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Investigator Initiated Research Projects (IIRP) and Innovative, Developmental or Exploratory Activities (IDEA) in Stem Cell Research (Rd 5) - 2016

RFA #: 1602230252

IIRP Awards

RFA #: 1206180230
Institution PI Amount Project Title
Albert Einstein College of Medicine Dawlaty, Meelad $1,080,000 Regulation of Embryonic Stem Cells by Noncanonical Functions of Tet Enzymes
Columbia University Medical Center Owens, David $1,072,475 Role of Epithelial Stem Cells in Age-Related Loss of Tactile Acuity
Columbia University Medical Center Tsang, Stephen $1,080,000 Comparative Effectiveness of CRISPRn, CRISPRd and CRISPRi in Patient-Specific Stem Cells
Columbia University Vunjak-Novakovic, Gordana $1,079,599 Optogenetic Regulation of Cardiac Arrhythmia Using Patient-Derived iPS Cell Models
Hunter College - CUNY Ortiz, Benjamin $845,858 Transforming T Cell Gene Therapy Via Locus Control Region-Driven Gene Regulation
Icahn School of Medicine at Mount Sinai Ding, Bi-Sen $1,080,000 Editing Hepatic Sinusoidal Vascular Niche to Induce Fibrosis-Free Liver Repair
Icahn School of Medicine at Mount Sinai Chaudhry, Hina $1,048,800 Novel Biomarkers and Placental Progenitors
Icahn School of Medicine at Mount Sinai Ghaffari, Saghi $1,061,238 Mitochondrial Heterogeneity in the Regulation of Human Leukemic Stem Cells
Icahn School of Medicine at Mount Sinai Moore, Kateri $1,074,900 Elucidating the Mechanisms of Hematopoietic Reprogramming
Icahn School of Medicine at Mount Sinai Papapetrou, Eirini $995,000 Reprogramming Leukemia to Study Human Pluripotent Stem Cell-Derived Hematopoiesis
Icahn School of Medicine at Mount Sinai Wang, Jianlong $1,077,840 Molecular Mechanisms of Naive and Primed Pluripotency
New York Medical College Chung, Sangmi $1,068,420 Stem Cell-Derived Human Cortical Interneurons for Cell Therapy for Epilepsy
The New York Stem Cell Foundation Fossati, Valentina $879,158 A Patient-Based Cellular Platform for Clinical Translation of Chemical Remyelinating Therapies
New York University Desplan, Claude $1,066,919 From Stem Cells to Differentiated Neurons: A Single Cell Transcriptional Approach
New York University School of Medicine Aifantis, Iannis $1,080,000 Enhancer-Promoter Dynamics Governing Hematopoietic Stem Cell Transcriptional Networks
New York University School of Medicine Bach, Erika $1,080,000 Signaling Mechanisms that Coordinate the Behavior of Stem Cells and Their Niche
New York University School of Medicine Maass, Karen $1,075,682 Generating Cardiac Conduction System Cells from Human Induced Pluripotent Stem Cells
New York University School of Medicine Placantonakis, Dimitris $993,892 Transcription Factor Network Reorganization by Mutant IDH1 in Gliomagenesis
New York University School of Medicine Salzer, James $1,056,232 Mobilizing Neural Stem Cells for Remyelination
The Rockefeller University Fuchs, Elaine $1,080,000 Identifying the Alterations in Skin Stem Function and Niche Interactions Responsible for Cancer
The Rockefeller University Greengard, Paul $986,000 Identification of Genetic Targets of a Genetic Master Regulator, a Neuroprotective Factor in Parkinson’s Disease
Sloan-Kettering Institute for Cancer Research Huangfu, Danwei $1,049,630 Novel Regulators of Endoderm Differentiation
Sloan-Kettering Institute for Cancer Research Studer, Lorenz $1,035,000 Human PSC-Derived Schwann Cells for Modeling and Treating Diabetic Neuropathy
University at Buffalo - SUNY Andreadis, Stelios $1,066,777 Skin-Derived Neural Crest Stem Cells for Treatment of Neurogenic Disorders
Weill Medical College of Cornell University Evans, Todd $1,074,420 Deriving Functional Cardiac Pacemaker Cells from Human Embryonic Stem Cells
Weill Medical College of Cornell University Rodriguez-Boulan, Enrique $1,053,409 Hedgehog Signaling in Adult Choroid: Implications for Stem Cell Therapy of Retinal Disease
Weill Medical College of Cornell University Wen, Duancheng $1,080,000 Contribution of H3.3-Mediated Chromatin Dynamics to Stem Cell Pluripotency and Developmental Potential

IDEA awards

Institution PI Amount Project Title
Albert Einstein College of Medicine Guo, Wenjun $330,000 Role of Novel Unipotent Stem Cells in ER-Positive Breast Cancer
Albert Einstein College of Medicine Hebert, Jean $330,000 Neovascularization for Optimal Survival of Neural Transplants
Columbia University Medical Center Egli, Dieter $324,551 Derivation and Functional Characterization of (Haploid) Androgenetic Human Pluripotent Stem Cells
Columbia University Medical Center Wichterle, Hynek $330,000 CRISPR Based System for Genetic Screening of Embryonic Stem Cell Differentiation
Cornell University Weiss, Robert $330,000 Molecular Determinants and Therapeutic Sensitivity in Testicular Germ Cell Cancers
Icahn School of Medicine at Mount Sinai Huang, Alice $330,000 Identification of Tendon Stem Cells Using Genetic Lineage Tracing
Icahn School of Medicine at Mount Sinai Sahoo, Susmita $330,000 Epitranscriptomic Mechanisms of Human CD34+ Stem Cell-Derived Exosomes
Icahn School of Medicine at Mount Sinai Wang, Jianlong $330,000 Pursuing Totipotency with miRNAs and MERVL
The New York Stem Cell Foundation Johannesson, Bjarki $327,654 Metabolic Maturation of Stem Cell Derived Beta Cells
New York University School of Medicine Aifantis, Iannis $330,000 Understanding the Impact of 3D Genome Topology in Early Hematopoietic Stem Cell Transformation
New York University School of Medicine Bach, Erika $330,000 Identifying New Germ Line Stem Cell Maintenance Factors
New York University School of Medicine David, Gregory $330,000 Transcriptional Regulation of Hematopoietic Stem Cells Functions
New York University School of Medicine Neel, Benjamin $330,000 Targeting TET2-Deficiency in Hematopoietic Stem Cells with High-Dose Vitamin C
New York University School of Medicine Park, Christopher $330,000 Identification of Novel Regulators of Hematopoietic Stem Cells
Sloan-Kettering Institute for Cancer Research Joyner, Alexandra $307,945 Angiocrine Functions of Endothelium in Regulating Stem Cell Driven Cerebellar Repair
University at Albany - SUNY Wang, Jun $275,445 Optimizing Replacement Cell Therapies Through Microchip Analysis of Cell Cooperativity
University of Rochester Small, Eric $327,039 Harnessing the Regenerative Potential of the Epicardium
Weill Medical College of Cornell University Min, Irene $311,000 Engineering Induced Pluripotent Stem Cells for "Off the Shell" Adoptive T Cell Therapy

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IIRP awards

Regulation of embryonic stem cells by noncanonical functions of Tet enzyme
Meelad Dawlaty, PhD
Albert Einstein College of Medicine

Induced pluripotent stem cells are stem cells that can be produced from any cell of the body and hold large promise to cure a wide variety of diseases. Because these induced pluripotent stem cells can be generated from older people, they may contain genetic mutations that accumulate as part of the normal aging process of all cells. These genetic mutations are a potential obstacle to the use of these stem cells for clinical applications. One goal of this project is to use whole genome sequencing to determine which cell type accumulates the least number of mutations during aging and is therefore optimal for the production of induced pluripotent stem cells. We will test the hypothesis that adult stem cells have evolved mechanims to avoid the accumulation of mutations during aging and are thus a good source of cells to be reprogrammed into induced pluripotent stem cells. To do this, we will compare blood and adult muscle stem cells to skin cells. Another goal of this project is to try to understand the consequences of the genetic and epigenetic mutations that are found in induced pluripotent stem cells using novel techniques that allow us to distinguish chromosomes from the mother from those from the father. Minimizing the number of mutations in induced pluripotent stem cells is important because these aging-related mutations might lead to cancer or reduce the functionality of cells, thereby reducing their therapeutic efficiency and usefulness.

Role of Epithelial Stem Cells in Age-Related Loss of Tactile Acuity

David Owens, PhD
Columbia University Medical Center

As we age, our skin gradually loses its sense of touch and this deficit is thought to contribute to the decline of postural stability and hand grip, and the resulting increase in falling frequency, which is a major factor for determining quality of life and the ability to live independently for the elderly. However, the cellular and genetic factors responsible for age-related loss of touch sensitivity are largely unknown. Our proposal focuses on a particular cellular touch receptor called a Merkel cell, which lives in our skin and whose numbers dramatically diminish as we age. Our understanding of the genetic regulators of Merkel cell homeostasis remains very poor, which has largely been due to the lack of available reagents to target and manipulate the stem cells that maintain this lineage. We have designed a mutant mouse model that specifically targets a small population of stem cells that reside in the Merkel cell niche, termed a touch dome. This model is the first of its kind to uniquely target this population of cells. Our approach will utilize a cadre of genetically engineered mutant mouse models that specifically targets the touch dome stem cell niche in the skin that is responsible for maintaining Merkel cells in healthy skin. We will use this experimental mouse model to disrupt genes that participate in a specific signaling pathway we believe is responsible for maintaining mature Merkel cells in young skin and is lost in aging skin. If proven, our application will unlock how stem cells in our skin maintain a differentiated lineage of sensory cells that perceive light touch responses and how this process is perturbed as we age.

