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Investigator Initiated Research Projects and Innovative, Developmental or Exploratory Activities (IDEA) in Stem Cell Research - 2009

RFA #: 0812220315

RFA #: 0812220315
IIRP Awards
Sponsoring Institute PI Amount Title
Albert Einstein College of Medicine Jayanta Roy-Chowdhury $1,078,000 Liver Repopulation with Hepatocytes Derived from Induced Pluripotential Cells for Treament of Alpha-1 Antitrypsin Disease
Columbia University Gordana Vunjak-Novakovic $1,019,668 Phenotypic Maturation of Human Cardiomyocytes by Electrical Field Stimulation
Columbia University Medical Center Robert Kass $999,709 Mechanistic Study of an Inherited Arrhythmia in a Complex Genetic Background using iPS Cell Derived Cardiomyocytes
Columbia University Medical Center Michael Shen $1,080,000 Identification and Analysis of Prostate Tumor-Initiating Cells
Columbia University Medical Center Michael Shen $1,080,000 Identification of Master Regulators for Stem Cell Pluripotency and Self-Renewal
Columbia University Medical Center Richard Sloan $1,043,955 Exercise and Neurogenesis
Columbia University Medical Center Stephen Tsang $990,210 Functional Analyses of Embryonic Stem Cell Derived Retinal Cells
Cornell University John Schimenti $544,130 Genome Maintenance in Germline Stem Cells
Masonic Medical Research Laboratory Charles Antzelevitch $1,080,000 Induced Pluripotent Stem Cell-Derived Cardiomyocytes as in Vitro Models of Early Repolarization Syndrome and Sudden Cardiac Death
Mount Sinai School of Medicine James Bieker $1,080,000 Redirecting Hemoglobin Expression During Human ES Cell Differentiation
Mount Sinai School of Medicine Ronald Hoffman $1,080,000 Ex Vivo Expansion of Cord Blood Hematopoietic Stem Cells
Mount Sinai School of Medicine Ihor Lemischka $1,056,229 Analyzing Pluripotency in Human Embryonic Stem Cells and Stem Cells from Reprogrammed Differentiated Cells
Mount Sinai School of Medicine Xiajun Li $1,024,200 ZFP57, a Link Between Genomic Imprinting and Stem Cells
Mount Sinai School of Medicine Jose Mario Wolosin $865,357 Dual Specificity Phosphatases in Ocular Surface Side Population Stem Cells
Mount Sinai School of Medicine Chen-Leng Cai $1,080,000 The Multipotency of Epicardial Cells and Mammalian Cardiac Repair and Regeneration
New York State Psychiatric Institute René Hen $1,075,647 Regulation of Dentate Gyrus Function by Hippocampal Stem Cells and Their Progeny: Relevance to Cognition and Depression
New York University School of Medicine James Salzer $728,189 Role of SHH-Responsive Stem Cells in Remyelination
The Rockefeller University Elaine Fuchs $1,025,000 Harnessing Keratinocyte Stem Cell Growth for Regenerative Medicine
Sloan-Kettering Institute Lorenz Studer $1,031,000 Modeling Pathogenesis and Treatment of Familial Dysautonomia in Patient Specific Human Induced Pluripotent Stem Cells
Sloan-Kettering Institute Lorenz Studer $1,080,000 Patient-Specific Human ESCs and iPSCs for Modeling Schwann Cell Differentiation and Charcot-Marie-Tooth Disease
SUNY – University at Buffalo Richard Gronostajski $1,060,681 Role of Nfix in Neural Stem Cells and Glioblastoma
University of Rochester Abdellatif Benraiss $787,934 Induction of Sustained Medium Spiny Neuronal Addition from Sub-Ependymal Stem Cells of Adult Monkey Brain as Therapeutic Strategy for Huntington's Disease
University of Rochester Steven Goldman $1,029,124 Atlas of Gene Expression by Progenitor Cells of the Human Brain and its Tumors
University of Rochester Mark Noble $1,080,000 Reversing Chemoresistance: Novel Pathways and Novel Treatment Targets
Weill Medical College of Cornell University Shahin Rafii $1,079,866 Generation of Abundant Clinical Grade Vascular Endothelium for Therapeutic Vasculoplasty Using a Highly Efficient Feeder Based Co-Culture Platform Free of Animal Contamination
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IDEA Awards
Sponsoring Institute PI Amount Title
Albert Einstein College of Medicine Ulrich Steidl $330,000 Dynamic Genome-Wide Transcription Factor-Chromatin Interactions During Hematopoietic Stem Cell Differentiation
Columbia University Medical Center Fiona Doetsch $327,822 Cerebrospinal Fluid Regulation of Adult Neural Stem Cells
Columbia University Medical Center Boris Reizis $299,700 Directed Differentiation of Embryonic Stem Cells Into Plasmacytoid Dendritic Cells
Cornell University Siu Lee $321,242 Roles for fhe Putative Longevity Determinants HCF1 and HCF2 in Stem Cell Function
Cornell University Robert Weiss $321,699 Investigating How DNA Damage Response Mechanisms Regulate the Tumorigenic Potential of Pluripotent Cell Types Using Mouse Models of Testicular Germ Cell Tumors
Mount Sinai School of Medicine Michael Rendl $330,000 Reprogramming the Hair Follicle Stem Cell Niche in Uncommitted Skin Fibroblasts
Mount Sinai School of Medicine Michael Rendl $330,000 Dermal Papilla Cells: an Accessible and Highly Reprogrammable Source for the Generation of Pluripotent Stem Cells
Mount Sinai School of Medicine Jianlong Wang $329,808 An Extended Protein Interaction Network for Stem Cells and Induced Pluripotency
Rensselaer Polytechnic Institute Deanna Thompson $308,780 Investigation of Adult Neural Progenitor Fate in Response to Endothelial Produced Factors
Sloan-Kettering Institute Stuart Chambers $274,744 Translational Control of Human Pluripotent Cell Maintenance and Neural Differentiation
Sloan-Kettering Institute Anna-Katerina Hadjantonakis $329,844 Pdgfra Signaling and the Stem Cells of the Early Mammalian Embryo
Sloan-Kettering Institute Gabsang Lee $251,232 Disease Modeling of Congenital Insensitivity to Pain and Anhidrosis (CIPA) Disease Using Human iPSC and Rescue of its Phenotypes
Sloan-Kettering Institute Stephen Nimer $330,000 Modulating Hematopoietic Stem Cell Quiescence to Improve Leukemia Treatment
Sloan-Kettering Institute Mark Ptashne $329,987 Use of Modified Transcriptional Regulators to Probe Mechanism and to Enhance Efficiencies of iPS Cell Formation
Sloan-Kettering Institute Viviane Tabar $330,000 Multipotential Cancer Stem Cells in Gliomas
SUNY - Stony Brook University Howard Sirotkin $316,609 Regulation of Stem Cell Pluripotency by REST/NRSF
SUNY - Stony Brook University Holly Colognato $240,000 Extracellular Matrix Regulation of the Neural Stem Cell Niche
SUNY – University at Buffalo Gail Seigel $253,994 Reversible Differentiation of Cancer Stem Cells
SUNY – University at Buffalo Fraser Sim $319,907 Induction of Oligodendrocyte Fate from Human Neural Stem Cells
SUNY – University at Buffalo Michal Stachowiak $330,000 INFS - New Module for Stem Cell Transcription Programming
University of Rochester Archibald Perkins $330,000 Development of a Polyamide-Based Targeted Therapy for Leukemia Stem Cells
University of Rochester Tirumalai Rangasamy $330,000 Pre-Clinical Evaluation of Nrf2 Overexprexpressing Mesenchymal Stem Cells in Pulmonary Emphysema
University of Rochester Jun Sun $330,000 Enteric Bacterial Regulation of the Intestinal Stem Cells
University of Rochester Medical Center Xinping Zhang $314,552 Multiphoton Imaging for Nanofiber Based MSC-Mediated Bone Tissue Repair
University of Rochester Yi Zhang $319,792 Targeting MDS-EVI1 Complex in MLL Leukemia Stem Cell Function

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IIRP Abstracts

 Liver Repopulation with Hepatocytes Derived from Induced Pluripotential Cells for Treament of Alpha-1 Antitrypsin Disease

Jayanta Roy-Chowdhury, MD
Albert Einstein College of Medicine
IIRP

Inherited alpha-1 antitrypsin (AAT) deficiency results in uncontrolled activity of protein-cleaving enzymes, causing severe lung disease.  Additionally, in the most common form of the disease, the abnormal AAT (AAT-Z) accumulates in liver cells, which may cause liver injury, cirrhosis and cancer.  AAT deficiency is the most common inherited disorder leading to liver transplantation.  Recently, we discovered that isolated normal liver cells, when transplanted into the liver of mice expressing AAT-Z, outgrow the diseased host cells, gradually replacing the AAT-Z-containing liver cells.  However, clinical application of liver cell transplantation for AAT deficiency is hindered by the scarcity of high quality donor livers, from which to isolate the cells, and the need for prolonged immunosuppression to prevent rejection of hepatocytes obtained from other individuals.  Our objective is to overcome these barriers by culturing cells obtained by skin biopsy from patients with AAT deficiency, reprogramming them into induced pluripotent stem cells (iPSCs), correcting their genetic defect and then differentiating them into liver cells by manipulating cell culture conditions.  To correct the AAT deficiency, we will introduce the normal AAT gene into the iPSCs and express shRNAs against AAT-Z to inhibit its production.  To test whether the resulting cells can spontaneously replace the AAT-Z-containing host liver cells, we will transplant the genetically corrected liver cells into immunosuppressed mice expressing human AAT-Z.  We will determine the extent of liver repopulation by the transplanted cells using tissue analysis.  We will also evaluate the safety of transplanting these cells in long-term experiments.  If successful, this study will provide a virtually unlimited supply of genetically corrected liver cells for the treatment AAT deficiency.  Furthermore, because the recipient of the cells will also be the donor, immune suppression will not be required.

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 Phenotypic Maturation of Human Cardiomyocytes by Electrical Field Stimulation

Gordana Vunjak-Novakovic, PhD
Columbia University
IIRP

In the developed world cardiovascular disease takes more lives than all cancers combined.  In most cases the loss of cardiomyocytes (for example, due to cardiac infarction) is the reason for heart failure.  Due to the inability of heart muscle to regenerate itself following injury, cardiac function can only be restored by repopulation of the infarct bed by new cardiomyocytes and supporting vascular cells.  The only cells with the proven capacity to form cardiomyocytes are embryonic-type stem cells.  For clinical utility, stem cell-derived cardiomyocytes need to be stable and develop functional features of native cardiomycytes.  Because electromechanical signals are inherent to heart development and function, we hypothesized that the combined application of molecular factors (inducing stem cell differentiation) and physiological electrical signals (stabilizing and maturing the cells) will result in mature, fully functional cardiomyocytes.  Our recent studies support this hypothesis and motivate this proposed study of the impact of electrical conditioning on the functional development of cardiac phenotype.  This is a fundamentally new approach that employs regulatory pathways involved in early heart development, and as such it represents a major advance over conventional cell derivation.  This project is a unique collaboration of three laboratories with expertise in human stem cell biology, cellular electrophysiology and tissue engineering.  The two specific aims of the project are: 1) to determine the effects of electrical stimulation on the development of ion channels at the cell level, and 2) to investigate the development of functionality for signal propagation and force generation at the tissue level.  The novelty and significance of these studies are substantial, in a way directly relevant to advancing the ESSCB’s mission.  We expect to generate significant new knowledge about the development of functional cardiomyocytes from human stem cells, with benefit to both heart therapy and fundamental stem cell research.

