho-genic factors produced by stem and progenitor cells that have immunomodulatory and regenerative/rejuvenative effects on somatic cells and tissues.

AccomplishmentsOver the past decade, McDevitt and his team defined scalable and robust technologies for enhanced control and consistency of stem cell differentiation and microtissue engineering. Using these platform technologies, they developed different heterotypic models of cardiac, neural, and hepatic tissues from human stem cell sources. One of their goals is to determine the specific effects of 3D multicellular heterotypic interactions and cell-derived extra-cellular matrix on individual cell phenotypes and the resulting physiological properties of engineered microtissues.

The researchers have found that tissue-specific stromal cells (i.e., fibroblasts) significantly impact parenchymal cell (i.e., cardiomyo-cyte) phenotype through several different mechanisms, which they continue to pursue in more depth. They also showed that the phenotypic changes affect the relative maturity of the human induced pluripotent stem cell (iPSC)–derived cells.

The team was the first to report the differentiation of excitatory spinal interneurons from human pluripotent stem cells. They are now examining the functional and potential therapeutic efficacy of using these cells to repair spinal cord injury and develop novel organoid models of the central nervous system.

Lab Members*indicates current lab membersJessica Butts*Ana De Andrade E Silva*Amy FoleyTracy HookwayDavid Joy*Michael Kang*Ariel Kauss*Ashley Libby*Ronald Manlapaz*

Oriane Matthys*Dylan McCreedy*Nik Mendoza-Camacho*Eszter Mihaly*Vaishaali Natarajan*Jessica SepulvedaDiwakar Turaga*Jenna WilsonJoshua Zimmermann

HighlightsGenerated excitatory spinal interneurons from human pluripotent stem cells capable of forming synaptic connections with other neuronsEngineered heterotypic cardiac microtissues that promote the phenotypic and functional maturation of human iPSC-derived cardiomyocytesCreated predictable and robust multicellular human iPSC patterns via manipulation of intrinsic cell mechanisms

Todd C. McDevitt PhDSENIOR INVESTIGATOR

19

Gladstone Institutes

Future DirectionThe team will continue to engineer robust methods to control organoid development from human pluripotent and post-natal stem cells, and use these novel tissue models to study mecha-nisms of human development and disease ex vivo. Furthermore, they aim to innovate new microphysiological systems and func-tional assays that enable the exploration of epigenetic effects of environmental factors on human tissue structure and function.

In addition, they will use the systematic and comprehensive anal-ysis of the molecules uniquely produced by stem cells to lead to new molecular therapies and engineered materials that stimulate regeneration and repair of somatic tissues.

They also devised a high-density perfusion bioreactor system for human iPSC culture that produces enhanced yields and concen-trations of morphogenic factors. They are identifying and examin-ing the rejuvenative effects of human iPSC-factors on aged cells and tissues in several mouse models.

Research ImpactHuman tissue constructs and organoids derived from stem cells offer novel model systems for probing mechanisms of embryonic development. Furthermore, engineered human microtissues offer tractable substrates to interrogate infectious and genetic diseases and the effects of exposure to other environmental factors. McDevitt’s laboratory uses a bottom-up approach to understand how cells cooperatively interact to form complex tissues and dictate multicellular organization and subsequent physiologic function. They also seek to determine the relative contributions of stromal cells to measurable physiological properties.

Stem Cells

NeuralCardiac Matrix Secretome

Tissue Molecules

The McDevitt laboratory focuses on the creation of tissue models and regenerative molecular therapies from stem cells. They are developing cardiac, neural, and other tissues to probe heterotypic impacts on development and disease, while in parallel exploring and exploiting the unique and complex cadre of molecules produced by stem cells.

Selected Recent PublicationsKhalil et al. Functionalization of microparticles with mineral coatings enhances non-viral transfection of primary human cells. Scientific Reports. 2017.

Jackson-Holmes EL et al. A microfluidic trap array for longitudinal monitoring and multi-modal phenotypic analysis of individual stem cell aggregates. Lab on a Chip. 2017.

Butts JC et al. Differentiation of V2a interneurons from human pluripotent stem cells. Proceedings of the National Academy of Sciences. 2017.

