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Investigator Initiated Research Projects and Innovative, Developmental or Exploratory Activities (IDEA) in Stem Cell Research - 2008

RFA #: 0804180400

IIRP Awards
Institution PI Amount Title
Albany Medical College Russell Ferland 1,080,000 The Role of Filamins and Formins in Stem Cell Development
Albert Einstein College of Medicine Jeffrey Pessin 1,080,000 Role of Adipocyte Progenitor Cells in Adipose Tissue Turnover and Obesity
Albert Einstein College of Medicine Charles Rogler 1,069,157 Roles of MicroRNAs in Hepatic Stem Cell Differentiation
Albert Einstein College of Medicine Jayanta Roy-Chowdhury 1,080,000 Amelioration of Hepatic Metabolic Defects by Stem Cell-Derived Human Hepatocytes
Albert Einstein College of Medicine Carl Schildkraut 1,080,000 Differential Regulation of DNA Replication During Neural Lineage Specification in Human ES Cells and Human iPS Cells
Albert Einstein College of Medicine Ulrich Steidl 961,499 Transcriptional Control of Cancer Stem Cells in Acute Myeloid Leukemia
Cold Spring Harbor Laboratory Grigori Enikolopov 1,079,996 Regulation of the Life Cycle of Adult Neural Stem Cells
Columbia University Christopher Rae Jacobs 1,049,875 Mechanosensitive Primary Cilia in Osteogenic Differentiation of Stem Cells Due to Loading
Columbia University Helen Lu 719,890 Stem Cell-Mediated Integrative Rotator Cuff Repair
Columbia University Medical Center Angela M. Christiano 1,080,000 Stem Cell Therapy for Epidermolysis Bullosa
Columbia University Medical Center Thomas Jessell 1,080,000 The Molecular Logic of Embryonic Stem Cell-Derived Motor Neurons
Columbia University Medical Center Jeremy Mao 1,079,985 Functional Synovial Joint Replacement by Stem Cell Homing
Columbia University Medical Center Tarique Perera 1,053,458 The Role of Endogenous Hippocampal Stem Cells in Treating Non-Human Primate Models of Depression and Anxiety
Columbia University Medical Center Michael Rosen 1,023,800 Stem Cell-Based Platform Therapy for Lethal Cardiac Arrhythmias
Columbia University Medical Center Stephen Goff 1,002,632 Characterization of the Retroviral Silencing Machinery in Embryonic Stem Cells
Cornell University Tudorita Tumbar 1,033,979 Control of Hair Follicle Stem Cell Proliferation and Epithelial Skin Tumorigenesis by Concerted RUNX1 and CDKN1a Action
Memorial Sloan-Kettering Cancer Center Elizabeth Lacy 1,007,280 Mechanisms by Which Nuclear Pore Composition Regulates Stem/Progenitor Differentiation
Memorial Sloan-Kettering Cancer Center Urs Rutishauser 1,070,964 Use of Polysialic Acid to Improve Integration of ES-Derived Cells into the Brain
Mount Sinai School of Medicine Stuart Aaronson 1,080,000 Role of Wnt Signaling in Sarcomas Initiated From Human Mesenchymal Stem/Progenitor Cells
Mount Sinai School of Medicine Julio Aguirre-Ghiso 1,080,000 Plasticity of Head and Neck Cancer Initiating Cells
Mount Sinai School of Medicine Margaret Baron 1,080,000 Red Blood Cell Development in Differentiating Embryonic Stem and Induced Pluripotent Stem Cells
Mount Sinai School of Medicine Hans-Willem Snoeck 1,048,941 Generation of Thymic Epithelial Cells From Embryonic Stem Cells
Mount Sinai School of Medicine Ming-Ming Zhou 1,080,000 Molecular Deciphering of Stem Cell Epigenetic Silencing
New York State Psychiatric Institute René Hen 1,080,000 Contribution of Hippocampal Stem Cells to the Action of Antidepressants: From Mice to Men
New York University College of Dentistry Louis Terracio 1,002,134 Skeletal Muscle-Derived Stem Cells
New York University School of Medicine Lisa Dailey 933,689 Functional Identification of Transcriptional Determinants of the Embryonic Stem Cell State and Early Lineage Commitment
New York University School of Medicine Gordon Fishell 900,000 The Directed Differentiation of Embryonic Stem Cells Into Specific Cortical Interneuron Subtypes
New York University School of Medicine Glenn Fishman 1,080,000 Embryonic Stem Cell-Derived Cardiac Conduction System Cells
Regenerative Research Foundation Sally Temple 1,049,036 Changes in RNA Synthesis and Timing of Cortical Development
Rensselaer Polytechnic Institute Robert Linhardt 1,080,000 Stem Cell Glycomics in Microarray Format
SUNY - Stony Brook University Wadie Bahou 1,080,000 Therapy of Hemophilia A Using Megakaryocyte-Targeted Stem Cell Delivery
SUNY - Stony Brook University Soosan Ghazizadeh 591,420 Immune Responses to Allogeneic Stem Cell Transplantation
SUNY - Stony Brook University Hsien-yu Wang 813,496 Novel Chimeric Frizzleds as Tools to Program hESC Differentiation
SUNY - University at Buffalo Stelios Andreadis 1,055,958 High-Throughput, Real Time Dynamic Monitoring of Stem Cell Differentiation
SUNY - University at Buffalo Stelios Andreadis 1,010,490 Hair Follicle Stem Cells for Cardiovascular Tissue Regeneration
SUNY - University at Buffalo Gen Suzuki 1,022,300 Modification of Resident Cardiac Stem Cells by Circulating Hematopoietic Stem Cells in Ischemic Cardiomyopathy
SUNY - University at Buffalo Emmanouhl Tzanakakis 589,686 Scalable Expansion and Directed Differentiation of Human Embryonic Stem Cells to Pancreatic Progeny
SUNY - Upstate Medical University Gerold Feuer 1,069,779 HTLV Infection of Human Hematopoietic Stem Cells: Induction of Novel Lymphoma in Humanized SCID Mice
University of Rochester Dirk Bohmann 1,050,872 Nrf2 as a Regulator of Stem and Progenitor Cell Function
University of Rochester Laura Calvi 358,500 Therapeutic Stimulation of the Hematopoietic Stem Cell Niche
University of Rochester Di Chen 1,001,308 Canonical Wnt Signaling Controls Mesenchymal Stem Cell Differentiation
University of Rochester Craig Jordan 1,079,790 Therapeutic Targeting of Leukemia Stem Cells
University of Rochester James Palis 1,049,110 Erythroid Precursor Self-Renewal
University of Rochester Xinping Zhang 756,732 Gli2-Activated MSCs for Bone Regeneration and Reconstruction
Wadsworth Center Randall Morse 1,040,161 Epigenetic Control of Murine Neural Stem Cell Self-Renewal and Differentiation Mediated by Bmi-1
Weill Cornell Medical College Margaret Elizabeth Ross 1,080,000 Neuronal Specification, Expansion and Self-Renewal of hESC-Derived Precursors
IDEA Awards
Institution PI Amount Title
Albany Medical College Jihe Zhao 240,000 Transcriptional Control of Stem Cell Metastasis of Breast Cancer
Albert Einstein College of Medicine Mukesh Kumar 240,000 Manipulation of Stem Cells for Treating Viral Hepatitis
Albert Einstein College of Medicine Ulrich Steidl 240,000 Identifying Epigenomic Determinants of Hematopoietic Stem Cell Commitment
Barnard College Matthew Wallenfang 227,452 Transplantation of Adult Male Germline Stem Cells of Drosophila melanogaster
Cold Spring Harbor Laboratory Marja Timmermans 239,134 The Role of the Chromatin-Remodeling Factor HIRA in Recruitment of Polycomb Complexes and Regulation of Pluripotency
Columbia University Tulle Hazelrigg 240,000 Maintenance of Adult Germ Stem Cells by Histone Modification
Columbia University James Manley 240,000 Alternative Splicing of mRNA Precursors: Links to Pluripotency of Human Embryonic Stem Cells
Columbia University Medical Center Fiona Doetsch 240,000 Molecular Profiling and Differentiation Potential of Purified Adult Neural Stem Cell Lineages
Columbia University Medical Center Edward Laufer 240,000 Differentiation of ES Cells Into Adrenocortical Lineages
Columbia University Medical Center David Owens 240,000 Epithelial Progenitor Cells as Targets For Cutaneous Neoplasia
Columbia University Medical Center Srikala Raghavan 240,000 Exploring the Function of MicroRNAs in Epidermal Stem Cells
Columbia University Medical Center Andreas Kottman 239,787 Does Graded Expression of Shh by Dopaminergic Neurons of the Mesencephalon Influence the Maintenance and Differentiation of Neural Stem Cells of the Adult Subventricular Zone?
Columbia University Medical Center Igor Matushansky 239,628 Using Mesenchymal Stem Cells to Deliver Tumor Environment-Activated Quantum-Dot Drug Conjugates Targeting Cancer Bulk and Cancer Stem Cells.
CUNY - Hunter College Benjamin Ortiz 224,103 TCR α Locus Control Region Activity During in vitro Stem Cell Differentiation: Application Towards Improving Lentiviral Gene Therapy Vectors
Memorial Sloan-Kettering Cancer Center Anna-Katerina Hadjantonakis 240,000 Characterization of Extraembryonic Endoderm (XEN) Cells
Mount Sinai School of Medicine Emily Bernstein 240,000 Investigation of Polycomb-Mediated Chromatin Alterations During Embryonic Stem Cell Differentiation
Mount Sinai School of Medicine Stuart Fraser 240,000 Copper Transporter-1, Ctr1: A Critical Regulator of Embryonic Stem Cell Differentiation
New York University School of Medicine Erika Bach 240,000 Elucidating the Role of the JAK/STAT Pathway in Stem Cell Self-Renewal
New York University School of Medicine Eva Hernando 240,000 Study of the Cell-of-Origin and Cancer Stem Cells in Melanoma
New York University School of Medicine David Levy 240,000 Derivation and Characterization of Dendritic Cell Lineages From Hematopoietic Stem Cells
New York University School of Medicine Jeremy Nance 240,000 Establishing the C. elegans Germline Stem Cell Niche
Regenerative Research Foundation Qin Shen 240,000 Novel Surface Markers for Neural Stem Cell Enrichment
Regenerative Research Foundation Jun Yan 240,000 Identification and Characterization of Endothelial Niche Factor(s) That Stimulate Self-Renewal and Neurogenesis of Neural Stem Cells (NSCs)
Roswell Park Cancer Institute Wendy Huss 240,000 High Transporter and Aldehyde Dehydrogenase Activity in Benign and Cancer Prostatic Stem Cells
Roswell Park Cancer Institute Bonnie Hylander 240,000 Role of Cancer Stem Cells in Resistance to Targeted Therapy and Tumor Recurrence
SUNY - University at Buffalo Sriram Neelamegham 240,000 Glycan Engineering of Stem Cells
University of Rochester Hani Awad 240,000 Modulating Stem Cell Differentiation Using Novel Allograft Scaffolds for Cartilage Repair
University of Rochester Gregory Tall 232,678 Deciphering the Role of Mammalian Ric-8 Proteins in Stem Cell (Asymmetric) Division
University of Rochester Lei Xu 240,000 Identifying the Stem Cells in Malignant Melanoma
Wadsworth Center Maria López 240,000 Hematopoiesis of the Gut Immune System and Gut Immunity in Immunodeficient Mice
Weill Cornell Medical College Stewart Anderson 240,000 Deriving Forebrain Interneurons From ES Cells by Inducible Expression of Lhx6
Weill Cornell Medical College Anthony Brown 240,000 Regulation of Mammary Stem Cells by Wnt Signaling

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Abstracts

 The Role of Filamins and Formins in Stem Cell Development

Russell Ferland, PhD

Albany Medical College
IIRP

 

The use of stem cells in the treatment of human disease will depend upon our ability to direct these cells into the proper locations and the proper physiological states. Understanding the mechanisms that are involved in these processes is critical to achieve this goal. We are identifying and studying human disease-causing genes that affect the properties of stem cells. We are studying two classes of genes, filamins and formins, that are important in controlling the state of both brain and bone stem cells, since mutations in these genes lead to abnormalities in brain and bone stem cell growth. This project has three aims: (1) we will test if one filamin gene (FLNA) is involved in regulating the growth and behavior of brain stem cells by studying mice that do not have the FLNA gene; (2) we will test if the other major filamin gene (FLNB) is involved in regulating the growth and behavior of bone stem cells by examining mice that do not have the FLNB gene; and (3) we will test how these genes cause their effects at the biochemical level by determining whom these genes "talk" to and interact with during the life of a stem cell. With our approach, we will begin to understand the role of these genes in stem cell physiology, and also to understand diseases affecting these tissues. Since many of the goals of stem cell biology are related to the potential use of stem cells as therapeutic agents, our work will help to determine the role of filamins and formins in controlling the functions of these stem cells so that the cells will function properly in patients.

 

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 Role of Adipocyte Progenitor Cells in Adipose Tissue Turnover and Obesity

Jeffrey Pessin, PhD

Albert Einstein College of Medicine
IIRP

 

Typical western diets are enriched in saturated fatty acids that either undergo oxidation for the generation of energy or, when consumed in caloric excess, are stored within adipocytes primarily as triglycerides. Recent data clearly demonstrate that increased lipid flux into adipocytes results in a chronic inflammatory state that has several negative consequences including the production of cytokines that produce insulin resistance leading to diabetes. In addition, associated with the inflammatory state, there is a recruitment of new adipocyte precursor cells that differentiate and contribute to an increase in adipocyte cell mass. It is not presently known where these precursor cells (adult adipocyte stem cells) reside, what their biochemical signature is, how they are recruited, and whether preventing their recruitment and/or differentiation will protect against obesity and co-morbidities of the inflammatory response. We will determine if, in a genetic mouse model, adipocyte cell death (that results from caloric excess) is the trigger responsible for the inflammatory response, or whether the inflammatory response occurs first and this triggers adipocyte cell death. Then we will use this information to identify the adult adipocyte precursor cells that are newly recruited into adipose tissue and subsequently differentiate into adipocytes, thereby resulting in a net increase in the number of adipocytes in obese individuals. Our novel preliminary data show that bone marrow provides a source of adult adipocyte progenitor cells. Understanding the source and mechanisms of adult adipocyte precursor recruitment and contribution to the increase in adipocyte cell number will provide new cellular and molecular insights into the pathophysiology and development of obesity. This knowledge may provide novel therapeutic targets to decrease obesity and/or the associated inflammatory processes responsible for many of the obesity-related complications including insulin resistance, diabetes and cancer.

 

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 Roles of MicroRNAs in Hepatic Stem Cell Differentiation

Charles Rogler, PhD

Albert Einstein College of Medicine
IIRP

 

Stem cells obtained from specific organs are often committed to specific lineages. This is the case for stem cells isolated from the fetal liver of mice. We isolated liver stem cells, called HBC-3 cells, that can be induced to become either hepatocytes (the main cell in the liver) or cholangiocytes (cells that produce bile ducts). The overall aim of our studies is to understand the mechanisms that control differentiation of these cells at the molecular level. Recently we began studying the role of a newly characterized set of genetic elements called microRNAs (miRNAs) and their control of differentiation in HBC-3 cells. These studies led us to identify a set of three miRNAs that may target a very important set of proteins that regulate differentiation of many cell types. These proteins, Smads, mediate the action of Tumor Growth Factor β (TGF-β). A deeper understanding of the mechanisms that the miRNAs use to affect differentiation of the liver stem cells, and the role of the TGF-β pathway in this process, will help in developing miRNAs for therapeutic uses. We will determine if these miRNAs can affect differentiation of liver stem cells into hepatocytes or cholangiocytes. We will then carry out molecular studies to identify specific miRNAs involved in cell fate regulation. Finally, using bioinformatics and mutagenesis approaches, we will identify the specific mechanisms by which the miRNAs work in the cell. Our studies, targeted towards identifying important miRNAs in liver stem cell differentiation and understanding the signaling pathways regulated by these miRNAs, will expand our conceptual knowledge of stem cell differentiation at the molecular level and may lead to practical applications in the area of liver stem cell transplantation. This project will have a wide impact in cell biology because it is the first study to link miRNAs to the control of TGF-β, a major signaling pathway with functions in a wide variety of processes. Additionally, the ability to easily transfer modified, stable miRNAs into cells will enable therapeutic applications of basic science discoveries to occur in a very rapid manner.