Comparative Effectiveness of CRISPRn, CRISPRd and CRISPRi in Patient-Specific Stem Cells

Stephen Tsang, MD, PhD
Columbia University Medical Center

Degeneration of the retina, the part of the eye that senses light, enabling it to be converted to a visual image by the brain, cause loss of activities of daily living in people with macular degeneration. Many macular degeneration patients lose independence and productivity, experience falls and resultant fractures, are subject to depression, and lose the ability to care for themselves and others. Best vitelliform macular degeneration (VMD), a juvenile form of macular degeneration, affects the ability of retinal cells to export chloride. It is caused by a dominant mutation, meaning that even if only one of the two copies of the gene has the mistake, it will cause macular degeneration. The goal of this study is to validate therapeutic gene editing to repair mutations in the BEST1 gene in stem cells from patients with the disease, then generate the parts of the retina that are affected in patients using the corrected cells. To do this we will: 1) compare different strategies for therapeutic gene repair in stem cells from patients; 2) demonstrate that the gene editing strategy will specifically target the BEST1 gene and not other genes; 3) demonstrate that the retinal cells that have undergone therapeutic gene editing can now export chloride, showing that the repaired gene functions correctly. This proposed research is expected to be significant because our approach, if successful, will open the door to treatment of a wide range of conditions that cause the death of nerve cells and/or are caused by similar types of mutations. The impact of these studies is that this is a first step toward development of a treatment to prevent loss of vision in patients who will develop macular degeneration, and thus will make an important contribution to public health.

Optogenetic Regulation of Cardiac Arrhythmia Using Patient-Derived iPS Cell Models

Gordana Vunjak-Novakovic, PhD
Columbia University

Heart disease remains the leading cause of death in the United States, and one of the most critical medical problems worldwide. Cardiac tissues generated from human induced pluripotent stem cells (iPSCs) are an ideal model for patient-specific studies of physiology and disease. However, the predictive power of these tissues remains limited by their highly immature state, which lacks the structural and functional hallmarks of adult myocardium. We recently overcame this fundamental limitation by developing protocols for maturing cardiac tissues formed from iPSC-derived cardiomyocytes. We now propose a highly advanced optogenetic approach for using human cardiac tissues derived from iPSCs in studies of arrhythmia, towards developing new treatment modalities. We will create light-sensitive cardiomyocytes by gene editing of iPSCs obtained from healthy individuals and patients born with genetic arrhythmias, and bioengineer heart tissues from these cells. Advanced optical platforms will be used for patterned illumination and high-speed imaging of the light-sensitive cardiac tissues, allowing us to measure and control the propagation of electrical activity through the heart. This approach will allow us to better understand how arrhythmias form, and how to improve current treatment strategies. The use of cells from patients will allow us to model disease and develop therapies in a personalized way. We propose to study the emergence and control of cardiac arrhythmias in an advanced model of light-sensitive human cardiac tissue derived from iPSCs. The completion of the proposed work will result in (i) new methods for optical stimulation of heart tissue; (ii) new approaches to modeling cardiac arrhythmia; (iii) better understanding of the relationship between genetic mutations and arrhythmias; (iv) a fundamental and technical basis for developing patient-specific therapeutic strategies for heart disease.

Transforming T Cell Gene Therapy Via Locus Control Region-Driven Gene Regulation

Benjamin Ortiz, PhD
Hunter College, CUNY

The limited longevity, and treatment stop/restart inflexibility, of cancer immunotherapy via chimeric antigen receptor (CAR)-gene engineered T cells potentially limits its long-term clinical efficacy. This approach involves genetic modification of mature T cells taken out of a patient. These cells display a limited life span after transplant. Current efforts to extend the functional lifespan of these T cells employ pharmacological approaches that add to the unnatural experiences T cells would bring with them into a patient. Current treatment-halting “suicide gene” strategies to limit side effects end up eliminating the engineered T cells, preventing resumption of therapy without another transplant. Here we aim to produce gene regulatory tools derived from the T-cell receptor (TCR)-α gene locus control region (LCR) designed to surmount these limitations. A TCRα mini-LCR would direct physiological, high-level, silencing-resistant CAR gene expression selectively to T-cell progeny of engineered stem cell transplants, thus providing a lifelong natural source of CAR+ T cells to a patient. It would also enable temporary interruption of treatment via suicide gene-based elimination of CAR-expressing T cells without stem cell transplant elimination. Our preliminary data also indicate that our efforts will yield a second mini-LCR that supports a distinct spatiotemporal activity pattern appropriate to other T-cell gene therapy applications. Our specific objectives are to: 1) develop two types of TCRα LCR-derived gene regulatory cassettes (mini-LCRs) that are applicable to a wide range of T cell gene therapy applications; and 2) test mini-LCR activity in the context of lentiviral vector transduced mouse and human stem cells. Collectively the tools to be developed here will transform treatment options for cancer, HIV, other viral infections and inherited immunodeficiency via genetically modified stem cell transplantation. Our technological arsenal positions us well to provide tools that will transform T cell gene therapy into a lifelong, curative, engineered stem cell-based intervention.

Editing Hepatic Sinusoidal Vascular Niche to Induce Fibrosis-Free Liver Repair

Bi-Sen Ding, PhD
Co-PI: Shahin Rafii, MD
Icahn School of Medicine at Mount Sinai and Weill Medical College of Cornell University

The self-regenerative capacity of the liver is frequently inhibited by injury or infection. Liver transplantation as a major therapy for end-stage liver diseases is hindered by clinical complications and lack of suitable donors. Transplanting stem cell-like hepatocytes holds promise for liver repair. However, expansion of transplanted hepatocytes is commonly prohibited by the environment of damaged livers, paradoxically exacerbating excessive scarring/fibrosis. Thus, coaxing the surrounding cells, such as vascular cells, to generate stem cell-friendly signals can enhance the growth of transplanted hepatoctyes and bypass scar formation. We previously showed that blood vessels express specialized molecules to increase stem cell expansion and reduce scar formation in the injured mouse livers, resulting in a complete regrowth of liver. The goal of this proposal is to identify how to stimulate liver vascular cells to generate beneficial signals to promote sustained hepatocyte transplantation and liver regeneration. We found that CXCR7 gene expression in the liver blood vessel stimulates production of a hepatocyte growth factor (HGF) molecule, and this molecule selectively increases liver regeneration and prevents scar formation. Thus, we will test whether stimulating CXCR7-dependent expression of HGF will enable a fibrosis-free liver repair in the damaged livers. Different compounds that activate this pathway will be tested in mouse models of liver injury, and their effect will be evaluated. The outcome of our study can enable stem cell-based therapeutic strategy to regenerate a diseased liver without scar formation.

Novel Biomarkers and Placental Progenitors

Hina Chaudhry, MD
Icahn School of Medicine at Mount Sinai

Our laboratory has discovered that stem/progenitor cells of fetal origin found in the placenta appear to repair the diseased heart and can form functioning heart muscle cells and blood vessels. We have found a novel cell type that can be isolated in the end-gestation mouse placenta that gives rise to the spontaneously beating cardiomyocytes and blood vessel cells. These cells express the protein Cdx2, and we have also found that CDX2 is expressed in the human term placenta. This discovery is of critical importance as the ideal cell type for cardiac regeneration has not yet been determined. Our goal is to develop these findings such that we can isolate these cells from otherwise discarded human placentas for broader use in regenerative medicine. In studying the potential of placenta cells for cardiac repair and blood vessel formation, we also hope to gain insights into other types of organogenesis that may be possible through the use of Cdx2 cells.

Mitochondrial Heterogeneity in the Regulation of Human Leukemic Stem Cells

Saghi Ghaffari, MD, PhD
Icahn School of Medicine at Mount Sinai

Less than 30% of patients with acute myeloid leukemia (AML) survive over five years. Conventional and targeted therapies have had little success in eradicating myeloid malignancies including AML and myeloproliferative neoplasms (MPNs) that designate a group of blood clonal stem cell disorders that have the potential to progress to leukemia. Leukemia therapies are based on targeting rapidly growing cells. The inability to reliably identify and target small clones of stem cell-like leukemic cells that are mainly inactive and therefore resistant to current therapies has contributed to this failure. To address this problem, we have focused on understanding how stem cells generate fuel to grow and how unhealthy stem cells may hide and escape therapies for a long period of time by generating low fuel. This approach has led us to devise a simple methodology that enables identifying stem cells with distinct capacities based on their levels of fuel generation. We propose to use this approach to identify human leukemic stem cells with the potential capacity to remain silent - in the absence of growth - for a long period of time. If successful this approach is likely to have a benefit for identifying human leukemia stem cells that initiate leukemia and for preventing leukemia progression.