Zimmerman JA et al. Enhanced immunosuppression of T cells by sustained presentation of bioactive interferon-γ within three-dimensional mesenchymal stem cell constructs. Stem Cells Translational Medicine. 2017.

Wang Y et al. Mineral particles modulate the osteo-chondrogenic differentiation of embryonic stem cell aggregates. Acta Biomaterialia. 2016.

Top Five Overall PublicationsSutha et al. Osteogenic embryoid body-derived material induces bone formation in vivo. Scientific Reports. 2015.

Murphy et al. Materials as stem cell regulators. Nature Materials. 2014.

McDevitt TC. Scalable culture of human pluripotent stem cells in 3D. Proceedings of the National Academy of Sciences. 2013.

Bratt-Leal AM et al. A microparticle approach to morphogen delivery within pluripotent stem cell aggregates. Biomaterials. 2013.

Singh A et al. Adhesion strength–based, label-free isolation of human pluripotent stem cells. Nature Methods. 2013.

Laboratory Reports

Deepak Srivastava’s laboratory focuses on the fundamental events involved in cell fate determination, differentiation, and organogenesis. Specifically, the team investigates the molecular events regulating cardiogenesis. They focus on signaling, transcriptional, and post-transcriptional networks in this process. They have leveraged knowledge of key gene networks to reprogram resident fibroblasts directly into cardiomyocyte-like cells for regenerative purposes. They also investigate the genetic causes of human cardiovascular disease and use human induced pluripotent stem cells (iPSCs) to reveal disruption of cardiogenic gene networks that lead to disease. By using these approaches, they are discovering the biology underlying cardiogenesis and cardiovascular disorders and beginning to find novel therapeutic interventions.

AccomplishmentsSrivastava’s team described complex signaling, transcriptional, and translational networks that guide early differentiation of cardiac progenitors and later morphogenetic events during cardiogenesis. By leveraging these networks, they repro-grammed disease-specific human cells to model human heart disease in patients with mutations in cardiac developmental genes. Deep epigenetic and transcriptome analyses revealed perturbations in pivotal gene networks, which contribute to disease that could be corrected by altering dosage of nodal points in the network. These studies revealed mechanisms of NOTCH1 and GATA4 haploinsufficiency, and the researchers showed the contribution of genetic variants inherited in an oligo-genic fashion in congenital heart disease.

They used a combination of cardiac regulatory factors and small molecules to directly reprogram resident cardiac fibroblasts into cardiomyocyte-like cells in vitro and in vivo to repair damaged hearts. Single-cell RNA-sequencing showed how heterogeneity of the reprogramming process may inhibit efficiency and could be manipulated. The small molecules appear to increase acces-sibility of reprogramming factors to their DNA-binding sites by opening chromatin at those sites genome-wide.

Lab Members*indicates current lab membersYen Sin AngBonnie Cole*Yvanka De Soysa*Aryé Elfenbein*Giselle GalangLaxmi GhimireCasey Gifford*Bárbara González Terán*Yu Huang*Isabelle N. KingWesley Kwong*Lei Liu*Elijah MartinKimberly R. Cordes MetzlerTamer MohamedShanelle Nebre*Karishma Pratt

Ethan RadzinskySanjeev Ranade*Janell RiveraGabriel RubioHazel Salunga*Ryan Samarakoon*Kaitlen Samse*Amelia Schricker*Nicole Stone*Joke van Bemmel*Vasanth VedanthamPengzhi Yu*Sarah Zambrano*Yu Zhang*Ping Zhou*Lili Zhu*

HighlightsDiscovered combination of tran-scription factors and small molecule inhibitors that optimally reprogram mouse and human cardiac fibroblasts to induced cardiomyocyte-like cells in vitro and in vivo and revealed mecha-nisms underlying the transitionIdentified a combination of genes that unlocks the proliferative poten-tial in cells that had permanently exited the cell cycleShowed a GATA4 missense muta-tion disrupts a transcription factor

“code” at cardiac enhancers, leading to abnormal cardiac septation and dysfunction

Deepak Srivastava MDPRESIDENT AND SENIOR INVESTIGATOR

21

Gladstone Institutes

Future DirectionThe Srivastava laboratory is demonstrating how complex inher-ited mutations in transcription factors can cause human disease in an oligogenic fashion. They are also revealing the mechanisms by which such factors dictate cardiac cell fate and how disruption causes disease. Finally, they will build on the novel approach to unlock the cell cycle in post-mitotic adult cardiomyocytes in vivo to induce cell division in other post-mitotic cell types that are lost in human disease.