 

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Amelioration of Hepatic Metabolic Defects by Stem Cell-Derived Human Hepatocytes

Jayanta Roy-Chowdhury, MD

Albert Einstein College of Medicine
IIRP

 

Cell therapies using hepatocytes, the most abundant cell type in the liver, are being developed for the treatment of acute liver failure and many inherited disorders that affect the synthetic and detoxification functions of the liver. These approaches, including hepatocyte transplantation and construction of bio-artificial liver assist devices, are minimally invasive alternatives to liver transplantation for such diseases. However, since there is a worldwide shortage of donor organs and only high-quality donor livers can be used for organ transplantation, human hepatocytes for transplantation and research are prepared from the poor-quality, rejected livers. The ability to generate functional hepatocytes from human embryonic stem cells (hESCs) could overcome this limitation to the realization of the full potential of hepatocyte-based therapies of liver diseases. We will determine if human hepatocytes, derived by differentiation of hESCs, can function in the liver of experimental animals, proliferate under appropriate stimulation, correct metabolic diseases in animal models and whether our screening of hepatocytes before transplantation is adequate to prevent tumor formation in the body. Transplantation of hepatocytes into animal models of metabolic disease will provide the first direct evaluation of the ability of the differentiated hepatocytes to correct inherited liver-based metabolic disorders. We are applying our novel liver repopulation method for the first time in the context of human hepatocytes. Successful transplantation of human hepatocytes in an animal model in conjunction with a clinically-approved immunosuppressive drug is innovative. These experiments will be an essential step in refining our methods to obtain an unlimited supply of functional human hepatocytes for the development of cell-based therapies for the treatment of liver diseases.

 

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 Differential Regulation of DNA Replication During Neural Lineage Specification in Human ES Cells and Human iPS Cells

Carl Schildkraut, PhD

Albert Einstein College of Medicine
IIRP

 

Each human cell type, in theory, duplicates its DNA through the same steps, or "program." In fact, however, these programs are significantly different; when tissue-specific gene loci are compared in different cell types there are often differences in replication timing, replication initiation sites and direction of replication fork progression. As cells differentiate, it is necessary to reset the program controlling the timing of replication. The DNA replication program includes a number of key parameters, such as when and where DNA replication initiates and in what direction the replication fork proceeds. SMARD (Single-Molecule Analysis of Replicated DNA) is a technique developed in our lab that reveals the dynamic changes in the replication program. We will use SMARD and other analyses of human embryonic stem cell (hESC) populations and neural cells derived from hESCs, to examine changes during cell differentiation. We will also employ state-of-the-art single-strand DNA mapping to analyze replication programs in a systematic manner. Furthermore, we will test whether failure to reset the replication program during embryonic development leads to epigenetic problems affecting development and differentiation. The results of this study should greatly increase our understanding of hESCs. Our studies should play a major role in making it possible to obtain cells to replace damaged human cells by providing criteria for obtaining accurately reprogrammed induced pluripotent stem cells that could be used to generate suitable differentiated cells for use in patients.

 

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 Transcriptional Control of Cancer Stem Cells in Acute Myeloid Leukemia

Ulrich Steidl, MD, PhD

Albert Einstein College of Medicine
IIRP

 

Relapse continues to be the most common cause of death in acute myeloid leukemia (AML) patients, despite the established use and optimization of regimens applying polychemotherapy and the development of new agents that are effective at reducing tumor burden. Based on recent experimental evidence, we propose a novel model of AML development in which rare cancer stem cells/leukemia stem cells (LSCs) give rise to functionally heterogeneous bulk tumor cells with limited life-span. Similar to normal blood stem cells, LSCs are not responsive to common cell-toxic agents, thereby contributing to treatment failure. As a consequence, future treatments must be directed against those LSCs in order to cure AML. Thus, defining the characteristics of LSCs is critical to understanding the genesis of leukemia and to developing strategies to eradicate these cells. In order to identify functionally critical LSC genes, fundamentally novel experimental approaches need to be established. We demonstrated a critical role for transcription factors in the genesis and function of cancer stem cells in leukemia and showed that, even in early stem cells, some of these factors are misregulated. We will now identify the misregulated transcription factors in specific stem and progenitor cells that are functionally critical for LSCs and the formation of leukemia, thereby providing a basis for targeted, LSC-directed, therapeutic approaches. We will apply the novel experimental approach of gene analysis in precisely defined stem and progenitor cells, which we originally developed in a mouse model of leukemia, to human patients with leukemia. This study is aimed at providing the basis for the development of targeted therapies specifically directed at LSCs rather than the bulk tumor population. Our identification of surface markers for LSCs in different subtypes of AML will have important implications for future applications in research as well as the clinic. The possibility of prospective isolation of LSCs will foster research on these rare cells and permit clinical monitoring of LSCs in human individuals with AML during the course of disease and treatment.

 

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 Regulation of the Life Cycle of Adult Neural Stem Cells

Grigori Enikolopov, PhD

Cold Spring Harbor Laboratory
IIRP

 

Adult brain retains the ability to produce new neurons. These neurons are produced from neural stem cells that reside in selected regions of the adult brain. One such region is the hippocampus, a brain structure important for memory and mood regulation. Neural stem cells of the hippocampus may be important for learning, memory, brain repair and the action of therapeutic drugs. The number of stem cells in the brain dramatically decreases with age and this decrease may contribute to age-related decrease in memory and mood. Understanding the basic mechanisms that regulate stem cells in the adult brain will provide means to manipulate production of new neurons in the adult and aging brain, and may direct us towards new types of treatments for cognitive disorders. We developed a new approach to analyze brain stem cells and to describe the basic rules of their division, differentiation, and disappearance. We will now use this approach to determine how the aging brain copes with the loss of stem cells and how it responds to therapies that increase production of new neurons. We will combine genetic and computational methods to determine how neural stem cells are regulated. We will use reporter transgenic mouse lines to identify, visualize, count and isolate neural stem cells and their progeny. We will also apply computational modeling to determine the specifics of how stem cells divide, produce progeny and disappear. All of our animal lines and software will be freely available to the research community. Our studies will provide a general framework for studying neural stem cell regulation. Furthermore, our studies will clarify the modes of action of potent antidepressant treatments, such as fluoxetine (Prozac) and electroconvulsive shock, thus providing insights into the mechanism of their therapeutic effect. Ultimately, our studies may help in designing new therapies to treat memory and mood disorders and to delay age-related cognitive impairment.

 

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 Mechanosensitive Primary Cilia in Osteogenic Differentiation of Stem Cells Due to Loading

Christopher Rae Jacobs, PhD

Columbia University
IIRP

 

Osteoporosis is a disease in which loss of bone tissue leads to increased risk of fracture at loads that would not normally be traumatic. It is a major health risk for 44 million Americans, 68% of whom are women. One out of every two women and one out of every four men over the age of 50 are predicted to experience an osteoporotic fracture in their lifetimes. In fact, more women die of osteoporosis than breast cancer. Osteoporosis occurs when stem cells in the bone marrow no longer supply new bone forming cells in sufficient numbers. One factor that stimulates the creation of new bone cells is physical loading of the skeleton. However, virtually nothing is known about how stem cells sense their mechanical environment. Our lab showed that the primary cilium, a cellular antenna-like structure, acts as just such a mechanical sensor in bone cells. The focus of this project is to determine if primary cilia act as mechanical sensors in stem cells. We will also determine what modifications to cellular DNA occur in stem cells in response to mechanical stimulation that turn off certain genes and turn on other genes, which allows the stem cells to differentiate into other cell types including bone cells. These research questions will be addressed through a combination of cell culture experiments and bone loading experiments in live mice. Determining how stem cells sense and respond to their mechanical environment is a highly innovative research direction. Clear evidence that primary cilia function as mechanical sensing antennae in stem cells would be extremely innovative. This project will provide critically needed knowledge of how the mechanical environment can be harnessed to guide stem cells to form desired tissue. Thus, the potential impact of our work is likely to reach beyond bone applications and advance many other stem cell therapeutics.

 

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 Stem Cell-Mediated Integrative Rotator Cuff Repair

Helen Lu, PhD

Columbia University
IIRP

 

The objective of our research is to evaluate the potential of utilizing stem cells for repairing shoulder injuries. The most common shoulder injury involves the rupture or tear of rotator cuff tendons at their interface with underlying bone, resulting in severe pain and disability to the patient. Due to the high failure rates and the lack of functional tendon-to-bone integration associated with current treatments, there exists a significant demand for integrative alternatives for the repair of these debilitating shoulder injuries. Our primary research question centers on how to maximize the regeneration potential of stem cells to restore shoulder function. We hypothesize that by designing implants, or scaffolds, which can directly stimulate desired stem cell differentiation, in particular into the cell type inherent at the transition between tendon and bone, these differentiated cells can then work to promote integration and facilitate functional tendon repair. We will first use state-of-the-art implant design and nanotechnology to make nanofiber-based scaffolds and substrates that exhibit a gradient of properties mimicking the native tissue. We will seed stem cells onto these scaffolds to optimize their response, then determine their regeneration potential at the interface of bone and connective tissue. Successful completion of our planned studies will harness the regenerative potential of stem cells to treat a debilitating clinical condition. The sophisticated design methodologies devised here have broader impact as they can also be applied to treat other soft tissue injuries in the body. The research approach proposed will lead to the development of a new generation of nanotechnology-driven orthopedic devices that can promote seamless integration between soft and hard tissues, and is anticipated to have a significant impact on rotator cuff repair as well as the field of soft tissue fixation to bone.

 

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 Stem Cell Therapy for Epidermolysis Bullosa

Angela M Christiano, PhD

Columbia University Medical Center
IIRP

 

Our research is aimed at developing therapeutic approaches for the treatment of epidermolysis bullosa (EB), a fatal genetic skin disease. In the past few years, mounting evidence pointed to the ability of circulating adult stem cells to take up residence in the skin and secrete skin proteins. We hypothesize that bone marrow cells taken from immunocompatible (human leukocyte antigen-matched) unaffected individuals can provide a source of genetically correct cells for therapy of EB. By using cells from a donor without EB, the cells would by definition contain functional genes encoding EB proteins, thereby obviating the need to introduce the gene exogenously. Moreover, the incorporation of bone marrow cells into wounds, and their persistence there, suggests that they may provide a life-long source of healthy stem cells in the dermis. We will perform bone marrow transplantation on mice engineered to have different types of EB. We will measure the benefit of stem cell therapy on extending their lifespan and healing the blisters on their skin, esophagus and internal organs. Based on our preliminary studies, we expect this approach to have significant positive benefits. The concept of using circulating bone marrow cells to cure a skin disease in a mouse or human subject has never been attempted before. Our lab was the first to do so earlier this year in a collaborative study in mice with a form of EB, and in one patient family with a severe form of the disease. In this proposal, we will extend this work to other forms of EB. This approach could lead to the treatment and cure of fatal skin diseases using transplantation of circulating adult stem cells.

 

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 The Molecular Logic of Embryonic Stem Cell-Derived Motor Neurons

Thomas Jessell, PhD

Columbia University Medical Center
IIRP

 

Stem cells hold great promise as reagents that can accelerate cures for human neuro-degenerative disorders and traumatic injury. Realizing this promise, however, will require that we obtain a more complete understanding of the molecular properties of stem cells and their developmental potential. We also need to optimize the ability to produce neuronal cell types suitable for high throughput drug screens and capable of replacing dying neurons. This project will study the conversion of stem cells into spinal cord motor neurons and explore the possibility that stem cell-derived motor neurons can help in the development of new therapies for motor neuron disease. The major question we will address is whether embryonic stem cells (ESCs) can be converted into highly specialized classes of motor neurons that are capable of innervating appropriate muscle targets in an animal. This is a crucial issue in the quest for cell-based therapies for motor neuron disease in particular, and neurodegenerative diseases in general. For motor neuron diseases, normal motor function will be restored only if neuronal connectivity is re-established. Defining optimal conditions for neuronal production and connectivity may therefore have widespread implications for treatment of neurodegenerative disorders. This project aims to rigorously define the molecular and functional properties of stem cell-derived motor neurons. We will combine mouse molecular genetic manipulation and gene expression array analysis with stem cell differentiation methods to generate motor neurons from stem cells. Then we will compare the properties of such neurons with their embryo-derived counterparts. With this foundation we anticipate that it will be possible to devise efficient drug screens to identify chemicals that promote the survival of human ESC-derived motor neurons carrying relevant disease genes. These studies should also help to evaluate the potential of cell replacement therapies in motor neuron diseases.

 

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 Functional Synovial Joint Replacement by Stem Cell Homing

Jeremy Mao, DDS, PhD

Columbia University Medical Center
IIRP

 

About 70 million people in the United States suffer from arthritis and related disorders and we spend over 75 billion dollars per year on treating arthritis and related conditions. New York State alone has approximately five million arthritis patients. Osteoarthritis is a chronic, degenerative disease. Currently, total joint prosthesis, with metal and plastic joints (total knee, total hip), is the primary surgical treatment choice for individuals with late-stage arthritis. One of the critical shortcomings of the procedure is that the prostheses last only 10 years on average, during which wear debris accumulates. In New York State, 3.5 out of the five million arthritis sufferers are 65 or younger. A biologic-based joint replacement therapy would circumvent the limitations of metal and plastic joints. Over the past five years we made substantial progress in replacing joints with stem cells and biomaterial scaffolds. Our most recent data demonstrate that rabbits with biologically replaced joints are able to walk virtually as well as normal rabbits. The bioengineered joints consist of normal articular cartilage ingredients and integrate with engineered subchondral bone and host bone. In the present study we will optimize our strategies to regenerate synovial joints that replace and remodel with the host´s native structures. A great deal of scientific study on the efficacy and safety of bioengineered joints needs to be performed before we or others move this to human clinical trials. We are currently at the stage of small animal studies, but we project that bioengineered joint analogs will replace current metal and plastic joints in the coming years.

 

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 The Role of Endogenous Hippocampal Stem Cells in Treating Non-Human Primate Models of Depression and Anxiety

Tarique Perera, MD

Columbia University Medical Center
IIRP

 

Millions of people worldwide suffer from debilitating neurological and psychiatric disorders associated with the loss of brain cells. Current available treatments fail to address the issue of neuron loss and consequently provide only symptomatic relief or slow progression of the disease without curing the underlying pathology. Curing these disorders may be possible if lost neurons can be replaced by inducing stem cells to differentiate and become neurons or glia. Embryonic tissue containing stem cells can be grafted into the damaged brain, however this approach is invasive and needs to overcome technical difficulties in precisely and adequately targeting damaged areas and avoiding rejection by the host brain. An alternative approach involves using a reservoir of endogenous stem cells from within the adult brain. As a possible treatment for major depression, these cells can be non-invasively induced to become new neurons (neurogenesis) and replace lost tissue. Our prior data showed that electroconvulsive therapy (ECT) robustly stimulates stem cell proliferation and neurogenesis in the brain. Our experimental design combines three interrelated studies: evaluation of whether stem cell induction is necessary for treating or preventing depression; detection of stem cells via brain scanning; and identification of genes that control stem cell induction. This innovative triple approach has the potential to fast-track knowledge on adult stem cells in a highly economical manner. Our aims will elucidate the basic mechanisms needed to treat and prevent depression, which could potentially lead to new treatments that are faster and more effective. Detecting stem cells with brain scans will significantly improve our abilities to apply stem cell-based treatments for humans. Identifying genes that regulate stem cells may allow us to use novel methods, such as gene therapy, to effectively manipulate these cells for therapeutic benefit.

 

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 Stem Cell-Based Platform Therapy for Lethal Cardiac Arrhythmias

Michael Rosen, MD

Columbia University Medical Center
IIRP

 

There are about 400,000 sudden deaths annually (CDC data) caused by lethal cardiac arrhythmias, most of which result from ventricular tachycardia and fibrillation (VT/VF). To prevent and treat these arrhythmias, physicians have used (1) antiarrhythmic drugs (often ineffective and/or cause other lethal arrhythmias); (2) devices that deliver shocks to terminate VT/VF (cumbersome, can give inappropriate shocks, not necessarily effective); or (3) combinations of drugs and devices (incidence of shocks may be reduced, but the risk of proarrhythmia remains). We used mathematical modeling and studies in mammalian cell lines to identify ion channel constructs whose antiarrhythmic mechanism of action is novel and which apparently do not cause arrhythmias. We administered them via adenoviral vectors in proof of concept studies that showed significant reduction of arrhythmias in a canine heart attack model that mimics the human condition. Because viral vectors are themselves of concern in human populations, we now propose to use adult human mesenchymal stem cells (hMSCs) as a platform to deliver these constructs to hearts at risk. We propose to advance stem cell biology by (1) taking advantage of the immunoprivileged status of hMSCs as demonstrated in humans and dogs; (2) exploring hMSCs as platforms for delivering unique ion channel constructs to provide potential therapies long known to be theoretically useful, yet inaccessible, via drug development; and (3) providing a direct track from modeling to cell testing to animal testing using hMSCs as the platform. If we are successful with our constructs, we will have developed a truly novel antiarrhythmic therapy and the means to deliver it to infarcted, arrhythmic myocardium. The result would be a reduction in need for drug or device therapies, and improved well-being in a population that contributes actively to the work force.