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 Mechanistic Study of an Inherited Arrhythmia in a Complex Genetic Background Using iPS Cell Derived Cardiomyocytes

Robert Kass, PhD
Columbia University Medical Center
IIRP

Heritable cardiac rhythm disturbances due to mutations in ion channels or ion channel associated proteins (channelopathies) constitute an important arrhythmia class that places mutation carriers at high risk of sudden cardiac death.  The Long QT Syndrome (LQTS) was the first reported and is now the most thoroughly studied cardiac channelopathy.  Association between specific ion channel mutations and dysfunction in heart electrical activity, monitored non-invasively via the electrocardiogram, provided great insight into mechanisms of and specific therapeutic strategies to manage LQTS.  However, to date, studies have fallen short in providing methodologies to evaluate directly the consequences of disease-causing gene mutations against what may be complex genetic backgrounds of patients harboring the mutations.  We propose using cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs), produced from skin fibroblasts, as a novel platform to investigate mechanisms underlying inherited arrhythmias in patients with complex genetic backgrounds and to use these cells to screen for drugs to treat the disorder.  This approach cannot be undertaken using any other current methodologies.  We will generate iPSC-CMs from family members of a LQTS patient who not only carries a severe mutation in the cardiac sodium channel that can underlie the disease phenotype, but also a polymorphism in a critical potassium channel that may complicate the disease phenotype and also alter the patient’s response to potential therapeutic agents.  The proposed experiments will provide, for the first time, a methodology for direct investigation of the cellular physiology and pharmacology of mutations that cause LQTS within a background that reproduces the complex genetic background of a patient.  The cells we obtain and study will enable drug screening to identify compounds that will minimize off target drug interactions (side effects) and maximize the therapeutic response of the patient.

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 Identification and Analysis of Prostate Tumor-Initiating Cells

Michael Shen, PhD
Columbia University Medical Center
IIRP

Stem cells are rare cells that can give rise to all the cell types present in an adult tissue and can continue to grow and divide for the life of the organism.  Similar stem cells may be present in many cancers and these cancer stem cells represent desirable targets for effective therapies.  However, it is unknown whether such cancer stem cells exist in all cancers, or whether they originate from normal stem cells during tumor formation.  To address these questions, we identified a new type of stem cell (called CARNs) in the prostate gland of mice and showed that CARNs can give rise to prostate cancer.  Using genetically engineered mice, in which CARNs are labeled with a fluorescent marker, we showed that individual CARNs can form prostate-like tissues and tumors when transplanted into other mice or when grown in a culture dish.  Based on our findings, we propose that CARNs can give rise to cancer stem cells during prostate tumor formation.  We will investigate this process in mice and in culture, and will identify important genes that are involved.  Furthermore, we will test a large number of possible therapeutic compounds against these cancer stem cells for the ability to affect growth of these cells.  Our proposal is extremely innovative in using genetically engineered mouse models to identify and follow cancer stem cells, which can be difficult to isolate from human tumors, and in pursuing a large-scale testing methodology to find potential drugs targeting prostate cancer stem cells.  Finally, our team unites investigators from multiple institutions with exceptional expertise in these experimental approaches. 

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 Identification of Master Regulators for Stem Cell Pluripotency and Self-Renewal

Michael Shen, PhD
Columbia University Medical Center
IIRP

Studies of embryonic stem cells (ESCs) have attracted considerable attention due to the remarkable ability of ESCs to form all the cell types of the developing embryo.  Interestingly, there are several different types of stem cells that can arise from the early embryo, including epiblast stem (EpiSC) cells, which closely resemble human ESCs.  The identification of master genes that control the ability of ESCs to grow in culture and form the entire repertoire of embryonic and adult cell types is of central importance for harnessing their potential for regenerative medicine, and for enhancing the reprogramming of non-stem cells into stem cell types.  We propose a computational approach, based on experimental assays, to understand the network of genes that control the central properties of stem cells and to identify the master genes involved.  Understanding these genes and how they interact will allow the manipulation of stem cell properties through alterations in the expression of specific combinations of master genes.  By analyzing the behavior of every known gene, our unbiased computational approach will identify candidate genes that may be master controllers of stem cells.  We will confirm the computational analyses with laboratory experiments using stem cells.  Our proposal is extremely innovative as it utilizes novel computational methods pioneered by the Califano laboratory, combined with our own stem cell expertise.  These approaches use new and powerful computing methods, requiring the resources of a supercomputer, to analyze large sets of data on levels of gene activity in stem cells.  These methods should provide important insights into stem cell biology that would be difficult to obtain by any alternative methodology. 

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 Exercise and Neurogenesis

Richard Sloan, PhD
Columbia University Medical Center
IIRP

In the US, reduced morbidity and mortality have resulted in a growing number of older adults, the demographic “time bomb” often referred to in public policy discussions.  According to Project 2015, an initiative of the New York State Governor’s office to assess the impact of an aging population, members of the baby boom generation will represent 24% of the State’s population by the year 2015.  This increased length of a healthy life for older adults is an undeniable societal benefit, but it comes at the cost of an epidemic of aging-related cognitive decline.  Public health efforts must address this problem.  We propose to test whether a widely accessible activity, aerobic exercise training, can improve cognitive function in older adults.  Moreover, we propose to examine whether this improvement in cognitive function is related to neurogenesis, the development of new neurons, in a part of the brain called the dentate gyrus (DG).  The DG is a subregion of the hippocampal formation that is associated with cognitive function.  The DG is one of the few brain regions of the adult brain withactive neurogenesis.  Animal studies show that neurogenesis declines as age increases and that physical exercise increases neurogenesis in aging rats, thereby attenuating age-related memory decline.  Taken together, these observations suggest that physical exercise can ameliorate age-related memory decline in aging humans by increasing neurogenesis in the DG.  Until recently, testing this hypothesis in humans required post-mortem brain samples.  We recently showed that it is possible to use magnetic resonance imaging (MRI) to detect a correlate of neurogenesis in living animals and humans.  In a small study, we also found that aerobic exercise training leads to an increase in this correlate, but this study was performed only in a small sample of young subjects.  Here we propose to apply this imaging approach to a larger sample of older subjects to test whether exercise training enhances cognitive function by increasing neurogenesis in the DG.

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 Functional Analyses of Embryonic Stem Cell Derived Retinal Cells

Stephen Tsang, MD, PhD
Columbia University Medical Center
IIRP

Many cases of early-onset retinal dystrophies, retinitis pigmentosa (RP) and age-related macular degeneration (AMD) leading to blindness are caused by dysfunctional retinal pigment epithelium (RPE) and photoreceptor loss.  AMD alone afflicts 9 million Americans, and its incidence is expected to double by 2020.  RPE-associated degenerative diseases are incurable, severely impair daily activities and often lead to psychological depression.  We generated a mouse model of retinal degeneration by inactivating the Rpe65 isomerase gene.  These mutant mice exhibit an early onset of blindness characterized by profoundly reduced activity on the electroretinogram (ERG).  Thus this is an ideal animal model in which to study the efficacy of stem cell therapy for RPE-related diseases.  Here we propose to induce embryonic stem cells to differentiate into RPE in culture, transplant them into the subretinal space of Rpe65 mutant mice and test for restoration of RPE function.  Recently, we carried out effective stem cell transplantation into Rpe65 mutant mice, as assessed by electrophysiological measurements (ERG).  In our proposed experiments, we will determine if such stem cell-based therapies can halt progressive photoreceptor death once photoreceptor degeneration initiates.  One innovative aspect of this proposal lies in the use of different fluorescent reporters to facilitate high-throughput, non-invasive imaging to document the survival and differentiation of stem cell transplants in a living host.  Success in our research will provide proof of principle that stem cell transplantation can improve neuronal function in the context of early-onset retinal dystrophies, as well as other forms of degeneration such as RP and AMD.  Our approach is a logical first step in advancing stem cell-based therapies as a remedy for neuronal dysfunction generally, and thus our findings will be applicable to many other degenerative diseases of the central nervous system.

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 Genome Maintenance in Germline Stem Cells

John Schimenti, PhD
Cornell University
IIRP

The germline, those cells that ultimately form sperm and eggs, is the guardian of our species because it contains the genetic information that is passed from generation to generation.  Therefore, it is important that germ cells have sensitive mechanisms for preventing mutations that could be transmitted to offspring.  Germ cells are actually formed during embryogenesis.  They proliferate, colonize the primitive gonads, and ultimately form cells that can become oocytes or sperm.  We and others discovered that mutations in genes that are important for DNA repair and DNA replication can deplete the reservoir of germ cells, often leading to infertility.  This project uses two unique mouse models of germ cell depletion, each with mutations in a single gene, to study the DNA repair systems in germline stem cells.  One gene is involved in replicating DNA (Mcm9), and the other in repairing DNA (Fanconi anemia M, or Fancm).  Both cause loss of germ cells in males and females.  We hypothesize that self-destruction is a mechanism used by germline stem cells to prevent the transmission of mutation-containing genomes to the next generation when the level of damage is beyond effective repair.  We will identify the exact stage of development that germ cell death occurs and perform a variety of molecular analyses to better characterize the genome maintenance and self-surveillance mechanisms that are operating in these cells.  This will give insight into the cells holding the keys to our species, the germline, and how genetic and environmental factors can impact fertility and the well-being of offspring.

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 Induced Pluripotent Stem Cell-Derived Cardiomyocytes as In Vitro Models of Early Repolarization Syndrome and Sudden Cardiac Death

Charles Antzelevitch, PhD
Masonic Medical Research Laboratory
IIRP

Abnormal rhythms of the heart, known as cardiac arrhythmias, are a major cause of mortality and morbidity in the United States and throughout the world.  Progress in the development of drugs for the treatment of life-threatening cardiac arrhythmias over the past couple of decades is disappointing.  The development of drugs and other therapeutic approaches is hampered by the lack of suitable in vitro human models.  We propose to create a novel human experimental model of cardiac arrhythmia disease using induced pluripotent stem cells (iPSCs) with which to evaluate drugs for the treatment of cardiac arrhythmias.  We will generate these iPSCs from skin fibroblasts and hair follicle keratinocytes isolated from of patients with Early Repolarization Syndrome, which was recently highlighted as a significant cause of inherited sudden cardiac arrhythmic death.  We will then differentiate the iPSCs into cardiomyocytes, heart muscle cells, to generate in vitro models of the disease.  These in vitro models should provide valuable new insights into the mechanisms underlying these disorders and offer a unique platform with which to improve diagnosis and delineate novel therapeutic strategies.  The novel concepts and methodological knowledge developed in this project could be extended, in the future, to establish disease-specific human models for a plurality of genetic disorders, thus improving the prospect for better diagnosis and, ultimately, patient-specific treatments that could find their way to the bedside.