Elucidating the Mechanisms of Hematopoietic Reprogramming

Kateri Moore, DVM
Icahn School of Medicine at Mount Sinai

Just 10 years ago seminal studies by Yamanaka and colleagues demonstrated that just four transcription factors (TFs) could change skin cells into pluripotent cells (iPSCs). These cells could then be differentiated into all cell types. Those studies caused a sea change and established the field of regenerative medicine. Other investigators have now used TFs to reprogram one somatic cell type into another cell type, e.g. skin into neural cells. Until our studies in 2013, no one had reprogrammed skin cells into a cell type with the degree of multipotency found in hematopoietic stem and progenitor cells (HSPCs) that make up all the cellular elements of our blood and immune systems. These studies also revealed the possibility of an alternative source for transplantable HSPCs. Directly programmed HSPCs would provide an unlimited patient-specific source for cell replacement and genetic correction therapies as well as a platform for the future generation of patient specific therapeutics and blood products. In order to make these possibilities a reality, it is necessary to understand the molecular and epigenetic mechanisms mediating this process. One objective of the proposed studies is to understand how TFs bind DNA and initiate a molecular program that changes the epigenetic landscape to allow a change in cell fate. The studies proposed here offer unprecedented opportunities to understand how a stem cell state is established and how key regulatory machinery is put into place. The second objective is to use computational biology to reveal patterns and a network of key elements in the data and develop a readily accessible interface for data extraction into testable hypotheses. These key elements will be studied to determine their loss and subsequent effects on reprogramming, ultimately bringing us closer to translating this technology for clinical use.

Reprogramming Leukemia to Study Human Pluripotent Stem Cell-Derived Hematopoiesis

Eirini Papapetrou, MD, PhD
Michael Kharas, PhD
Icahn School of Medicine at Mount Sinai and Sloan-Kettering Institute for Cancer Research

Bone marrow transplantation is the only curative treatment for a number of cancers and other blood diseases. While over 50,000 transplantations are performed annually, still many patients do not have a matched donor and therefore their needs cannot be met with current supplies. Human pluripotent stem cells (hPSCs) could provide an alternative source of transplantable blood cells that would be unlimited. However, despite efforts by many investigators over several years, the derivation of transplantable blood cells from hPSCs has not been feasible. Whereas blood cells can now be made from hPSCs very efficiently, these cells - unlike natural cells harvested from the bone marrow of donors - do not have the ability to engraft. Finding ways to make these cells engraftable in adult bone marrow thus remains a "holy grail" in stem cell research. A major obstacle towards attaining this goal is our incomplete understanding of the requirements for blood cells to have engraftment potential. We have recently developed hPSCs from patients with leukemia and found that the blood cells generated from them do have the ability to engraft. This is the first time that this has ever been observed. Based on this original finding, we propose that studying these cells with intrinsic engraftment capability can help us understand what gives them this ability and use this knowledge to finally be able to make engraftable blood cells for transplantation. The proposed work can help accelerate all ongoing efforts towards this goal.

Molecular Mechanisms of Naive and Primed Pluripotency

Jianlong Wang, PhD
Icahn School of Medicine at Mount Sinai

Stem cells derived from early embryos, known as embryonic stem cells (ESCs), are capable of propagating indefinitely in culture and also differentiating to all other types of cells in our body. These two so-called self-renewal and pluripotency properties make ESCs an ideal cellular system to study basic stem cell biology and offer an unlimited cell source for disease therapeutics and regenerative medicine. It is now known that pluripotency is not a singular state, but rather spans a spectrum encompassing different pluripotent states ranging from ground-state naive pluripotency to developmentally late primed pluripotency. The most well-studied naive pluripotent cells are mouse ESCs, whereas the best characterized primed pluripotent stem cells are mouse epiblast-derived stem cells (EpiSCs), although human ESCs with naive and primed features are much under-studied due to the technical difficulties. Such alternative pluripotent states directly correlate with cells’ capacities in self-renewal and differentiation to other cell lineages; therefore, understanding how these distinct pluripotent states, i.e., naive and primed, are molecularly controlled is important for better application of these cells, particularly human ESCs, in regenerative medicine. The objectives of this proposal are to dissect the molecular mechanisms controlling naive and primed pluripotency in both mouse and human systems. The goal is to understand how several molecular players we recently discovered, including Zfp281 and Tet1/2, regulate primed versus naive pluripotency of mouse and human ESCs, with an overarching goal of devising an efficient approach to make naive human ESCs without the abnormalities associated with existing naive human ESCs. Our project is of high scientific significance owing to the discovery of novel functions of these factors and also to our detailed mechanistic inquiries. It will have a great impact on regenerative medicine where both conventional primed human ESCs and their naive counterparts can be optimally derived and maintained without any inherent defects.

Stem Cell-Derived Human Cortical Interneurons for Cell Therapy for Epilepsy

Sangmi Chung, PhD
New York Medical College

Epilepsy, a debilitating neurological disorder characterized by unpredictable and abnormal electrical discharges and recurrent seizures, affects more than 65 million people worldwide. In this study, we will optimize the critical parameters for successful translation of stem cell-based therapy for epilepsy, including cell preparation methods and the ideal dose of grafted interneurons, not only to maximize integration of grafted human interneurons in epileptic brain circuitry but also to ensure the safety of the stem cell-derived grafts. This work will be pivotal for accelerating the translation of basic scientific discoveries into clinical applications.

A Patient-Based Cellular Platform for Clinical Translation of Chemical Remyelinating Therapies

Valentina Fossati, PhD
The New York Stem Cell Foundation

Nerve cells are an important cell type that function to carry signals form the brain to the rest of the body. These nerve cells require a layer of insulation around them in order to function properly and remain healthy. This insulating layer, myelin, is generated by another cell type, oligodendrocytes, present in the brain. In multiple sclerosis (MS), myelin is damaged by inflammatory cells, which leaves the nerve cells unprotected and unable to function – ultimately leading to patient disability. There is a clear need to identify therapeutic strategies that enhance myelin repair to restore normal function to patients. Recent work in rodents has identified drugs with the capacity to stimulate stem cells in the brain to make new myelin. However, it remains unknown how these medications function in humans. In this proposal we have united technologies from two leading laboratories to directly address the clinical potential of these myelin medicines in human patients. We have developed a technology to non-invasively generate brain stem cells in the laboratory from any patient. In this grant we will test these cells from both healthy and MS patients to evaluate whether candidate drugs for remyelination show efficacy in human cells. Moreover, this platform may reveal patient-specific differences that could be utilized in selecting patients most likely to respond in the clinical phase. This will result in higher success rates and reduction in costs for bringing a new drug to patients.

From Stem Cells to Differentiated Neurons: A Single Cell Transcriptional Approach

Claude Desplan, PhD
Co-PI: Rahul Sajita, PhD
New York University

Is it possible to produce different neuron types in order to repair brain or spinal cord injury, replace retinal cells damaged by diseases, or correct the loss of neurons in neurodegenerative diseases? The advent of induced pluripotent stem cells (iPSCs), where cells of a patient can be reprogramed to become neurons, has made it a possibility. However, it is essential that we understand how stem cells generate different neurons. Indeed, we only barely comprehend how to generate the huge diversity of neurons that populate brain structures affected by diseases. We offer to address this issue in a relatively simple nervous system where we can establish basic concepts that may be used in all neural systems to generate neuronal diversity. The visual brain center of a genetic model organism, the fruit fly Drosophila, contains a limited number of neurons (less than 100,000 vs. billions in mammals) and 100 cell types (vs. thousands in mammals). We have already established basic concepts that explain how a limited number of stem cells generate diverse Drosophila neuron types. We now want to match the rules established early during development with the final differentiation of adult neurons. For this purpose, we will use a new, revolutionary technique called Drop-seq that takes advantage of microfluidics and next generation mRNA sequencing. We can generate a complete description of the gene expression pattern (‘transcriptome’) of single cells with extremely high throughput; a single experiment produces individual transcriptomes of more than 100,000 cells. Thus, with Drop-seq we will be able to follow and model the complete developmental trajectory of a cell. Through integration of large-scale data and computer modeling, we hope to reconstruct the formation of a neuron and its integration into a neural network.

Enhancer-Promoter Dynamics Governing Hematopoietic Stem Cell Transcriptional Network

Iannis Aifantis, PhD
New York University School of Medicine

Hematopoietic stem cells (HSCs) are a rare population of cells that reside in the bone marrow and are responsible for maintaining and replenishing all blood and immune cells. The hallmarks of HSCs are self-renewal (ability to divide into new stem cells) and differentiation potential (the ability to turn into all types of mature immune and blood cells). HSC function must be tightly regulated to produce the billions of new hematopoietic cells required daily and prevent hematologic malignancies in the form of bone marrow failure or leukemia. This balance relies on an exquisite control of the transcriptional networks and gene expression programs that define each cell type of the hematopoietic system, from stem cell through all stages of differentiation until maturity. Gene expression is governed by stage-specific transcription factors that bind to promoters and enhancers, two distant DNA regulatory regions that communicate through transcriptional regulators to control gene expression. A key regulator of promoter-enhancer interactions is the Mediator, a large complex that functions as a bridge between enhancers and promoters, thus regulating cell-specific gene signatures. We recently demonstrated that Med12, a member of Mediator, is required for HSC survival and that Med12 loss causes bone marrow failure and rapid mouse lethality. We also demonstrated that Med12 is required to preserve enhancer activity, thus dictating the expression of essential HSC genes. Recent studies are revealing that MED12 is mutated in human blood malignancies, including chronic lymphocytic leukemia, myeloproliferative neoplasms and acute myeloid leukemia. In this proposal, we interrogate Med12 function in committed blood precursors (stem cells with lineage-restricted differentiation potential). We will also explore potential gain-of-function mechanisms of Med12 mutants in leukemia by modeling Med12 mutations. A better understanding of novel regulators of self-renewal, differentiation and malignant transformation, such as Med12, will contribute to the prevention or treatment of blood diseases.