 

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 Characterization of the Retroviral Silencing Machinery in Embryonic Stem Cells

Stephen Goff, PhD

Columbia University Medical Center
IIRP

 

Embryonic stem cells (ESCs) have the remarkable ability to block or "silence" incoming viruses, protecting the precious germ line from infection and preventing deleterious mutations. This block is mediated by a large, molecular machine that recognizes and binds a specific conserved DNA sequence present on viral and mobile element DNAs, silencing the DNA and preventing its transcription into RNA. We propose that this silencing machinery is a critical part of pluripotency, an ESC´s ability to give rise to differentiated cells in an efficient way. We recently succeeded in identifying two key components, and will extend this work to identify and characterize all the components of the system in detail, both in mouse and human ESCs. We will study its mechanism of action by studying viral DNA after viral infection of ESCs. We will determine the consequences of removing the machinery from ESCs to test for its importance in ESC function. We will purify the silencing machinery, and clone, express and knock-out the genes to alter the levels of the machinery in various settings. We will infect these cells in culture with retroviruses to monitor their ability to block viral replication. Although the ability of ESCs to silence incoming viruses has been known for more than thirty years, how they accomplish this is not understood. We are the first to finally isolate this machinery and to initiate its characterization. We will now learn for the first time how virus infection is prevented, providing first-ever knowledge of this important machinery. These studies should help define the unique properties of ESCs and may provide the tools to screen ESC clones for the best ones to use for therapeutic purposes.

 

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 Control of Hair Follicle Stem Cell Proliferation and Epithelial Skin Tumorigenesis by Concerted RUNX1 and CDKN1a Action

Tudorita Tumbar, PhD

Cornell University
IIRP

 

The basic mechanisms controlling the dynamics of adult stem cell function for maintenance of normal adult tissues are poorly understood. Understanding these mechanisms is important for designing future therapies for diseases that arise when an imbalance in the dynamics occurs. A special case in point is cancer initiation and progression, in which enhanced self-renewal of stem or progenitor cells, and inhibited differentiation to more committed cell lineages, can have an important impact. We use the mouse hair follicle as a model system to study this question because the precise tissue location of the epithelial stem cells is known and many genetic tools allow insight into mechanistic, basic science questions. The hair follicle is useful for analysis of the effect of targeted mutations in essential genes since it is not critical for an animal´s survival. Yet many factors regulating normal and malignant behavior of stem cells of essential tissues, such as blood, operate similarly in the hair follicle. Recently we found that Runx1, also known as the acute myeloid leukemia gene (AML1), exerts its function by repressing a tumor suppressor gene (CDKN1a) already known to be involved in skin cancer. The involvement of Runx1 in leukemia is well documented, but its link with skin tumorigenesis has not been addressed. Our genetically modified mice allow modeling of stem cell behavior in hair follicles, thereby permitting a greater understanding of the mechanisms by which known tumor suppressor genes perturb stem cell behavior. Our work will shed light on basic science mechanisms that regulate hair follicle stem cell function. Uncovering the basic molecules involved in cancer progression, their complex network of interaction and the potential link with stem cell regulation, is a great endeavor of the stem cell field. Our newly developed tools will allow more in-depth elucidation of how cancer prone mutations in complex model systems perturb the behavior of stem cells.

 

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 Mechanisms by Which Nuclear Pore Composition Regulates Stem/Progenitor Differentiation

Elizabeth Lacy, PhD

Memorial Sloan-Kettering Cancer Center
IIRP

 

Cells of higher organisms, including mammals, are structurally compartmentalized: the nuclear envelope surrounds the nucleus, separating it from the cytoplasm, while the cell membrane separates the cytoplasm from the exterior. This compartmentalization reflects a separation of functions. The nuclear pore (NP) is a structure in charge of bi-directional transport of molecules between the cytoplasm and nucleus. NPs are large complexes consisting of many proteins called nucleoporins (Nups). Because they mediate all transport in and out of the nucleus, NPs play a crucial role in every cellular process. We discovered that NPs adapt or respond to different stimuli by changing their composition in a tissue and developmental stage-specific manner. We found that one nucleoporin, Nup133, is present predominantly in the NPs of rapidly dividing, relatively undifferentiated cells, such as embryonic stem and embryonic neural stem cells (neuronal precursors). Without Nup133, stem cells are unable to generate functional neurons. We will now determine how Nup133 controls the balance between the pluripotent and the differentiated states of a cell. NPs represent a newly recognized molecular player in the field of stem cell biology. Thus the mechanism by which NP composition modulates stem cell differentiation is unknown. Additionally, although fundamental to embryonic development and tissue homeostasis, the mechanisms coordinating changes in cell proliferation with differentiation remain poorly defined. Studies on NPs promise to provide a new paradigm for elucidating how cell number/tissue size is coordinated with the differentiation of distinct cell types to form functional tissues. This study will contribute to our understanding of which basic mechanisms and which molecules are necessary for a stem cell to differentiate properly. In addition, Nup133 mutations have been found in human breast cancers, making it a breast cancer susceptibility candidate gene.

 

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 Use of Polysialic Acid to Improve Integration of ES-Derived Cells into the Brain

Urs Rutishauser, PhD

Memorial Sloan-Kettering Cancer Center
IIRP

 

Embryonic stem cells (ESCs) provide a ready source of dopamine-producing neurons for treatment of Parkinson´s Disease. However, when grafted into the brain, the cells show a limited ability to disperse and integrate into their new surrounding environment. While functional improvement is obtained under these conditions, incomplete integration raises concerns about functionality and side effects of the grafted cells, such as the severe dyskinesia observed in several patients with fetal dopamine neuron grafts. Polysialic Acid (PSA) is a large carbohydrate whose expression at the cell surface during embryonic development facilitates cell migration and axon outgrowth by reducing the strength of cell-cell interactions. Engineering enhanced levels of PSA in adult tissues, which normally express little or no PSA, promotes tissue repair by producing an environment more suitable for changes in cell position and shape. We propose that the engineered expression of PSA in transplanted ESC-derived dopamine neurons and/or the brain environment into which they are introduced will augment the dispersal of the cells and the extent of outgrowth obtained by their emerging axonal processes. We anticipate that this improvement in graft/host integration will improve the therapeutic potential of the transplantation approach. The use of PSA to enhance the integration of grafted cells is a new approach, used in unpublished preliminary studies of Schwann cells introduced into the site of a spinal cord lesion. Using PSA to promote tissue repair was recently applied successfully in several contexts: growth of severed spinal cord axons through scar tissue, recruitment of neuronal precursors into the injured brain and the dispersal of grafted cells at the site of a spinal cord injury. This proposal is the first application of the PSA technology to ESC-derived cells of any type; if the previous results can now be extrapolated to the grafting of ESC-derived dopamine neurons, there is good reason to expect an improved, clinically relevant outcome. Moreover, this approach could be applied generally to any type of cell transplantation therapy involving solid tissues.

 

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 Role of Wnt Signaling in Sarcomas Initiated From Human Mesenchymal Stem/Progenitor Cells

Stuart Aaronson, MD

Mount Sinai School of Medicine
IIRP

 

The Wnt family of secreted signaling proteins plays a critical role in embryonic development: the renewal of normal tissues. However, when chronically activated due to genetic aberrations, Wnt signaling contributes to the development of a variety of human cancers. Recent studies demonstrated that Wnt signaling is involved in regulating differentiation of human mesenchymal stem cells (hMSCs), which give rise to bone and adipose tissue, cartilage and muscle. We found that endogenous Wnt signaling is highly active in a small subpopulation of hMSCs with biological properties consistent with those reported for self-renewing, multi-potent tissue stem cells that are involved in maintaining normal tissues. Moreover, we showed that high Wnt levels in hMSCs inhibit their ability to undergo osteogenic and adipocyte differentiation in vitro and in vivo. These findings led us to investigate whether aberrations resulting in chronic activation of Wnt signaling might contribute to the development of human sarcomas. Our preliminary evidence indicates that this is indeed the case as we observe a very high frequency of Wnt signaling activation in human osteogenic sarcomas, as well as some soft tissue sarcoma cell lines. We will now determine if chronic upregulation of Wnt signaling plays an important role in the development of human sarcomas by expanding an undifferentiated hMSC/progenitor cell population. Our research will contribute important insights as to whether the concept of cancer stem cells or tumor initiating cell is applicable to human sarcomas. Sarcomas generally arise in younger individuals and are the cause of tremendous morbidity and death. The proposed studies should elucidate a novel mechanism involved in human sarcoma development. These studies also offer the potential for new sarcoma therapies through inhibition of the Wnt signaling pathway activated in these tumors.

 

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 Plasticity of Head and Neck Cancer Initiating Cells

Julio Aguirre-Ghiso, PhD

Mount Sinai School of Medicine
IIRP

 

Head and neck cancer is an aggressive disease for which survival rates have improved only marginally in the last 30 years. Surgery allows clearing the initial tumor, however, more than 50% of patients develop recurrent cancer lesions near the area of the original tumor or in separate organs, including the lung. Although cancer recurrences can appear at a rate of 5% per year after treatment, some patients remain without signs of disease for up to 10 years. This suggests that residual cancer cells may remain in a quiescent state. To prevent recurrences, it is crucial to understand the behavior of cancer cells remaining after treatment. Current evidence in the stem cell field suggests that normal stem cells in human tissues are in a quiescent state and are only called into action when needed. It is proposed that cancer stem cells in general may have adopted this state by remembering some of the functions they possessed before becoming cancerous. We discovered that cancer stem cells from head and neck tumors also adopt a quiescent state when they find themselves in appropriate conditions. In this state they become resistant to current therapies and in due time become active and aggressive. We will now isolate cancer stem cells using cell surface markers and study the signals controlling the behavior of these cells during quiescence and growth. Our ability to identify cancer stem cells and study the way they reprogram into quiescent and non-aggressive behavior will allow us, for the first time, to gain insight into this process in patients. These studies might have a major impact on how patients in remission are treated. In the long term our studies may lead to strategies to identify and attack residual dormant tumor stem cells, favoring the eradication of the disease before it resumes growth.

 

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 Red Blood Cell Development in Differentiating Embryonic Stem and Induced Pluripotent Stem Cells

Margaret Baron, MD, PhD

Mount Sinai School of Medicine
IIRP

 

A mainstay of many medical therapies is blood transfusion, but blood supplies are limited. There is an urgent need to develop additional sources of red blood cells for use in patients. Embryonic stem cells (ESCs) have gained increasing attention as a potentially inexhaustible source of cells for the development of new therapies for human diseases. Induced pluripotent stem cells (iPSCs) offer the possibility of serving as a source of patient-specific cells for customized therapies. Primitive erythroid cells (EryP) are the first differentiated cells to form in the mammalian embryo and in ESC-derived "embryoid bodies". EryP are an outstanding model for erythroid differentiation because they develop in a synchronous and stepwise manner. A major impediment to studying embryonic red blood cell formation is the inability to reliably separate embryonic and adult type red cells at stages when both are present in the blood. We recently overcame this obstacle using a genetically engineered mouse line in which expression of a green fluorescent protein (GFP) is used to specifically mark EryP so these cells can be purified. Our studies of these cells clearly indicate that embryonic and adult red blood cells display common and distinguishing features. We will apply our tools and insights to ESC and iPSC systems to define better conditions for directed differentiation along the red cell lineage. We generated mouse ESC lines in which GFP is expressed exclusively within EryP and propose to create analogous human ESC and mouse iPSC lines. We will examine the differentiation of these cells in vitro, determine if antibodies can be used to identify, isolate and characterize EryP precursor cells, and reprogram immature red blood cells to a stem cell-like state. This work could open up a new field of study focused on inducing maturing red blood cells to divide and replacing red cells lost to injury or disease without the need for stem/progenitor cells. The ability to generate patient specific erythroid cells or precursors for transfusion or transplantation would make it possible to avoid the significant risks that are currently associated with these therapies.

 

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 Generation of Thymic Epithelial Cells From Embryonic Stem Cells

Hans-Willem Snoeck, MD, PhD

Mount Sinai School of Medicine
IIRP

 

T lymphocytes, which are produced in the thymus, are critical for immune defense against microorganisms and tumors. Stem cells from the bone marrow colonize the thymus and produce T cells that are capable of attacking invading microorganisms while sparing the cells of the body. A major health problem is that with age, the function of the thymus declines exponentially, leading to defective immunity in the elderly. Furthermore, after bone marrow transplantation, the function of the thymus is severely affected. Finally, in a number of genetic diseases, the thymus is absent. Thus, there is a critical need for thymic replacement therapy. Embryonic stem cells (ESCs) can be propagated in culture and have the potential to differentiate into all cells of the body. Therefore, we hypothesize that it is possible to generate functional thymic tissue from ESCs. It is difficult to generate mature tissues from ESCs such that they function in an adult. Thymic tissue may be an exception, as fetal thymic tissue can function in the adult. We will, attempt to coax ESCs to develop along the initial stages of thymic development. In addition, we will use a high-throughput approach to optimize strategies to generate thymic cells from ESCs. It is widely hypothesized that thymic replacement in the elderly will improve immunity, enhance the efficacy of vaccination (vaccine failure in the elderly is a major public health concern) and may even enhance life span. In the setting of bone marrow transplantation, thymic replacement could play a major role in preventing some of the most life-threatening complications of this procedure. For patients with congenital absence of a thymus, as thymus donors are rare, ESC-derived thymus tissue may be the only way to reestablish a functioning immune system.

 

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 Molecular Deciphering of Stem Cell Epigenetic Silencing

Ming-Ming Zhou, PhD

Mount Sinai School of Medicine
IIRP

 

Recent rapid advances in stem cell biology research generated a tremendous amount of enthusiasm and hope for the development of novel stem cell therapies for human diseases through tissue or organ repair/replacement and regeneration. However, our knowledge of how stem cells maintain a pluripotent state, and what instructs stem cells to transform into different types of cells, is still limited. While it is generally believed that epigenetic control of gene expression holds the key to these questions, the details underpinning such biological processes remain elusive. Our research aims to fill this important scientific gap in stem cell biology. Our research is built on our recent discovery of several key molecules involved in regulating the biological process of gene repression, which determines stem cell fate between replicative self-renewal and differentiation. Our central hypothesis is that a stem cell´s self-renewal is dependent upon silencing of genes that would otherwise result in cell lineage commitment. This gene silencing process is controlled through close coordination between non-coding RNA transcripts of a target gene subject to silencing, and factors that function to change the chromatin structure where the target gene resides in the human genome. We specifically propose to study the stem cell biology of epigenetic gene silencing to attain mechanistic insight. We also propose to design small-molecule chemicals that target proteins that play an important role in gene silencing. Our emphasis is on the role of non-coding RNA in gene expression control in stem cells. We expect that the results will enhance our current understanding of transcriptional programs that balance stem cell replicative self-renewal and lineage commitment, and also facilitate the future development of novel tools and technologies to maintain and manipulate stem cells for the emerging field of regenerative medicine.

 

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 Contribution of Hippocampal Stem Cells to the Action of Antidepressants: From Mice to Men

René Hen, PhD

New York State Psychiatric Institute
IIRP

 

Depression is a devastating illness that is characterized by some or all of the following symptoms: depressed mood, sleep and appetite disturbances, anhedonia, worthlessness, psychomotor slowing or agitation, fatigue and thoughts of death. In the US, 10-14 million people are depressed in any year. During their lifetime one in eight persons may require treatment for major depression. In any year, one in 10 depressed persons attempts suicide, which makes depression the single largest risk factor for suicide. The most common treatments for depression are the serotonin reuptake inhibitors, or SRIs, such as Prozac, and psychotherapy, which can be administered separately or in combination. About 70% of patients respond to these SRIs. However, many responders experience side effects (often sexual) and a delayed onset of full therapeutic efficacy, which can be as long as six weeks. Adult neurogenesis, the generation of new neurons in the adult brain, is 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, leading to the hypothesis that increases in neurogenesis may contribute to the behavioral effects of enriched environment and antidepressants. We are developing a non-invasive imaging strategy to detect neurogenesis in humans based on biomarkers. We propose to further validate these biomarkers in rodents and also analyze changes in these biomarkers in patients treated with antidepressants. Our proposal is the first attempt to image neurogenesis in depressed patients and to determine if there is a relation between increases in neurogenesis and an antidepressant response (a mood elevation). We will also assess whether neurogenesis is lower in depressed individuals than in normal controls. This proposal may provide ideas for the generation of novel antidepressants that would directly target neurogenesis or mimic the properties of the young hippocampal neurons. Due to the anatomic specificity of neurogenesis, such agents might be expected to have fewer side effects and possibly a faster onset of therapeutic efficacy.