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 Redirecting Hemoglobin Expression During Human ES Cell Differentiation

James Bieker, PhD
Mount Sinai School of Medicine
IIRP

Hemoglobinapathies are blood diseases such as sickle cell disease and b-thalassemia.  These are anemias of varying intensity that have a deleterious impact on the health of millions of people in the US and worldwide.  We have an interest in developing novel approaches to alter the normal hemoglobin switching mechanism so that patients with hemoglobinapathies might benefit from derepression of fetal or embryonic globin expression.  Briefly, using human embryonic stem cells (hESCs) engineered to express detectable reporters from their fetal and adult globin promoters, we will screen for small molecules that either induce adult globin expression or reactivate fetal globin expression.  The major advantages of this approach is that it avoids the historical use of the mouse to address what is a human gene regulatory pattern and it takes advantage of the normal hematopoietic process observed during hESC differentiation, a process that cannot be recapitulated within human leukemic cell lines.  Our major innovation is that we will establish a unique hESC line with a characterized molecular baseline of marked b-like globin gene switching that will then provide a rigorous and directly relevant avenue for testing inducers of adult globin or reactivators of fetal globin expression.

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 Ex Vivo Expansion of Cord Blood Hematopoietic Stem Cells

Ronald Hoffman, MD
Mount Sinai School of Medicine
IIRP

Cord Blood (CB) is collected from the placentas of women after the normal birth of a child.  CB specimens are frequently discarded, but they contain sufficient numbers of blood stem cells to provide a graft for a child with a blood cancer who is receiving a stem cell transplant.  Unfortunately, there are frequently too few stem cells in most CB collections to serve as a stem cell source for adults, since adults are larger than children.  In this proposal we plan to develop the means to effectively expand the number of blood stem cells in CB collections so that they can be used in adult transplant recipients in a routine fashion.  We will use a group of drugs called chromatin modifying agents to treat the blood stem cells so they retain the characteristics of the original CB stem cells.  Chromatin modifying agents alter the structure of DNA in stem cells, thereby controlling the expression of genes critical for cell function.  We will also examine the pattern of genes expressed by CB stem cells prior to and following expansion.  We hypothesize that the expression patterns will be similar, thereby confirming that the expanded stem cells closely resemble the original CB stem cells.  Furthermore, we will create a means of enumerating the numbers of blood stem cells in these expanded stem cell cultures in order to determine an effective dose of stem cells for adults.  The completion of such studies will greatly expand the use of CB as an alternative source of stem cells for transplantation of adults with blood cancers and genetic blood diseases.

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 Analyzing Pluripotency in Human Embryonic Stem Cells and Stem Cells from Reprogrammed Differentiated Cells

Ihor Lemischka, PhD
Mount Sinai School of Medicine
IIRP

Stem cell research has the ability to revolutionize 21st century medicine.  There are two broad classes of stem cells: adult and pluripotent.  The former are dedicated to producing cell types characteristic of a given tissue, while the latter can produce all 200 or so cells in the body.  Pluripotent embryonic stem cells (ESCs) are derived from early embryos, a process accompanied by numerous bioethical considerations.  Recently it became possible to “reprogram” adult cells to a pluripotent ESC-like state using only a few proteins.  These induced pluripotent stem cells (iPSCs) can be derived from a patient’s skin cells and closely resemble their ESC counterparts.  The patient-specific iPSCs are genetically identical to the patient and can be used to model specific inherited diseases.  The exact degree of ESC/iPSC equivalence is currently unclear.  In this proposal we aim to comprehensively compare ESCs and iPSCs that are genetically identical.  We hypothesize that ESCs and iPSCs will share most biological and molecular features.  We will employ “systems biology” approaches to globally measure gene and protein expression patterns, and will seek to functionally identify regulatory components responsible for controlling cell fate decisions made by these cells.  We will construct sophisticated computational strategies in order to extract important biomedical insights, then provide the complete data set to the community via accessible and unrestricted on-line resources.  This information will inform the future applications of iPSC methodologies in studying disease etiology, developing novel diagnostic and pharmacological compounds, and eventually producing cell populations for transplantation-based therapeutic regimens.  As a final goal, we will explore strategies from the newly emerging field of “synthetic biology” as a means to “program” ESCs and iPSCs towards clinically important cell types such as insulin secreting pancreatic cells, neurons, cardiac cells and many others.  Taken together, our systems and synthetic biology approaches represent new ways to understand and manipulate stem cell functions.

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 ZFP57, a Link Between Genomic Imprinting and Stem Cells

Xiajun Li, PhD
Mount Sinai School of Medicine
IIRP

Genomic imprinting is essential for mammalian development.  Dysregulation of genomic imprinting is associated with a variety of human diseases including metabolic diseases, cardiovascular diseases and cancer.  Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) reprogrammed from adult cells are candidates for cell-based therapies for various degenerative diseases.  However, ESCs and iPSCs all have genomic imprinting defects that may cause cancer and other human diseases.  Therefore, a requirement for attaining proper cells for therapeutic applications is a thorough understanding of the underlying mechanisms of genomic imprinting.  We can now achieve this goal since we discovered an important regulator in genomic imprinting, ZFP57.  We will analyze the role of ZFP57 in acquisition and maintenance of genomic imprinting in ESCs.  In addition, we will probe its functions in governing the generation of various cell types from ESCs.  The results from the proposed research will set the stage for future studies aimed at deriving proper cells from ESCs, iPSCs and other pluripotent stem cells for cell replacement therapies.

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 Dual Specificity Phosphatases in Ocular Surface Side Population Stem Cells

Jose Mario Wolosin, PhD
Mount Sinai School of Medicine
IIRP

The eye surface is protected by unique lining (or epithelium) consisting of 5-6 layers of quickly dividing and regenerating cells totally dependent on stem cells for survival.  These cells are located mostly in a narrow outer rim called the limbus.  This segregation has critical clinical consequences.  Damage to the limbus by chemical or thermal injuries, microbial infection, or autoimmune reactions results in limbal stem cell deficiencies (LSCD).  In the most severe cases of LSCD the corneal lining fails, leading to blindness.  Corneal disease typically impacts young individuals that, unless cured, face a long life of blindness or highly reduced visual function.  Transplants with limbal cells incorporating stem cells can restore full vision.  The highest success rates are seen in transplants that use small amounts of cells obtained from an immunocompatible eye.  However, the number of donor cells that can be removed from these sources without risk to the donor organ eye is very small.  Thus, the cells need to be pre-expanded in culture.  In vivo, these stem cells divide with low frequency.  To expand these cells in culture, we need them to proliferate rapidly.  However, the slow division rate of the stem cells prevents them from maintaining their proportion of the total population.  Thus, finding the factors that regulate stem cell proliferation and duplication is critical to improve these vision-restoring transplants.  We isolated limbal stem cells and found that they over express certain proteins (phosphatases) that block proliferation.  We hypothesize that the phosphatases are involved in the slow cycling of stem cells and interfere with the efficiency of expansion in culture.  We propose experiments to test this hypothesis and the value of inhibition of these phosphatases for improving the cultures used for transplants.

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 The Multipotency of Epicardial Cells and Mammalian Cardiac Repair and Regeneration

Chen-Leng Cai, PhD
Mount Sinai School of Medicine
IIRP

Cardiovascular disease is the leading cause of death in the world.  While a wide array of medical and surgical therapies exist, few are curative.  Stem cell therapy that replaces diseased, dysfunctional cells with healthy, functioning ones would provide more effective treatment for many forms of cardiovascular disease.  To facilitate this, it is important to identify an optimal source of stem cells for achieving cardiac repair and regeneration.  The purpose of our study is to determine if cells covering the heart (called epicardium or epicardial cells) can be used in heart repair and regeneration.  We recently found that epicardial cells possess developmental plasticity enabling them to differentiate into several types of cardiac cells, including cardiomyocytes (contractile cells), coronary vascular cells and cardiac fibroblasts (that form connective tissue) during mouse heart formation.  In zebrafish, which have an amazing regeneration capacity and can "re-grow" their heart after major injuries, epicardial cells play very important functions during heart regeneration.  Cardiomyocyte hyperplasia in these animals is accompanied by re-activation of epicardial cells after the cardiac injury.  The epicardial cells surround and vascularize the wounded region, thus contributing to re-growth of the heart.  These discoveries suggest that epicardial cells may be curative for injured hearts in humans.  In this study we will first use mice as a model to determine if epicardial cells can differentiate into all types of cardiac cells during different stages of heart formation.  We will then determine if the human epicardial cells have similar differentiation potential.  Finally, we will transplant epicardial cells into mouse hearts and determine if the transplanted cells can improve cardiac function of the injured hearts.

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 Regulation of Dentate Gyrus Function by Hippocampal Stem Cells and Their Progeny: Relevance to Cognition and Depression

René Hen, PhD
New York State Psychiatric Institute
IIRP

Adult neurogenesis refers to the generation of new neurons in the adult brain.  In mammals, there are only two regions in the adult brain where stem cells give rise to neurons throughout life: the olfactory bulb and the hippocampus.  Although this phenomenon was discovered several decades ago, it started receiving more attention when it became clear that neurogenesis was regulated by a number of environmental and pharmacologic manipulations.  Specifically, stress and aging decrease neurogenesis while enriched environment, exercise, learning and antidepressants stimulate hippocampal neurogenesis.  Furthermore, we showed that hippocampal neurogenesis is required for some of the behavioral effects of antidepressants, as well as for certain forms of learning and memory.  This led us to speculate that stimulating neurogenesis may produce antidepressant and pro-cognitive effects.  Thus, we hypothesized that stimulation of young neurons in the hippocampus will enhance cognition and mood.  We will now test this by selective activation of young hippocampal neurons and determine if this results in a decrease in the activity of mature granule cells.  Furthermore, we will test the hypothesis that activation of young neurons in the dorsal hippocampus results in an improvement in certain forms of learning, while activation of young neurons in the ventral hippocampus results in an antidepressant–like response.  This proposal is the first attempt to assess the impact of strategies aimed at stimulating neurogenesis.  Application of our findings could lead to pharmacological intervention aimed at modulating hippocampal neurogenesis, with beneficial effects on the treatment of mood or cognitive disorders. 

