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Investigator Initiated Research Projects and Innovative, Developmental or Exploratory Activities in Stem Cell Research - 2013

RFA #: 1206180230

IIRP Awards

RFA #: 1206180230
Institution PI Amount Project Title

Albert Einstein College of Medicine

Bouhassira, Eric

$1,080,000

Allele-Specific Characterization of iPS cells

Albert Einstein College of Medicine

Frenette, Paul

$1,080,000

Function of NG2+ Mesenchymal Stem Cells in the Hematopoietic Stem Cell Niche

Albert Einstein College of Medicine

Guo, Wenjun

$1,080,000

Role of Mammary Stem/Progenitor Cell Regulator Sox9 in Basal-Like Breast Cancer

Cold Spring Harbor Laboratory

Enikolopov,Grigori

$1,078,363

Redox Regulation of Adult Neural Stem Cells

Columbia University Medical Center

Kass, Robert

$1,023,841

Calcium-Dependent Spontaneous Activity as a Novel Therapeutic Target of Inherited Arrhythmia Studied in Human Induced Pluripotent Stem Cell-Derived Cardiac Myocytes 

Columbia University Medical Center

Liu, Kang

$1,020,607

Cellular and Molecular Events Underlying Human Dendritic Cell Differentiation from Hematopoietic Stem Cells

Columbia University Medical Center

Tsang, Stephen

$1,080,000

Comparative Effectiveness of Embryonic and Induced Pluripotent Stem Cell-Based Therapies

Icahn School of Medicine at Mount Sinai

Bernstein, Emily

$1,078,896

Investigating Epigenetic Barriers to Somatic Cell Reprogramming

Icahn School of Medicine at Mount Sinai

Chaudhry, Hina

$1,073,000

Fetal-Derived Placenta Stem Cells for Cardiac Repair

Icahn School of Medicine at Mount Sinai

Hoffman, Ronald

$1,080,000

Epigenetic Regulation of Symmetrical Hematopoietic Stem Cell Renewal Divisions

Icahn School of Medicine at Mount Sinai

Rendl, Michael

$1,080,000

Stem Cell Niche Control of Hair Regeneration

Icahn School of Medicine at Mount Sinai

Xu, Pin-Xian

$1,080,000

Exploring New Strategies for Potential Regenerative Treatments Based on Transcriptional Reprogramming or Transdifferentiation in the Mammalian Auditory System

New York State Psychiatric Institute

Karayiorgou, Maria

$954,414

Making Headway in Schizophrenia by Combining the Power of iPSC Technology with New Genetic Knowledge

New York University School of Medicine

Aifantis, Iannis

$906,150

TET Family Proteins and the Regulation of Hematopoietic Stem Cell Self-Renewal and Differentiation

New York University School of Medicine

Basilico, Claudio

$1,065,042

Regulatory Networks Determining Sox2 Dependence of Osteosarcoma Stem Cells

New York University School of Medicine

Nance, Jeremy

$898,578

The Role of Adhesion and Nutrition in Stem Cell Activation

The Rockefeller University

Fuchs, Elaine

$1,080,000

Identifying the Alterations in Skin Stem Cell Function Responsible for Cancer

Sloan-Kettering Institute

Hadjantonakis, Anna-Katerina

$1,067,063

The Emergence of Pluripotency In Vivo

Sloan-Kettering Institute

Huangfu, Danwei

$995,183

Precision Genetics in hESCs to Define the Roles of GATA6 in Human Development and Disease

Sloan-Kettering Institute

Shi, Song-Hai

$1,080,000

Clonal Production and Organization of hPSC-Derived Cortical Excitatory Neurons Under Normal and Disease Conditions

University at Buffalo - SUNY

Feng, Jian

$1,080,000

Understand the Pathophysiology of Parkinson's Disease Using Genetically Modified iPS Cells

University at Buffalo - SUNY

Gronostajski, Richard

$1,073,068

Role of Nfi Genes in Neural Stem Cell Homeostasis

University of Rochester

Bulger, Michael

$1,080,000

Self-Renewing Erythroblasts from Human ES Cells as a Source of Blood

University of Rochester

Hsu, Wei

$1,061,020

Stem Cell-Mediated Craniofacial Skeletogenesis in Health and Disease

University of Rochester

Noble, Mark

$1,080,000

Lysosomal Storage Disorder-Induced Precursor Cell Dysfunction in the CNS

IDEA awards

Institution

PI

Amount

Project Title

Columbia University

Lu, Helen

$330,000

Stem Cell-Mediated Integrative Cartilage Repair

Columbia University Medical Center

Christiano, Angela

$330,000

Stem Cell and iPSC Therapy for Epidermolysis Bullosa

Columbia University Medical Center

Leibel, Rudolph

$330,000

The Role of Endoplasmic Reticulum Stress in Diabetic Beta Cell Failure 

Cornell University

Buchon, Nicolas

$328,498

Impact of Microbes on Intestinal Stem Cells in Drosophila

Cornell University

Lee, Siu Sylvia

$326,071

Using C. elegans Germline as a Model to Study the Functions of HCF-1, a Critical Factor in Stem Cell Pluripotency