 

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 Skeletal Muscle-Derived Stem Cells

Louis Terracio, PhD

New York University College of Dentistry
IIRP

 

Loss of skeletal muscle may evolve as a consequence of a developmental anomaly or as a result of traumatic injury or surgery to remove a tumor. Structural defects that affect striated tissues range from functionally benign to profoundly debilitating. In either circumstance, the condition can affect a patient on a number of different levels. For example, structural defects to the musculature of the face may have a negligible impact on the ability of a patient to survive; yet, even minor cosmetic defects in the muscles of the face can have profound psychological implications. Defects in the structure of muscle in the arms or legs can limit mobility and greatly compromise quality of life. An overlooked area of research, especially for facial repair, is the field of tissue engineering of skeletal muscle prostheses. For prostheses, skeletal muscle has an advantage over other tissues in that it has a readily available source of stem cells that can be isolated from the patient, eliminating the need for embryonic stem cells or adult-derived stem cells that must be induced to turn into muscle. We will isolate, expand and induce these muscle stem cells to differentiate with the long-range goal of developing muscle prostheses for transplantation. The innovative elements of the project are the application of stem cells to a unique system of tissue-engineered components designed to allow the stem cells to become muscle cells, which will then organize themselves into a structure that looks like skeletal muscle. If successful, our porcine studies will demonstrate the feasibility of the proposed approach to treat humans, and the successful isolation, expansion and purification of stem cells from human biopsy specimens will set the stage for development of muscle prostheses to treat the loss of skeletal muscle in patients.

 

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 Functional Identification of Transcriptional Determinants of the Embryonic Stem Cell State and Early Lineage Commitment

Lisa Dailey, PhD

New York University School of Medicine
IIRP

 

Embryonic stem cells (ESCs) have the remarkable ability to give rise to nearly any type of specialized cell found in adult tissues and organs, opening the possibility that new therapies could be developed for the treatment of debilitating conditions resulting from diseased or damaged tissues. However, any potential use in human therapies requires that the nature of these cells be fully understood, both to ensure safety and to develop efficient methods for creating desired cell types. Since the different properties of ESCs and their derivatives are the result of the distinct sets of genes they expressed, we will gain a great deal of insight into the nature of these cells by identifying the mechanisms that control gene expression in ESCs, and how these change as the cells differentiate. Transcription factors proteins (TFs) bind to specific DNA sequences near genes to either enhance or repress expression of the genes. Different cells contain different sets of TFs, resulting in the expression of different groups of genes. A number of TFs have been identified in ESCs that are required for maintaining the integrity of the ESC state. However, roles for many additional ESC TFs remain unknown, and little is known regarding TFs that are important for converting ESCs into specialized cell lineages (i.e. "differentiation"). We developed a new method to isolate hundreds of regulatory DNA fragments, sequences that TFs bind to, that are either only active in ESCs or become active as the cells differentiate to neural progenitors. Since we can predict which TF is binding based on the DNA sequence, isolation of these fragments will tell us which TFs are critical to each cell type. Our experiments will rely on a novel high-throughput functional assay. Identification of these components will provide valuable basic insights into the nature of ESCs, but may also facilitate additional efforts at reprogramming mature cells, such as skin cells, into patient-compatible induced pluripotent stem cells. Knowledge of key lineage-specific TF determinants will facilitate efforts to direct ESC differentiation to specific cell types.

 

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 The Directed Differentiation of Embryonic Stem Cells Into Specific Cortical Interneuron Subtypes

Gordon Fishell, PhD

New York University School of Medicine
IIRP

 

Neurons in the brain can be broadly grouped into two classes: excitatory cells (also known as pyramidal cells) that increase electrical activity and inhibitory cells (also known as interneurons) that act as brakes by reducing electrical activity. Both are vital in order for the brain to function properly. Too much excitation leads to giant waves of electrical activity that can lead to seizures, a disorder known as epilepsy, affecting one in every 250 adults. Stem cells are a very promising avenue of research for disorders like epilepsy because they have the ability to turn into many different cell types (including inhibitory interneurons) and provide a limitless supply of that cell type. Our first goal is to develop a method for preferentially making inhibitory interneurons from stem cells. We will try to make large numbers of inhibitory interneurons from stem cells in two ways. First, we will change the environment that the stem cells grow in by adding different factors that act as instructive cues to tell stem cells what kinds of cells to turn into, in this case inhibitory interneurons. Second, we will change the programming going on inside the stem cells, again, to tell stem cells to become inhibitory interneurons. Once we establish our method and show that the inhibitory interneurons work properly, we will test the ability of inhibitory interneurons to control seizures in a mouse model of epilepsy by transplantation of the cells. This study, and others like it, is an important first step for translating stem cell research from the bench to the clinic.

 

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 Embryonic Stem Cell-Derived Cardiac Conduction System Cells

Glenn Fishman, MD

New York University School of Medicine
IIRP

 

During a normal lifespan our hearts beat over two billion times. The ability of our hearts to beat in an exceedingly reliable and regulated manner is dependent upon a specialized network of cardiac cells that possess unique electrical properties, known as the specialized cardiac conduction system. Abnormalities in heart rhythm represent an enormous public health problem affecting more than three million Americans and accounting for almost 500,000 deaths annually. In 2001, $2.7 billion was paid to Medicare beneficiaries for cardiac arrhythmia-related diseases. Our current therapies for patients at risk for heart rhythm disorders are less than ideal. Anti-arrhythmic drugs are not particularly effective, especially for the most life-threatening forms of arrhythmias, and disappointingly, the use of these drugs sometimes paradoxically increases arrhythmic risk. Implantable electronic devices such as pacemakers and defibrillators are effective, but these approaches require surgical implantation, which may be complicated by infection, the need for battery changes or even device failure. Thus, there is a clear and compelling need for novel medical treatments for patients at risk of heart rhythm disorders. Unfortunately, cells of the specialized cardiac conduction system comprise a very small proportion of total cells in the heart and they are exceedingly difficult to study. Accordingly, our ability to study these cells and test potential new therapies for conduction system disorders is severely hampered. Embryonic stem cells (ESCs) have the capacity to differentiate into virtually any cell type in the body, including the specialized network of cells within the heart that regulate heart rhythm. In this study we will use modern biological methods to coax stem cells into heart conduction system cells. Through the generation of ESC-derived cardiac conduction system cells, we expect to gain novel information on the developmental biology of the heart´s specialized conduction system, new knowledge about the electrical properties of the cells within this system, and ultimately, discover novel targets for the treatment of heart rhythm disorders.

 

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 Changes in RNA Synthesis and Timing of Cortical Development

Sally Temple, PhD

Regenerative Research Foundation
IIRP

 

Nervous system stem cells produce many different subtypes of cells that are important for brain function. Each cell subtype arises on a precise time-schedule during normal development. Understanding this process will help us generate specific cell types from stem cells in tissue culture, providing cells that can be used as disease models or in replacement therapies. We will determine how gene expression changes during development for stem cells and the blood vessel cells that they interact with in the cerebral cortex, a major region of the brain that controls movement, sensation and thought. The nervous system and vascular cells in the cerebral cortex are damaged in diseases such as stroke, Alzheimer´s disease and during formation of brain tumors. Hence understanding the genes involved in cortical development has relevance to these conditions. We propose to test the hypothesis that the development of stem cells in their normal environment occurs through expression of particular genes at particular times. We will determine which genes are responsible for generating the diverse cells present in the cerebral cortex, and what coordinated changes occur in the developing blood vascular system as they form the stem cell environment, or ´niche.´ Our novel approach will use a new strain of mice engineered to express Uracil phosphoribosyl transferase (UPRT), an enzyme that allows specific tagged molecules to be made and incorporated into nascent RNA molecules (RNA molecules represent gene expression). By turning the UPRT enzyme on at specific times during development in the nervous system stem cells, we will identify which RNA molecules are newly synthesized. This will provide a dataset of genes important for making each major cortical neuron subtype, as well as the developing vasculature, at specific times during development. The datasets will include target genes that can be investigated in future studies to determine their role in the stem cell niche and in nervous system diseases and disorders.

 

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 Stem Cell Glycomics in Microarray Format

Robert Linhardt, PhD

Rensselaer Polytechnic Institute
IIRP

 

Our understanding of the genomics (the genes encoded by DNA) of human embryonic stem cells (hESCs) was advanced by the sequencing of the human genome in 2001. Today, scientists are struggling to determine the human proteome, the structure and function of proteins encoded by genes. 80% of all human proteins are post-translationally modified in ways not easily predicted from the gene sequence. The most common and perhaps the most important post-translational modification is glycosylation, the addition of sugars to proteins. Glycosaminoglycans (GAGs) are a major group of sugars with great structural complexity and are of critical importance in developmental processes, such as hESC renewal and differentiation. We will examine the change in the structure of all GAGs (GAGome) on hESC differentiation and the impact of GAGs on the growth, maintenance and differentiation of hESCs. This research will provide a new technology allowing researchers to print stem cells on a slide, which will aid in their understanding of stem cell biology. We will determine the GAGome of hESCs and differentiated cells, print these cells in 3-dimensional spots on microscope slides containing various mixtures of GAGs, then determine the impact of GAGs in the surrounding environment on the growth and differentiation of the cells. This research is the first examination of the GAGome of hESCs and differentiated human stem cells. An understanding of the hESC GAGome and its impact on stem cell biology will help in understanding how to better grow, maintain and differentiate hESCs along desired pathways. Our research should also provide new GAG markers that will help scientists identify the exact type of human stem cell line they are working with. Finally, this research should afford a new platform, a 3D-microarrary, for the rapid screening of hESCs and human stem cell lines.

 

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 Therapy of Hemophilia A Using Megakaryocyte-Targeted Stem Cell Delivery

Wadie Bahou, MD

SUNY - Stony Brook University
IIRP

 

Hemophilia is a genetic bleeding disorder that requires lifelong treatment with infusions of the deficient clotting factors. Deficiency of clotting protein Factor VIII causes hemophilia A, the most common serious bleeding disorder in the world. The incidence of the disease is near one in 5,000 males, with estimates that it affects almost 2,000 New York residents. Not only is the disease crippling, but the requirement for lifelong treatment is associated with high incidence of HIV, hepatitis and insurance restrictions because of the continuous costs. We recently showed that Factor VIII protein can be made in genetically engineered blood platelets and stop bleeding in mice (normally, Factor VIII is synthesized in the liver). Based on these studies, we propose that delivery of small amounts of Factor VIII can be achieved by using hematopoietic stem cells and delivered by bone marrow transplantation. Since this would be autologous [i.e. self] transplantation, it would not be expected to require lifelong immunosuppressive treatments. We will generate viruses carrying the Factor VIII gene that can infect bone marrow stem cells and remain dormant in a safe region of the human genome, only to be expressed in platelets. Until recently, most investigators used viruses to directly deliver genes to liver cells, which required highly invasive approaches. Our proposed approach is safer, since the bone marrow cells will be removed, genetically modified, and re-infused back into the same patient. The innovative aspects involve the use of unique designer viruses that can deliver the Factor VIII gene into a silent area of the genome, coupled with the use of stem cells for long-term bone marrow delivery into normal blood cells. Hemophilia A is crippling in nature and has devastated the hemophiliac community, which developed a high incidence of HIV disease from contaminated blood. The ability to develop a safe and effective means of constantly delivering Factor VIII by a safe, generally available procedure has the potential for high therapeutic impact.

 

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 Immune Responses to Allogeneic Stem Cell Transplantation

Soosan Ghazizadeh, PhD

SUNY - Stony Brook University
IIRP

 

As our understanding of the role of stem cells in normal tissue maintenance improves, the use of these cells to restore tissue function following damage or in degenerative diseases becomes more appealing. Although the use of autologous, reprogrammed stem cells for patient-specific therapies is an attractive option, it is likely to be difficult to implement practically on a large scale. The alternative approach, the use of allogeneic stem cells has a major hurdle to overcome, namely host immune-mediated rejection. Currently there is a major gap in our understanding of host responses to stem cell transplantation. Clearly, host responses to stem cell-based therapy must be addressed before fundamental stem cell research can be translated into new therapies. A clear understanding of immunological responses to different types of stem cells will allow development of new strategies for controlling immune responses to the presence of allogeneic stem cells. Our overall hypothesis is that different classes of stem cells are likely to induce different types of immune responses. We also hypothesize that stem cells can be modified to protect them from destructive immune responses. Using our well-characterized skin regeneration model, we will analyze engraftment of two distinct populations of stem cells and track stem cell engraftment in live animals by monitoring surface fluorescence. Stem cells are labeled with a fluorescent protein to allow monitoring of their engraftment in live animals over a long period of time. We will analyze host responses to allogeneic stem cell engraftment and determine the phenotype of those stem cells that survive immune attack. In addition, we will genetically engineer stem cells to express a factor that reduces immunogenic reaction and assess the effect of this strategy on protection of different types of stem cells. Development of a successful strategy for engineering a stem cell with universal donor capability that can evade host immune responses will have a significant impact on adoption of stem cell-based therapies for degenerative disorders.

 

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 Novel Chimeric Frizzleds as Tools to Program hESC Differentiation

Hsien-yu Wang, PhD

SUNY - Stony Brook University
IIRP

 

Human embryonic stem cells (ESCs) are capable of yielding all of the differentiated cells found in the adult human. ESCs can be propagated and differentiated artificially in culture into complex mixtures of various cell types, not homogeneous cell types like the pancreatic beta-cell that secretes insulin. In normal development, a system exists whereby activating molecules (Wnts) bind to cell receptors (Frizzleds) and guide cell differentiation. For optimizing the study of these cells and stimulating them toward well-defined differentiated cell types, we will exploit the Wnt-Frizzled system using molecular tools. The central question of our research is how the natural activators (Wnts) control differentiation of ESCs to useful terminal states (e.g., muscle, nerve, bone cells) through Frizzleds? We hypothesize that the Wnt-Frizzled pathways are activated individually, in combination and in a precise order to yield very specific terminal cells of therapeutic potential. Having successfully created novel, chimeric Frizzled proteins that are easy to activate, we propose to systematically express these receptors (alone and in combination) and provoke the ESCs into well-defined, terminal cell populations. We will express our engineered Frizzled molecules in ESCs, then use DNA microarrays and other techniques to establish which differentiated cell populations are produced. The ability to manipulate the Frizzled pathways individually and in combination is a powerful and novel strategy for which there is no comparable alternative. It is important to recognize that the perennial inability to produce homogeneous, well-characterized, differentiated clones derived from ESCs remains a major stumbling block to the full realization of the potential of such cells to treat human diseases.

 

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 High-Throughput, Real Time Dynamic Monitoring of Stem Cell Differentiation

Stelios Andreadis, PhD

SUNY - University at Buffalo
IIRP

 

Stem cells provide tremendous potential for understanding cellular differentiation and for discovering therapies to treat devastating diseases, e.g. heart and Alzheimer´s disease. To achieve differentiation of stem cells to the desired cell type or tissue, it is important to understand how different gene products contribute to stem cell differentiation. Although the sequencing of the human genome led to a rapid cataloguing of genes, the challenge still remains to understand the function of gene products in different biological contexts, including stem cell differentiation. To address this challenge we propose to develop a novel microarray of recombinant gene transfer carriers to monitor simultaneous expression of multiple genes in living stem cells undergoing differentiation to desired cell types. We will construct a novel lentivirus microarray (LVA) of gene promoters driving green fluorescent protein, which will be taken up by stem cells and cause the cells to appear green when the promoter is active. By measuring the intensity of green fluorescence we will determine how a particular gene´s expression changes over time as bone marrow stem cells undergo differentiation. This novel microarray can be used to monitor gene expression in any type of adult or embryonic stem cell or any other cell type, e.g. tumor cells, under a variety of biological contexts such as biochemical or biophysical stimuli. This resource will be made available to other investigators, thereby increasing the potential impact of this technology beyond the boundaries of the proposed work.

 

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 Hair Follicle Stem Cells for Cardiovascular Tissue Regeneration

Stelios Andreadis, PhD

SUNY - University at Buffalo
IIRP

 

The mortality rate of heart disease in Western New York (WNY) is higher than the average rate in New York State and the United States overall. In addition, the high rate of obesity further increases the odds for high incidence of diabetes and heart disease in this area. In conjunction with a growing elderly population in the US, and WNY in particular, these data point to a growing need for cardiovascular therapies. In particular, there is increasing demand for small diameter blood vessels as replacement grafts. Although venous grafts are currently the gold standard, the ten-year failure rate approaches 35%. Bioengineered vascular grafts are capable of remodeling in response to host signals and offer a clear alternative to existing technologies. However, removing tissue for the graft from the patient´s vessels requires invasive surgery and can injure the donor site. In this regard, stem cells can provide a potential source of vascular cells that can be used for development of cardiovascular therapy. Hair follicle cells can differentiate into smooth muscle cells, and thus we hypothesize that hair follicles may provide a rich and easily accessible source of autologous vascular cells that can be used to engineer small-diameter, biological vascular grafts. We will isolate smooth muscle progenitor cells from the hair follicles of neonatal and adult animals to evaluate the effect of organismal aging on the quality of smooth muscle cells and the resulting bioengineered vessels. Finally, we developed a large ovine animal model that we will employ to evaluate the implantability of tissue-engineered vessels. The same cells may also be applied in engineering other cardiac tissues, such as heart valves and cardiac patches, further increasing the potential clinical impact of this work. In addition to smooth muscle, hair follicle stem cells can differentiate towards fat, bone and cartilage, suggesting that future studies may address regeneration of these tissues using similar tissue engineering approaches.