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 Role of SHH-Responsive Stem Cells in Remyelination

James Salzer, MD, PhD
New York University School of Medicine
IIRP

Multiple Sclerosis leads to progressive neurological disability due to immune-mediated damage to an insulating sheath around nerve fibers, termed myelin.  This sheath surrounds nerve fibers in the brain and spinal cord and is critical for their ability to conduct impulses.  A key therapeutic goal in multiple sclerosis research is to restore function by enhancing formation of new myelin sheaths to replace those that have been lost.  New myelin, in turn, requires the generation and differentiation of specialized cells in the brain, oligodendrocytes, which make the myelin sheath.  There are many precursors of oligodendrocytes in the adult brain and these are thought to be the primary source of new myelin sheaths that are made after injury.  However, our studies suggest that stem cells resident within the adult brain are an additional, and potentially major, source of remyelinating oligodendrocytes.  Our data also suggests that loss of myelin triggers the expansion of stems cells and their differentiation and myelination of injury sites via a signal called sonic hedgehog (SHH).  Finally, our data suggests that as animals age, they lose their ability to make myelin, potentially due to changes in stem cell responsiveness to SHH.  Our major goals are to elucidate the role of stems cells, and SHH, in repairing myelin in the adult brain by: 1) analyzing the precise contribution of stem cells to new myelin, 2) determining whether SHH expression and its signaling are indeed required for remyelination and 3) determining whether changes in stems cells, and their responsiveness to SHH, underlies the limited ability of the aged nervous system to repair itself by forming new myelin.  The focus on the role of stem cells rather than on oligodendrocyte progenitors as a source of remyelinating cells is novel; the role of demyelination as a trigger for stem cell expansion is also a novel foundational result. 

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 Harnessing Keratinocyte Stem Cell Growth for Regenerative Medicine

Elains Fuchs, PhD
The Rockefeller University
IIRP

The potential of adult epithelial stem cells (SCs) for regenerative medicine is enormous.  Adult skin SCs can be cultured, making them an outstanding model for SC research.  They are also effective for treating burn patients and generating induced pluripotent stem cells (iPSCs).  Similarly, cultured corneal SCs can restore vision.  Methods to expand the proliferative capacity of epithelial SCs in vitro, without compromising their tissue regenerative potential, would greatly enhance the success and number of patients for whom treatments would be feasible.  Such advances require understanding the factors that govern self-renewal, proliferation rates and undifferentiated states of skin and corneal SCs.  We propose to develop the basic biological framework necessary to achieve these clinical objectives.  With the necessary expertise, tools and groundwork, we will now address the following questions: How are corneal and skin SCs similar? What molecular features define their unique capacity to self-renew and maintain long-term regenerative potential? Can this information be used to enhance SC proliferative capacity in culture, without compromising their effectiveness in regenerative medicine?  Our laboratory has strength in mouse genetics, molecular biology and hair follicle (HF) SCs.  Our collaborator’s expertise is corneal SC biology and translational research.  Specifically, we will: 1) use our mice, in which epithelial SCs are fluorescently tagged, to isolate corneal SCs, determine which genes they express and define their relation to already characterized skin SCs; 2) test corneal SC capacity for proliferation in culture and tissue regeneration in engrafted mice; 3) identify genes intrinsically expressed by SCs irrespective of quiescent status; 4) use innovative lentiviral genetic screens to identify the genes that enhance the growth of HF and corneal SCs in culture without compromising their ability to function in tissue regeneration; and 5) evaluate whether this information can be exploited to improve the therapeutic potential of corneal SCs in regenerative medicine in humans.

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 Modeling Pathogenesis and Treatment of Familial Dysautonomia in Patient Specific Human Induced Pluripotent Stem Cells

Lorenz Studer, MD
Sloan-Kettering Institute
IIRP

Familial dysautonomia (FD) is a rare but fatal disorder in young children that primarily affects the peripheral nervous system.  We recently demonstrated that FD can be modeled in a petri dish using patient-specific induced pluripotent stem cells (FD-iPSCs).  Our data provided insights into what goes wrong in the disease and why the peripheral nervous system cells are particularly affected.  The cells also allowed us to test potential candidate drugs to see whether they can improve disease-associated behavior.  Based on these interesting findings, we will advance iPSC-based disease modeling further.  We will test whether FD-iPSC technology can distinguish between patients with different disease severity and carrier status.  We will also identify novel drugs to treat the disease using the FD-iPSC assay.  We propose three aims: 1) generate and characterize FD patient-specific iPSC lines from fibroblast of patients with mild disease or with carrier status; 2) test candidate drug treatments, in particular the importance of timing, and rescue the disease using a from of gene repair; and 3) use our culture system to test 5,000 drugs (most available drugs on the market – to see whether any of them are effective in our FD-iPSC system.  A positive result would have a good chance of translation into clinical trials in the future.  Many aspect of the proposal are highly innovative including: 1) the attempted correlative studies with disease severity in patient-specific iPSCs, which has never been attempted for any disease before; 2) the BAC technique for gene repair, which is quite innovative; and 3) the 5,000 compound screen is innovative in the context of patient-specific iPSCs.  No such screen has been performed for any disease to date.

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 Patient-Specific Human ESCs and iPSCs for Modeling Schwann Cell Differentiation and Charcot-Marie-Tooth Disease

Lorenz Studer, MD
Sloan-Kettering Institute
IIRP

Charcot-Marie-Tooth is a disease of the peripheral nervous system causing severe neuronal dysfunction and muscular weakness.  It is the most common genetic disease affecting the peripheral nervous system and currently there are no acceptable treatment options.  We are using two highly complementary approaches to model this disease: human embryonic stem cells (hESCs) and patient-specific induced pluripotent stem cells (iPSCs), combined with protocols for differentiation of these cells specifically into the affected cell types.  We propose to: 1) generate additional patient-specific iPSC lines to obtain robust data in disease modeling; 2) optimize protocols for generating the peripheral nervous system cells most affected in Charcot-Marie-Tooth (Schwann Cells); and 3) model the disease using cells generated in aim 1 and protocols optimized in aim 2.  To do so, we will carefully characterize iPSCs and hESCs to assure that they carry the correct mutation and that they are true human pluripotent stem cells.  We will then use BAC transgenesis, a novel tool we developed, to generate the Schwann cells.  Finally, we will explore gene expression, proliferation, differentiation and myelination (ensheathing of axons) in disease and control lines.  Ultimately, we will test candidate drugs for effectiveness in reversing disease associated symptoms in the petri dish.  Many aspects of the proposal are highly innovative including our cell differentiation protocols, the use of genetic reporter lines to optimize Schwann differentiation, and the first comparison of hESCs and hiPSCs in modeling human genetic disease.

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 Role of Nfix in Neural Stem Cells and Glioblastoma

Richard Gronostajski, PhD
SUNY – University at Buffalo
IIRP

Neural stem cells are essential for adult brain function and maintenance.  A single neural stem cell can produce both neurons and glial cells.  We previously inactivated the Nuclear Factor I X (Nfix) gene in mice and showed these mice have brains that are ~20% larger and heavier than normal.  Such brains have abnormal cells that express markers of neural stem cells.  The goal of our research is to understand the effects of inactivation of Nfix on brain development.  Previous studies found that viruses that cause certain brain tumors in mice, known as glioblastomas, often insert themselves into the Nfix gene, implicating Nfix in brain tumor formation in humans.  To identify the cells that require Nfix to prevent these brain abnormalities, we will delete Nfix only in specific types of brain cells.  We will grow abnormal cells in culture to understand how they proliferate and differentiate into neurons and glia.  We will mark mutant cells, inject them into the brains of normal mice and follow how these cells divide, migrate and become neurons or glia.  We will inject tumor-inducing viruses into Nfix-inactivated mice to see if lack of Nfix affects brain tumor formation.  We will determine which genes are expressed differently in these mutant cells compared to normal cells using microarrays.  Our results will, for the first time, define the role of Nfix in brain development in detail and will generate new insights into the function of this gene in both embryonic and adult brains.  This novel mouse model will yield important information on how Nfix controls the growth, differentiation and migration of stem cells in the brain and how glioblastomas form in the brain.

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 Induction of Sustained Medium Spiny Neuronal Addition from Sub-Ependymal Stem Cells of Adult Monkey Brain as Therapeutic Strategy for Huntington's Disease

Abdellatif Benraiss
University of Rochester
IIRP

The adult human brain is capable of generating new neurons from endogenous neural stem cells.  However, the human brain is not able to harness these new neurons to heal itself in the event of disease or trauma.  During development, the brain produces certain factors, such as brain derived neurotrophic factor (BDNF) and noggin that control cell proliferation and fate.  By reintroducing genes for these factors in the adult ventricular wall, by way of transiently active adenoviruses injected into the ventricles (AdBDNF/AdNoggin), we effectively mobilized endogenous neural stem cells to generate new neurons.  Previously, we showed that new neurons can be induced to migrate into the striatum in adult rats, mice and, more recently, in squirrel monkeys.  Since the new neurons integrate as medium spiny neurons into the network of the striatum, we hypothesized that they might provide functional benefit in Huntington’s disease (HD), a neurodegenerative disease of the striatum.  We found that AdBDNF/AdNoggin co-treatment resulted in significant improvement in both the motor behavior and life span of HD mice.  We now propose a preclinical assessment of AAV4-mediated gene delivery in the adult primate brain.  Specifically, we will determine whether long-lasting BDNF and noggin expression, enabled by adeno-associated virus (AAV4), induce sustained neuronal addition to the adult primate brain sufficiently as to indicate the potential utility of this strategy for the treatment of HD.  Before proceeding to clinical trials, the therapeutic strategy described here must be demonstrated effective in a primate model of HD.  The success of these studies will also prove the effectiveness of the mobilization of endogenous neural stem cells for treatment of other neurodegenerative diseases.  In broader terms, this may serve as a conceptual basis for designing strategies to promote cellular regeneration in a host of other neurodegenerative and traumatic-ischemic disorders.

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 Atlas of Gene Expression by Progenitor Cells of the Human Brain and its Tumors

Steven Goldman, MD, PhD
University of Rochester
IIRP

The goal of this study is to define, archive and atlas the genes differentially expressed by each of the major progenitor cell types of the normal adult human brain, their mature progeny and their cancerous derivatives in primary brain tumors.  Full characterization of adult human neural and glial progenitor cells by mRNA and miRNA profiling will enable us to specifically regulate their activation, proliferation and differentiation.  This information should also allow us to direct endogenous progenitor cells to repair the effects of brain damage and degenerative disease, as well as to treat brain tumors that may arise from dysregulated stem and progenitor cells.  The data provided in our analyses should therefore be of immediate interest to investigators in fields as diverse as multiple sclerosis (remyelination from endogenous glial progenitors); spinal cord injury (prevention of glial scar formation after traumatic cord injury); stroke (induction of neurogenesis); epilepsy (suppression of post-ischemic and post-traumatic gliosis and secondary epilepsies); brain tumor biology and neuro-oncology (identification of tumorigenic genes and pathways not shared by normal progenitors); and neurodegenerative diseases (induction of restorative neurogenesis, as in Huntington’s Disease).  As such, the fundamental importance of this project lies in its provision of a resource, a novel and hitherto unavailable adult human brain genomics database, upon which a plethora of disease targets may then be more effectively studied with therapeutic endpoints.  We will make our data broadly available to the basic and translational neuroscience communities via an interactive web site linked to the NIH/NCBI GEO database; we expect that this genomics database should quickly develop as a critical resource to investigators, both within and beyond New York State, studying virtually all major categories of neurological disease and drug discovery.