Cornell University

Shen, Xiling

$323,042

Versatile MicroRNA Regulation For Cell Fate Decision

Icahn School of Medicine at Mount Sinai

Dubois, Nicole

$330,000

Exploring Pathophysiology and Therapy Strategies of Duchenne Muscular Dystrophy Cardiac Dysfunction in Human iPSC-Derived Engineered Heart Tissue

Icahn School of Medicine at Mount Sinai

Ehrlich, Michelle

$329,954

Terminal Differentiation of Striatal Medium Spiny Neurons from H9 Cell Line

New York University School of Medicine

Littman, Dan

$330,000

Control of the Intestinal Stem Cell/Progenitor Pool by IL-23-Dependent Lymphocyte Signaling During Homeostasis and Repair

Mount Sinai School of Medicine

Sfeir, Agnel

$330,000

Identifying Telomerase Regulators in Pluripotent Cells

New York University School of Medicine

Stadtfeld, Matthias

$329,950

Molecular Roadblocks for In Vitro Hematopoietic Stem Cell Specification

University of Rochester

Mayer-Proschel, Margot

$330,000

Precursor Cells As Viral Targets: a New Approach to Understanding Failure of Repair in the Human CNS

University of Rochester

Que, Jianwen

$330,000

Modulation of Intestinal Differentiation In Esophageal Basal Stem Cells

University of Rochester

Xing, Lianping

$330,000

Proteasomal Regulation of Osteoblasts

University of Rochester

Zhang, Yi

$330,000

Role of MECOM in Leukemia Stem Cell Quiescence

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IIRP awards


Allele-Specific Characterization of iPS cells

Eric Bouhassira, 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.


Function of NG2+ Mesenchymal Stem Cells in the Hematopoietic Stem Cell Niche

Paul Frenette, MD
Albert Einstein College of Medicine

Stem cells maintain the tissues of the body throughout life through tightly controlled decisions about survival vs. death, proliferation vs. quiescence. Blood stem cells (also called “hematopoietic stem cells”, HSCs) can form all blood cells of the circulation. To ensure lifelong replenishment of all blood lineages, HSCs must keep a constant renewing pool. Rare cellular division (i.e. “cell cycle quiescence”) is a critical feature contributing to stem cell maintenance. Recent studies have highlighted the importance of specific bone marrow microenvironments, also referred to as niches, in HSC behavior. There has been considerable interest and debate about whether or not quiescence and proliferation of HSCs are regulated by distinct niches. Previous reports have suggested that quiescent HSCs reside near bone cells (called osteoblasts) whereas proliferative HSCs were found near blood vessels. However, this popular concept has not been supported experimentally. To gain more insight into the spatial localization of HSCs, we developed a staining technique that allows precise measurements of tridimensional distances of endogenous HSCs from niche structures, and we combined it with computational simulation to define the significance of these interactions. This novel approach allowed us to uncover two distinct types of vessels associated with quiescent or proliferative HSCs. Indeed, we found that small arteries supported HSC quiescence, whereas veins (called sinusoids in the marrow) were associated with proliferation. In this proposal, we will define better the niche cells that comprise these different microenvironments. Using genetic markers, we will investigate the hierarchical organization of these niche cells. We will also explore the concept that quiescence of the niche cell may regulate HSC quiescence through crosstalk between these two cells. These results will provide novel conceptual and technical advances, and contribute to define further the microenvironments that regulate stem cell behavior in living mammals.