As an alternative approach, the team identified a combination of cell-cycle regulators that efficiently induce adult cardiomyocytes to divide in a stable fashion, resulting in sufficient new muscle to regenerate infarcted hearts in vivo.

Research ImpactThe scientists leveraged the body of knowledge from cardiac developmental biology to directly reprogram non-muscle cells in the mouse heart into cells that function like heart muscle cells, effectively regenerating heart muscle after damage. This new paradigm of harnessing endogenous cells to regenerate organs may be broadly applicable to other organs.

Similarly, they used knowledge of developmental cell-cycle regulators to unlock the post-mitotic state in adult cardiomyo-cytes and stimulate stable cell division sufficient to regenerate cardiac muscle in the adult. They also revealed the mechanisms underlying human disease caused by mutations in cardiac developmental regulators using human iPSCs and used these to screen for novel therapeutic approaches to disease.

The Srivastava laboratory develops knowledge of critical gene networks that regulate cardiogenesis, explores how those networks are disrupted in the setting of human genetic disease, and leverages the developmental networks to reprogram non-muscle cells into cardiomyocyte-like cells for regenerative purposes.

Selected Recent PublicationsMohamed et al. Regulation of cell cycle to stimulate adult cardiomyocyte proliferation and cardiac regeneration. Cell. In press.

Ribeiro et al. Multi-imaging method to assay the contractile mechanical output of micropatterned human iPSC-derived cardiac myocytes. Circulation Research. 2017.

Theodoris et al. Long telomeres protect against age-dependent cardiac disease caused by NOTCH1 haploinsufficiency. Journal of Clinical Investigation. 2017.

Mohamed et al. Chemical enhancement of in vitro and in vivo direct cardiac reprogramming. Circulation. 2017.

Ang et al. Disease model of GATA4 mutation reveals transcription factor cooperativity in human cardiogenesis. Cell. 2016.

Top Five Overall PublicationsTheodoris CV et al. Human disease modeling reveals integrated transcriptional and epigenetic mechanisms of NOTCH1 haploinsufficiency. Cell. 2015.

Qian L et al. In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes. Nature. 2012.

Ieda M et al. Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell. 2010.

Zhao Y et al. Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis. Nature. 2005.

Garg V et al. GATA4 mutations cause human congenital heart defects and reveal an interaction with TBX5. Nature. 2003.

Laboratory Reports

The goal of Shinya Yamanaka’s laboratory is to understand the mechanisms underlying pluripotency and transcription factor–induced somatic cellular reprogramming. They have studied how cell fate can be changed by RNA-binding proteins, such as LIN41 and NAT1, in mouse and human pluripotent stem cells. By analyzing these RNA-binding proteins, they concluded that post-transcriptional regulation is important for the mainte-nance of pluripotency and precise differentiation.

AccomplishmentsMouse and human pluripotent stem cells are distinguishable in their characteristics, such as differentiation potential and epigen-etic status. Understanding how these two types of pluripotency—naive and primed—are determined is important. The scientists developed a novel and efficient method for converting mouse primed pluripotent stem cells to naive pluripotency.

They examined induced pluripotent stem cell lines derived from patients with fibrodysplasia ossificans progressive, who carry a missense mutation in ACVR1 (R206H) that leads to hyperactiva-tion of BMP-SMAD signaling. They found that the BMP-SMAD-ID axis promoted human cellular reprogramming toward pluripo-tency by inhibiting p16INK4A expression.

They previously showed that LIN41 improved human cellular reprogramming, but the mechanisms were poorly understood. They have since found novel roles of MYC and LIN41 in the early stage of human cellular reprogramming. They showed that post-transcriptional activation of LIN41 expression by MYC is crucial for efficient expansion of reprogramming cells.