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 Reversing Chemoresistance: Novel Pathways and Novel Treatment Targets

Mark Noble, PhD
University of Rochester
IIRP

Our research program is focused on discovering mechanisms to eliminate the tumor initiating cells (TICs, also known as cancer stem cells) in brain tumors, with a particular focus on glioblastoma multiforme (GBM), the most malignant of brain tumors.  As cancer treatments require combinatorial treatment strategies, our research also provides strategies relevant to optimization of combination therapies for GBM and identification of patients most likely to benefit from specific combinations of therapies.  Elimination of TICs, and of cancers in general, requires a mechanistic understanding of how tumors become resistant to multiple chemotherapeutic agents and discovery of compounds that target the mechanisms of resistance.  We recently discovered that a normal regulatory pathway is disrupted in GBMs.  This pathway has dual roles: it is involved in TIC maintenance and its disruption provides a previously unknown path to chemoresistance.  This discovery allowed identification of 10 new compounds suitable for rapid pre-clinical evaluation so far.  Moreover, our discoveries offer a means of prospectively identifying patients likely to benefit from particular drug combinations.  Thus, this research offers the prospects of enabling rational formulation of combinatorial treatment strategies designed to reduce chemoresistance in TICs and other cancer cells, and increase sensitivity to multiple treatments.  In addition, it is essential that treatment strategies enable selective targeting of tumor cells.  Our efforts are unique in their attempts to identify means of enhanced killing of GBM cells without also causing unacceptable CNS damage.

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 Generation of Abundant Clinical Grade Vascular Endothelium for Therapeutic Vasculoplasty Using a Highly Efficient Feeder Based Co-Culture Platform Free of Animal Contamination

Shahin Rafii, MD
Weill Medical College of Cornell University
IIRP

An unlimited supply of vascular endothelial cells (VECs) is among the most promising dividends of human stem cell application in regenerative medicine, yet the signaling pathways and transcriptional networks that govern primary vascular differentiation in human embryogenesis are unknown.  We generated an hESC reporter line that expresses GFP exclusively in cells committed to a VEC fate using a promoter from the VE-cadherin gene, which is expressed specifically in vascular endothelium.  Using this reporter line, we screened for signaling pathways and transcriptional networks that promote and/or augment VEC differentiation in hESC cultures.  We showed that under serum-free conditions, co-culture of hESCs with a mature human VEC feeder layer, but not other non-VEC cell types, induced abundant expression of the reporter and produced VEC committed cells with more than 80% of the population differentiating to cardiovascular cell types.  This method for vascular differentiation from hESCs presents an unprecedented resource for pre-clinical study of cell-based therapies of cardiovascular disease.  We also isolated a diversity of subpopulations from hESC/VEC feeder co-cultures and demonstrated in vivo functional potential in mice.  The use of animal derived serum for production of cells for human therapies is problematic for many reasons.  Thus, we now propose to scale up the serum-free differentiation platform to test the ability of discreet subpopulations of vascular cell types to restore blood vessels in mouse models of tissue ischemia (reduced blood flow), as a precursor for direct application of this knowledge to clinical treatments.

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IDEA Abstracts

 Dynamic Genome-Wide Transcription Factor-Chromatin Interactions During Hematopoietic Stem Cell Differentiation

Ulrich Steidl, MD, PhD
Albert Einstein College of Medicine
IDEA

Hematopoietic stem cells (HSCs) ensure the life long production of the entire spectrum of mature blood cells.  The balance between self-renewal, pluripotency and differentiation is controlled by transcription factors, proteins that regulate gene expression.  The large amount of material required for conventional assays generally precludes the use of stem cells from transcription factor binding studies because stem cells numbers are limiting.  Furthermore, most studies focus on individual target genes and binding sites close to the start of genes. To overcome these limitations we propose to 1) establish a novel methodology for genome-wide identification of transcription factor-chromatin (packaged DNA) interaction sites in hematopoietic stem cells and their progeny, and 2) delineate dynamic chromatin-binding patterns of one important stem cell master regulator (PU.1) during hematopoietic stem and progenitor cell commitment.  We will accomplish this by combining two key technologies: high-speed multi-parameter cell sorting (FACS) and chromatin-immunoprecipitation followed by genome-wide DNA sequencing (ChIP seq).  We will isolate hematopoietic stem cells (HSCs) and the earliest lineage-committed blood progenitor cells, then analyze genome-wide chromatin-binding patterns of PU.1 as a paradigm of a stem cell master regulator.  This will provide a map of binding patterns and target regions that are characteristic of the multipotent stem cell state, as well as HSC commitment towards the myeloid lineages.  This research proposal is aimed at filling a critical methodological gap in the study of HSC biology.  Establishing this novel methodology will produce an unbiased map of transcription factor binding sites in one type of adult stem cell, and its earliest progenitors, thus helping to identify regulatory regions that are important for stem cell self-renewal, clonogenic potential and differentiation that could not be identified otherwise.  This study will greatly enhance our knowledge of gene regulation processes in early stem cells and identify novel key regions and genes critical for stem cell function.  Ultimately, this will provide us with a new experimental tool to delineate disrupted transcription factor binding in stem cell-related diseases such as leukemia and bone marrow failure.

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 Cerebrospinal Fluid Regulation of Adult Neural Stem Cells

Fiona Doetsch, PhD
Columbia University Medical Center
IDEA

Adult neural stem cells continuously generate neurons, a process termed neurogenesis, in restricted parts of the brain and may represent an important source of endogenous cells that can be stimulated for brain repair.  Stem cells reside in specialized niches, or microenvironments, that support their self-renewal and maturation into different cell types.  The subventricular zone (SVZ) is the largest germinal area in the adult brain.  Stem cells in this brain region are in direct contact with cerebrospinal fluid (CSF), which fills the brain ventricles.  However neither the factors present in the CSF, nor its role in supporting adult brain stem cells are known.  We hypothesize that signals from the CSF regulate SVZ stem cell behavior.  We propose to identify the factors in the CSF that modulate SVZ stem cells and to characterize how CSF affects stem cells in culture and in a mouse model using several innovative approaches.  We found that CSF promotes stem cell activity in culture.  We have now set up a system to isolate CSF from the brain that will allow us to identify novel components present in it.  We will test the effect of interesting candidates we identify both in culture and in vivo by manipulating their levels and defining the ensuing the functional consequences.  Our proposed methodology will allow us to comprehensively dissect the role of the CSF in the adult brain and in neurogenesis.

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 Directed Differentiation of Embryonic Stem Cells into Plasmacytoid Dendritic Cells

Boris Reizis, PhD
Columbia University Medical Center
IDEA

Embryonic stem cells (ESCs) and their artificial counterparts, induced pluripotent stem cells (iPSCs), can give rise to all cell types of the adult body, raising the hope of stem cell-based cell replacement therapies.  Plasmacytoid dendritic cells (PDCs) represent a blood cell type specialized in virus recognition and rapid antiviral immune responses.  Because the transfer of PDCs would boost antiviral resistance in patients with decreased immunity, the generation of PDCs from ESCs/iPSCs would have broad therapeutic applications.  However, this has not yet been achieved.  We propose to generate PDCs from mouse ESCs and to confirm their proper function in immune responses against viruses.  Recent studies from our lab defined key regulatory signals required for PDC development.  We will use these findings to optimize the conditions for generation of PDCs in vitro.  The resulting PDCs will be injected into PDC-deficient animals and tested for function in response to virus-associated molecules.  Our proposals will pave the way for the production of functional PDCs from human ESCs/iPSCs.  In the long run, this will enable the generation of functional PDCs for subsequent transfer to patients, a desirable therapeutic option to increase antiviral resistance in immunocompromised populations such as the elderly, transplant recipients and AIDS patients.

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 Roles for the Putative Longevity Determinants HCF1 and HCF2 in Stem Cell Function

Siu Lee, PhD
Cornell University
IDEA

Two major unresolved questions in stem cell and aging research are whether a decline in stem cell function causes aging and whether a boost in stem cell function can reverse the toll of aging under normal physiological conditions.  One way to address these important questions is to study whether the molecules that control how long an organism lives also control how well their stem cells work.  Ongoing research identified a key role for the FOXO family of proteins in both aging rate and adult blood stem cell function.  Interestingly, we recently identified a new factor named HCF-1 in the nematode that influences aging rate by ensuring proper function of FOXO protein.  The HCF-1 proteins in evolutionarily diverse species are highly similar, and our preliminary results show that the mammalian counterparts of HCF-1 (HCF1 & HCF2) also help to ensure that FOXO proteins work properly in mammals.  Our proposed research will determine whether the mammalian counterparts of HCF-1 also affect blood stem cell function.  Because some proteins that affect blood stem cells also affect embryonic stem cells, we will test whether HCF1 and HCF2 are required for embryonic stem cells to work properly.  These are two key questions at the intersection of aging and stem cell biology.  Results from our research will provide important insights into the understanding of how stem cell function and aging is linked.  This has important implications for future drug development to improve the quality of life of the elderly and to treat age-dependent diseases such as cancer, diabetes and neurodegeneration.

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 Investigating How DNA Damage Response Mechanisms Regulate the Tumorigenic Potential of Pluripotent Cell Types Using Mouse Models of Testicular Germ Cell Tumors

Robert Weiss, PhD
Cornell University
IDEA

Stem cells hold great promise for treating many diseases.  However, stem cell therapies also are associated with a considerable cancer risk.  This proposal seeks to determine how one of the main mechanisms that cells use to prevent cancer operates in stem cells.  The DNA damage response (DDR) is a system that responds to genome damage and suppresses the mutation accumulation and uncontrolled proliferation characteristic of cancers.  To study DDR functions in stem cells we will focus on testicular germ cells, which share many biological properties with the stem cells being developed for therapeutic applications.  Moreover, testicular germ cells are amenable to experimental analysis and are the precursors to testicular germ cell tumors (TGCTs), the most common cancers of young men.  Evidence suggests that the DDR operates differently in testicular germ cells and other stem cells than in other tissues.  Accordingly, mutations in genes involved in the DDR, which are common in cancers, are rare in TGCTs, and advanced TGCTs remain highly sensitive to anti-cancer therapies, unlike most malignancies.  We hypothesize that these unique characteristics originate from the stem cell-like properties of the germ cells from which TGCTs originate and that these properties influence how these cancers develop and respond to therapies.  We will test this hypothesis using mouse models, which afford the opportunity to fully interrogate the underlying molecular and genetic mechanisms.  Current mouse TGCT models do not entirely recapitulate the human disease, so an important innovative aspect of the proposal is to develop novel mouse models that more closely resemble human TGCTs.  Additional studies will use cultured cells to directly compare DDR mechanisms in testicular germ cells, other stem cells and non-stem cells.  These experiments will reveal how a critical regulatory process operates in stem cells, with implications for the prevention and treatment of cancers originating from stem cells.