 

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 Modification of Resident Cardiac Stem Cells by Circulating Hematopoietic Stem Cells in Ischemic Cardiomyopathy

Gen Suzuki, MD, PhD

SUNY - University at Buffalo
IIRP

 

Heart failure from narrowed coronary arteries continues to be the most common cause of death and disability in the United States. Adult bone marrow-derived stem cells have emerged as a therapeutic alternative to cardiac transplantation that could be applicable to patients affected by heart failure. Early clinical trials demonstrated the safety, feasibility, and efficacy of a variety of bone marrow-derived stem cell therapies in humans with ischemic heart disease. Nevertheless, chronic effects on function are controversial and a mechanistic understanding of bone marrow components injected into the coronary arteries or myocardium is lacking. Preclinical large animal studies in models resembling human ischemic cardiomyopathy are needed to understand better the cellular mechanisms of heart and vascular regeneration in vivo and to extend this regenerative therapy to therapeutic applications in humans. We hypothesize that adult stem cells derived from a patient´s own heart (cardiosphere-derived stem cells, CDCs) can repair damaged heart muscle. Furthermore, the amount of heart muscle regenerated can be amplified by simultaneously using high doses of the drug pravastatin to mobilize a second stem cell population from the bone marrow to the heart. We will conduct our studies in pigs with chronic heart failure. We will inject cardiogenic cells from CDCs directly into the heart muscle and compare this to a similar amount of cells filtered and injected into the blood supplying the heart or coronary arteries. Once we identify the best approach to give the CDCs, we will determine whether administering CDCs along with high-dose statins, to mobilize bone marrow stem cells, will have a greater effect on heart function than CDCs or pravastatin therapy alone. This work could provide a foundation for a clinical trial evaluating CDCs in ischemic heart failure. Since the cells can be obtained from a heart biopsy and are patient-matched, the approach circumvents difficulties encountered in transplanting mismatched cells from a donor. The studies will also provide a basis with which to compare other therapeutic stem cell approaches using embryonic pluripotent and induced pluripotent stem cells.

 

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 Scalable Expansion and Directed Differentiation of Human Embryonic Stem Cells to Pancreatic Progeny

Emmanouhl Tzanakakis, PhD

SUNY - University at Buffalo
IIRP

 

Stem cells can proliferate to large numbers and become specialized cells such as liver cells, blood cells, nerve cells and pancreatic insulin-producing cells. Thus far, studies on stem cell proliferation and differentiation to various cell types were performed in small volume cultures. Bioreactor systems suitable for culturing stem cells and their specialized progeny in large quantities must be developed before stem cell-based therapies can become reality. We propose to explore the culture of stem cells in a bioreactor. Cells grown on spherical beads have a high surface-to-volume ratio, thereby allowing the production of more cells than in dish cultures. Furthermore, stem cells can acquire biochemical and functional attributes of pancreatic cells when treated with factors engaged in embryonic pancreas development. Our aim is to induce large numbers of cells in a microcarrier bioreactor to become pancreatic cells, ultimately for use in diabetes therapies. The main innovative elements of the proposed work are the use of a scalable microcarrier bioreactor for: (1) the generation of a large number of human embryonic stem cells without loss of their ability to give rise to multiple cell types of the body; and (2) the differentiation of stem cells to pancreatic cells. Our findings will have a significant impact on the development of systems for the culture of human stem cells in large quantities and the production of specialized cell types for therapeutic use. Although the present study focuses on the generation of pancreatic cells, scalable systems, such as proposed herein, will be essential for producing sufficient amounts of stem cell material for cell therapies against a wide spectrum of maladies that were previously considered incurable.

 

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 HTLV Infection of Human Hematopoietic Stem Cells: Induction of Novel Lymphoma in Humanized SCID Mice

Gerold Feuer, PhD

SUNY - Upstate Medical University
IIRP

 

Human T cell leukemia/lymphoma virus type 1 (HTLV-1) is a human virus that is linked to the development of malignant adult T cell leukemia (ATL). ATL has an extremely high fatality rate and does not respond well to treatment. We recently established that HTLV-1 infects human hematopoietic stem cells. We speculate that HTLV-1 infects stem cells in the human bone marrow and creates an infected "Leukemic Stem Cell" (LSC). These LSCs persist over the lifetime of an individual and ultimately give rise to full-blown ATL. Injection of human stem cells, infected with HTLV-1 in culture, into severe combined immune deficient (SCID) mice ("Humanized SCID" mice) results in reproducible induction of lymphoma of human cell origin, with many of the features displayed by ATL cells. This now establishes a very unique small animal model that recapitulates human malignant disease development. We will use this mouse model to understand how HTLV-1 infection induces mutations in human stem cells that result in the eventual development of leukemia/lymphoma. We will also decipher the role of individual viral genes in development of disease by using leukemia/lymphoma induction as a read-out. Our preliminary results suggest that HTLV-1 induces a LSC similar to what is reported for acute myeloid leukemia. Development of an animal model for HTLV-1 replication that recapitulates ATL is a major advance in understanding the link between human viral infections and induction of cancer. Using our Humanized SCID mouse model, we have the ability to discern the molecular events induced in human stem cells that predispose these cells to become malignant.

 

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 Nrf2 as a Regulator of Stem and Progenitor Cell Function

Dirk Bohmann, PhD

University of Rochester
IIRP

 

Somatic, or adult, stem cells persist in many organs into adulthood. These cells are unique in that they can self-renew indefinitely and generate several or all cell types of the organ in which they reside, thus potentially regenerating organ structures or entire organs from a few cells. As opposed to human embryonic stem cells, the use of adult stem cells in research and therapy is not ethically controversial. A key to using adult stem cells for any therapeutic application is the ability to control tightly how often they divide and at what rate they generate specific cells intended to benefit the patient. Ideally, strategies should be developed to control the balance between self-renewal and differentiation of these cells. Our preliminary data identified Nrf2, a gene regulatory protein, as a key component of the proposed mechanism that controls this balance. Interestingly, the principal Nrf2-based mechanism of stem cell regulation appears to operate both in mammals and in fruit flies, the two model systems we use. This conservation affords powerful experimental possibilities because of the availability of superior genetic approaches in the fly, and the ease of analysis of mouse progenitor cells in cultured cell systems. We will confirm the function of Nrf2 as a stem cell regulator in mice and flies, investigate the contribution of the proposed mechanism in aging and tissue regeneration, and explore possibilities to influence the activity of this switch using drugs that could be translated into therapies. From the basic science perspective, this project offers new information about principles of stem cell regulation. At the biomedical level Nrf2-targeting drugs are available and, through the research initiated here, these might become interesting candidates for modulating stem cell behavior for therapeutic benefit.

 

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 Therapeutic Stimulation of the Hematopoietic Stem Cell Niche

Laura Calvi, MD

University of Rochester
IIRP

 

The extraordinary potential of stem cells is limited by our inability to manipulate their fate at will, without compromising their developmentally primitive nature and their unlimited self-renewal, while at the same time avoiding the development of undesired tumorigenic effects. One novel strategy to manipulate stem cell fate is to target the stem cells indirectly through the microenvironmental components that drive their behavior. Our previous work identified bone-forming cells as key components of the hematopoietic stem cell (HSC) microenvironment, or "niche," that could be targeted specifically, increasing HSC numbers and improving survival after bone marrow injury. We will now determine if we can improve the ability of animals to survive bone marrow injury, by stimulating the microenvironment and using some of the known regulatory components of the bone marrow (specifically, activated bone forming cells and their product, Prostaglandin E2). The two components of the bone marrow that we selected specifically stimulate subpopulations of blood forming cells that are less quiescent (while still multipotent), and which should be best at accelerating recovery after bone marrow injury. We will specifically expand less quiescent blood-producing stem cells in animal models to determine if our intervention improves survival after bone marrow injury induced by radiation. Then we will use genetically engineered mice to study the mechanisms that are responsible for this important clinical outcome. The proposed research adopts several state-of-the-art methodologies and will confirm the existence of specialized components of the microenvironment that control rapid response of blood forming stem cells to subsequent bone marrow injury. These experiments will identify specific strategies aimed at improving medical care in a number of diseases for which treatments result in bone marrow destruction following chemotherapy and radiation.

 

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 Canonical Wnt Signaling Controls Mesenchymal Stem Cell Differentiation

Di Chen, MD, PhD

University of Rochester
IIRP

 

As the number of elderly Americans increases, age-related osteoporosis is becoming a major public health threat. It is characterized by low bone formation rates with normal or slightly increased bone degradation. Formation of new bone by osteoblasts is reduced in the aged skeleton in favor of fat cell (adipocyte) formation in bone marrow cavities. The molecular mechanisms controlling mesenchymal stem cell (MSC) differentiation into osteoblasts or adipocytes remain poorly understood. Recent studies suggest that β-catenin protein plays a key role in MSC differentiation during embryonic limb development, but its role in mesenchymal cell differentiation in postnatal and adult mice is unknown. Our preliminary data demonstrate that there is significant bone loss and accumulation of fat cells in bone marrow cavities of mice lacking β-catenin in MSCs, suggesting that β-catenin controls osteoblast/adipocyte differentiation in postnatal mice. We will use comprehensive molecular and genetic methods to determine the role of β-catenin in MSC differentiation and investigate if bone morphogenetic protein 4 (Bmp4) is the critical downstream target gene of β-catenin signaling in these cells. Our use of a variety of genetically altered mice in which the β-catenin gene is either over-expressed or deleted specifically in MSCs in trabecular bone will allow us to determine if β-catenin: (1) promotes MSCs to differentiate into osteoblasts rather than adipocytes, thus promoting new bone formation in adult mice; and (2) regulates the interaction of MSCs with osteoclasts and thus modulates osteoclast formation and bone degradation. These studies will help us define a novel molecular pathway controlling osteoblast differentiation and facilitate the development of new drugs for the treatment of osteoporosis and other metabolic bone diseases associated with reduced bone formation.

 

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 Therapeutic Targeting of Leukemia Stem Cells

Craig Jordan, PhD

University of Rochester
IIRP

 

Any tissue that relies on the activity of stem cells for normal function is also at risk when stem cells behave in an abnormal fashion. This scenario appears particularly clear in the case of human cancer, where numerous studies have demonstrated that tumors may arise when normal stem or progenitor cells are damaged. To date, the relative contribution of so-called "cancer stem cells" is most clear for the blood cancer, acute myeloid leukemia (AML). While most AML cells can be destroyed by standard chemotherapy, AML stem cells are resistant to conventional drugs. This observation may explain the frequent clinical course of AML, where remission is achieved for the majority of patients, but then the disease relapses. We propose to identify new drugs that can directly eradicate AML stem cells, an urgent priority for research. The main goal of this project is to define the key molecular features that control survival of AML stem cells, and to use this knowledge to develop drug screening and discovery processes that will identify more efficacious drugs. In a pilot study we used this strategy to identify one new drug (known as parthenolide), which will enter clinical trials this year. The proposed studies will greatly expand our knowledge of AML stem cells, and thereby create much more effective means of finding promising new drugs. This study represents the first comprehensive effort to create drug discovery processes directly focused on AML stem cells. Previous drug development work has not considered the special challenge inherent in targeting the AML stem cell population, and the result is drugs that are generally not effective for destroying this distinct cell type. The proposed studies have the potential to directly effect outcomes in AML by developing agents that can more effectively eradicate AML stem cells.

 

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 Erythroid Precursor Self-Renewal

James Palis, MD

University of Rochester
IIRP

 

Humans make more than 2 million red blood cells every second in our bone marrow to keep from becoming anemic. We recently discovered a red blood precursor cell, present only in the mouse embryo but not the adult, with the amazing ability to divide every day to form two identical daughter cells, a process termed "self-renewal." It was thought that stem cells are the only blood cells that can self renew, however, it is now clear that these immature red blood cells can be cultured for months and yet are still able to become normal mature red blood cells. We want to understand better how these embryonic red blood cell precursors are able to keep dividing while maintaining their potency. Furthermore, we want to learn how to culture equivalent human cells. Since the mouse cells come from the embryo, we think that the equivalent human cells can be found in cultures of human embryonic stem cells (ESCs). We will study when and where these red blood cell precursors arise in the mouse embryo. We will also grow these cells from mouse and human ESCs in the laboratory, study how they become mature red blood cells, and compare them to red blood cells from our bodies to insure they are equivalent. In the United States, 10% of the population suffers from anemia and 38,000 units of blood are transfused every day to treat these people. Currently, all that blood must come from blood donors. The ability to grow large numbers of immature human red blood cells (from ESCs) that have the same characteristics as those we discovered in mice will provide us with the ability to produce universal donor red blood cells to treat children and adults who have life-threatening anemia. Accomplishing our goal will bring us closer to being able to make red blood cells to transfuse into people.

 

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  Gli2-Activated MSCs for Bone Regeneration and Reconstruction

Xinping Zhang, MD, PhD

University of Rochester
IIRP

 

Mesenchymal stem cells (MSCs) can be readily isolated from various tissues and are considered to be the most important building block for bone tissue regeneration. We aim to engineer a replacement tissue mimicking the potent regeneration capacity of periosteum. Periosteum is a well-vascularized tissue that covers the outer surface of the bone. Periosteum contains a large number of MSCs that can form bone at the site of injury. We will construct tissue containing a biodegradable scaffold, MSCs and selected genes that induce bone formation at the site of compromised periosteum. The key to success will be to identify adequate MSCs and critical genes that direct the MSCs to form bone at the site of injury. To this end, we isolated MSCs from periosteal tissue at the site of injury and identified a gene, Gli2, that effectively drives periosteal MSCs to form excessive bone in an animal model. We will now use cell culture models and a mouse femur bone defect model to determine the effects of combining MSCs and Gli2 in regeneration of a functional replacement tissue for repair of large bone defects. We will determine if Gli2 is effective in directing various MSCs to form bone in repairs, and if combining active Gli2 with MSCs can create a functional periosteum replacement for repair of large bone defects. To do so, we will manipulate the level of active Gli2 in MSCs in mice, and use MSCs with activated Gli2 in our bone repair model. The success of our current approaches could provide the foundation for development of a novel stem cell-based therapy for bone repair and reconstruction.

 

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 Epigenetic Control of Murine Neural Stem Cell Self-Renewal and Differentiation Mediated by Bmi-1

Randall Morse, PhD

Wadsworth Center
IIRP

 

In order that stem cells may eventually be used for therapeutic purposes, it is important to understand how genes that regulate the ability of stem cells to self-renew and to differentiate into specific types of cells are controlled. Adult neural stem cells (NSCs) can self-renew or differentiate into distinct subtypes of cells found in the nervous system, such as glial cells and neurons. These cells can be isolated from the brains of adult mice and grown in culture but gradually lose their ability to differentiate. Our prior studies showed that overexpression of one protein, Bmi1, increases the ability of these isolated cells to self-renew and to differentiate into neural subtypes; conversely, inhibiting Bmi1 expression reduces the ability of NSCs to differentiate properly. These findings indicate that Bmi1 is important for regulating "stemness"-the ability of stem cells to propagate themselves or to give rise to various other cell types. We hypothesize that Bmi1 regulates genes critical to the regulation of adult neural stem cells. We propose that this regulation occurs by alterations in chromatin (the complex of DNA with histone proteins that comprises chromosomal DNA) that in turn prevent transcription factors from inducing expression of these specific gene targets. We intend to test these hypotheses and identify genes that are critical for NSC regulation using methods that examine large numbers of genes at once (genomic methods) and by using standard molecular biological techniques to interrogate specific genes. The proposed studies will provide critical new insights into the mechanisms by which adult neural stem cells are able to self-renew while retaining the ability to differentiate into distinct subtypes of nerve cells. The knowledge gained will be important in the eventual use of stem cells, not only of neural but potentially of other (e.g. hematopoietic) cell types as well, in therapeutic cell replacement to treat human disease.