Twenty years ago, Yamanaka’s team found that NAT1-deficient mouse pluripotent stem cells impaired differentiation potentials, but the mechanism was unclear. They revisited the function of NAT1 in mouse pluripotency and identified NAT1’s target gene, which can explain the phenotype of NAT1-deficiency. Also, they identified the NAT1-interacting proteins using immunoprecipita-tion and mass spectrometry analyses.

Lab Members*indicates current lab membersDaeun Jeong*Maaz Asher Khurram*Mariselle Gay Lancero*Sean OkawaPrakriti PaulSamuel Perli*Alexis Rand

Timothy Rand*Kazutoshi Takahashi*Emi TomodaKiichiro TomodaSongnan Wang*

HighlightsGenerated a novel and efficient method for the conversion of primed mouse pluripotent stem cells to naive pluripotencyGained understanding of the role of MYC and LIN41 in early stages of human cellular reprogrammingIdentified NAT1’s interacting proteins and target RNAs in mouse pluripotent stem cells

Shinya Yamanaka MD, PhDSENIOR INVESTIGATOR

23

Gladstone Institutes

Future DirectionThe team led by Yamanaka recently started to expand its research to the discovery of novel translation mechanisms and the full understanding of the mechanisms of translation initiation. Thus, the future direction of the laboratory is trying to understand the specific and common roles of each eIF4G family in the trans-lation process in pluripotency and cell fate determination.

Research ImpactTextbooks have said that eukaryotic translation initiation factor gamma 1 (eIF4G1) plays major roles in the initiation of protein translation in eukaryotic cells. However, the Yamanaka labora-tory hypothesizes that other members of the eIF4G family, such as NAT1 (also known as eIF4G2) and eIF4G3, also have import-ant roles. This, in part, is because NAT1 expression, at least, is much higher than other eIF4Gs in various tissues and cell types. Actually, the result of loss-of-function of the NAT1 gene in mouse embryogenesis and embryonic stem cells suggested the essen-tial roles of NAT1 in mouse early embryonic development and differentiation. In this period, the team could identify the NAT1-interacting proteins. Some of them, such as eIF3 and eIF4A, were common in eIF4G1 and NAT1.

The researchers’ result also suggested that NAT1 has unique interacting partners, such as FXR and PRRC2. Encouraged by these data, they began to think about the unique role of NAT1 in non-canonical-translation rather than canonical cap-dependent translation driven by eIF4G1. Through the functional analyses of all eIF4Gs in parallel, they expect to find novel translation mecha-nisms, such as cap-independent translation.

CAP-dependentTranslation

CAP-independentTranslation?

Cell FateDetermination?

Survival/Proliferation

eIF4G Family

NAT1(eIF4G2) eIF4G1 eIF4G3

The goal of the Yamanaka laboratory is to discover novel translation mechanisms such as cap-independent translation and their roles in pluripotency and cell-fate determination by comparison analyses of eukaryotic translation initiation factor gamma (eIF4G) family and their interacting proteins.

Selected Recent PublicationsSugiyama H et al. NAT1 promotes translation of specific proteins that induce differentiation of mouse embryonic stem cells. Proceedings of the National Academy of Sciences. 2017.

Hayashi Y et al. BMP-SMAD-ID promotes reprogramming to pluripotency by inhibiting p16/INK4A-dependent senescence. Proceedings of the National Academy of Sciences. 2016.

Kim C et al. Autotaxin-mediated lipid signaling intersects with LIF and BMP signaling to promote the naive pluripotency transcription factor program. Proceedings of the National Academy of Sciences. 2016.

Top Five Overall PublicationsOkita K et al. A more efficient method to generate integration-free human iPS cells. Nature Methods. 2011.

Hong H et al. Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. Nature. 2009.

Miura K et al. Variation in the safety of induced pluripotent stem cells. Nature Biotechnology. 2009.

Takahashi K et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007.

Takahashi K et al. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006.