Role of Mammary Stem/Progenitor Cell Regulator Sox9 in Basal-Like Breast Cancer

Wenjun Guo, PhD
Albert Einstein College of Medicine

Several types of aggressive breast cancer have been shown to originate from breast stem cells or progenitor cells. However, the exact mechanism by which these normal stem or progenitor cells are preferentially transformed into malignant cancer cells is not clear. It is agreed upon that the unique properties of stem/progenitor cells may render them prone to become cancerous. Our previous studies have identified an important gene called Sox9 that is required for governing breast stem and progenitor cell properties. In this proposal we will investigate the function of this stem cell gene and breast stem/progenitor cells in the formation and progression of a particularly aggressive breast cancer, the so-called triple-negative or basal-like breast cancer. We will assess whether Sox9 is required for the cancer formation and whether inhibiting this gene and its associated molecules can block the growth of already formed tumors. In addition, we will investigate whether the tumor cells expressing Sox9 are cancer stem cells, the rare cell population that is responsible for tumor resistance to chemotherapy. Our studies are likely to provide insights into the mechanisms by which stem and progenitor cells contribute to malignant cancer formation and progression. Through identifying key genes and cell types that are required for these processes, our studies will help discover therapeutic targets that can be exploited for cancer treatment.


Redox Regulation of Adult Neural Stem Cells

Grigori Enikolopov, PhD
Cold Spring Harbor Laboratory

Contrary to long standing belief, a large number of new neurons are generated in the adult brain of humans and animals. These neurons are important, among their other roles, for certain features of memory, brain repair after injury, and therapeutic action of antidepressants. New neurons arise from stem cells, a population of cells that reside in restricted regions of the adult brain and produce neurons throughout the life of the animal, albeit in ever diminishing numbers. How these stem cells are regulated, what drives their age-related decrease, and how they can be activated to fight disease or aging are some of the most promising and yet most challenging questions of biology, neuroscience, and medicine. We recently presented a new model of regulation of neural stem cells that explains their age-related decline and has important implications for biology and medicine. One of the main outstanding issues is regulation of neural stem cells in the adult brain. It is increasingly clear that changes in the oxidation of certain proteins (so called redox regulation) may play a crucial regulatory role. However, proper animal models are required to test different hypotheses on the role of redox regulation in stem cells. To address this issue, we generated several mouse lines in which neural stem and progenitor cells are marked by the expression of various fluorescent proteins, thus making it possible to identify and track these cells. Furthermore, we generated another series of recombinant proteins and mouse lines, where changes in the redox status are sensed and reported by other types of fluorescent proteins. Together, the combination of these genetically modified animal lines provides a unique opportunity to visualize the changes in the redox status in the brain and to determine the role of these changes in stem cell function, aging, and brain repair.


Calcium-Dependent Spontaneous Activity as a Novel Therapeutic Target of Inherited Arrhythmia Studied in Human Induced Pluripotent Stem Cell-Derived Cardiac Myocytes

Robert Kass, PhD
Columbia University Medical Center

Inherited cardiac rhythm disturbances due to alterations (mutations) in genes that generate proteins in the heart called ion channels are a unique class of diseases (channelopathies) that place mutation carriers at high risk of heart attack and death. The Long QT Syndrome (LQTS) was the first reported and now the most thoroughly studied cardiac channelopathy. A great deal has been learned about LQTS mechanism and mutation-specific therapeutic strategies using methodologies that have relied on animal models or on indirect in vitro assays. Although important, these approaches have not addressed clinical problems presented by patients with complex genetic backgrounds. This limitation is being overcome by new technologies that have enabled the derivation of heart cells (cardiomyocytes) derived from induced pluripotent stem cells (hiPSC-CMs) obtained directly from patients with the inherited gene defects. hiPSCs are cells that have been reprogrammed into a pluripotent state from small skin samples obtained from mutation carriers and from family members who are not affected. This approach permits investigation of disease mechanism and therapeutic approaches to disease management, in a patient-specific manner. In a first study, our laboratory used hiPSC-CMs from members of an LQTS family in which the patient at risk had a complex genetic background. This successful study provided direct insight into one mechanism underlying the electrical dysfunction in the patient as well as methods for optimizing therapeutic strategies to manage the disease, which correlated directly with improved clinical regimens. We now propose to study the role of an additional arrhythmogenic mechanism which we hypothesize is due to altered intracellular calcium dynamics changed indirectly by the primary gene mutation in the patient to identify compounds that will maximize the therapeutic response of patients presenting similar disease phenotype by targeting calcium handling proteins in the heart.