Signaling Mechanisms that Coordinate the Behavior of Stem Cells and Their Niche

Erika Bach, PhD
New York University School of Medicine

Stem cells are uniquely endowed with the ability to self-renew and produce specialized cells. In some tissues, two or more different stem cell populations reside in the same niche, and their self-renewal and differentiation must be coordinated to optimize tissue function. How this occurs is not known. In addition, while it is known that stem cells regulate the function of their niche, the signals controlling this process have not been identified. Understanding these regulatory mechanisms holds great potential for breakthroughs in the treatment of cancer, diabetes, infertility and spinal cord injuries. The Bach lab is focused on finding the signals that coordinate self-renewal and differentiation of distinct stem cell pools within the same niche using the Drosophila testis as a model system. We have discovered a secreted protein that promotes the co-differentiation of germline stem cells (GSCs) and somatic cyst stem cells (CySCs). We have also found an inhibitor of this signal produced by the stem cells that protects niche cells from the deleterious effects of the “differentiation” signal. This study is designed to determine how one signal controls the co-differentiation of GSCs and CySCs and how niche cells are protected from this signal. Aims 1 and 2 are focused on how this signal regulates differentiation of somatic and germline lineages, respectively. Aim 3 examines how the inhibitor of the differentiation signal protects niche cells. 70% of human disease genes have counterparts in Drosophila, highlighting the relevance of Drosophila to human health. We have identified a factor conserved in humans that controls the co-differentiation of distinct stem cells in the same niche and that, if not properly countered, causes niche dysfunction. The work in this proposal may shed light on stem cell-niche function and stem cell differentiation in mammals.

Generating Cardiac Conduction System Cells from Human Induced Pluripotent Stem Cells

Karen Maass, PhD
Co-PI: Glenn Fishman, MD
New York University School of Medicine

Within the heart, a small population of specialized cells forms the cardiac conduction system and regulates each heartbeat. Diseases compromising these cells can cause life threatening arrhythmias, a major healthcare burden in developed countries. Unfortunately, pharmacological treatment of arrhythmias has been very ineffective. Due to the rarity of cardiac conduction system cells called Purkinje cells, mechanisms triggering fatal arrhythmias remain poorly understood. We have recently described a mouse stem cell model, allowing insight into development and specification of conduction system cells in the dish. Here, we propose experiments that will allow characterization, enrichment and maturation of human conduction system cells. Our major goal is to generate and isolate human cardiac conduction system cells from induced pluripotent stem cells. These cells, generated from tissue biopsies, can turn into any cell type in the dish. With our existing human conduction system reporter model, we will study normal development, the pathways increasing yield and maturity of this cell type, and the mechanisms leading to arrhythmogenic diseases. Combining our knowledge of the mouse conduction system and the proposed detailed analysis of human fetal heart cardiac conduction system tissue, we will generate a human stem cell model allowing efficient enrichment and cryopreservation of cardiac conduction system cells. These cells will provide a valuable tool for translational applications, including anti-arrhythmic drug screens and regenerative tissue engineering. Human cardiac conduction system cells are the best model available to study the precise basics of pro-arrhythmogenic mechanisms in inherited or acquired cardiac disease and to translate the findings into efficient therapies. The generation of pure working cardiomyocytes and pure conduction system cell cultures provides refined tools for the study of anti-arrhythmic drugs. Finally, Purkinje cell fibers generated in the dish might very well have high therapeutic potential in regenerative tissue engineering.

Transcription Factor Network Reorganization by Mutant IDH1 in Gliomagenesis

Dimitris Placantonakis, MD, PhD
New York University School of Medicine

Low-grade gliomas (LGGs) are slow-growing primary brain tumors that invariably progress to deadly high-grade gliomas. LGGs are histologically classified as astrocytomas or oligodendrogliomas, both of which contain gain-of-function isocitrate dehydrogenase 1 (IDH1) mutations in ~80% of cases. To test the hypothesis that mutant IDH1 is a driver of LGG formation and generate an experimental model, we have used neural stem cells derived from human embryonic stem cells to express the IDH1 mutation along with two other common loss-of-function genetic changes found in low-grade astrocytomas. We found that mutant IDH1 blocks normal differentiation of human neural stem cells and promotes their invasion of normal brain tissue, a histologic hallmark of LGGs, in mice. This novel tractable model allows a step-by-step characterization of molecular and cellular changes in neural stem cells after the IDH1 mutation is introduced, and provides an in vivo platform to study salient features of LGGs, such as brain invasion. We hypothesize that the differentiation block induced by mutant IDH1 is essential to LGG formation and is mediated by downregulation of transcription factor Sox2, which is normally required for the capacity of neural stem cells to differentiate to neurons and glia. Here, we propose to elucidate in vitro and in vivo molecular mechanisms that underlie this downregulation of Sox2 and to develop drug screening platforms to reverse the effects of mutant IDH1 on neural stem cell differentiation. The proposed experiments will not only advance our understanding of LGG formation but will also provide insights into future treatment strategies.

Mobilizing Neural Stem Cells for Remyelination

James Salzer, MD
New York University School of Medicine

An important therapeutic goal in various neurological disorders is to enhance nervous system repair. In particular, there is great interest and an urgent need to repair myelin, the protective insulation that surrounds nerve fibers. Myelin is lost in a number of neurological diseases – most notably in multiple sclerosis – resulting in abnormal conduction of electrical impulses, nerve degeneration, and severe neurological deficits. We have recently found and reported that neural stem cells, resident within the adult brain, are recruited to lesion sites where they form new myelin. Moreover, by inhibiting a specific protein expressed in these stem cells, we greatly enhanced their ability to form new myelin sheaths and significantly improved functional recovery in experimental mouse models of myelin damage. We now propose to further elucidate how neural stem cells repair myelin damage. To this end, we will: i) characterize potential competition between stem cells and parenchymal progenitors in remyelination to see if these are truly separate pools and ii) determine if the inhibitory protein in stem cells blocks repair by a direct effect on the stem cells themselves or indirectly via other cells in the brain. Finally, and unexpectedly, we have obtained evidence that neural stem cells in the brain rely on signals from a brain immune cell to expand and potentially for recruitment into lesion sites - we will begin new studies to elucidate how this occurs. Our focus on the role of stem cells, rather than on the more well characterized oligodendrocyte progenitors, highlights their unexpectedly important role in myelin repair. Further, by characterizing how neural stem cells repair the brain and how their interactions with other cell types in the brain impact their effectiveness, these studies may guide efforts to develop rational, reparative therapies for neurological disorders.

Identifying the Alterations in Skin Stem Function and Niche Interactions Responsible for Cancer

Elaine Fuchs, PhD
The Rockefeller University

Adult stem cells (SCs) are natural units of tissue repair and homeostasis. They self-renew and also generate differentiated tissue. Tumor-initiating cells (cancer stem cells, CSCs) are similar in concept in that they too propagate the tumor and also generate its differentiating cells. Effective cancer therapies are predicated upon targeting CSCs without altering their normal tissue counterparts. A prerequisite to achieving a clinical-basic science interface is to elucidate how CSCs differ from normal adult SCs, understand the differences arising during tumor progression and determine which changes are most critical to cancerous and not normal SC growth. Lineage tracing shows that SCs of hair follicle (HF-SCs) and epidermis (epi-SCs) are major sources of squamous cell carcinomas (SCCs), prevalent world-wide. We have purified and characterized mouse skin CSCs. Representing ~1% of SCCs, these CSCs initiate tumors when transplanted at single-cell level. In NYSTEM-funded research, we have shown that, surprisingly, transcriptional and chromatin profiles of SCC-CSCs bear little resemblance to their parental HF-SCs or epi-SCs, with SOX2 and ETS2/ELK3 transcription factors playing key roles in SCC-CSCs but not normal SCs. We identified two interchangeable SCC-CSC states differing in cycling rates: slower-cycling CSCs arise when juxtaposed to TGFβ-rich perivasculature. Most importantly, these CSCs invade the stroma, evade chemotherapy and regrow the cancer following treatment. Based upon this progress, we will now explore how TGFβ-responding SCC-CSCs acquire chemo-resistance and why they become refractory to immune surveillance. Dissecting both upstream and downstream events in malignancy and metastasis, we will tackle the physiological relevance of key gene differences and identify those responsible for the progressive transformation of normal skin SCs into SCC-CSCs. Finally, we will explore the relation of our findings to human diseases, including hyerproliferative and premalignant disorders, malignant and metastatic SCCs. This proposal focuses on these global objectives, with the ultimate goal of identifying new cancer targets.