24

Findings 2018

Programs and InitiativesRoddenberry Stem Cell CenterThe Roddenberry Stem Cell Center at Gladstone, directed by Deepak Srivastava, is an international powerhouse in the study of stem cell biology. Established through a generous gift from the Roddenberry Foundation, it honors the legacy of Eugene Roddenberry, the creator of Star Trek. The center provides Gladstone researchers with resources that advance efforts to harness the tremendous potential of stem cell biol-ogy and regenerative medicine to transform the treatment of human disease.

Stem cell and regenerative medicine research at Gladstone embody the organization’s spirit of deep discovery and inno-vation to solve a wide range of diseases. Supported by this center, investigators have made tremendous scientific strides by creating a unique and well-rounded program that is poised to fundamentally change the future of medicine.

Most of Gladstone’s scientists use approaches based on induced pluripotent stem cells. They use this technology to explore and model diseases, test potential new drugs, and leverage cellular reprogramming for regenerative medicine. They have also built upon this technology to develop direct cellular reprogramming approaches, find ways to reprogram cells through chemical biology, and even engineer three-dimensional tissues from stem cells.

Tenaya TherapeuticsIn 2016, Gladstone launched a spin-off company, Tenaya Therapeutics Inc., to build on its discoveries in cardiovascular disease research, concentrating on regenerative medicine and drug discovery for heart failure. The new biopharmaceutical company is the first formed out of BioFulcrum, an entrepre-neurial initiative within Gladstone to accelerate the discovery of cures by bringing together scientists, nonprofit institutions, and industry partners.

Supported by a $50-million Series A financing led by David Goeddel with The Column Group, Tenaya Therapeutics aims to leverage Gladstone’s pioneering work in cellular reprogramming to develop novel therapies for heart failure and advance them towards clinical translation. Currently, the only possible cure for heart failure is a heart transplant. This new science-focused venture will hopefully lead to a more scalable cure for patients.

Scientists at Tenaya Therapeutics combine Gladstone’s basic science expertise with the resources and translational know-how of the biotechnology industry. They aim to use cellular reprogramming technology to regenerate heart muscle cells in patients with heart failure and develop cellular models of heart disease from stem cells to identify potential new drug targets.

Ogawa-Yamanaka Stem Cell PrizeEvery year, Gladstone presents the Ogawa-Yamanaka Stem Cell Prize to a scientist whose original translational research advances cellular reprogramming technology for regenerative medicine. The prize was established in 2015 through a gener-ous gift from the late Hiro and Betty Ogawa and recognizes the importance of induced pluripotent stem cells (iPSCs), discovered by Gladstone Senior Investigator Shinya Yamanaka.

The first winner of this prize was Masayo Takahashi, project leader at the Laboratory for Retinal Regeneration at the RIKEN Centre for Developmental Biology. In 2015, she was honored for her trailblazing research that led to the first clinical trial to use iPSCs in humans. Her work is paving the way for using stem cells to treat retinal diseases, including macular degeneration.

Douglas Melton received the 2016 Ogawa-Yamanaka Stem Cell Prize. He is co-director of the Harvard Stem Cell Institute, a Xander University Professor at Harvard University, and an investigator of the Howard Hughes Medical Institute. Melton was honored for his research that led to a novel way of reprogram-ming human stem cells into insulin-producing beta cells. His work provides the foundation for the ultimate goal of transplant-ing patient-specific beta cells to treat diabetes.

The latest prize was awarded in 2017 to Lorenz P. Studer, direc-tor of the Center for Stem Cell Biology and member of the Developmental Biology Program at the Memorial Sloan Kettering Cancer Center. A distinguished stem cell biologist, Studer was selected for his transformative contributions to the field of cellular reprogramming and the application of human iPSCs to human disease. His groundbreaking research has advanced the therapeutic potential of stem cell–based therapies in Parkinson’s disease and other neurological disorders.

Gladstone Institutes

25

Research InfrastructureThe Gladstone Institutes is at the epicenter of biomedical and technological innovation in the San Francisco Bay Area, a location that provides the ideal environment to conduct transformative science. To support bold projects and unique research needs, Gladstone maintains robust research infrastructure.

Gladstone’s cutting-edge research facilities serve as a shared resource for the entire scientific community, offering technologies and services that may be too expensive for a single laboratory to purchase.