Cellular and Molecular Events Underlying Human Dendritic Cell Differentiation from Hematopoietic Stem Cells

Kang Liu, PhD
Columbia University Medical Center

In humans, one trillion new blood cells are generated everyday in the bone marrow. These blood cells with distinct functions mediate immune responses that protect us against tumors and infections. Despite their difference in distribution and function, these blood cells all descend from hematopoietic stem cells (HSCs) through a highly organized process called hematopoiesis that includes many cellular and molecular events. Among these blood cells, a unique population with stellate morphology, called dendritic cells (DCs), play a critical role in detecting infection and initiating immune responses. DCs are constantly replenished through hematopoiesis. It was recently recognized that patients with few or no DCs, caused by aberrant hematopoesis, suffer from recurrent infections and ultimately develop malignancies. However, prompt diagnoses and therapy of DC-deficiency are lacking, because the precise cellular and molecular events of human DC hematopoiesis in health and disease are unclear. We hypothesize that human DCs form a unique branch in the roadmap of human hematopoiesis, and this branch is comprise of distinct progenitor cells. Normal DC development depends on transitions between precursor cells orchestrated by the transcriptional program. This project characterizes the precise cellular progenitors and reveals the molecular mechanisms underlying hematopoiesis of DCs from HSCs, thereby providing diagnostic tools and cellular and molecular targets for therapeutic treatment.


Comparative Effectiveness of Embryonic and Induced Pluripotent Stem Cell-Based Therapies

Stephen Tsang, MD, PhD
Columbia University Medical Center

Age-related macular degeneration (AMD) and some forms of retinitis pigmentosa (RP) are blinding diseases characterized by loss of the retinal pigment epithelium (RPE). Recent advances suggest that stem cells can replace damaged RPE. One such therapy using embryonic stem cells (ESCs) was recently approved for clinical trials. However, these ES cell-based therapies require immunosuppression and are restricted to a limited, expensive, and controversial source of tissue. One alternative option, treatment with induced pluripotent stem cells (iPSCs), would involve use of stem cells derived from the patient’s own skin, rendering them unlikely to promote rejection. Gene therapy is a third possible approach to treating RP. The proposed research will explore iPSC-based transplantation in a mouse model of RP and compare the effectiveness of iPSC- and ESC-based therapies for RP. Unlike tissues transplanted from another individual, iPSC-derived RPE should not introduce a risk of graft vs. host disease. This would be the first-ever study to directly compare iPSC and ESC-based therapies for RP. We will also investigate the promise of gene therapy techniques, which directly compensate for mutations by providing a new copy of the gene. In future trials, RPE could be derived from patients themselves and used for transplant therapy. Overall, the eye is an ideal proving ground for developing and testing any kind of stem cell therapy because one eye can be treated and compared to an untreated fellow eye, and the blood-retina barrier grants the eye relative immune privilege. The eye is also optically transparent, making it possible to non-invasively monitor stem cell grafts. We will use live imaging techniques that make it possible to view a cross-section of the eye after treatment without sacrificing the animals.


Investigating Epigenetic Barriers to Somatic Cell Reprogramming

Emily Bernstein, PhD
Icahn School of Medicine at Mount Sinai

Recent technologies have allowed for the generation of stem cells from committed adult tissues. This is referred to as somatic cell reprogramming. Reprogramming can be achieved by the expression of four transcription factor proteins, which together reorganize gene expression programs and alter the chromatin landscape. Chromatin is the complex of our DNA and the proteins that package and regulate our genome. It is possible to envision that by changing the chromatin structure itself, cell fate may be manipulated. Recently, we demonstrated that the deposition of specialized macroH2A histone variants into the chromatin of somatic cells creates a barrier to reprogramming. These histone variants, which are involved in gene silencing, cooperate with another silencing mark in chromatin called H3K27me3, to prevent activation of stem cell genes in somatic cells and upon reprogramming. Here we propose to identify the mechanisms by which macroH2A acts as a barrier to reprogramming. We will perform extensive genomic analyses, with a focus on a subset of stem cell genes that are reactivated early in the reprogramming process by the ‘eraser’ of H3K27me3 (called Utx). We will also examine the role of macroH2A at the newly described ‘super-enhancer’ genomic elements that appear to be important in defining cell fates. Finally, we will use a unique genetic screen to identify novel chromatin regulators in the reprogramming process that may cooperate with or antagonize macroH2A function. Collectively, these studies will enhance our understanding of the chromatin barriers that impede reprogramming, and allow improved reprogramming methodologies. The ability to change cell fates also has tremendous potential for patient-specific cell treatments and drug development, with the ultimate goal of counteracting disease.