Identification of Genetic Targets of a Genetic Master Regulator, a Neuroprotective Factor in Parkinson’s Disease

Paul Greengard, PhD
Co-PI: Lorenz Studer, MD
The Rockefeller University and Sloan-Kettering Institute for Cancer Research

Parkinson’s disease (PD) is the second most common age-related neurodegenerative disorder. Its prevalence in industrialized countries is almost 2% among individuals 65 years of age and older. A pathological hallmark of PD is the progressive degeneration of a subset of neurons located in a defined area of the midbrain, the so-called substantia nigra (SN). These cells play an important role in the control of movements. Thus, the loss of these neurons causes the typical motor symptoms observed in PD patients. However, in close proximity exists a population of neurons which is biologically very similar and functionally related but not very affected in PD. These cells are located in the so-called ventral tegmental area (VTA). Recently, we used a mouse model for PD and investigated the gene activity in the two different brain regions. Eventually, we were able to identify a genetic factor that can act neuroprotectively in the cells which are affected in PD. The identified factor acts as a genetic master regulator (MR) and can regulate the activity of several genes. In the proposed project we will characterize the MR specifically in human neurons. To generate human neurons in vitro, we will make use of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), which can be differentiated in the neurons of interest. Using these specific cells is the best approach to investigate biological processes which take place in the human living brain. With these cells we will model PD and be able to monitor the activity of the MR. Finally, we aim to understand the mode of action of the MR to identify the downstream factors which are involved in the neuroprotection. Understanding the cellular mechanism of neuroprotection will eventually lead to the identification of novel potential drug targets for the treatment of PD.

Novel Regulators of Endoderm Differentiation

Danwei Huangfu, PhD
Co-PI: Anna-Katerina Hadjantonakis, PhD
Sloan-Kettering Institute for Cancer Research

Genetic screens in model organisms have been essential to our understanding of embryonic development and causes of human diseases. Although such screens have been successfully conducted in mice, the scales have been limited, and might miss finding genes important in humans. To overcome this challenge, we propose performing genetic knockout screens in human pluripotent stem cells (hPSCs) to discover novel regulators of human development. We are specifically focused on the formation of the early cell type endoderm, which gives rise to most cells in our respiratory and digestive organs including the lung, thymus, stomach, pancreas, liver, intestine and colon. Understanding how endoderm forms will lay the logical groundwork for understanding the progression of diseases that affect these endoderm-derived organs. It could also facilitate the development of improved hPSC directed differentiation protocols for the generation of endoderm-derived cells for cell replacement therapy. Our approach is ambitious and innovative, as no genome-wide knockout screen to study human development using hPSCs has been reported. We have strong preliminary data supporting the success of the project, and have assembled a unique team of collaborative scientists with complementary expertise in stem cell biology and endoderm differentiation. Thus, we foresee successful completion of the screen with the identification of novel hits. We will select some of the hits for detailed analysis, and perform cross-species and platform comparison while conducting mechanistic studies making use of strengths for several complementary experimental systems.

Human PSC-Derived Schwann Cells for Modeling and Treating Diabetic Neuropathy

Lorenz Studer, MD
Sloan-Kettering Institute for Cancer Research

The Peripheral Nervous System (PNS) is a vast communication network that transmits signal from the body to the brain and spinal cord and modulates the function or organs. Schwann cells are an important PNS cell type that supports nerve function and provides the insulating myelin sheet, which is important for rapid conduction of nerve signals. Schwann cells are likely involved in diabetic neuropathy, which is a disease of the PNS that is debilitating and affects millions of patients. The study of diabetic neuropathy has been challenging due to the difficulty in obtaining access to the human cell types involved in the disease. Here we propose to develop a technology to generate Schwann cells in a dish from human pluripotent stem cells (hPSCs). The resulting Schwann cells will be tested for their ability to promote nerve cell function and to produce the insulating myelin sheet important for fast conduction. Schwann cell-mediated myelination will be tested by growing the cells together with sensory nerve cells in a dish and by transplanting them into a rat model of peripheral nerve damage. We obtained exciting preliminary data demonstrating that Schwann cells are particularly vulnerable to high glucose levels, such as the levels found in patients suffering from diabetic neuropathy. Therefore, we carried out a screen of >1,000 FDA approved drugs on hPSC-Schwann cells to find an existing drug that may protect Schwann cells and thereby prevent or treat diabetic neuropathy. Here we propose to further test promising compounds and to understand their mechanism of action. Finally, we will test whether such drugs, identified in our stem cell-based model of the disease, can rescue diabetic neuropathy in mice, which may set the stage for the future application of those compounds in diabetic patients.

Skin-Derived Neural Crest Stem Cells for Treatment of Neurogenic Disorders

Stelios Andreadis, PhD
Co-PI: Fraser Sim, PhD
University at Buffalo, SUNY

We recently discovered that human epidermal keratinocytes (KC) from the skin could be reprogrammed into a neural crest (NC) fate without genetic modification or reprogramming to pluripotency. The KC-derived NC (KC-NC) could be coaxed to differentiate into all functional NC derivatives including peripheral neurons, melanocytes, Schwann cells and mesenchymal stem cell derivatives (osteocytes, chondrocytes, adipocytes and smooth muscle cells). Upon transplantation into chicken embryos, KC-NC gave rise to multiple NC derivatives indicating that these cells were indeed NC cells. Here we propose to extend our findings to study the mechanism of this transition from epidermal to NC fate. We will also examine whether adult keratinocytes have the potential to be reprogrammed into NC fate, thereby opening new possibilities for disease treatment. To this end, we will differentiate KC-NC into myelin-producing cells (Schwann cells) that will be implanted into the brain of a mouse model that lacks myelin and suffers from Parkinson-like symptoms, such as shivering (the shiverer mouse). These experiments will determine whether KC-NC derived Schwann cells can be used to treat devastating diseases, which are difficult to treat with cell therapies due to scarcity of autologous cells. Our work has demonstrated, for the first time, the plasticity of human epidermal cells to be reprogrammed into NC cells under defined culture conditions. As a result, this work represents a paradigm shift in stem cell biology as well as in regenerative medicine as it has the potential to provide a novel, autologous source of abundant, readily accessible stem cells for treatment of devastating neurodegenerative diseases, for which cell sourcing remains a severe impediment hampering cell therapy approaches.

Deriving Functional Cardiac Pacemaker Cells from Human Embryonic Stem Cells

Todd Evans, PhD
Weill Medical College of Cornell University

Cardiovascular disease is the leading cause of death in the US, and each year, of the more than 300,000 sudden cardiac-based deaths, nearly all are caused by cardiac arrhythmias. The rhythmic heartbeat reflects a highly coordinated wave of electrical activity, initiated and propagated throughout the working heart muscle by a specialized inter-connected network of cells called the cardiac conduction system (CCS). At the top of the CCS hierarchy are the cardiac pacemaker cells (CPCs) that generate the electrical impulses that trigger each heartbeat. The CPCs are unique cells based on their ability to initiate a heartbeat, while remaining regulated by the autonomic nervous system. Loss of CPCs and degeneration of the pacemaker are major causes of rhythmic dysfunction and heart disease, and there are no cellular or pharmacological therapies to treat such disease. Compared to working muscle, pacemaker cells are rare, which has limited research in understanding their biology. In principle, hESCs should be an outstanding source for generating pacemaker cells. We propose to screen for signaling pathways and genes that help to generate human pacemaker cells from differentiating ESCs. Through the generation of hESC-derived CPCs, we expect to gain new insight into the developmental biology of the heart’s specialized conduction system, new knowledge about the electrical properties of the cells within this system, and ultimately, the discovery of novel targets for the treatment of heart rhythm disorders. If successful, our studies, coupled with tissue engineering approaches, may even lead to cellular therapies, and diminish the profound burden of cardiovascular disease.

Hedgehog Signaling in Adult Choroid: Implications for Stem Cell Therapy of Retinal Disease

Enrique Rodriguez-Boulan, MD
Weill Medical College of Cornell University

Age-related macular degeneration (AMD) is the leading cause of vision loss among the elderly in developed countries, and its most common “dry” or “atrophic” form is currently incurable. A main cause of AMD is believed to be the degeneration of underlying choroid blood vessels that provide nutrients and other support to the retina. Recently, we made the exciting discovery that choroid endothelial cells (ECs) lining the inside of these vessels express Indian Hedgehog (IHH), a factor involved in the maturation and maintenance of body organs. Our preliminary data suggest that defective production of IHH by adult choroid, or defects in this signaling pathway, result in drastic retinal defects. As our data also indicate that the main target of IHH in adult choroid is a population of adjacent stem cells, they suggest the innovative hypothesis that a cause of atrophic AMD is disrupted HH signaling that leads to choroid atrophy. We predict that reconstituting the HH pathway by introducing IHH-expressing ECs derived from stem cells or by administering drugs that mimic IHH function may be a useful treatment for AMD.

Contribution of H3.3-Mediated Chromatin Dynamics to Stem Cell Pluripotency and Developmental Potential

Duancheng Wen, PhD
Co-PI: Shahin Rafii, MD
Weill Medical College of Cornell University

Stem cells hold great potentials for regenerative medicine, because they are able to differentiate into many different cell types to repair and replace damaged tissues in the body. It is critical to determine the potential of stem cells before they are used for the cell-based therapies. Effective assessment of stem cell potential developed in mouse models using chimeras is not applicable to human stem cells. Much evidence has shown that stem cells with similar gene expression may not have the same potential to differentiate into other cell types. We previously found that histone variant H3.3 is an important factor regulating stem cell pluripotency and developmental potential, and it is required for maintenance of bivalency for developmental genes in mouse embryonic stem cells. In this proposal, we will investigate how H3.3 regulates stem cell pluripotency and developmental bivalent genes. We will determine whether the epigenetic states in developmental bivalent genes are associated with the stem cell developmental potential, and develop a novel approach to assess stem cell developmental potential that may be used for human stem cells. We expect this work will significantly enhance our understanding of stem cell potential as defined by epigenetic mechanisms and offer new strategies to evaluate the efficiency and safety of cell-based therapies.