The core facilities and technology center bring scientists together to share expertise and encourage collaboration. In addition to consultation services, training, and expert technical support, users can access over 100 pieces of highly specialized laboratory equipment.

To keep up with the latest advances, Gladstone continually updates its research infrastructure and the services offered. Since 2016, the Assay Development and Drug Discovery core facility was created, and significant resources in imaging and data analysis were added to microscopy and bioinformatics facilities, respectively.

Core FacilitiesAssay Development and Drug Discovery

Behavioral

Bioinformatics

Flow Cytometry

Genomics

Histology and Light Microscopy

Stem Cell

Transgenic Gene Targeting

Technology CenterMass Spectrometry

Findings 2018

26

Recent DiscoveriesStudy Reveals How to Reprogram Cells in Our Immune SystemScientists revealed, for the first time, a method to reprogram certain immune cells, called T cells. More precisely, they discovered how to turn pro-inflammatory cells that boost the immune system into anti-inflammatory cells that suppress it, and vice versa. By manipulating the function of T cells, they could help restore the immune system’s balance and create new potential therapies. Their findings could have a significant impact on the treatment of autoimmune diseases, as well as on stem cell and immuno-oncology therapies.

Sheng Ding | Nature | August 2017

New Study Helps Solve a Great Mystery in the Organization of Our DNAAfter decades of research aiming to understand how DNA is organized in human cells, a team of researchers shed new light on this mysterious field by discovering how a key protein helps control gene organization. They discovered that the key to organizing topologically associating domains—large domains into which chromosomes are pack-aged together—is a protein called CTCF. Their insights will allow the scientific commu-nity to reevaluate the cause of diseases such as cancer and developmental defects including congenital heart disease.

Benoit Bruneau | Cell | May 2017

Cancer-Cardiac Connection Illuminates Promising New Drug for Heart FailureResearchers found that a small molecule called JQ1 can effectively treat severe, pre-established heart failure by blocking inflammation and fibrosis (scarring of the heart tissue). Drugs derived from JQ1, currently under study in early phase human cancer trials, act by inhibiting a protein called BRD4, which directly influences heart failure. The team thus uncovered a new strategy to treat heart failure that goes to the root of the problem and blocks destructive processes in the cell’s nucleus, in contrast to currently available drugs that work at the surface of heart cells.

Saptarsi Haldar | Science Translational Medicine | May 2017

Discovery Offers New Hope to Repair Spinal Cord InjuriesA team of scientists was the first to produce V2a interneurons from human stem cells. These special neurons transmit signals in the spinal cord to help control movement and can also reroute after spinal cord injuries, which makes them a promising therapeutic target. The researchers’ goal is to rewire the impaired circuitry by replacing damaged interneurons and creating new pathways for signal transmission around the site of the injury. This, ultimately, could help restore movement after damage has occurred. Interneurons could also potentially play a role in models of neurodegenerative move-ment disorders such as amyloid lateral sclerosis.

Todd McDevitt | Proceedings of the National Academy of Sciences | April 2017

Longer Telomeres Protect against Diseases of Aging: A Tale of Mice and MenA group of scientists discovered a key mechanism that protects mice from developing a human disease of aging called calcific aortic valve disease. They found that shortening telomeres—protective caps on the ends of chromosomes that erode with age—in mice carrying a human genetic mutation linked to heart disease results in a deadly buildup of calcium in heart valves and vessels. This innovative model allows the researchers to test potentially viable drug therapies, in the hopes of discovering the first medical treat-ment for the disease.

Deepak Srivastava | Journal of Clinical Investigation | March 2017

Spot

light Todd McDevitt

Appointed to Lead the UC Berkeley-UCSF Graduate Program in BioengineeringSenior Investigator Todd C. McDevitt was appointed to two new positions within the joint Graduate Program in Bioengineering from UC Berkeley and UC San Francisco. He was selected in fall 2017 as the new program director to lead all matters related to the graduate program. In addi-tion, he was elected to co-chair the executive commit-tee and, after serv-ing a 2-year term, he will rotate to the role of committee chair for an addi-tional 2 years.