Fetal-Derived Placenta Stem Cells for Cardiac Repair

Hina Chaudhry, MD
Icahn School of Medicine at Mount Sinai

Fetal cells are known to enter the maternal circulation during pregnancy and may persist in maternal tissue for decades as microchimeras. Based on clinical observations of peripartum cardiomyopathy and the high rate of recovery from heart failure such patients experience, we were inspired to examine whether fetal cells migrate to the maternal heart and differentiate to cardiac cells. We have recently reported that fetal cells selectively home to injured maternal myocardium and undergo differentiation into diverse cardiac lineages. Using enhanced green fluorescent protein (eGFP)-tagged fetuses, we demonstrated engraftment and cardiac differentiation of multipotent fetal cells in injury zones of maternal hearts. In vitro, fetal cells isolated from maternal hearts recapitulate these differentiation pathways, forming vascular tubes and spontaneously beating cardiomyocytes in a fusion-independent manner. A significant proportion (~40%) of fetal cells in maternal hearts express Caudal-related homeobox 2 (Cdx2), previously associated with trophoblast stem cells. Based on these results and additional RNA array data with respect to pluripotency genes expressed in fetal-derived placenta cells, we hypothesize that Cdx2 cells or other pluripotent cells capable of cardiac differentiation can be isolated from end-gestation placentas and be utilized for allogeneic stem cell transplantation. Our goal is to translate these studies for eventual clinical use, and we propose two specific aims in order to achieve these goals. In the first specific aim, we aim to isolate Cdx2 cells and other fetal-derived placenta cells from end-gestation mouse placentas and test their differentiation properties in vitro. We will employ novel mouse models for lineage-tracing in selecting Cdx2 cells, and will utilize established live-imaging approaches in our laboratory to carry out this aim. Furthermore, we have set up collaborations with world-reknowned stem cell biology expert Dr. Ihor Lemischka and established placenta biologist Dr. Soumen Paul who will both provide intellectual support for our project. With regard to the second specific aim, we plan to test the ability of Cdx2 cells (or other fetal-derived placenta cells that may undergo the most efficient in vitro differentiation) to form cardiomyocytes and vascular cells in vivo and restore cardiac function via transplantation experiments in a murine infarction model.


Epigenetic Regulation of Symmetrical Hematopoietic Stem Cell Renewal Divisions

Ronald Hoffman, MD
Icahn School of Medicine at Mount Sinai

Hematopoietic stem cells (HSCs) are the mother cells for all blood cells. They are responsible for rescuing patients with blood cancers or genetic blood disorders who receive high doses of chemotherapy and/or radiation therapy in preparation for a potentially curative allogeneic stem cell transplant. Blood stem cells are able to generate additional stem cells, a property called self-renewal. Umbilical cord blood is an alternative source of stem cells for patients who need a transplant but do not have a family member who is genetically matched. The success of cord blood transplantation in adults is limited since cord blood collections contain limited numbers of stem cells, resulting in an unacceptably high rate of graft failure or an excessive amount of time being required for the patient’s blood counts to recover. These limitations can be overcome by infusing greater numbers of stem cells. A great deal of research has been performed to expand the numbers of cord blood stem cells in the laboratory but these efforts have met with limited success. We have developed a new method to expand human cord blood stem cells in the laboratory by treating them with a drug (valproic acid - VPA) which increases the expression of genes required for a stem cell to symmetrically divide and produce additional functional stem cells. In this project, we will study the characteristics of HSC expanded with VPA and learn how this drug acts to promote the expansion of the number of stem cells. We will determine the changes in chromatin structure and resulting gene expression pattern required to instruct these HSC to undergo self-renewal. These studies will provide new information about stem cell division which will be useful in developing additional effective strategies to expand stem cell numbers for use as grafts for transplant patients.


Stem Cell Niche Control of Hair Regeneration

Michael Rendl, MD
Icahn School of Medicine at Mount Sinai

Understanding the regulation of stem cell activation and self-renewal by supporting niche cells is a crucial requirement for future tissue-specific regenerative therapies, but how niche cells communicate with the stem cells in most tissues is largely unknown. In this application we explore the control of stem cells in the hair follicle, a well-defined and highly accessible model system for studying stem cell functions right at the surface of the skin. Although many insights have been gained about hair follicle stem cells in general, little is known about their molecular control by neighboring dermal papilla niche cells. Here, we will establish genetic tools to study for the first time gene functions specifically in the adult dermal papilla niche, and test the stem cell activating, hair regenerating capacity of these cells. We will then systematically define their molecular features at a genomic scale and functionally test the role of PDGF signaling and other newly identified signaling molecules for SC activation during hair follicle regeneration. Our work will provide essential new tools and insights for the hair follicle stem cell niche. They will greatly improve our ability to manipulate adult skin stem cells for future hair regenerative therapies. Finally, new knowledge about niche control of adult stem cells will also have global relevance for other regenerative tissues, where stem cell niches operate to maintain tissue homeostasis.