 

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 Neuronal Specification, Expansion and Self-Renewal of hESC-Derived Precursors

Margaret Elizabeth Ross, MD, PhD

Weill Cornell Medical College
IIRP

 

In order for stem cell therapies to succeed in the repair of brain tissues damaged by stroke, trauma or disease, we must find ways to direct these precursors to take on the characteristics of a diverse range of neuronal types in different parts of the brain. Recent data suggest that neuronal subsets may differ in the expression of hundreds of key genes that distinguish one neuronal subtype from its neighbors. Thus, better ways of defining the genetic signatures of specific neurons are needed to guide us in the differentiation of neuronal precursors before they are ready to be introduced into brain. In addition, it is necessary to control their proliferation (cell division) in order to produce enough of the desired cell type at the appropriate stage of differentiation. This must be accomplished in a way that produces the needed cells, but then allows the cells to stop dividing, exit the cell cycle and differentiate into the desired neuronal type. We found that neural precursors in developing brain and adult stem cell niches use certain proteins to drive the cell cycle when they self-renew, and other proteins when they differentiate into mature neurons. Specifically, cyclin D1 (cD1) and cyclin D2 (cD2) regulators may be used for cell divisions with different outcomes. Our overall goals are to use novel genetic approaches to compare gene expression profiles of differentiated mouse and human embryonic stem cells with gene expression profiles of purified neurons at specific stages of brain development. We will test the hypothesis that manipulation of cD1 and cD2 expression will influence whether a cell division gives rise to two identical daughter cells (symmetric division, promoted by cD2 expression) or results in one neuron and one renewed precursor (asymmetric division, promoted by cD1) or leads to two daughter cells that exit the cell cycle and differentiate into two neurons or two glia (both cyclins turned off). We will further test the requirement of these division modalities for neuronal differentiation. This project will provide essential insights for treatment of neurological disorders affecting cerebellum, and the approach will be applicable to all other brain regions.

 

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 Transcriptional Control of Stem Cell Metastasis of Breast Cancer

Jihe Zhao, MD, PhD

Albany Medical College
IDEA

 

Despite the advances in surgical, radio- and chemotherapies in the past decades, breast cancer remains one of the most lethal cancers for women. Lethality is due to the continued existence of "cancer stem cells" (CSCs) that remain in the body even after complete removal of the affected breast, which are resistant to therapy and can form new tumors. These CSCs can remain undetected and quiescent for more than 10 years before attacking distant, vital organs and initiating new tumor growths that eventually kill the patients. Therefore, effective treatment must be aimed at eliminating these cells. CSCs are very rare, early precursor cells that produce the bulk of differentiated tumor cells, and they are the only sources of all the cell types within a tumor. CSCs are therapy-resistant, whereas the bulk tumor cells are easily eliminated by surgery or other therapies. We propose a new concept: cancer metastasis stem cells (CMSCs) that are derived from but different than primary CSCs. Our central hypothesis is that CMSCs express genes critical for CSC-to-CMSC transition. The goal of this project is to identify the breast CMSCs, determine their potency in forming metastatic tumors in the lungs, and identify the critical regulatory genes. We believe that one such gene encodes a transcription factor, KLF8, as we found that KLF8 regulates cellular processes important for stem cell pluripotency, normal-to-tumor cell transformation and initiation of metastasis. Importantly, KLF8 belongs to the KLF family of proteins, which play a key role in induction and maintenance of induced pluripotent stem (iPS) cells. We also found that KLF8 controls the expression of a majority of the key transcription factors essential for iPS cell formation from breast cells. All these facts support our novel hypothesis that KLF8 plays a critical role in the regulation of CMSCs during breast cancer metastasis.

 

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 Manipulation of Stem Cells for Treating Viral Hepatitis

Mukesh Kumar, PhD

Albert Einstein College of Medicine
IDEA

 

Hepatitis C is a liver disease caused by the hepatitis C virus (HCV) and transmitted through direct contact with blood from infected individuals. There is no vaccine available for this disease and only a small percentage of people respond to the combined therapy of interferon alpha and ribavirin. A majority of infected individuals develop chronic liver disease, and, over a period of time the disease advances requiring liver transplantation and often leading to eventual death. Our hypothesis is that liver cells generated from human stem cells or fetal tissue, when made resistant by expressing genes that will inhibit virus replication or infection, and when transplanted, will replace diseased liver cells with healthy cells leading to liver repopulation. We will modify a cell line (Huh7.5) by introduction of genes that will interfere with virus infection and replication, and then attempt to infect the cells with HCV. In this way, we will identify the genes that inhibit virus infection and replication. Once the most effective genes are identified, we will express them either alone or together in liver cells derived from human stem cells or fetal liver stem/progenitor cells, and test their ability to inhibit virus infection and replication. We will also ensure that our genetic manipulations have not changed the liver properties of these cells and, using mice, that they are safe for engraftment. Our use of stem and fetal-derived liver cells to express these sequences and replace diseased liver cells is a novel approach to finding a cure for chronic liver diseases. We anticipate these studies will provide a basis for applications of stem cells in overcoming chronic viral hepatitis by liver repopulation.

 

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 Identifying Epigenomic Determinants of Hematopoietic Stem Cell Commitment

Ulrich Steidl, MD, PhD

Albert Einstein College of Medicine
IDEA

 

A rare population of blood stem cells ensures the life-long production of all mature blood cells in the human body, including white blood cells, red blood cells and platelets. Blood stem cells, called hematopoietic stem cells (HSCs), are capable of differentiating into all different types of blood cells, but at the same time maintain a constant pool of multipotent stem cells through a process termed self-renewal. The fate of an individual stem cell, to either self-renew or differentiate to a certain cell lineage, is determined by networks of genes controlled in a coordinated manner. The gene regulatory processes are themselves controlled by enzymatic modifications of the DNA or histones, the proteins around which the DNA is wrapped. These so-called "epigenetic" processes do not change the underlying DNA sequence. Although histone modification has an important role in regulating the differentiation of stem cells, modification of the DNA itself, through methylation, the adding of a methyl group, cannot yet be studied in a comprehensive manner. This is due to lack of an appropriate assay and technical limitations of conducting genome-wide studies with the scarcity of HSCs. We will develop a novel assay to study methylation changes as human HSCs self-renew or differentiate, thus filling a critical methodological gap. Our novel approach will provide an epigenetic signature of HSCs and reveal new target regions that regulate HSC commitment towards the different blood cell lineages. Thereby, this study will greatly enhance our knowledge of epigenetic processes and regulation at the early stem cell level, and identify novel key epigenetic changes critical for stem cell function. It will provide us with a novel tool to study other types of human stem cells, and also enable us to study disrupted epigenetic pathways in cancer and leukemia. Most important, these novel assays will provide a new map of targets that can potentially be manipulated by epigenetically-targeted drugs in the future.

 

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 Transplantation of Adult Male Germline Stem Cells Of Drosophila melanogaster

Matthew Wallenfang, PhD

Barnard College
IDEA

 

Adult stem cells maintain most tissues in the body, particularly those that undergo rapid turnover, including skin, gut, and blood. Because these cells persist throughout the lifetime of an organism, they may play a prominent role in aging. We study stem cell aging using the fruit fly, Drosophila melanogaster, specifically looking at male germ line stem cells (GSCs), the stem cells that produce sperm throughout adulthood. In contrast to most stem cells found in other tissues or organisms, Drosophila GSCs are easily identified directly within the tissue. Flies have a relatively short lifespan (~2 months), which facilitates the study of aging in this system, and Drosophila has long been used as a model for cellular processes common to most multicellular organisms including humans. A wealth of genetic and molecular tools are available to study Drosophila GSCs, and it is likely that principles of stem cell function discovered in flies will be applicable to stem cells in humans. We previously found that GSCs do in fact alter their activity during aging, most notably in that they divide less frequently as flies age. Stem cells are known to rely on a number of external cues that direct their behavior. We will determine if age-related changes in stem cells are due to aging of the stem cells themselves, to aging of the cells´ environment, the niche, or a combination of both. Stem cell transplantation-based therapies may be used as treatments for a number of diseases, including diabetes, Parkinson´s, and Alzheimer´s. As many such diseases show increasing incidence with age, it is important to understand how an aged environment affects the function of stem cells. The research proposed herein will answer fundamental questions about how aging impacts stem cells, knowledge which should be applicable to a wide range of stem cell research in other systems.

 

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 The Role of the Chromatin-Remodeling Factor HIRA in Recruitment of Polycomb Complexes and Regulation of Pluripotency

Marja Timmermans, PhD

Cold Spring Harbor Laboratory
IDEA

 

A major question in the study of higher organisms is how the fertilized egg develops into an embryo and then into an adult with heart, liver, skin and all the other organs and tissues. The early embryo contains a unique population of cells, embryonic stem cells (ESCs), that have the capacity to take on many different cell fates. However, this is a one-way street: skin cells cannot normally go back and become ESCs again. The ability to do so would make it possible to regenerate, for instance, a new liver from skin cells, a prospect of great therapeutic importance. We use plants to study one key aspect of how organisms develop from single cells, with the understanding that this knowledge is useful for future work in humans. Although most people do not usually think that way, plants can regenerate very efficiently from adult cells; grass grows back after it is mowed, and a plant that is cut back will sprout new shoots. In fact, scientists showed in the 1970s that single cells can be isolated from a grown leaf and made to develop into a root or shoot or even into an embryo that then forms a complete living organism. Surprisingly, the basic developmental processes are very similar in plants and animals and many of the regulatory factors are the same. We focus on two such highly conserved factors, PRC2 and HIRA. PRC2 ensures the regulatory genes that induce cell differentiation are off in ESCs but turn on when needed later. However, in developed tissues, PRC2 ensures stem cell genes are off and thereby helps safeguard cells from becoming cancerous. Understanding how PRC2 knows which genes to silence, and in which cell types, forms a long-standing question in biology. Our studies in plants suggest that HIRA helps direct PRC2 to the correct targets. The aim of this proposal is to study how HIRA interacts with PRC2 and other proteins to carry out this function.

 

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 Maintenance of Adult Germ Stem Cells by Histone Modification

Tulle Hazelrigg, PhD

Columbia University
IDEA

 

Stem cells in adult tissues are the source for differentiated cells produced by these tissues (e.g. bone marrow stem cells produce many types of blood cells), and also for the regeneration of damaged tissues (for instance the cells for liver regeneration and skin regeneration are produced from stem cells residing in each of these tissues, respectively). One problem when studying adult stem cells is that they are often difficult to identify because they are embedded in tissues and represent a small fraction of the cells in a given tissue. These problems can be overcome in studies of adult stem cells in the ovaries of the fruit fly, Drosophila melanogaster. One important challenge faced by adult stem cells is their need to be maintained for long periods of time as animals age. We will determine how adult stem cells are maintained in tissues for long periods of time. We are particularly interested in determining how changes in the packaging of DNA by modified histones contribute to this maintenance. We previously found that histone methylation by the Eggless protein in Drosophila ovaries maintains germ stem cells, the cells that produce oocytes. We will determine if Eggless works by allowing the correct association of the stem cells with surrounding tissue, by keeping them from committing cellular suicide and/or by preventing them from differentiating into other cells. We will also test whether the effects of Eggless on stem cell maintenance involve a new class of small RNAs, called piRNAs, possibly by repressing expression and mobilization of retrotransposons (DNA elements derived from retroviruses). Our experiments will allow us to identify components that are needed for the process of stem cell maintenance. Understanding how animals maintain stem cells in adult tissues will impact our ability to manipulate these cells for therapeutic purposes.

 

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 Alternative Splicing of mRNA Precursors: Links to Pluripotency of Human Embryonic Stem Cells

James Manley, PhD

Columbia University
IDEA

 

Properties of proteins can to be radically altered at multiple points in their production from genes. One of the main steps where such changes can occur is in RNA splicing, which can include or remove significant portions of the RNA message that is ultimately used to guide protein synthesis. The vast majority of cellular mRNAs are subject to splicing, and splicing patterns contribute (through generation of specific proteins) to a cell developing a unique set of properties that distinguish it from other specialized cells. The ability of stem cells to change into any cell type results from the unique set of proteins produced in these cells. Discovering the exact contents of this set is essential for understanding the origins of this valuable property of stem cells. To identify the unique RNA splicing events, which produce a unique set of proteins in stem cells, we will identify and quantify all known segments of mRNAs that can be included or skipped during the splicing process. We will compare the relative number of these segments in the mRNA population of stem cells to that of several other cell types. Previous studies of this kind compared stem cells to only one other cell type, in effect defining differences between two cell types. In contrast, we intend to compare stem cells to several cell types in order to identify the splicing events that are unique to stem cells. Our study is aimed not only at identifying the unique mRNAs produced in stem cells, but also at establishing the composition of these RNAs to the maximum extent possible with current technology. This will greatly expand our understanding of the unique protein content of stem cells and is essential for understanding their properties, knowledge that may be of great help in custom derivation and engineering of stem cells for therapeutic purposes.

 

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 Molecular Profiling and Differentiation Potential of Purified Adult Neural Stem Cell Lineages

Fiona Doetsch, PhD

Columbia University Medical Center
IDEA

 

Adult neural stem cells (NSCs) continuously generate neurons in restricted parts of the brain and may represent an important source of endogenous cells that can be stimulated for brain repair. It is unknown whether cells, such as adult NSCs and their daughter transit amplifying cells (which are slightly more mature), have the capacity to generate a broad spectrum of neurons and glia cells or only a limited subset. This is a fundamental question to be answered if these cells are eventually to be used for brain repair. It was not possible to obtain pure populations of stem cells to test their potential until recently. However, we developed tools that allow us to do this for the first time. We propose to directly test the capacity of purified stem cells and transit amplifying cells to generate a broad spectrum of neurons and glia by isolating adult mouse NSCs based on the combination of markers they express. We will purify adult NSCs and their more differentiated progeny and transplant them into the developing mouse brain, at a time point when many signals are present that allow the formation of a broad variety of neurons, to test their potential in vivo. Our results will reveal whether adult NSCs are restricted to generating one class of neurons or have the capacity to give rise to a broad spectrum of neurons. We will also evaluate whether transit-amplifying cells have stem cell potential in vivo. This is important for determining the potential of adult NSCs and their progeny for brain repair, either by stimulating resident stem cells or by cell replacement therapy. Furthermore our purification method will enable us for the first time to screen for pharmacological agents that affect stem cell behavior and to perform functional assays.

 

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 Differentiation of ES Cells Into Adrenocortical Lineages

Edward Laufer, PhD

Columbia University Medical Center
IDEA

 

The outer part of the adrenal gland, the adrenal cortex, is an endocrine organ that produces steroid hormones. These hormones are required throughout life to regulate kidney function and help the body respond to stress. Inappropriate function of the adrenal cortex is a significant risk factor for hypertension, and some genetic diseases of the cortex are lethal without hormone replacement therapy. Embryonic stem cells (ESCs) have the potential to provide an unlimited source of adrenal cells that could be used both for studying adrenal function and in cell replacement therapies. However, there are currently no defined procedures for generating adrenal cells from ESCs. We will generate adrenocortical cells from mouse ESCs by recapitulating the normal differentiation steps that occur when the adrenal gland develops in the embryo. Because our approach is to recapitulate the normal developmental progression of adrenal cell formation, we hypothesize the resulting cells will function normally. This project has the potential to provide techniques for generating normal adrenocortical cells, essentially at will. These cells will be useful for studying normal adrenocortical function and how this is disturbed in endocrine diseases. The techniques developed in this project are also important prerequisites for developing normal human adrenocortical cell cultures, which would be an even better model for similar types of studies. In the longer term, these cells and approaches might also be applied towards cell replacement therapies in patients with misfunctioning adrenal glands.

 

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 Epithelial Progenitor Cells as Targets for Cutaneous Neoplasia

David Owens, PhD

Columbia University Medical Center
IDEA

 

Adult stem cells are distinguished by their high capacity for self-renewal and their ability to give rise to daughter cells that are committed to differentiation. These unique properties are essential to ensure that integrity is maintained in tissues that exhibit high rates of cell growth, such as the skin, gastrointestinal tract and bone marrow. The outer layer, or epithelium, of human skin contains permanently residing stem cells that maintain multiple differentiated cell types, including the skin, hair and sebaceous gland. Recent evidence suggests that, as opposed to a single stem cell population, multiple classes of stem cells may exist in adult skin that are responsible for maintaining distinct regions of the skin epithelium. We are interested in the implications of these new stem cell populations and what role they may play in maintaining the integrity of skin, and in skin diseases such as wounding and cancer. While there is compelling evidence to support a role for stem cells in the skin as the target cells for skin cancers, the precise location of these cells remains largely unknown. To determine the cells from which cancers originate, we engineered a mutant mouse model that specifically targets the stem cells residing in the upper isthmus region of hair follicles in adult skin. We will use this model in combination with tissue culture systems, surgical implantation and skin cancer assays to determine whether these stem cells are the origins of skin cancer. Determining the function of genes that are essential for stem cell maintenance is a powerful approach to further our understanding of normal skin development, as well as disease states such as chronic wounds and cancer. Our ability to isolate and manipulate stem cells is a critical step towards better understanding of skin biology, and is of significant impact for tissue regeneration and the genesis of cancer stem cells.