Gladstone Institutes

27

Scientists Discover Drug that Increases “Good” Fat Mass and FunctionThe research team identified an FDA-approved drug that can create beneficial brown fat. In contrast to the more commonly known white fat, which stores energy, brown fat helps the body burn energy through heat. Mice treated with the anti-cancer drug called bexarotene (Bex) had more brown fat, faster metabolisms, and less body weight gain, even after being fed a high-calorie diet. The researchers say the technique, which uses cellular reprogramming to convert muscle precursor cells and white fat cells into brown fat cells, represents an exciting new approach to treat obesity and associated metabolic diseases, such as diabetes.

Sheng Ding | Cell Reports | January 2017

One Gene Mutation, Two Diseases, Many Insights into Human Heart FunctionScientists linked a single gene mutation to two types of heart disease: one causes a hole in the heart of infants, and the other causes heart failure. The study involved a family of patients who suffer from congenital heart disease and carry a mutation in GATA4. This gene is essential for normal heart development and healthy heart function, and mutations cause septal defects (or the formation of holes in the heart). As a result, the researchers gained insight into congenital heart disease, human heart development, and healthy heart function.

Deepak Srivastava | Cell | December 2016

Scientists Create Heart Cells Better, Faster, StrongerA team of researchers identified two chemicals that improve the speed, quantity, and quality of direct cardiac reprogramming, a method to regenerate muscle cells in the heart. After a heart attack, connective tissue forms scar tissue at the site of the injury, contributing to heart failure. The two chemicals improve the scientists’ ability to trans-form this scar tissue into healthy, beating heart muscle. This brings the technology one step closer to regenerating damaged hearts in patients and could lead to new and effective treatments for heart failure.

Deepak Srivastava | Circulation | November 2016

Research on Rare Genetic Disease Reveals New Stem Cell PathwayScientists discovered a way to increase the efficiency of stem cell reprogramming with a gene mutation. By attempting to create a cellular model to study an extremely rare genetic disease called fibrodysplasia ossificans progressiva (FOP), they found that they could create more induced pluripotent stem cells (iPSCs) from cells taken from FOP patients than those taken from healthy individuals. This is the first reported case show-ing that a naturally occurring genetic mutation improves the efficiency of iPSC genera-tion. And although it may not be very useful for disease modeling, it offers valuable new insights into the reprogramming process.

Bruce Conklin, Shinya Yamanaka | Proceedings of the National Academy of Sciences | October 2016

How to Engineer a Stronger Immune SystemA research team discovered a biomaterials hack that can boost cells’ ability to combat inflammation and potentially treat autoimmune diseases. They improved mesenchy-mal stromal cells (MSCs), which secrete anti-inflammatory proteins that help regulate the immune system. However, they must be triggered by pro-inflammatory proteins to produce their immune-suppressing effects, so many clinical trials using MSCs have failed. The scientists engineered the MSCs to ensure they are consistently activated and can reliably dampen the immune response for longer. Their work could one day benefit patients who suffer from inflammatory bowel disease or organ transplant rejection.

Todd McDevitt | Stem Cells Translational Medicine | August 2016

Deepak Srivastava to Lead the International Society for Stem Cell ResearchIn June 2017, Deepak Srivastava was elected vice president of the International Society for Stem Cell Research (ISSCR), the voice of the global stem cell research community. Following his 1-year term as vice presi-dent, he will serve as president-elect for 1 year. Then, beginning in June 2019, he will become ISSCR president for 1 year.

Spot

light

28

Findings 2018

Scientists Turn Skin Cells into Heart and Brain Cells Using DrugsIn a scientific first, researchers used a combination of chemicals to convert skin cells into heart and brain cells. All previous work on cellular reprogramming required adding external genes to the cells, making this accom-plishment an unprecedented feat. In two studies, the team identified chemical cocktails to gradually coax skin cells to transform into organ-specific stem cell-like cells and, ultimately, into heart or brain cells. This discov-ery offers a more efficient and reliable method to reprogram cells and avoids medical concerns surrounding genetic engineering. It also lays the groundwork for one day being able to generate new cells at the site of injury in patients.