Exploring New Strategies for Potential Regenerative Treatments Based on Transcriptional Reprogramming or Transdifferentiation in the Mammalian Auditory System

Pin-Xian Xu, PhD
Icahn School of Medicine at Mount Sinai

Hearing loss is one of the most widespread disabilities in the world. The primary cause is damage to the inner ear sensory hair cells and their associated spiral ganglion neurons. In mammals, cochlear hair cells and their associated neurons do not regenerate, resulting in irreversible deafness. To date, many molecular strategies have failed to induce a true post-trauma regeneration of hair cells and neurons. Transplantation of progenitor cells in damaged cochlea represents a potential cell therapy for deafness. However, the maintenance and propagation of progenitors is a challenge. We do not yet have a definitive strategy for auditory regeneration. The only therapy currently available is the use of hearing aids and cochlear implants, which do not fully replace the complexity of the biological organ. Thus, there is a desperate need for innovative strategies for auditory repair. The goal of this application is to explore new strategies for potential regenerative treatments based on transcriptional reprogramming or transdifferentiation in the mammalian auditory system. We recently found that reprogramming of cochlear nonsensory cells toward a hair-cell or neuronal fate can be induced by a set of inner ear neurosensory cell-specific transcription factors in combination with different cofactors. Furthermore, overexpression of this set of factors in combination with a chromatin-remodeling complex in fibroblast cells can convert the cells to neurons. However, direct reprogramming by these factors in vivo remains to be shown. In this application, we will test whether misexpression of these factors in damaged cochlea is sufficient to induce nonsensory cells to regenerate functional hair cells and whether the converted fibroblast-neuronal cells are functional for neuronal repair. This study represents a major step forward for reprogramming or transdifferentiation in the auditory field and has the potential to provide alternative or complementary strategies to cell replacement or cell transplantation-based approaches for auditory repair and regeneration.


Making Headway in Schizophrenia by Combining the Power of iPSC Technology with New Genetic Knowledge

Maria Karayiorgou, MD
New York State Psychiatric Institute

There is considerable promise in generating induced pluripotent stem cells (iPSCs) and neurons from patients afflicted with central nervous system (CNS) diseases, and in particular mental illnesses. The inaccessibility of the human CNS renders the availability of neurons derived from patients with mental illness very valuable. Major mental illnesses, such as schizophrenia, are highly heterogeneous but focusing on a small number of mutations and looking for common alterations in patient-derived neurons may provide important insights into disease mechanisms. Recent genomic advances have identified a number of mutations that unequivocally increase vulnerability to schizophrenia and provide a unique opportunity to implement such a research strategy. We propose to focus on a set of three specific mutations robustly associated with schizophrenia and characterize in detail their effects on neuronal structure and function. Our studies will provide a wealth of information toward the pathophysiology of the disease and a useful platform for testing potential therapies.


TET Family Proteins and the Regulation of Hematopoietic Stem Cell Self-Renewal and Differentiation

Iannis Aifantis, PhD
New York University School of Medicine

In adults, the continuous replenishment of cells in the blood is controlled by a small population of stem cells known as hematopoeitc stem cells (HSCs). The hallmarks of a stem cell, the ability to self-renew and differentiate, are the same features that underscore many blood malignancies. Indeed, leukemia is initiated by genetic events occurring in small stem or progenitor cell populations, termed leukemia-initiating cells. The molecular mechanisms controlling normal HSC self-renewal and differentiation are now issues of intense study. In patients with leukemia, a novel class of mutations have recently been identified that perturb blood stem cell differentiation. Specifically, deletions and mutations in the TET2 gene were identified in almost 30% of all myeloid malignancies and were associated with decreased patient survival. The TET family proteins (TET1-3) are enzymes that modify DNA epigenetically to promote DNA demethylation and removal of gene silencing, in this way regulating normal gene activation. TET2 mutant proteins observed in leukemia patients have been shown to be deficient in this enzymatic function. Mutant forms of the other family members, such as TET1, are also being discovered in patients with leukemia and both of these enzymes are expressed in blood stem cell populations. Taken together, these data suggest that alterations in genes that regulate the epigenetic state of HSCs are a common pathogenic event in leukemia. Our laboratory has recently developed Tet2-deficient mice and these animals develop myeloid leukemia that mirrors the human disease. We also demonstrated that Tet2 inactivation leads to alterations in DNA methylation, enhanced stem cell self-renewal and enlargement of a pool of putative leukemia initiating cells. We have performed similar studies with Tet1-deficient mice and find that both genes have the potential to be tumor suppressors of blood stem cells. Our current proposal introduces the hypothesis that loss of TET function affects DNA methylation patterns and transcriptional networks essential for stem cell quiescence, self-renewal and differentiation during hematopoieisis. We will continue to test this hypothesis using Tet-deficient mouse models to identify gene networks that regulate self-renewal in HSCs. The gene expression networks regulated by the Tet proteins may also point toward novel therapeutic targets to treat these stem cell-driven blood malignancies.