IDEA Awards

Role of Novel Unipotent Stem Cells in ER-Positive Breast Cancer

Wenjun Guo, PhD
Albert Einstein College of Medicine

Breast cancers remain the leading cause of cancer death in women despite significant improvement in early detection and treatment. About 70% of human breast cancers are classified as positive for estrogen receptor (ER+). Although many ER+ cancers respond to hormonal therapy, a substantial fraction of them will relapse and become resistant to the therapy. Consequently, ER+ cancers account for the majority of breast cancer death. Thus, understanding the etiology of ER+ breast cancers will have major implications for cancer prevention and treatment. However, which cell type gives rise to ER+ cancers is still unclear. We recently identified a novel population of stem cells that is specialized in maintaining the normal ER+ mammary cells. We hypothesize that ER+ breast cancers preferentially originate from these novel ER+ stem cells. We will use state-of-the-art cell fate tracing technologies to determine the role of ER+ stem cells in mammary tissue repair after injury. We will also determine the effect of obesity, a major breast cancer risk factor, on disrupting proper functions of ER+ stem cells. Finally, we will test whether ER+ breast cancers are preferentially induced in the ER+ stem cells or other stem cell populations. Our studies will fill an important knowledge gap in our understanding of ER+ breast cancer etiology and the role of stem cells in cancer development. Identifying the cell-of-origin for ER+ breast cancer and the mechanisms by which cancer mutations transform these cells will aid the development of cancer prevention and treatment strategies.

Neovascularization for Optimal Survival of Neural Transplants

Jean Hebert, PhD
Albert Einstein College of Medicine

In certain tissues of the body, such as the brain’s neocortex, when cells are lost due to damage or disease, they are not replaced. This is due to the lack of active resident stem or precursor cells. Therefore, the transplantation of exogenous stem or precursor cells provides an attractive approach for tissue regeneration. However, when neural precursor cells are transplanted into the brain, most of them do not survive. Although rejection by the immune system can in part be responsible for the removal of transplanted cells, a lack of blood supply to and within the transplant site may also contribute to the death of transplanted cells. In this proposal, using transplants of embryonic mouse neocortical cells into the adult neocortex, we will test whether having blood vessel precursor cells mixed with neural precursor cells is necessary for the ensuing transplant to become vascularized. We will also examine whether blood vessels in the transplants form a normal blood-brain-barrier and whether these blood vessels sustain the survival and function of transplant-derived neurons. Finally, we will examine whether in addition to blood vessel precursors, other cells, namely microglia, are required for fusing new blood vessels in the transplant with existing vessels in the host neocortex. Together these studies will address key gaps in our understanding of what it will take to promote optimal survival and functionality of neural stem cell transplants as the therapeutic uses of such transplants increasingly move toward clinical trials.

Derivation and Functional Characterization of (Haploid) Androgenetic Human Pluripotent Stem Cells

Dieter Egli, PhD
Columbia University Medical Center

All cells of the human body contain both a maternal and a paternal genome, constituting 46 chromosomes. Only nondividing germ cells contain a single set of 23 chromosomes. The contribution to development of paternal and maternal genomes is unequal, neither genome alone can result in a human being. These differences are relevant to human health: when a single chromosome is present in two copies from the same parent, it can cause severe disorders, such as metabolic defects and obesity. Studies in human cells have thus far been challenging, because functional differences in developmental potential cannot readily be evaluated with available stem cells containing both maternal and paternal genomes. The goal of this propoosal is to investigate these differences and to provide a novel tool for genetic studies. A central goal of biomedicine is to better understand human gene function. Cells that contain only one set of the human genome provide a distinct advantage for genetic studies. All genetic changes will be functionally penetrant, because the cell does not also have a second ‘back-up’ copy that would mask the effect of a genetic variant. We have recently shown that pluripotent stem cells with a single genome of 23 chromosomes can be derived from human oocytes. Thus far only haploid human pluripotent stem cell lines of maternal origin (parthenogenetic) have been reported. The goal of this proposal is to generate haploid stem cells derived from a single sperm, providing the paternal counterpart. We will characterize DNA methylation, gene expression in pluripotent and in differentiated cells, and compare differentiation potential to parthenogenetic ES cells and IVF-ES cells. We will also test the suitability of haploid stem cells for genetic studies in the context of diabetes. We anticipate that the cells generated here will provide an important platform for future genetic studies.

CRISPR Based System for Genetic Screening of Embryonic Stem Cell Differentiation

Hynek Wichterle, PhD
Columbia University Medical Center

Stem cells in developing animals begin with unlimited potential to multiply but can also exit the cycle of self-renewal and commit to forming restricted types of cells through a process called specification. Harnessing the ability of stem cells to expand to vast numbers and then guide their specification into cell types of interest is one of the most powerful transformative technologies in modern biology. We use this system to understand how the developing organism generates motor neurons – the cells that control muscle contraction and therefore movement. Historically, such studies were done by perturbing single genes and asking whether those alterations cause a change in motor neuron production. Very few attempts to comprehensively map gene requirements in vertebrate motor neuron specification have been made. Using a highly efficient method for producing motor neurons from stem cells, we propose a strategy to do this using a new technique for the precise manipulation of gene expression called CRISPR. The CRISPR system can be used to either eliminate, turn down, or turn up the levels of specific parts of the genome- and this can be done systematically across the entire genome. Here we propose to generate specific tools and reagents that will make motor neuron differentiation compatible with this new technology. These include the development of new methods for labeling and purifying stem cell derived motor neurons, the validation of the CRISPR approach with a panel of robust controls, and ultimately the adaptation of the system to virally delivered collections of gene targeting elements. Collectively these aims will produce a working system for interrogating the genetic determinants of motor neuron development and function with unparalleled power. The impact of this technology will extend to studies of neuronal degeneration with a potential to yield new therapeutic targets for currently untreatable conditions such as amyotrophic lateral sclerosis (Lou Gehrig's disease).

Molecular Determinants and Therapeutic Sensitivity in Testicular Germ Cell Cancers

Robert Weiss, PhD
Cornell University

Testicular Germ Cell Tumors (TGCTs) are the most common cancers in young men and are composed of cells that share many functional properties with stem cells. Although many TGCTs can be effectively treated, current therapies utilize chemotherapeutics with significant acute and long-term toxicities and which are ineffective in a subset of patients with resistant cancers. We seek to understand the genetic events that lead to the formation of TGCTs and will also investigate alternative therapies, called differentiation therapies, which may be less toxic and potentially also effective against cancers that are resistant to traditional chemotherapies. We will accomplish these aims using a genetically engineered mouse model of TGCT recently developed in our laboratory. This animal model accurately recapitulates many aspects of the human disease and will allow us to comprehensively map the genes and biological pathways whose alteration contributes to TGCT development. We also will create additional mouse models in which human TGCTs are directly implanted in mice, allowing for the direct analysis of human testicular cancers in a living host. We will use both mouse models to test the effectiveness of alternative therapies designed to target the stem cell nature of these cancers. The knowledge gained from this work will reveal how to: 1) further improve TGCT treatment while minimizing toxicity, 2) effectively treat the small fraction of therapy-resistant TGCTs and, most importantly, 3) use the understanding of the origins and therapeutic sensitivity of TGCTs to detect and treat other cancers, including stem cell therapy-associated neoplasms as well as cancers that do not respond favorably to conventional chemotherapy.

Identification of Tendon Stem Cells Using Genetic Lineage Tracing

Alice Huang, PhD
Icahn School of Medicine at Mount Sinai

Tendon injury is a common clinical problem characterized by slow recovery and high recurrence after treatment. Adult tendons heal via fibrosis (scarring), and this failure to re-establish native tendon structure is likely the leading cause of injury recurrence. Improving tendon healing to a regenerative, tenogenic state is therefore a crucial research priority. Toward that end, our broad objective is to identify the cells and signaling pathways that distinguish tenogenic regeneration vs fibrosis to improve tendon healing. Although several groups have now isolated putative tendon stem cells, these cells were isolated based on simple adherence to tissue culture plastic and much of this work relied solely on in vitro characterization. To date, definitive markers for tendon stem cells have not been identified; the source and activity of the cells during tendon growth and healing remain completely unknown, thus limiting clinical application of these cells. Elucidating the markers, sources, and functions of stem cells that may mediate tendon healing is therefore critical to the development of novel therapies to treat tendon injuries or degeneration. Here we propose to use genetic mouse models to identify tendon stem cells in vivo. The objective is to locate these cells in their native environment and trace the activity of these cells in the context of regenerative (tendon forming, restoration of function) and non-regenerative (scar and abnormal cartilage/bone formation in tendon), and establish the functional roles of these cells in both types of healing. Successful completion of this project will provide definitive markers for tendon stem cells and identify their role in regenerative and non-regenerative tendon healing, thus advancing the field of tendon biology toward clinical translation using these cells.