Sheng Ding | Cell Stem Cell | April 2016

Micro Heart Muscle Created from Stem CellsA group of researchers invented a new way to create three-dimensional human heart tissue from stem cells. Using a special dish shaped like a tiny dog bone, they bioengineered micro-scale heart tissues, which will enable scientists in stem cell biology and the drug industry to study heart cells in their proper context and test potential drugs. The new method dramatically reduces the number of cells needed, making it an easier, cheaper, and more efficient system. It also opens the door for a precision medicine approach to treating heart disease.

Bruce Conklin | Scientific Report | April 2016

Modified Form of CRISPR Acts as a Toggle Switch to Control Gene Expression in Stem CellsScientists manipulated the genome of stem cells using a modified form of CRISPR. The new method, called CRISPR interference (CRISPRi), inactivates genes in induced pluripotent stem cells (iPSCs) and heart cells created from iPSCs. The method significantly improves the original CRISPR-Cas9 system by allowing genes to be silenced—or turned off—more precisely and efficiently. CRISPRi also offers the flexibility of reversing and carefully controlling the amount of gene suppression. Using this technology, they can mimic disease in a homogenous population of heart cells created from iPSCs, as well as study genetic diseases more easily and potentially identify new therapeutic targets.

Bruce Conklin | Cell Stem Cell | March 2016

New Method for Producing Heart Cells May Hold the Key to Treating Heart FailureA research team discovered how to make a new type of cardiac stem cell in the laboratory that may hold the key to treating heart disease. These induced expandable cardiovascular progenitor cells (ieCPCs) can organ-ically develop into heart cells, while still being able to replicate. The scientists used pharmaceutical drugs to catch and maintain heart stem cells at the cardiac precursor state before they developed into fully functional heart cells. When injected into a mouse after a heart attack, the cells improved heart function dramatically. With this new technology, billions of ieCPCs could potentially be created in a dish and transplanted into patients to treat heart failure.

Sheng Ding | Cell Stem Cell | March 2016

True Love: How Transcription Factors Interact to Create a HeartScientists discovered how three transcription factors that are crucial for heart development—NKX2-5, TBX5, and GATA4—interact on a genomic and physical level. They revealed for the first time how these factors interact with each other and the genome to influence how a heart forms in an embryo. Without these protein interactions, severe congenital heart defects can occur. By understanding how the transcription factors work together during heart development, researchers may discover new ways to treat heart disease.

Benoit Bruneau | Cell | February 2016

Insulin-Producing Pancreatic Cells Created from Human Skin CellsResearchers successfully converted human skin cells into fully functional pancreatic cells. The new cells produced insulin in response to changes in glucose levels, and, when transplanted into mice, they protected the animals from developing diabetes. The study presents significant advancements in cellular reprogram-ming technology, which will allow scientists to efficiently scale up pancreatic cell production and manufacture trillions of the target cells in a step-wise, controlled manner. This accomplishment opens the door for disease modeling and drug screening and brings personalized cell therapy a step closer for patients with diabetes.

Sheng Ding | Nature Communications | January 2016

Inherited Neuropathies/Charcot-Marie-Tooth

Intractable andRare Diseases

ImmuneDisorders

Glucocorticoid/Hormone Disorders

Diabetes

Cancer

Birth Defects

AgingTissueFibrosis

Stroke

Spinal CordInjury

Sepsis

Hypertension

RetinitisPigmentosa

Neurodegeneration

MetabolicSyndrome

Atherosclerosis

CongenitalHeart Disease

Cardiac RegenerationGenetic Forms ofCardiac FailureEpigenetics of

Cardiac DiseaseTissue EngineeringStem Cell Biology

The Gladstone Institute of Cardiovascular Disease may focus on unraveling the deepest biological mysteries of the cardiovascular system, but its research has potential applications for a large number of approaches, conditions and diseases that affect organs other than the heart.

It is vital to recognize and encourage outstanding scientists who conduct groundbreaking explorations of translational regenerative medicine using reprogrammed cells.

Every year, Gladstone is proud to present the Ogawa-Yamanaka Stem Cell Prize to a deserving researcher. The awardee also receives an unrestricted personal cash prize of $150,000 USD.

Learn more at gladstone.org/stemcellprize

1650 Owens Street, San Francisco, CA 94158 | 415.734.2000 | gladstone.org