Regulatory Networks Determining Sox2 Dependence of Osteosarcoma Stem Cells

Claudio Basilico, MD
Co-PI: Alka Mansukhani, PhD
New York University School of Medicine

Osteosarcomas are the most common type of bone tumors that arise in children and adolescents and the long-term survival rate has not improved much during the last 30 years. Like other cancers, osteosarcomas contain a population of stem cells capable of initiating and spreading tumors as well as causing relapse. Such cells have been termed tumor initiating cells or cancer stem cells. The study of such cancer stem cells is of great importance for effective tumor therapy, since these cells are highly resistant to conventional chemotherapy. We have identified a gene called Sox2, whose best-known function is to regulate the expression of “stemness” genes, as a distinct marker of cancer stem cells in osteosarcomas. When Sox2 is inactivated in human or mouse osteosarcoma cells, they can no longer form tumors and acquire some properties of normal cells. In the present project, we aim to identify the genes regulated by Sox2 in osteosarcomas and the properties of the stem, and non-stem tumor cells. We also want to study how Sox2 exerts its influence on other pathways whose normal function is to suppress tumorigenicity and the importance of such influence in the maintenance of cancer stem cells in osteosarcomas. Finally we will determine whether inactivating Sox2 in the bones of mice will prevent the formation of osteosarcomas. These studies will pinpoint the cells from which these cancers originate. By identifying the genes controlled by Sox2 and determining how it maintains cancer stem cells, we could develop new strategies for innovative therapies to cure osteosarcomas and possibly other tumors.


The Role of Adhesion and Nutrition in Stem Cell Activation

Jeremy Nance, PhD
Co-PI: E. Jane Hubbard, PhD
New York University School of Medicine

A major goal of stem cell research is to manipulate stem cells to become specific cell types, so that these cells can be used to replace damaged or missing cells following injury or disease. In the body, stem cells receive local instructions on what to become from neighbors called niche cells. Stem cells also receive systemic signals, such as hormones and metabolites, that allow them to respond to changes in organismal environment. It is poorly understood how niche and systemic signals affect stem cells in the body, and learning these mechanisms will provide critical information on how to manipulate stem cells. In addition, defects in niche-stem cell interactions could explain some diseases that involve defective stem cells, such as certain cancers. Most niche-stem cell interactions are difficult to study directly in humans. Their study is also difficult in mammalian model organisms, where niche cell identity is not known or when tissues are not easily accessible. Fundamental principles of human stem cell biology have been discovered using invertebrate model organisms. We propose to investigate niche-stem cell and systemic signals using the simple model organism C. elegans, a small worm. Specifically, we will investigate how interactions between niche cells and stem cells in the worm gonad influence whether the stem cells divide. We will also investigate how nutritional signals control stem cell division. We have developed tools to observe and manipulate these cells in living embryos, and will use C. elegans genetic tools to learn how these events take place and what genes regulate them. This project will provide new insights into how stem cells are controlled by local and systemic signals, leading to a better understanding of how to manipulate stem cells for regenerative therapies.