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 Reprogramming the Hair Follicle Stem Cell Niche in Uncommitted Skin Fibroblasts

Michael Rendl
Mount Sinai School of Medicine
IDEA

Regulation of stem cell activation and self-renewal is necessary for the formation of many organs and for their coordinated maintenance throughout life.  In many tissues this is achieved by signals from supporting cells within the stem cell vicinity, called the stem cell niche.  How cells in mammalian stem cell niches, such as the bone marrow, brain, intestine and hair follicles, acquire their specialized status is largely unknown due to the lack of methods to isolate and characterize the cells.  This project aims to unravel the molecular mechanisms of how the fate of cells in the niche is acquired using mouse hair follicle formation as a model system.  We developed novel genetic tools to study niche fate characteristics in hair follicle stem cell niche cells, called dermal papilla (DP) cells.  This allowed us to purify these cells and define their molecular identity through their specific gene signature.  The DP signature contained a battery of highly enriched transcription factors (TFs).  TFs are instrumental for coordinated gene expression in all organisms, thus suggesting that the enriched TFs in DP cells could have a specialized function in programming the stem cell activating, hair inducing fate.  In this project, we will test this hypothesis and address two major goals: 1) defining which aspects of the molecular fate are essential for niche function and 2) determining which TFs can reprogram the niche fate in regular fibroblasts in vitro and in vivo.  We will use molecular biological, biochemical and genetic techniques, combined with molecular and functional fate switch analysis.  In summary, this research will identify the core transcriptional regulation of the stem cell niche fate of hair follicle DP cells.  Since many tissues control cellular morphogenesis through similar mechanisms, these findings will have global relevance for other regenerative tissues, where stem cell niches operate to maintain tissue homeostasis.

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 Dermal Papilla Cells: an Accessible and Highly Reprogrammable Source for the Generation of Pluripotent Stem Cells

Michael Rendl
Mount Sinai School of Medicine
IDEA

The generation of pluripotent stem cells by direct reprogramming of non-stem cells from patients has great potential for the development of regenerative therapies, eliminating the ethical issues surrounding the use of embryonic stem cells (ESCs) and the immune rejection problems of using foreign cells.  The simple viral overexpression of only four transcription factors can convert skin, blood and other cells into induced pluripotent stem cells (iPSCs) that closely resemble ESCs at the molecular level and in their functional capacity.  Problems surrounding the introduction of viral genetic material are addressed by reducing the number of reprogramming factors in specialized cells.  Indeed, mouse brain cells already express three of the four reprogramming factors and can be reprogrammed into iPSCs by overexpressing one, Oct4, alone.  However, the relative inaccessibility and difficulty of obtaining human brain cells remains an unresolved issue.  We have recently discovered that readily accessible dermal papilla (DP) cells from mouse hair follicles in the skin also express high levels of three of the four reprogramming factors, suggesting that DP cells represent a prime candidate cell type for easier and efficient iPSC generation.  To test this hypothesis we will reprogram DP cells into iPSCs by omission of the three factors already expressed.  Importantly, we will also determine the reprogramming capacity of human DP cells using the full range of established pluripotency assays.  In summary, our work will establish human DP cells as an easily accessible source for reprogramming with fewer factors, which will lead to the development to a generation of reprogrammed patients cells that can be safely used in regenerative therapies.

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 An Extended Protein Interaction Network for Stem Cells and Induced Pluripotency

Jianlong Wang, PhD
Mount Sinai School of Medicine
IDEA

Mouse embryonic stem cells (mESCs) are derived from cells of the early embryo that give rise to all cell types of the developing embryo, a property known as pluripotency.  Many cell types of adult animals can also be returned to an embryonic state, creating induced pluripotent stem cells (iPSCs).  iPSCs are generated by introduction of a set of genes known as reprogramming factors (RFs), resulting in induced pluripotency.  Pluripotency is governed by a number of important factors, including transcription factors such as the RFs Nanog, Oct4, Sox2, Klf4, that directly act on a cell's DNA to control expression of other genes.  Others factors, called epigenetic factors, alter gene expression by modifying the accessibility of DNA to transcription factors.  How these transcription factors and epigenetic factors interact to maintain and induce pluripotency is not well understood.  We are interested in dissecting the inter-relationships of these factors for controlling stem cell identity and their function in conversion of somatic cells to iPSCs.  In this study, we will undertake biochemical and molecular analyses to understand how each RF and other critical epigenetic factors interact with one another.  We will also determine what other proteins are associated with the RFs and epigenetic factors to set up and maintain the unique pluripotent program in mESCs (and iPSCs).  By mapping a protein interaction landscape of these RFs and epigenetic factors, we will be able to uncover novel RFs, identify potential epigenetic barriers to reprogramming, and develop novel strategies to make reprogramming more efficient for use in regenerative medicine.

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 Investigation of Adult Neural Progenitor Fate in Response to Endothelial Produced Factors

Deanna Thompson, PhD
Rensselaer Polytechnic Institute
IDEA

Rare multi-potential populations of neural stem cells (NSCs) are present in the adult brain and possess the ability to self-renew or differentiate into neurons or glia.  Stem cell fate is influenced by local environment (niche).  In adults, neural stem cells are concentrated around blood vessels, where components of the local microenvironment (vascular niche) include the local extracellular matrix (ECM), vascular and non-vascular cells, their products and products of their interaction.  Endothelial cells (EC) are a key element; however specific mechanisms controlling neural stem cell fate in the vascular niche remain unknown.  Our long-term goal is to explore the mechanisms of endothelial-neural stem cell interactions to elucidate pathways controlling cell fate in adult neural tissue.  Our working hypothesis is that EC phenotype, characteristic of the vasculature surrounding pools of NSCs, controls the composition and local microenvironment of the neural stem cell niche.  We propose three aims: 1) to investigate neural progenitor cell (NPC) self-renewal and differentiation in response to EC release of soluble factors, 2) to investigate NPC self-renewal and differentiation in response to EC produced ECM, and 3) to investigate the role of NPC-EC interactions on NPC fate in the vascular niche.  This project is innovative in the use of adult NPC and cell culture models that regulate EC phenotype to deconstruct the influence of more physiologically relevant EC-produced soluble factors, ECM and EC-NPC interactions on NPC fate.  NPC expression of self-renewal or differentiation markers will be assessed and functional correlations to microenvironment will be made.

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 Translational Control of Human Pluripotent Cell Maintenance and Neural Differentiation

Stuart Chambers, PhD
Sloan-Kettering Institute
IDEA

Regenerative medicine offers great promise for curing diseases such as Parkinson’s and spinal cord injury.  Human pluripotent cells represent the greatest avenue for cell-replacement therapies, since embryonic and induced pluripotent stem cells act as an unlimited source of all adult tissues in the human body.  However, an important hurdle in using stem cell-based therapies is controlling stem cell differentiation, or making the correct cell type needed for a given therapy.  Understanding what guides a human pluripotent cell to differentiate to a particular cell type will allow greater control over the type of cell produced.  We focus on neural differentiation, a key step in development that creates cells of the skin and nervous system, as a model system because of our experience in making this tissue in great abundance and purity.  A large body of literature in primitive animals suggests the timing of protein synthesis from messenger RNA (mRNA translation) is crucial for development and cell differentiation, yet, little attention has been paid to mRNA translation in human pluripotent stem cells.  We hypothesize that two factors known to control mRNA translation in other model systems, SAMD4A and GW182, are important for controlling differentiation of pluripotent cells.  We have begun to characterize the mRNA targets of both proteins and what impact removing these factors (knock-down) has on pluripotent cell maintenance and differentiation.  Moreover, using an innovative method to identify translationally-reduced or enhanced mRNA across the whole genome as pluripotent cells undergo neural differentiation, we aim to identify and characterize other potential regulators of mRNA translation during neural development.  Understanding how pluripotent stem cells control mRNA translation at steady-state and as the cells differentiate may lead to mechanistic insight into stem cell neural differentiation and the generation of neural derivatives for treating human disease.

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 Pdgfra Signaling and the Stem Cells of the Early Mammalian Embryo

Anna-Katerina Hadjantonakis, PhD
Sloan-Kettering Institute
IDEA

Regenerative medicine holds enormous promise for patients with degenerative and other diseases.  But the foundations for safe and effective treatment depend on a deep understanding of the mechanisms of lineage specification, cellular reprogramming and the optimal environment for maintaining the phenotypic stability of stem cells.  These foundations will, in large part, be defined through experimental investigation of factors affecting pluripotency, specification and commitment of these lineages during embryogenesis in animal models.  This high-risk high-impact project is based on a fundamental observation made in our laboratory that suggests that tissues previously thought to be exclusively extraembryonic, meaning that they form the placenta and extraembryonic membranes during pregnancy, are incorporated into the developing embryo, and may eventually give rise to cells of the respiratory and digestive tracts and associated organs, such as lungs, liver and pancreas.  These unprecedented observations increase our awareness of extraembryonic tissues (namely the extraembryonic endoderm, ExEn), and mandate that the ExEn be investigated in greater detail.  As a first step we identified Platelet derived growth factor receptor-alpha (Pdgfra) as being specifically expressed by the ExEn.  Furthermore, our preliminary data suggest that Pdgfra may be required in the ExEn and its stem cells.  The goal of this research proposal is to mechanistically define the role of Pdgfra, both in early mammalian embryos, and in stem cells types derived from them.  This project is innovative both in the preliminary observations that led to the hypothesis being tested, and in the experimental tools and methods used.  This study will use the mouse as a genetically tractable mammalian model and exploit novel tools from embryology, genetics, pharmacology, molecular and cell biology, and live imaging, many of which were developed in our laboratory. 

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 Disease Modeling of Congenital Insensitivity to Pain and Anhidrosis (CIPA) Disease Using Human iPSC and Rescue of its Phenotypes

Gabsang Lee, DVM
Sloan-Kettering Institute
IDEA

Congenital Insensitivity to Pain and Anhidrosis (CIPA) disease is caused by various types of inactivating mutations in the TrkA gene.  Clinically, CIPA is associated with failure of sensory and autonomic neuron populations, leading to severe peripheral neuron dysfunction.  The disease is fatal and has a high mortality rate, particularly in the early postnatal stage.  CIPA is a particularly interesting disorder for induced pluripotent stem cell (iPSC) technology, as there are currently no effective treatments and no suitable mouse models available, mainly due to perinatal death of mice with mutations in the TrkA gene.  We propose to isolate CIPA-iPSC lines from patient fibroblasts, then differentiate these cell lines toward disease-relevant cell types such as neural crest and peripheral neurons.  This will enable us to identify genetic and cellular signatures of this disease.  Moreover we will attempt to rescue CIPA-specific phenotypes with genetic and pharmacological approaches.  We believe our proposed study has three significant points.  First, we will determine abnormal cellular aspects during CIPA-hiPSC differentiation and identify the most impaired genes, possibly elucidating how CIPA peripheral neurons are affected.  Moreover, the disease mechanism of CIPA is closely related with pain sensation.  Therefore our study may offer novel insights into the mechanisms controlling pain, a huge medical problem for human welfare and quality of life.  Second, we will optimize a novel methodology for specification of sensory neurons and autonomic neurons from human pluripotent stem cells.  This will be a valuable tool for stem cell biology, basic neuroscience and regenerative medicine.  Finally, we will validate candidate chemicals/molecules that activate the TrkA pathway in CIPA patient-specific neurons, which may be clinically useful for future therapies.