Epitranscriptomic Mechanisms of Human CD34+ Stem Cell-Derived Exosomes

Susmita Sahoo, PhD
Icahn School of Medicine at Mount Sinai

Myocardial ischemia leads to lack of oxygen supply to heart muscle causing tissue damage. Overtime, myocardial tissue damage causes reduced blood pumping ability of the heart. Stem cell-based therapies, including the recent CD34+ cell therapy, are one of the most promising approaches to enhance healing of the damaged tissue. The regenerative efficacy of transplanted CD34+ stem cells is limited by their poor viability and retention within the damaged tissue. Therefore, to address these limitations and to develop novel alternate approaches, we must seek to understand the mechanisms that lead to therapeutic recovery. Recent research from my laboratory demonstrated that human CD34+ stem cells secrete miniature vesicles called exosomes (CD34Exo) that have similar beneficial effects to the stem cells to repair a damaged heart. Further, the beneficial effects of CD34Exo are mediated by small RNAs, such as miRNA-126, carried by CD34Exo that are delivered to different cell types in the heart. Interestingly, we discovered for the first time that CD34+Exo treatment induces a chemical change in the cellular mRNA content of the treated heart, known as N6methyladenosine (m6A) modification of mRNA. We also discovered that this novel process regulates individual cardiomyocyte function and possibly cardiac function in diseased condition. Here we propose to understand the cellular and molecular mechanism of CD34Exo-induced m6A modifications of mRNA using a mouse model of myocardial ischemia. Our approach will lead to a better understanding of the function of a novel cell-free therapeutic entity for patients with myocardial ischemia. It may also open up m6A modifications as a new perspective for cardiovascular scientists studying cardiac regeneration. This innovative study is important to unlock the transformative potential of cell-free human stem cell-derived exosomes and can advance cell-based therapies by exploiting many practical and technical advantages of exosomes relative to cells for application in cardiovascular regenerative medicine.

Pursuing Totipotency with miRNAs and MERVL

Jianlong Wang, PhD
Icahn School of Medicine at Mount Sinai

Following fertilization, the totipotent zygote created by fusion of terminally differentiated sperm with oocyte undergoes several cell divisions to give rise to the blastocyst, which is composed of the inner cell mass from which pluripotent embryonic stem cells (ESCs) are derived, and the trophectoderm that mainly gives rise to the placental tissue. Zscan4 is sharply expressed in the late two-cell (2C) state of the mouse embryo, and is also transcribed transiently in sporadic ESCs, marking the totipotent population in conventional ESC culture. In addition, many endogenous transposable elements are activated in the 2C embryos and 2C-like cells in ESC culture, particularly the MERVL subfamily retroelements. Despite the well-recognized totipotent states of 2C embryos and 2C-like cells in ESCs, the molecular mechanisms underlying the totipotent state remain elusive due to the lack of a robust culture condition for totipotent cells. This has become a bottleneck in our understanding of early development and the potential use of totipotent cells as an alternative source in regenerative medicine. This research proposal is designed to address these urgent needs. We propose to dissect miRNA-mediated post-transcriptional control in totipotent cells and explore the causative effect of MERVL activation in the establishment and maintenance of totipotency. The significance of this work lies in the first such attempt at a systematic characterization of functional genomic, epigenetic, and post-transcriptional regulators underlying totipotency. This proposal will be the first work that defines a totipotency-specific miRNA in post-transcriptional control of the totipotent 2C-like cell state in ESCs and addresses the cause or effect of MERVL/2C gene activation in totipotency. Our work will have an overarching impact in achieving stable totipotent cell culture for the advancement of cell-based therapies and regenerative medicine using totipotent cells and in understanding early embryo development.

Metabolic Maturation of Stem Cell Derived Beta Cells

Bjarki Johannesson, PhD
Co-PI: Scott Noggle, PhD
The New York Stem Cell Foundation

The differentiation of stem cells into pancreatic beta cells is a powerful tool for studying diabetes and has enormous potential for future cell replacement therapies. However, current methods are inefficient as only a small fraction of the produced cells can effectively secrete insulin upon glucose stimulation, the central function of beta cells. Our research, along with growing observations in the field, indicates that this inefficiency stems from an incomplete maturation of stem cell-derived beta cells into a fully functional state. In particular, our results point towards a fetal-like mode of energy production as a major obstacle to achieving functioning cells. We hypothesize that shifting cell metabolism towards an adult-like state will yield the functional switch necessary for generating mature beta cells. We propose to achieve this by treating cells with compounds capable of modifying cellular metabolism, aimed at stimulating maturation. Because this strategy involves the testing of multiple compound combinations and concentrations, it will not feasible on a traditional, manual basis. We thus propose to automate this process using our unique robotics platform, the New York Stem Cell Foundation Global Stem Cell Array. This will allow us to test thousands of compound combinations and systematically devise an optimized procedure. Differentiation protocols are generally developed on a single cell line, which leads to unpredictable results when extended to diverse samples. This creates a major limitation for using stem cells for the study of diseases such as diabetes. To generate a protocol that performs equally well across cell lines, we will apply the robotics platform to fine-tune the protocol using 12 independent lines. Our efforts should yield a robust method for generating fully functional stem cell-derived beta cells across individuals, with transformative effects on stem cell-based diabetes research.

Understanding the Impact of 3D Genome Topology in Early Hematopoietic Stem Cell Transformation

Iannis Aifantis, PhD
New York University School of Medicine

Chromosomes represent heritable and dynamic carriers of genetic information that are constantly looping to shape the gene expression pattern of a cell. This three-dimensional genome landscape, known as the genome topology, provides the physical structure required to inform the identity and function of a cell. However, this DNA looping is not random, but is instead tightly regulated by specific proteins that act to compartmentalize the expression of genes in specific tissue. The key players in establishing the genome topology include the cohesin complex, which physically “wraps” around DNA to establish looping events, as well as the CCCTC-binding factor (CTCF), a protein that acts to bind DNA and establish the boundary of genome topological domains. Interestingly, it has been shown that regulators of genome topology are commonly mutated in various diseases, including cancers such as acute myeloid leukemia (AML). Acute myeloid leukemia (AML) is the most common adult leukemia and is characterized by excessive proliferation of abnormal immature white blood cells. AML continues to have a dismal survival rate amongst all subtypes of leukemia (<50% five-year overall survival rate). To date, however, there have been limited insights into how maintaining genome integrity halts AML formation. Notably, mutations in the cohesin complex, for example, have been shown to be an early step in AML formation, suggesting that controlling DNA looping and genome topology is a critical function to prevent cancer. This proposal focuses on understanding how regulators of the genome’s 3D structure protect blood stem cells from forming leukemia. We believe this work will shed new light on the earliest steps in leukemia formation with the potential to inform new treatment strategies targeting the root genetic causes of leukemia development.

Identifying New Germ Line Stem Cell Maintenance Factors

Erika Bach, PhD
New York University School of Medicine

Stem cells hold great potential for advances in treating cancer and in regenerative medicine and in stem cell-based therapies. In the Drosophila testis, one of the best characterized paradigms for investigating stem cell behavior, germline stem cells (GSCs), which give rise to gametes, are supported by niche cells and somatic stem cells called CySCs. Prior work has shown that niche cells and CySCs secrete factors called BMPs that promote GSC self-renewal. However, little is known about other signals that control GSC self-renewal. My lab has amassed preliminary data showing that CySCs must produce one of more factors in addition to BMPs that promote GSC maintenance, but their identity is not known. We have comprised a list of 25 genes as potential candidates for these unknown GSC maintenance factors. This proposal outlines a genetic screen to assess these candidates and then to characterize further the ones selected from the screen for their roles in regulating processes required for GSC maintenance. Aim 1 is focused on the genetic screen to identify new factors produced by CySCs that induce GSC self-renewal. Aim 2 will test the role of the candidate gene in the maintenance of GSCs and determine if the candidate also impacts CySC self-renewal. Aim 3 is focused on elucidating the function of the candidate gene in processes required for GSC maintenance, including compensating for BMP signaling, repressing differentiation genes and promoting niche adhesion or gap junctions. 70% of human disease genes have counterparts in Drosophila, highlighting the relevance of Drosophila to human health. We will identify one of more factors conserved in humans that are required for the maintenance of GSC. The work in this proposal may shed light on stem cell-niche function in mammals.

Transcriptional Regulation of Hematopoietic Stem Cells Functions

Gregory David, PhD
New York University School of Medicine

A healthy individual needs to produce several millions of blood cells per second to maintain blood homeostasis. This constant production of new cells depends on a rare subpopulation of cells that reside in the bone marrow and are called hematopoietic stem cells (HSCs). As the individual ages, these hematopoietic stem cells become impaired in their ability to give rise to all blood cells, potentially resulting in anemia and defective responses to viruses. It is believed that HSCs need to be dormant and non-proliferative in the bone marrow in order to keep their properties and remain functional. However, the mechanisms that allow these cells to stay dormant and functional are largely elusive. We have identified a novel protein, Sin3B, which is required for HSC functions in vivo. In addition, we have shown that HSCs that do not have the Sin3B protein exhibit hallmarks of old HSCs, thus providing us with a unique opportunity to understand how HSCs age. We propose here to dissect at the molecular level how the Sin3B protein regulates HSC function and aging, in order to identify therapeutic intervention to promote HSC function and treat age-associated blood diseases.