 

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 Exploring the Function of MicroRNAs in Epidermal Stem Cells

Srikala Raghavan, PhD

Columbia University Medical Center
IDEA

 

Understanding the molecular regulation of skin epidermal stem cells holds great potential for developing treatments for a variety of skin diseases, including potentially fatal skin cancers, as well as for skin regeneration for treating victims of burns and other skin blistering diseases. One key to unlocking the secrets of stem cells is to understand the regulation of genes that control their fates. The recent discovery of small RNAs, microRNAs (miRs), as key regulators of gene expression, and major advances in the field of high-throughput screening (HTS) using RNA-interference (RNAi) technology, provide us with a unique opportunity to characterize and test the function of miRs that are specifically expressed in skin stem cells. The use of such miRs as novel molecular tools and their development for clinical application could be crucial to modulate the activity of genes involved in stem cell homeostasis, both in normal development and disease. Using a variety of genomic tools, including microarray analysis of purified cells, we will identify miRs expressed in skin stem cells. We will then use HTS technologies to test the function of the miRs (identified as present or absent in the stem cells) in regulating stem cell behavior. The systematic analysis of the expression and function of miRs in follicular stem cells will provide us with critical insights into the mechanism of gene regulation in the stem cell compartment that is crucial for its maintenance. Moreover, results from this study will provide novel molecular tools that may be employed to treat human disease, such as skin cancers, that arise from the dysregulation of stem cells.

 

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 Does Graded Expression of Shh by Dopaminergic Neurons of the Mesencephalon Influence the Maintenance and Differentiation of Neural Stem Cells of the Adult Subventricular Zone?

Andreas Kottman, PhD

Columbia University Medical Center
IDEA

 

In translational stem cell research, particular interest is devoted to neural precursor/stem cells resident in regions that display neurogenesis in adult mammals. This is based on the theory that neuronal stem cells resident in the adult brain can be coaxed into replenishing brain tissue with functional neurons and glia that are lost in neurodegenerative diseases. In support, many neurodegenerative diseases lead to changes in the cytoarchitecture and qualitative outcome of neurogenesis in the subventricular zone (SVZ), pointing to pathological as well as adaptive and possibly corrective functional alterations in the SVZ dependent on the specific disease. We will analyze neuronal stem cells and their differentiation potential in the SVZ of adult animals as a function of the expression of the cell signaling molecule sonic hedgehog (Shh) in dopaminergic neurons. We will make use of genetically altered mice that are either rendered unable to express Shh or that over-express Shh as a result of inducing neuronal dysfunction in cholinergic neurons. Our experiments will help to assess the dynamic range of potential outcomes of neurogenesis in vivo in the adult brain. Data derived from our studies will be of direct physiological relevance for devising methods that could alter the differentiation path of new neurons produced in the adult brain. Hence, these studies could contribute to finding novel approaches to stimulate in vivo resident stem cells to give rise to particular cells that need to be replaced in neurodegenerative diseases.

 

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 Using Mesenchymal Stem Cells to Deliver Tumor Environment-Activated Quantum-Dot Drug Conjugates Targeting Cancer Bulk and Cancer Stem Cells

Igor Matushansky, PhD

Columbia University Medical Center
IDEA

 

Over the last decade, research in cancer biology determined that cancers are composed of two types of cells. The first, referred to as tumor bulk, or differentiated cells, refers to those cancer cells that make up the majority of the tumor. They are capable of rapid growth and to a varying degree respond to cytotoxic chemotherapy. However, chemotherapy often cannot be delivered fast enough or at high enough doses (due to systemic toxicity) to affect these rapidly growing cells. While the tumor bulk cells grow rapidly they cannot form secondary tumors at distant sites. This latter property is a defining feature of cancer stem cells (CSCs). CSCs are believed to be slow growing, are innately resistant to chemotherapy and have the unique property of initiating tumors. It is these latter CSCs that are believed to be responsible for metastases and recurrences. Effective treatment for cancer must take both of these types of cancer cells into account. Mesenchymal stem cells (MSCs), progenitors of connective tissue (bone, fat, etc.), migrate to and participate in many inflammatory processes including tumor initiation and tumor growth. We will use the innate tumor homing properties of MSCs to deliver drugs that are capable of targeting both tumor bulk and tumor stem cells. Our approach will use the MSCs as a "Trojan Horse" to maximize drug concentrations at tumor sites via site-specific delivery of chemotherapy. This will produce chemotherapy concentrations at tumor sites exceeding what is possible via conventional systemic delivery systems, resulting in complete tumor bulk elimination. Elimination of the tumor bulk would leave CSCs more accessible to stem cell differentiation modifying agents delivered via a similarly targeted mechanism. Our research will create the means with which to deliver cytotoxic chemotherapy in sufficiently high doses to completely eradicate tumor bulk cells, and to effectively target the CSCs using the novel approach of differentiation therapy.

 

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 TCRα Locus Control Region Activity During in vitro Stem Cell Differentiation: Application Towards Improving Lentiviral Gene Therapy Vectors

Benjamin Ortiz, PhD

CUNY - Hunter College
IDEA

 

Embryonic and adult stem cells can self-renew and continually produce new cells of many types as needed within tissues. Thus, in principle, gene therapy strategies in which genes are introduced into stem cells that are then transplanted into a patient can lead to continual production of therapeutic gene products in the desired subset of differentiated stem cell progeny within an organ. This is a promising approach to correcting genetic deficiencies and combating diseases such as cancer and AIDS. Viral carriers (called lentiviruses) have unique properties making them capable of delivering therapeutic genes into the chromosomes of stem cells. However, the stem cell differentiation process leaves lentiviruses vulnerable to silencing by negative regulatory influences at their location on the chromosome. Furthermore, conventional lentiviruses cannot produce developmentally controlled or cell type-specific expression of the therapeutic genes they carry. These limitations can cause transferred genes to be expressed at the wrong place, wrong time, or not at all, raising safety and efficacy concerns. A combination of regulatory DNA sequences must be identified for inclusion in lentiviruses that can properly direct gene expression and that is independent of regulatory influences at their site of integration on the chromosome. We study a large regulatory DNA sequence called a locus control region (LCR) that regulates mouse T cell receptor α (TCRα) gene expression. We identified DNA sequences within this LCR that direct developmentally controlled and cell type-restricted expression in T cells, and that is protected from the influence of nearby regulatory DNA. Using a novel embryonic stem cell-to-T cell differentiation system, coupled to lentiviral constructs containing the LCR, we will apply our knowledge to improving the safety, specificity and efficacy of lentiviral constructs used in stem cell gene therapy.

 

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 Characterization of Extraembryonic Endoderm (XEN) Cells

Anna-Katerina Hadjantonakis, PhD

Memorial Sloan-Kettering Cancer Center
IDEA

 

Regenerative medicine using cell-based therapies holds enormous promise for patients with degenerative and other diseases. Cell-based therapies rely on methods to direct the differentiation of stem cells, usually embryonic stem cells (ESCs), grown in culture. Our preliminary observations suggest that ESCs, the near-universal stem cell type for directed differentiation experiments, may not be able to form all cell types of the respiratory and digestive tracts and their associated organs, which include the lungs, liver and pancreas. If this is the case, alternative stem cell types need to be identified that can be grown in culture in unlimited quantities and can be differentiated into these cell and tissue types. Our central hypothesis is that XEN cells, an early mammalian embryo-derived stem cell type that can be grown in culture, can be differentiated into cell types of the respiratory and digestive tracts and their associated organs, including the lungs, liver and pancreas. XEN cells may, therefore, represent an alternative stem cell source to ESCs for certain cell types. We will derive XEN cells from early mouse embryos, propagate and characterize them in culture, and analyze the cell types they give rise to when directed to differentiate both in culture (ex vivo studies) and in the context of a developing embryo or adult organism (in vivo studies). These studies will have important bearings on the basic biology of XEN cells, a little understood stem cell type, that may potentially be used as an alternative to ESCs for the production of cells and tissue of the respiratory and digestive tracts and associated organs.

 

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 Investigation of Polycomb-Mediated Chromatin Alterations During Embryonic Stem Cell Differentiation

Emily Bernstein, PhD

Mount Sinai School of Medicine
IDEA

 

The sequencing of the human genome provided researchers with a blueprint of our genetic code and revolutionized molecular biology research. However, understanding how the genetic code is interpreted and functionally translated still remains a great challenge. There are multiple factors that affect the readout of the genome without directly changing the underlying DNA sequence. These are referred to as ´epigenetic´ mechanisms and include chemical modifications to both the DNA itself and the histone proteins that package the DNA into chromatin. Histones comprise the major protein component of chromatin and are chemically modified in numerous, diverse and dynamic ways that can have drastic consequences on the regulation of genes (activation or repression). This is achieved, in part, through the binding of proteins that bring about changes to the chromatin template. Polycomb is one such histone-modifying factor originally identified in flies; five Polycomb proteins are reported in mammals. We are interested in understanding the role of the mammalian Polycomb proteins during embryonic stem cell (ESC) differentiation, as their unique functions remain poorly understood. Based on our previous data, we will focus on one of the Polycomb proteins, Cbx7. We will investigate how this protein is targeted to chromatin during the course of ESC differentiation and we will identify potentially novel classes of RNA molecules that mediate this Polycomb protein´s specificity and targeting. We will differentiate female ESCs into neural progenitors and examine the chromatin changes taking place over the course of one week. We will utilize biochemical and cell biology techniques that allow us to purify cellular Polycomb complexes as well as associated nucleic acids. Unraveling the mysteries of chromatin changes that take place during stem cell differentiation will facilitate our understanding of how we can manipulate these processes more efficiently, with the ultimate goal of developing and improving stem cell therapies.

 

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 Copper Transporter-1, Ctr1: A Critical Regulator of Embryonic Stem Cell Differentiation

Stuart Fraser, PhD

Mount Sinai School of Medicine
IDEA

 

Copper is an essential trace element in our diet and is transported into cells by a protein on their surface named copper transporter-1 (Ctr1). Embryonic stem cells (ESCs) that have both copies of the Ctr1 gene inactivated are remarkable in that they fail to form mature cell types under conditions that induce differentiation in normal ESCs: Ctr1 null ESCs remain ESCs. This makes Ctr1 null ESCs a very useful system for identifying the requirements for differentiation. ESCs that have lost one copy of Ctr1 are remarkable in that they do not form tumors when injected into adult mice, unlike normal ESCs, which form teratomas. This tumor-forming potential of ESCs has prevented them from fulfilling their promise as a major source of material for patients requiring transplants, such as pancreatic β-cells, neurons or skin cells. Our system is unique in that these latter ESCs can differentiate, but do not form tumors, and may help us understand these different potentials and design methodologies to prevent tumor formation. We will determine the signals transmitted to ESCs via Ctr1, why loss of one Ctr1 gene impairs tumor formation, whether this can be translated into human ESC systems, whether inhibition of Ctr1 can function in the reprogramming of adult cells to make induced pluripotent stem cells, and finally, whether or not Ctr1 functions in adult stem cells.

 

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 Elucidating the Role of the JAK/STAT Pathway in Stem Cell Self-Renewal

Erika Bach, PhD

New York University School of Medicine
IDEA

 

Stem cells have the unique property of producing multiple types of specialized cells and they can regenerate damaged tissues throughout the lifetime of an individual. A stem cell can divide such that one descendent remains a stem cell while the other one becomes a specialized cell type, such as skin or hair. Understanding how a stem cell chooses between remaining a stem cell or becoming a specialized cell holds great potential for breakthroughs in the treatment of cancer, diabetes and spinal cord injuries. JAK and STAT signaling proteins are important regulators of stem cell numbers in mammals and in the fruit fly, Drosophila. In normal cells, the activities of JAKs and STATs are regulated, and they work together in a coordinated manner to relay signals from the outside of a stem cell to its interior. However, when these proteins are turned on constantly in humans they cause blood cancers and they are implicated in more than 50% of breast, prostate and lung cancers, 50% of multiple myelomas and 95% of head and neck cancers. We previously showed that activating JAK and STAT proteins in Drosophila tissues leads to dramatic overgrowths that consist of stem cells and resemble human tumors. Furthermore, when these proteins are inactivated, stem cells are lost. We are interested in finding the genes that are targeted by JAK and STAT signaling pathways and then discovering the functions of these genes in stem cells. One such gene we previously identified is Chinmo, which must be present in stem cells in order for them to remain stem cells. When Chinmo protein is absent from stem cells, they differentiate into specialized cells. We will discover how Chinmo controls the numbers of stem cells in flies and mice, and our results will likely provide critical insights into the functions of the Chinmo counterpart in humans, which may have implications for cancer.

 

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 Study of the Cell-of-Origin and Cancer Stem Cells in Melanoma

Eva Hernando, PhD

New York University School of Medicine
IDEA

 

Melanoma development is classically perceived as a stepwise process in which mature melanocytes in the epidermis progressively acquire genetic mutations in oncogenes and/or tumor-suppressor genes, ultimately leading to metastatic melanoma. Yet clinically, only 26% of melanomas observed evolve from nevi (skin lesions including moles and birthmarks), and less than 50% are associated with dysplastic (abnormal) nevi. This indicates that the majority of melanomas arise from normal-appearing skin and not from dysplastic nevi, suggesting that melanoma development may not follow the classic linear mode of progression. Thus, although the apparent target of transformation is differentiated melanocytes, melanomas may also be derived from transformed melanocytic stem or progenitor cells. These cells, once transformed, may be responsible for the highly infiltrative and metastatic behavior of these tumors. We are engineering transgenic mouse models to understand if the initial oncogenic transformation occurs in a progenitor or a more mature cell. We will use these models to study the differentiation potential and metastatic behavior of different populations of tumor cells. The identification of a ´melanoma stem cell´ population from mouse melanomas will provide insights into the mechanisms of tumor maintenance, recurrence and chemoresistance, and may contribute to a better understanding of the pathogenesis of melanoma, the identification of prognostic biomarkers and help identify potential new targets for therapeutic intervention. This project will increase our understanding of the cellular and molecular mechanisms underlying melanoma pathogenesis.

 

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 Derivation and Characterization of Dendritic Cell Lineages From Hematopoietic Stem Cells

David Levy, PhD

New York University School of Medicine
IDEA

 

Dendritic cells (DCs) are key to controlling immunity in response to infectious diseases and to preventing autoimmune disease. Although they are of hematopoietic (blood cell) origin, the identity and potential heterogeneity of their progenitors among the hematopoietic stem cell population have remained enigmatic. Furthermore, it is unclear if different types of DCs originate from the same or different precursor stem cells. Due to the great importance of these cells for immune regulation, through their role in orchestrating almost all aspects of immune responses and immune homeostasis, there is a compelling need to understand their derivation from progenitors and their functional heterogeneity. However, studying these cells has been hampered by their rarity and the difficulty of culturing them in vitro. Although a few leukemia cell lines that retain DC properties have been isolated, no one has found a reliable method for maintaining and expanding DC progenitors in culture in order to provide the raw materials for molecular and biological analysis. Our goal is to develop and optimize a method to grow DC progenitors in vitro in a manner that will promote their proliferation and expansion indefinitely, yet allow them to retain the ability to differentiate into growth-arrested and functional end-stage cells. We will study the mechanisms and processes that regulate the commitment of stem cells to DC progenitors, as well as the differentiation of progenitors into functional DCs. This project will create a number of valuable DC lines and will uncover important information concerning the process of stem cell commitment and progenitor cell differentiation. This is a high risk/high payoff exploratory project that should be considered a proof of principle for validating the concept of producing DC lines that are viable in culture. This research could provide the impetus to develop similar approaches for DC propagation for therapeutic purposes.

 

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 Establishing the C. elegans Germline Stem Cell Niche

Jeremy Nance, PhD

New York University School of Medicine
IDEA

 

Stem cells possess the ability to form the many different types of cells found in the body. A principal goal of stem cell research is to manipulate stem cells in culture and force them to become specific cell types that could then be used to replace damaged or diseased cells within the human body. Understanding how to manipulate stem cells in vitro requires that we learn how stem cells are controlled within their normal environment. Tissue stem cells often reside in a structure, or microenvironment, called the "niche," from which they receive important regulatory signals. How the niche-stem cell interaction is established during development, such that these regulatory signals are properly received, is poorly understood in any tissue and organism. We will examine how a simple niche-stem cell interaction is established during development in a model organism, the nematode worm C. elegans. C. elegans offers many advantages for these studies, such as a short life cycle, transparent body where the niche can be viewed in live animals, and tools to study the genes that control niche-stem cell formation and signaling. Moreover, the niche-stem cell system (the gonad primordium) we study is very simple, it consists of two niche cells and two stem cells. We are in the process of analyzing how the gonad primordium is formed in live embryos. Our preliminary results suggest that the gonad primordium develops through a series of regulated interactions between specific groups of cells. Our experiments will allow us to identify the cell-cell interactions and the genes that are important for the establishment of a niche-germline stem cell system in a live animal. Our results will provide a foundation for understanding how cultured stem cells can be best manipulated for regenerative medicine applications.