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 Modulating Hematopoietic Stem Cell Quiescence to Improve Leukemia Treatment

Stephen Nimer, MD
Sloan-Kettering Institute
IDEA

For acute leukemia to develop, a normal but immature bone marrow cell must acquire or retain the ability to renew itself indefinitely (a process we call self-renewal) and it must be blocked from maturing to the point where it loses the ability to live indefinitely (which we call a block in differentiation).  It is these two features that define an acute leukemia cell, and they generally occur when the bone marrow cells accumulate mutations in their DNA or acquire other inheritable alterations.  Leukemia-initiating cells (LICs) are generally malignant versions of normal hematopoietic stem cells (HSCs) and because many of the LICs are in a quiescent state, they are resistant to chemotherapy or targeted therapies.  The goal of this project is to develop new therapeutic approaches that can target LICs; we believe our findings will have a profound impact on our ability to eradicate leukemia.  Recently, we demonstrated that p53 plays a critical role in regulating HSC quiescence and identified necdin as an important p53 target gene in HSCs.  We will now attempt to stimulate quiescent LICs to enter the cell cycle, in order to sensitize them to chemotherapy or radiotherapy and improve leukemia treatment.  Our specific aims are to: 1). Define the role of necdin in the initiation of acute myelogenous leukemia (AML) and 2). Investigate the impact of lowering necdin levels acutely on the response of LICs to chemotherapy or radiotherapy, using two mouse models of AML that are established in our lab.  The proposed studies will provide valuable insight into normal and leukemic hematopoietic stem cell biology and the pathogenesis of human cancer. 

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 Use of Modified Transcriptional Regulators to Probe Mechanism and to Enhance Efficiencies of iPS Cell Formation

Mark Ptashne, PhD
Sloan-Kettering Institute
IDEA

The introduction of just four transcription factors can induce a fully differentiated human cell to reprogram to form a pluripotent stem cell.  Such cells, called induced pluripotent stem cells (iPSCs), do not require destruction of embryos and hold great promise for regenerative medicine.  Current strategies for iPSC formation are slow and inefficient.  We propose a simple alternative to enhance the speed and efficiency of this process based on our knowledge of how transcription factors work.  Our proposal is a tripartite collaboration between two labs with excellence in the field of iPS cell formation and our own, with excellence in mechanisms of gene regulation.  The reprogramming necessary for the generation of iPSCs is slow and inefficient.  We will generate chimeric proteins, fusing the reprogramming factors to known transcriptional activators and repressors, then use these chimeric proteins to measure the frequency and rate of iPSC formation.  In parallel we will measure specific effects of these chimeric proteins on gene activity in the population as a whole.  It is possible that our approach will directly produce high efficiency reprogramming.  If not, we anticipate that the information gleaned from use of these reagents will provide insights into the mechanisms of iPSC formation, and that information, in turn, will help us design protocols for high-efficiency reprogramming.  The experiments will exploit our extensive experience generating and characterizing human iPSCs, including newly developed methods that monitor expression of the introduced transcription factors during the reprogramming process. 

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 Multipotential Cancer Stem Cells in Gliomas

Viviane Tabar, MD
Sloan-Kettering Institute
IDEA

Brain tumors are among the most aggressive cancers in adults, leading to life expectancy of about one year following diagnosis.  There has been relatively little progress in our ability to improve prognosis over the past decades.  Recent work suggests that stem-like cells within these tumors are responsible for their aggressive behavior and ability to recur following treatment.  Our lab has analyzed cancer stem cells isolated from malignant brain tumors obtained from patients during surgery.  We found that, similar to normal brain stem cells, cancer stem cells can acquire a variety of fates including turning into endothelial cells, thus making up a proportion of the blood vessels that feed the tumor.  These findings are very interesting in view of the importance of blood vessel formation (angiogenesis) as a therapeutic target.  We propose to expand our findings to a larger number of malignant brain tumors, and to include lower grade tumors in an effort to understand what controls the transition from a benign tumor to a rapidly lethal glioblastoma.  In addition, we will analyze mature tumor blood vessels to confirm their tumor origin.  Our second goal is to demonstrate, using benchmark tests, that cancer stem cells are in fact multipotential, just as normal stem cells are.  Finally we will test drugs currently in clinical use in order to determine their impact on tumor blood vessel formation from cancer stem cells.  This proposal addresses a radically novel idea that has not been explored previously and that contends that cancer stem cells within a brain tumor contribute directly to the formation of their own blood vessels.  If confirmed, our hypothesis will dramatically impact our understanding of the role of cancer stem cells in tumors and, subsequently, our ability to treat them effectively.

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 Regulation of Stem Cell Pluripotency by REST/NRSF

Howard Sirotkin, PhD
SUNY - Stony Brook University
IDEA

Generation of the diverse cell types that comprise the vertebrate embryo depends on events that occur at early stages of development.  While much is known about the molecular mechanisms of this process, many questions remain.  This proposal addresses some of the mechanisms that govern the emergence of neural and non-neural cell types from stem cell pools.  We focus on the role of the REST/NRSF transcription factor, which plays a role in the regulation of transcription of a wide range of neural-specific genes.  REST/NRSF is involved in both short and long term repression of transcription.  Despite the wide range of studies on REST/NRSF in cultured cells, few studies have addressed the role of REST/NRSF in intact animals.  We seek to clarify the in vivo activity or REST/NRSF in stem cell populations.  The central question we will address is: What is the role of REST/NRSF in regulating growth and development of stem cells?  We will utilize a REST/NRSF zebrafish mutant that we generated to address this question in intact embryos.  Because the environment that stem cells are grown in is important, we will utilize intact zebrafish embryos, a powerful in vivo model system.  Fundamental aspects of stem cell biology are conserved between fish and humans.  However, as a model, zebrafish are cheaper and easier to use than mammalian systems.  Zebrafish embryos are transparent and embryonic development occurs rapidly.  These attributes foster detailed observation of normal and aberrant embryonic development.  In addition, zebrafish produce large numbers of offspring, which facilitates phenotypic characterization and enhances genetic analysis.  Most stem cell studies are performed on cultured cells.  While these studies are important, handling and growing cells in glass dishes can alter their properties.  To circumvent those difficulties we will perform our analysis on stem cells in their natural environments.  The major impact of these studies will be to examine the function of the REST/NRSF transcription factor on the ability of stem cells to maintain their properties in intact animals. 

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 Extracellular Matrix Regulation of the Neural Stem Cell Niche

Holly Colognato, PhD
SUNY - Stony Brook University
IDEA

Neural stem cells (NSCs) are specialized cells that produce all neural cells in the brain.  NSCs can be mobilized to a limited degree in adult brains to produce new cells in response to injury or disease.  If NSCs could be activated sufficiently, more newborn cells could be harnessed for repair.  Unfortunately understanding regulation and activation of NSCs, particularly to produce glial cells, is limited.  We recently found that a secreted protein, laminin, regulates glial cell production and resides in the NSC niche, the specialized region where NSCs reside and generate newborn neural cells, including glia.  Children with laminin gene mutations have severe brain malformations and functional deficits including seizures and mental retardation, suggesting that laminins likely regulate the human NSC niche.  Our goal is to find out whether laminin, or dystroglycan, a receptor that transmits laminin signals into cells, can alter the organization and function of the NSC niche, in particular to produce newborn glia from NSCs.  We hope to determine how laminin-dystroglycan interactions alter cell function in the niche by modifying gene expression in the brain, either globally or in individual cells, and monitoring cell responses such as cell division, cell survival, and cell lineage progression.  How NSC niche organization drives gliogenesis is a virtually unexplored area.  Yet, glial cell dysfunction is emerging as important player in developmental disorders such as autism and schizophrenia, as well as in neurodegenerative conditions such as Multiple Sclerosis and Alzheimer's disease.  The proposed experiments will provide important molecular insight into the poorly understood structure-function relationship of the niche during gliogenesis.  Because laminins and their receptors are cell surface molecules, their accessibility may render them particularly suitable to therapeutic modification.

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 Reversible Differentiation of Cancer Stem Cells

Gail Seigel, PhD
SUNY - University at Buffalo
IDEA

Retinoblastoma (RB) is a blinding eye tumor of childhood.  Our group was the first to identify subpopulations of RB cells that display stem cell characteristics, such as slow cell cycling, expression of stem cell markers, and invasive tumor formation.  These slow-cycling cancer stem cells (CSCs) are believed to be primarily responsible for tumor progression and metastasis.  A better understanding of these CSCs is necessary in order to develop new therapeutic strategies that will eradicate entire tumors.  We identified a unique compound called succinylated concanavalin A (SCA) that reversibly causes RB cells to stop growing and differentiate.  We propose to use RB cells and SCA treatment to study this phenomenon in CSCs.  Our overall goal is to identify the important changes involved in the maturation/growth arrest of CSCs, as well as the reversal/re-initiation of CSCs in retinoblastoma.  This, in turn, will provide a better understanding of how chemo-resistant CSCs might be rendered less harmful by differentiation treatment, as well as the steps that these mature cells might take as SCA is removed and the CSC cells return.  This information may lead us to new targets for cancer stem cell therapies.  There is currently no published reversible system in which to study CSC behavior.  We present a unique, non-toxic compound (SCA) and a novel strategy for identifying important elements involved in the behavior of CSCs.  Since CSCs are resistant to conventional therapies, new strategies are needed for their eradication.  Can we render chemo-resistant CSCs harmless by causing them to mature?  We plan to identify the key elements of CSC behavior that we can use to rehabilitate CSCs in human cells by indentifying new potential therapeutic targets for intervention.