Targeting TET2-Deficiency in Hematopoietic Stem Cells with High-Dose Vitamin C

Benjamin Neel, MD, PhD
Co-PI: Luisa Cimmino, PhD
New York University School of Medicine

Vitamin C is essential for maintaining healthy hair, skin, immune system and heart function. In addition to these health benefits, vitamin C has the potential to be a non-toxic anti-cancer agent for the treatment of patients with a variety of different tumors. The antioxidant properties of vitamin C protect us from damage to cells and DNA in the body. However, another important role of vitamin C is to enhance the activation of a group of enzymes called TET proteins that determine whether key genes are expressed in different cell types. The TET proteins are highly expressed in stem cells of the immune system, and deficient TET activity, due to mutations in the genes for these proteins, causes stem cells to lose control of their growth and become blocked in their ability to form blood normally. Up to 30% of patients with myeloid leukemias have mutations in the gene TET2, which leads to impaired TET2 function. Interestingly, only one of the two copies of the TET2 gene is defective in these leukemia patients. We hypothesize that treatment of TET2-deficient leukemia cells with high-dose vitamin C could enhance the activity of the remaining, non-mutant, TET2 protein and restore normal stem cell function. In addition, whether vitamin C deficiency in the diet effects TET protein function in stem cells of the immune system and leukemia progression has not been studied. We propose to use mouse models that mimic TET or vitamin C deficiency, and leukemia samples from patients with TET2 mutations to assess whether high-dose vitamin C treatment can restore deficiencies in stem cells that give rise to leukemia. Targeting disease-initiating stem cells with vitamin C might provide a safe and effective strategy to improve outcome for patients with leukemia.

Identification of Novel Regulators of Hematopoietic Stem Cells

Christopher Park, MD, PhD
New York University School of Medicine

The central dogma of biology holds that the flow of genetic information is unidirectional and runs from DNA to RNA through a process called transcription, and that RNA is converted to protein through a process called translation. Since the central dogma was first described in 1956, it has become increasingly clear that each of these steps in the flow of information - transcription and translation - is highly regulated. Thus, many factors determine whether or not genes encoded in the DNA eventually give rise to the proteins that support cellular processes. Given the specialized nature of the many cell types in multicellular organisms, perhaps it is not surprising that the complement of RNA and proteins made in each cell type is unique despite their shared DNA genetic code, including in the cells that are the focus of this proposal – blood stem cells. Unfortunately, prior studies have focused on identifying RNAs present in blood stem cells without assessing whether they are being actively converted into protein. Since we know that RNAs are not necessarily fated to give rise to protein, not understanding how this process is regulating in blood stem cells represents a significant gap in our understanding of how they function. We hypothesize that proteins actively synthesized in blood stem cells are likely to be important regulators of their function. Thus, we propose to utilize novel experimental tools developed in our lab to identify RNAs that are being actively translated into proteins. In order to accomplish this, we will evaluate this process in blood stem cells from both mice and humans. Through these comparisons, we expect to reveal novel insights regarding how RNA is utilized to make protein in blood stem cells as well as identify novel genes that regulate their function.

Angiocrine Functions of Endothelium in Regulating Stem Cell Driven Cerebellar Repair

Alexandra Joyner, PhD
Co-PI: Shahin Rafii, MD
Sloan-Kettering Institute for Cancer Research and Weill Medical College of Cornell University

A critical function for blood vessels that is beyond their well-known role in distribution of oxygen and nutrients to tissues, is that they secrete proteins that are necessary for development, homeostasis and repair of the rest of all organs. Blood vessels support these critical organ functions by creating niches for stem cell populations wherein the blood vessels supply the necessary proteins required both to maintain an adequate stem cell number throughout our lifetime and to support their mobilization to replenish lost cells following injury or normal wear and tear. We propose to study a recently appreciated stem cell population in the developing cerebellum, and determine the role of blood vessels in their ability to respond to injury and replenish lost cells. The cerebellum is critical for skilled motor performance and also influences cognitive and social functions. It is particularly vulnerable to clinical and environmental factors around birth, since much of its growth occurs in the third trimester and continues for a year after birth. Preterm babies are particularly vulnerable to cerebellar damage. We will utilize mouse models for in vitro stem cell-blood vessel coculture assays and studies of the roles of blood vessels in enabling cerebellar stem cells to replenish cerebellar cells lost due to damage around birth. The results of these studies will not only provide insights into how blood vessels provide a niche for cerebellar stem cells and aid in a regenerative process, but also lay the foundation for capitalizing on the instructive functions of blood vessels for therapeutic repair of compromised cerebella by expanding stem cell populations in vitro or in the brain of humans.

Optimizing Replacement Cell Therapies Through Microchip Analysis of Cell Cooperativity

Jun Wang, PhD
Co-PI: Janet Paluh, PhD
University at Albany, SUNY and SUNY Polytechnic Institute

Traumatic brain injury (TBI) is a top biomedical priority, affecting 1.5 million Americans per year. Recognized in importance as early as 1992 by establishment of the Defense and Veterans Brain Injury Center (DVBIC) and tracked statewide including In New York State, each year 150,000 new civilian cases occur, at an economic cost of $5 billion. TBIs are complex and distinct, and often impact the cerebral cortex of the brain, which is the regionally diverse outer layer of neural tissue involved in controlling movement, interpreting the senses, conscious thought and memory. There is limited neurogenesis to repair damage or replace the cells that die upon brain injury in adults. For mild to severe TBI, the prognosis is more critical than diagnosis for treatment and quality of life. Stem cell research with in vitro models of TBI holds great promise for rapid discovery to attain this goal. Our proposal addresses this gap by providing a detailed map of neural differentiation from progenitors to mature neurons of cell interacting microenvironments and injury responsiveness for critical mechanistic details. We will develop an in vitro high throughput technology for cortical neural differentiation that brings access to information on changing cell microenvironments and their released signaling factors that bathe and instruct these cells. The uniqueness of this neural cell-cell interaction microchip (NCCIM) is the ability to identify and study subpopulations of the whole in regard to signaling changes that instruct their destiny and injury responsiveness. We expect to advance in vitro models of neural differentiation that describe transiting subpopulations and their ability to respond to compressive forces to assist mechanistic understanding of compressive TBI and biomarkers. We expect the NCCIM to be extendable to other central nervous system injuries that dramatically impact the quality of human life.

Harnessing the Regenerative Potential of the Epicardium

Eric Small, PhD
University of Rochester

The heart has a surprising capacity to heal and support life even after catastrophic injury such as myocardial infarction (heart attack). However, the healing process in the adult heart proceeds primarily via scar formation, which results in reduced cardiac function that often transitions into heart failure. Current therapeutic strategies have not proven useful in stimulating regeneration of healthy cardiac tissue. Remarkably, fetal cardiac tissue displays "scarless" repair, which stems from a nearly unlimited regenerative potential. It is hypothesized that the epicardium, a single cell-layer sheet of multi-potent cardiac progenitor cells that surrounds the heart, contributes to cardiac regeneration in the fetal and early post-natal periods. This fetal epicardium stimulates cardiac cell proliferation and contributes to the formation of new coronary vessels. Unfortunately, although the epicardium becomes "reactivated" after a heart attack, this regenerative capacity is lost in the adult. We have identified a number of factors that are secreted from the fetal epicardium that might underlie the ability of this tissue to support cardiac repair. It is the goal of this proposal to define which of these "epicardium progenitor cell-derived regeneration factors" might improve cardiac function after a heart attack. To accomplish this goal, we will evaluate the ability of candidate factors to stimulate various processes that are required for efficient cardiac repair, including cardioprotective inflammation, new vessel growth, and cardiomyocyte regeneration. These studies will be conducted using "heart patches" or viral technologies to deliver the candidates to the heart in a standard model of heart attacks in mice. We believe these studies will identify genes that guide the unique capacity of the fetal epicardium to support cardiac repair. Finally, these studies will have significant impact on our understanding of epicardium-derived progenitor cell reactivation following heart attack and may provide novel therapeutic strategies for the treatment of ischemic heart disease.

Engineering Induced Pluripotent Stem Cells for "Off the Shell" Adoptive T Cell Therapy

Irene Min, PhD
Co-PI: John Lis, PhD
Weill Medical College of Cornell University and Cornell University

Adoptive cell therapy is a highly personalized cancer therapy that uses the patient’s own immune cells to mount cancer-killing activity. Adoptive transfer of T lymphocytes engineered to express chimeric antigen receptors (CAR) can recognize and kill tumor-associated molecules. This immunotherapeutic approach has shown great success against blood cancers and is currently in clinical trials for some solid cancers. The CAR-T cell therapy has the potential to be applied to a wide of range of cancer patients when the tumors express the same tumor-associated molecule. However, this versatility is limited to custom-made production because donor T cells are taken from the cancer patient directly to avoid attacking other healthy organs and tissues. Human induced pluripotent stem cells (iPSCs) that are made from adult cells have the ability to generate functional T cells, therefore iPSC-derived T cells have the potential to serve as universal donor source for CAR T cell therapy. Firstly, we propose to genetically engineer iPSCs to eliminate possibilities of immune rejection and harming the recipient by triggering an immune response. Secondly, we will direct the iPSC differentiation to T cells to use as a source for "off the shelf" CAR-expressing T cells. Thirdly, we will analyze unique molecular signatures associated with iPSC-derived CAR T cells and compare with conventional CAR T cells. This analysis will provide a ‘fingerprint’ informative of how iPSC-derived CAR T cells will respond to the tumor cells. Providing a universal source for “off the shelf” adoptive T cell therapy with an easy and fast access may lead to a breakthrough in cancer treatment.

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