 

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 Novel Surface Markers for Neural Stem Cell Enrichment

Qin Shen, PhD

Regenerative Research Foundation
IDEA

 

Neural stem cells (NSCs) are stem cells that are specialized to make nervous system cells. They are very rare in the body, making them difficult to locate and study. NSC markers would be of great value to research and therapeutic applications because such markers would allow substantial enrichment of these cells. We analyzed gene expression in a population of enriched neural stem and progenitor cells and identified several promising candidates as novel NSC markers. We propose to determine the efficacy of combining our novel markers and existing NSC markers to further enrich NSCs using fluorescence activated cell sorting. We will perform tissue culture and animal assays to determine the efficacy of the markers for enriching NSCs. The proposed work is innovative because our data set produced candidate NSC enrichment markers that have not been studied previously in this context. Additionally, a search of the NIH database revealed that currently no investigators are searching for new NSC surface markers, placing our research in a position to fulfill this unique need. Our results will be significant because identification of surface markers to improve isolation of NSCs will advance both basic research and potential therapeutic applications of these cells. The goal of this proposal is to develop a new technology to select for these rare cells and produce an enriched population that can be studied and grown for therapeutic purposes, including drug screening. Moreover, these studies will introduce these novel biomarkers to the field of NSC biology and thereby open up new avenues to research.

 

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 Identification and Characterization of Endothelial Niche Factor(s) That Stimulate Self-Renewal and Neurogenesis of Neural Stem Cells (NSCs)

Jun Yan, PhD

Regenerative Research Foundation
IDEA

 

Neural stem cells (NSCs) can reproduce themselves and can differentiate into all kinds of neurons and glial cells in the nervous system. Their growth and differentiation are influenced by their microenvironments. NSCs in embryos and adults are located very close to blood vessels and/or ventricular systems. Our lab demonstrated that endothelial cells, the cells that line the inner surface of blood vessels, secrete a factor(s) that stimulates mouse embryonic cortical NSCs to grow and generate more neurons. We tested many soluble growth factors, but none of them reproduced the effect of the endothelial cells. Thus, we hypothesize that blood vessel endothelial cells secrete either a novel protein or a known protein with previously unknown NSC-stimulating activity. We will use two major complementary methodologies to identify this factor. First, we will use microarray data to screen for known molecules, which will reveal genes expressed by a specific kind of cell. Second, we will purify the secreted putative novel NSC-stimulating activity from the cell medium in which the endothelial cells are cultured. This project will greatly advance the understanding of the basic biology of NSC-endothelia interaction. The incorporation of this NSC-stimulating molecule will improve NSC culture without the complications and the high cost related to co-culturing endothelial cells with NSCs. Reliable, abundant sources of NSCs are essential for basic research and for future clinical applications.

 

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 High Transporter and Aldehyde Dehydrogenase Activity in Benign and Cancer Prostatic Stem Cells

Wendy Huss, PhD

Roswell Park Cancer Institute
IDEA

 

Androgen deprivation therapy is the standard of care for advanced prostate cancer. The initial response, reduction of tumor volume and decrease in PSA levels, is ultimately followed by recurrent prostate cancer that is resistant to androgen deprivation therapy. The reproducible recurrence following therapy indicates that a common cell survives androgen deprivation and is the source of recurrent prostate cancer. Cancer stem cells (CSCs) are a likely source of both the initial prostate cancer and recurrent prostate cancer. Therapy against CSCs would decrease the risk of developing recurrent prostate cancer and ultimately reduce suffering and mortality due to prostate cancer. The CSC hypothesis contends that a single cell is capable of tumor generation, however few analyses are designed to examine the potential of single cells to cause cancer. We will select cells based on the protective functions associated with CSCs, test them for stem cell properties and determine potential therapeutic strategies. This proposal uses prostate cancer as a model of solid tumors but the results will be applicable to most solid tumors. Our preliminary data suggest cellular efflux of toxins and aldehyde dehydrogenase activity can be used to isolate benign or malignant stem cells that are capable of generating prostate tissue and tumor from a single cell. To address this, we will now isolate cells from human prostate specimens that were surgically removed in treating cancer. We will examine these cells using gene expression analysis, their ability to grow in 3-dimensional culture and their ability to generate prostate tissue and/or tumors in vivo. Functional properties are likely to be shared by CSCs of multiple organs, therefore these studies will determine if inhibition of these functions can provide a therapeutic option for cancer treatment. Additionally, we may also identify therapeutic targets associated with these functional properties.

 

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 Role of Cancer Stem Cells in Resistance to Targeted Therapy and Tumor Recurrence

Bonnie Hylander, PhD

Roswell Park Cancer Institute
IDEA

 

One of the most depressing aspects of cancer is the fact that many patients who experience the joy of a complete remission will later find that their cancer has returned. Cancer researchers deal with this problem by using stronger drugs and testing new drug combinations. Unfortunately, this approach often does not improve overall survival, but increases the toxicity to normal tissue caused by most chemotherapies and contributes to debilitating sickness in patients. A new generation of drugs is emerging that specifically "targets" tumor cells and it is hoped that these new drugs will be effective against tumors while greatly reducing side effects in patients. A new paradigm is emerging suggesting that tumors contain a subpopulation of rare "cancer stem cells" (CSCs) that can constantly renew themselves and also give rise to the cells which form the bulk of the tumor. Importantly, these CSCs appear to be resistant to chemotherapy and radiation, which kill the majority of the bulk tumor cells. Consequently, it is now hypothesized that the reason many tumors regrow is that even the strongest cancer drugs do not kill the CSCs. The identification of targets for new drug development is furthered by substantial progress in understanding the biology of tumors. However, whether CSCs will be resistant to these therapies remains unknown. We are using human pancreatic tumor CSCs to assess the targeted therapy, Apo2L/TRAIL. We will examine patients´ CSCs to understand the responses to Apo2L/TRAIL, which is 30% effective, and to conduct molecular analyses. Our model system will help generate important new information regarding the role of CSCs in tumor resistance to therapy, and this research will speed the identification of therapies, which will truly prolong survival after pancreatic cancer.

 

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 Glycan Engineering of Stem Cells

Sriram Neelamegham, PhD

SUNY - University at Buffalo
IDEA

 

The regenerative potential of stem cells can only be realized through the efficient delivery of these cells to the required sites in the human body. Our proposal examines ways to enhance this delivery process under conditions of fluid flow that are found in human circulation. The homing of white blood cells (leukocytes) to sites of vascular injury or inflammation has been under intense investigation for nearly 20 years, and methods to modulate leukocyte-endothelial cell binding properties (endothelial cells are the cells that line all blood vessels) are well developed. We will engineer stem cells to enhance their binding to sites of local inflammation, thereby improving delivery of stem cells to endothelial cells. We will engineer stem cells to incorporate all the carbohydrate structures and scaffold proteins that human blood cells normally employ to bind to the vascular endothelium at sites of inflammation. We will then test the degree to which alteration in stem cell carbohydrate structures enhances the ability of the stem cells to bind the endothelium under fluid flow and inflammatory stimulus conditions. If successful, this project has the potential to open a new method for targeting stem cells to local tissue, which will impact the practice of regenerative medicine.

 

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 Modulating Stem Cell Differentiation Using Novel Allograft Scaffolds for Cartilage Repair

Hani Awad, PhD

University of Rochester
IDEA

 

Over 50% of orthopedic injuries involve cartilage in articular joints. The economic burden associated with these injuries costs the U.S. health care system more than $40 billion annually. Despite early surgical interventions, such injuries often progress to degenerative osteoarthritis (OA). Therefore, any therapy that improves surgical outcome and slows OA progression will have significant clinical and economic impact. To address this problem, we recently developed a process, for which a patent application is pending. The process uses articular cartilage (allograft) tissue from deceased organ and tissue donors. The cartilage allograft is minced, decellularized, dissolved and then re-formed through freeze-drying as a porous, sponge-like construct for implantation to repair cartilage injuries. These allografts should promote articular cartilage regeneration by delivering mesenchymal stem cells (MSCs) to the site of injury and providing the MSCs with a native substrate onto which they can differentiate to produce new cartilage tissue. Initially, we will optimize the processing of porous cartilage constructs to maximize the differentiation of the MSCs into cartilage cells and tissue. Once optimized, we will use the constructs in a preclinical study with an established rabbit model of knee articular cartilage repair to generate proof-of-concept data. The porous cartilage constructs are engineered to contain growth factors known as cartilage-derived morphogenetic proteins. Therefore, these constructs are analogous to demineralized bone matrix (DBM) and similar bone derivatives that contain bone morphogenetic proteins and which are typically used to stimulate MSCs for bone repair and fusion. DBM and similar bone fillers are a major product line in the orthopedic implant industry. Porous cartilage-derived constructs can potentially have a similar impact in the cartilage repair market, once validated in preclinical and clinical studies, to truly realize the potential for MSC-based repair of articular cartilage.

 

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 Deciphering the Role of Mammalian Ric-8 Proteins in Stem Cell (Asymmetric) Division

Gregory Tall, PhD

University of Rochester
IDEA

 

Division of stem cells produces either two new stem cells (symmetric division), thereby increasing the pool of stem cells, or one new stem cell and a cell that will differentiate and contribute to tissue growth (asymmetric division). Asymmetric division fulfills the body´s need to grow or replace the cells that make up tissues and organs. The ´decision´ of whether to divide symmetrically or asymmetrically is brought about by the cellular machinery that mechanically drives cell division. In cancers of stem cell origin, data suggest the decision balance is shifted towards the overproduction of stem cells. It is not completely understood how this decision is made, since the identity of the signals sent to the cell division machinery that determine symmetric or asymmetric cell division are unknown, as are the malfunction(s) in this machinery that cause stem cell cancers. We study a collection of proteins that are thought to influence decisions of the cell division machinery. Specifically, we study two similar proteins, Ric-8A and Ric-8B, and consider them to be upstream activators of this process. We knocked out genes encoding both Ric-8A and Ric8-B in mice to determine if there are defects in cell division associated with the loss of either gene´s expression. Removing either gene results in death of the mice before birth. We are now generating embryonic stem cells from mutant embryos that we will use to assess the processes of cell division in the complete absence of either or both genes, and then compare the ability of these stem cells to form unique embryonic and brain cells in the laboratory. Our research will help scientists understand the mechanism by which stem cells divide normally and in defective genes. Our studies will further the use of stem cells to treat stem cell-derived cancers and other diseases.

 

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 Identifying the Stem Cells in Malignant Melanoma

Lei Xu, PhD

University of Rochester
IDEA

 

Cancer stem cells (CSCs) are tumor-initiating cells thought to be resistant to chemotherapy and therefore responsible for the recurrence of tumors after cancer treatment. Eliminating CSCs is crucial for effective cancer treatment and identifying which cells are CSCs is the first important step in attacking them. Based on the hypothesis that metastatic cells are similar to CSCs, because they can grow whole new tumors, we identified new markers of CSCs in malignant melanoma, a highly aggressive cancer that is resistant to therapy. Expression of these markers, four genes called BMPR1A, EDNRB, EDBB3 and FZD7 (abbreviated as BEEF), correlate with the metastatic states of the cells, and high expression levels of certain genes in human melanoma metastases correlate with shorter survival in patients. We will now isolate cells expressing high levels of BEEF, inject them into immunodeficient mice and assess the outcomes in comparison to cells expressing reduced levels of BEEF. We will also assess the stem cell properties of the BEEF-positive cells and test the effects of BEEF depletion in melanoma progression. Finally, we will use commercial inhibitors of BEEF proteins to inhibit melanoma progression in tumor-bearing mice. The proposed research aims to establish a new paradigm for identifying CSC markers and will contribute to the eradication of cancer cells in malignant melanomas. The results will provide important insights and research tools for understanding CSCs in malignant melanoma, lead to more effective treatment strategies for this deadly disease and contribute to more effective cancer treatments in general.

 

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 Hematopoiesis of the Gut Immune System and Gut Immunity in Immunodeficient Mice

Maria López, PhD

Wadsworth Center
IDEA

 

The gut is not only involved in the digestion and absorption of food, it is also a huge immune organ. In healthy subjects the intestinal wall separates the outside world of food and bacteria from thousands of immune cells that can produce antibodies and fight bacteria, fungi and viruses. Children born with severe immunodeficiency lack an immune system. Similarly, in patients undergoing chemotherapy and radiation for leukemias and lymphomas, all the immune cells in the gut die. It is not known if gut immune cells recover after patients are transplanted with either bone marrow or cord blood, and some of these patients suffer chronic gut infections and food intolerance. We will use "humanized" mice, immunodeficient mice in which the immune system is rebuilt by human cord blood transplants, to study whether or not human immune cells can establish lymphocytes (B and T cells) in the gut as efficiently as in the spleen and other lymphoid organs. After transplantation we will investigate ways to accelerate development of gut immunity and examine the function of new gut immune cells. This is the first time that immunodeficient mice will be humanized to study the ability of human cells to reconstitute the mouse gut. We will also be the first investigators to determine if human cells in the mouse gut can protect mice from a pathogen such as Cryptosporidium parvum, which causes deadly diarrhea in untreated AIDS patients. This project will provide a unique model to study how the gut immune system recovers after immunodeficiency followed by cord blood stem cell transplantation, and will help define the mechanistic problems that occur with development of gut immunity.

 

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 Deriving Forebrain Interneurons from ES Cells by Inducible Expression of Lhx6

Stewart Anderson, MD

Weill Cornell Medical College
IDEA

 

Inhibitory interneurons of the cerebral cortex have critical roles in brain function. These roles are served by subtypes of interneurons distinguished by their neurochemistry, morphology, connectivity and physiology. The abnormal development and function of cortical interneurons is implicated in the pathology of several major neurological and psychiatric disorders, including schizophrenia, autism and epilepsy. Particularly in the case of medication-resistant seizures, cell-based therapy has been proposed as an alternative to surgical intervention. Our major research focus is to understand how different types of cortical interneurons are generated during development. A second focus of this work is exploring the use of cortical interneuron transplants as a cell-based therapy for mediation-resistant seizures. Part of this effort involved developing methods for making cortical interneurons from embryonic stem cells (ESCs). We had initial success at generating cortical interneurons from human and mouse ESCs, bolstering our enthusiasm for the project. However, to use this system as a tool for the study of interneuron development, and potentially for a cell-based therapy for forebrain disorders such as medication-intractable seizures and Parkinson's disease, we need to greatly improve the efficiency of our protocol. We identified two transcription factors, Nkx2.1 and Lhx6, master regulator genes that activate or repress the expression of other genes, that function to specify the fate of most cortical interneurons. We will now use interneuron-competent ESCs, in combination with inducible Nkx2.1 or Lhx6, to drive differentiation specifically to interneurons. We will then test the function of these cells by transplantation in the cortex of mice. Success at driving interneuron fates in this proposal would greatly improve the rationale for future experiments in which ESCs are manipulated to become drug delivery vehicles or used directly for transplantation.

 

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 Regulation of Mammary Stem Cells by Wnt Signaling

Anthony Brown, PhD

Weill Cornell Medical College
IDEA

 

Stem cells in an adult tissue such as the breast are a small but critical population of cells that govern the production of diverse, specialized cell types in the tissue while retaining an ability to replicate almost indefinitely. Recent evidence indicates that stem cells with altered properties are frequently found in cancer where they may play important roles in maintaining the continuous production of new cells in the tumor. The Wnt signaling pathway is an important mechanism by which cells receive signals that regulate their biological behavior. Wnt signaling is frequently activated aberrantly in cancer and contributes to tumor formation. Current evidence suggests that it may do this by altering the behavior of stem cells in some way, but this is partly speculative and few details are known. We use a mouse model of activated Wnt signaling to understand the effects on mammary stem cells and how this leads to cancer. We will use the Wnt-induced breast cancer model to isolate the stem cell population, then study the biology of these cells by manipulating the levels of Wnt signaling and determining how their behavior is altered. Additionally, we generated a mouse model in which Wnt signaling is inhibited, rendering the mice resistant to Wnt-induced cancer, and we will also use this model to study the mammary stem cell population. Because the same signaling mechanisms are activated in human cancer, we expect our analysis of mammary stem cells in this mouse model to elucidate the biological mechanisms by which the altered stem cells lead to cancer. This information may be critically important for devising future methods of clinical intervention against cancer stem cells.

 

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