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 Induction of Oligodendrocyte Fate from Human Neural Stem Cells

Fraser Sim, PhD
SUNY - University at Buffalo
IDEA

Currently, there are no effective therapies for human demyelinating diseases such as multiple sclerosis and congenital leukodystrophies.  Development of cell therapeutics has been hampered in part by a lack of appropriate human cells.  Human neural stem cells derived from embryonic and induced pluripotent stem cells present an attractive and abundant cell source.  However, the induction of oligodendrocytic or glial progenitor cells from human pluripotent stem cells has proven problematic as our current knowledge is based primarily on rodent models of development.  Our primary goal is to identify novel drugs that induce oligodendrocyte development from human stem cells.  We believe this approach will advance the progress toward using cell therapeutics to treat human demyelinating disease.  We used novel cell sorting techniques to specifically isolate human glial progenitors and neural stem cells from the developing human brain, followed by whole genome microarrays to quantify the expression of all genes in both cell types.  We then compared these gene expression profiles and applied a novel pharmacogenomic approach to identify candidate drugs for the induction of glial progenitors from human neural stem cells.  We intend to test these drugs on sorted neural stem cells from both fetal brain and embryonic stem cell sources.  We will measure their effects on glial induction in tissue culture and following transplantation into a newly developed mouse model of leukodystrophy appropriate for the transplantation of human cells.  Our initial studies will utilize a high-throughput genomic approach that accurately measures the expression of several glial progenitor genes.  Importantly, we will also assess the ability of drug-induced cells to repair damaged brain following transplantation into a mouse model of myelin disease.

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 INFS - New Module for Stem Cell Transcription Programming

Michal Stachowiak, PhD
SUNY - University at Buffalo
IDEA

Therapeutic application of stem cells requires a fundamental understanding of the processes that control stem cell biology.  The defining features of stem cells include the ability to continuously proliferate, maintain pluripotency or multipotency (potential to develop into diverse types of cell) and to differentiate into mature specialized cells.  Each process is known to engage a plethora of cell factors organized into complex networks.  The regulatory network motifs that execute the cell cycle and pluripotency gene programs have been identified, at least in part.  Known proteins were found to perform new functions of molecular switches that trigger cell proliferation and maintenance of pluripotency and multipotency.  Targeting of these molecular switches offers new tools for the generation and maintenance of the natural and induced pluripotent cells.  Our studies have identified an analogous universal regulatory motif, Integrative Nuclear Fibroblast Growth Factor (FGF) Receptor-1 Signaling (INFS), that executes the stem cell transition from proliferating multipotent or pluripotent states into the differentiated neuronal phenotype.  In the proposed studies we will verify the role of INFS as a molecular switch that activates the “neural gene program” in human brain derived neural stem/progenitor cells and will determine the mechanisms of this activation.  Our research has the potential to fundamentally change the way we understand stem cell development.  It offers new tools for effective control of neuronogenesis and for neuronal replacement in disease.  Our results will set a standard for the future verification of strategies used for neuronal development by natural and induced pluripotent cells.

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 Development of a Polyamide-Based Targeted Therapy for Leukemia Stem Cells

Archibald Perkins, MD, PhD
University of Rochester
IDEA

Stem cell research encompasses not only their application for regenerative medicine, but also the study of cancer stem cells – those cells within a tumor that have the ability to regenerate it.  Effective eradication of cancer will require that we eliminate this population.  It is thought that our inability to completely eliminate certain cancers is due to the resistance of cancer stem cells to conventional chemotherapy.  We wish to develop drugs that target leukemia stem cells.  Our studies indicate a protein called EVI1 is essential for leukemia stem cell survival.  We hypothesize that EVI1 elimination from the tumor will result in tumor regression.  We further hypothesize that a chemical we have designed to inhibit EVI1 will be effective in tumor regression.  To address if EVI1 is essential for stem cell survival, we use a mouse in which we can delete the EVI1 gene within leukemic cells.  We will induce acute myeloid leukemia induced in the mouse, and, once established, we will delete EVI1.  We will then follow whether the function of the leukemia stem cells are affected.  To address whether the polyamide is effective in blocking EVI1 function, we will employ a series of straightforward biochemical and cell-based assays.  If successful, these experiments will provide effective new therapies for the elimination of cancer stem cells, as well as novel approaches for use by other investigators.  At present, no therapeutic agents are clinically available that specifically target cancer stem cells.  This study will hopefully provide the first compounds to specifically target leukemia stem cells and will provide better treatment for lethal cancers such as AML.

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 Pre-Clinical Evaluation of Nrf2 Overexprexpressing Mesenchymal Stem Cells in Pulmonary Emphysema

Tirumalai Rangasamy, PhD
University of Rochester
IDEA

Pulmonary emphysema is one of the major pathological abnormalities associated with Chronic Obstructive Pulmonary Disease (COPD) and is expected to become the third largest cause of death worldwide in 2020.  The damage to the lung in pulmonary emphysema is irreversible and there is no cure for COPD.  Hence, there is a strong need for new approaches that are capable of halting disease progression, and interventions that repair/regenerate alveolar structures in the lung.  Mesenchymal stem cells (MSCs) are prime candidates for the repair and regeneration of damaged tissues and organs and have been shown to engraft in the injured lung and even differentiate into type I and type II alveolar epithelial cells and bronchial epithelial cells.  These studies raise the possibility that bone marrow MSC transplantation may be developed as an effective intervention in pulmonary emphysema.  Recently, we discovered a novel transcription factor, nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) that plays a critical role in determining the susceptibility to cigarette smoke (CS)-induced emphysema.  Nrf2 plays an important role in the proliferation and survival of structural cells including alveolar epithelial cells, endothelial cells, smooth muscle cells, and fibroblasts.  However, very little is known about the role of Nrf2 in stem cell functions.  Our preliminary data indicate that cigarette smoke extract (CSE) significantly impaired the multiple physiologic functions of MSCs and activation of Nrf2 by sulforaphane or overexpression of Nrf2 using lentiviral particles, markedly rescued MSCs from CSE-induced oxidative stress and apoptosis.  We will investigate the therapeutic potential of Nrf2-overexpressing MSCs in CSE-induced pulmonary emphysema in a mouse model.  We will transduce MSCs with Nrf2 overespression vectors to create a new generation of oxidant-resistant MSCs that will be particularly effective in oxidant-rich environment in COPD.  Our results will demonstrate whether MSCs can be engineered and introduced into the body to counteract the harmful effects of cigarette smoke.

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 Enteric Bacterial Regulation of the Intestinal Stem Cells

Jun Sun, PhD
University of Rochester
IDEA

Bacteria are known to manipulate the pathways critical for the immune response and host defenses.  Recent studies reveal that bacteria target stem cells in the gut to enhance their long-term survival and function in fruit flies.  However, in mammalian animal models, little is known regarding the effects of bacterial infection on intestinal stem cells.  Moreover, the bacteria proteins that participate in the modulation of stem cells in the pathogenesis of digestive diseases are unknown.  More than 500 species of bacteria coexist in the human colon and the number of microbial cells within the gut lumen is about 100 trillion, 10 times larger than the number of human cells in the body.  Due to the complexity of gut flora, identification of the specific microbial agents that contribute to stem cell renewal and cancer remains challenging.  Our proposal aims at understanding the role of bacterial regulation of intestinal stem cell initiation, growth, and death, and exploring the molecular mechanism of this regulation in models of intestinal inflammation and colon cancer caused by bacterial infection.  We will determine the effects of bacteria such as Salmonella and E. coli on stem cells.  We will also identify bacterial proteins that contribute to the stem cell niche in a Salmonella-infected colon cancer model.  We will investigate the novel role and mechanism of bacteria in regulation of stem cells during intestinal inflammation and cancer development.  Our research will not only provide a better understanding of intestinal bacterial infection and stem cells, it will also bring us closer to understanding the processes that hamper cancer stem cells and colon cancer development.  Eventually, we will apply our findings to the therapy of infectious diseases and risk assessment and prevention of colon cancer.

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 Multiphoton Imaging for Nanofiber Based MSC-Mediated Bone Tissue Repair

Xinping Zhang, PhD
University of Rochester
IDEA

Mesenchymal stem cells (MSCs) have enormous potential for repair of bone defects.  Current efforts are directed at design and fabrication of suitable bioscaffolds to enhance MSC-based bone repair and regeneration.  Among available technologies, electrospun nanofiber-based scaffolds hold the greatest promise due to their versatility in creating an integrated platform for stem cell manipulations.  However, current studies to elucidate the functional significance of nanofiber-based tissue repair remain limited to in vitro studies that do not allow the evaluation of functional behavior of MSCs in a complex in vivo bone-healing environment.  To gain better understanding of the interaction of MSCs with nanofibers in vivo, we established a cranial bone defect model that allows real-time and high-resolution imaging of MSCs in a true defect-healing scenario.  With this proposal, we will use state-of-the-art multiphoton imaging technology to characterize nanofiber-based MSC-mediated bone repair and regeneration in vivo.  Our proposal will center on three important design elements to address the instructive role of nanofibers on MSC-mediated matrix organization, cell migration and vascular ingrowth.  Our current proposal represents the first attempt to establish an in vivo high-resolution imaging modality to address the interaction of MSCs with fibrous matrices in a critical bone defect repair model.  We further propose a multidisciplinary collaboration among stem cell biologists, biomedical engineers and material scientists to accomplish the goal.  The success of this project will open up research opportunities to study detailed molecular and cellular mechanisms underlying MSC-based bone repair and regeneration, further offering an experimental testing modality for therapeutic strategies.  The success of the proposed study will potentially offer a biomimetic flexible construct to treat patients who are at risk of impaired healing.

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 Targeting MDS-EVI1 Complex in MLL Leukemia Stem Cell Function

Yi Zhang
University of Rochester
IDEA

Stem cell research encompasses not only the application of stem cells for regenerative medicine, but also the study of cancer stem cells – those cells within malignant tumors that have the unique ability to completely regenerate the entire tumor mass from very few cells. Our inability to completely eliminate certain cancers is likely due to the resistance of cancer stem cells to conventional chemotherapy.  Our prior studies indicate that a protein called MDS-EVI1, when knocked out in mice, reduces the pool of functional blood, or hematopoietic, stem cells (HSCs).  The HSCs in these knockout mice are rendered non-functional after stress by chemotherapy drugs.  We hypothesize that MDS-EVI1 is also critical for leukemia stem cell resistance to chemotherapy.  By blocking this gene, we will similarly reduce the pool of leukemia stem cells, resulting in a better response to chemotherapy.  We will identify the molecules with which MDS-EVI1 interacts in leukemic stem cells, thereby providing new insights to improve chemotherapy responses in leukemia patients.  To identify molecules that interact with MDS-EVI1, we will employ two state-of-the-art technologies.  First, using a molecular “hook,” we will pull MDS-EVI1 out of leukemic cells, along with the molecules that interact with it, which we will then identify by mass spectroscopy.  This hook has already been incorporated into a mouse, providing us with a perfect biological model for our studies.  Second, we will employ gene suppression, isotope labeling, immunoprecipitation, and mass spectroscopy to identify molecules that interact with MDS-EVI1.  The use of two independent technologies to achieve the same goal will provide us with a rigorous proof that our conclusions are correct.  Targeting leukemia stem cell pool stress response is a novel and creative approach.  If successful, our experiments will provide critical groundwork for the development of effective new therapies for the elimination of cancer stem cells, and improve leukemia stem cell chemotherapy response. Ultimately, this study may provide the first compounds to specifically target leukemia stem cells. 

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