Identifying the Alterations in Skin Stem Cell Function Responsible for Cancer

Elaine Fuchs, PhD
The Rockefeller University

Adult stem cells (SCs) are natural units of tissue repair and homeostasis. They have the remarkable capacity to both self-renew and also generate the differentiated cells within the tissue. Tumor-initiating cells (cancer stem cells, CSCs) are similar in concept in that they too are responsible for propagating the tumor and also generating the differentiating cells of the tumor. Effective cancer therapies are predicated upon targeting CSCs without altering their normal tissue counterparts. A prerequisite to achieving this clinical-basic science interface is to elucidate how CSCs differ from normal adult SCs, understand the differences arising during tumor progression and determine which of the 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 the major sources of squamous cell carcinomas (SCCs), prevalent world-wide. We purified and characterized mouse skin SCC-CSCs. Representing as little as 1% of the tumor, these CSCs initiate SCCs when transplanted at single-cell levels. Surprisingly however, transcriptional profiles of SCC-CSCs bear little resemblance to their parental HF-SCs or epi-SCs: >740 genes change expression by ≥2X in CSCs relative to either of these two normal SC populations. We now want to identify which of these differences are unique to CSCs and elucidate when and how these changes arise during tumor progression. We will then tackle the physiological relevance of these gene differences to identify the ones functionally responsible for the progressive transformation of normal skin SCs into SCC-CSCs. Our studies should reveal the changes contributing to uncontrolled cancer growth but which do not affect normal SC homeostasis. Finally, we will explore the relation of our findings to humans, including hyerproliferative and premalignant disorders, malignant SCCs of different grades/types, and metastatic tumors. This proposal focuses on these global objectives, with the ultimate goal of unraveling new cancer targets.


The Emergence of Pluripotency In Vivo

Anna-Katerina Hadjantonakis, PhD
Sloan-Kettering Institute

Pluripotent cells possess the unique ability to reproduce themselves (self-renew), as well as to differentiate into all tissues of the adult organism. Pluripotent stem cells are maintained and predominantly studied in culture. However, pluripotency is a hallmark of a naturally occurring cell population present in early mammalian embryos. The experiments detailed in this proposal provide a strong basis for further progress using the mouse model to understand the establishment of pluripotency in its native environment, harnessing this knowledge for application to stem cell paradigms grown in culture, and future translation to understand these events as they occur in the human. Our laboratory is uniquely qualified to study early mouse embryos. In particular, we have pioneered the development of tools and methods for visualizing embryonic stem cells and mouse embryos. This project combines the use of live imaging for the visualization of cells and genes in living embryos, combined with the use of mouse genetics and embryological approaches.


Precision Genetics in hESCs to Define the Roles of GATA6 in Human Development and Disease

Danwei Huangfu, PhD
Co-PI: Todd Evans, PhD
Sloan-Kettering Institute

GATA6 belongs to a family of GATA factors that play critical roles in embryonic development, and mutations in GATA genes have been identified in a wide spectrum of human diseases including those that affect the pancreas and the heart. Studies using zebrafish and mouse models have contributed greatly to our understanding of the functions of GATA factors. However, despite overall conservation, emerging evidence suggests that significant differences exist between fish, murine and human GATA factor function. Accordingly, understanding the roles of GATA genes in neonatal or adult-onset disorders of the human pancreas and the heart will require novel approaches. To overcome limitations of classic non-human model organisms, we aim to unravel novel mechanisms of pancreatic and cardiac diseases by using human embryonic stem cells (hESCs) to recapitulate key steps of cellular differentiation “in a dish”. Additionally, we have developed precise genome-editing tools for interrogating gene function during hESC differentiation. Combining the expertise of the Huangfu and Evans laboratories, we will define the specificity for GATA6 in human development and disease by interrogating the sufficiency and necessity of GATA6 in specific steps of both pancreatic and cardiac development. Our findings will elucidate both conserved and human-specific developmental mechanisms, and contribute to the development of novel therapeutics for diseases such as diabetes and cardiovascular disease. Our experimental platform will provide the much-needed framework for revealing human-specific functions of disease-associated genes using hESCs as a powerful experimental system.


Clonal production and organization of hPSC-derived cortical excitatory neurons under normal and disease conditions

Song-Hai Shi, PhD
Co-PI: Lorenz Studer, MD
Sloan-Kettering Institute

The human cerebral cortex is one of the most complex brain structures, commanding all higher-order brain functions, including perception, language and cognition. Extensive loss of cortical neurons has frequently been observed during aging and in many neurodegenerative diseases. Abnormal development and function of cortical neurons have also been found in many neurodevelopmental disorders, such as Rett syndrome. Recent advances in stem cell-based therapy raise an intriguing possibility of restoring cortical function in the aging and diseased cortex via neuron transplantation. In this proposal, we aim to develop effective methods for fast generation of functional cortical excitatory neurons from human embryonic stem cells and PAX6-expressing human neural precursor cells. To achieve this, we propose to manipulate multiple signaling pathways that are critical for cortical neuron specification, differentiation and maturation using a combination of small molecules. Furthermore, our recent studies of mouse cortex development demonstrated that the lineage relationship of excitatory neurons guides their structural and functional organization. To gain insights into human cortex development, we will examine the lineage progression of individual PAX6-expressing human neural precursor cells at structural and functional levels in a 3D culture system, which recapitulates many aspects of native organization of the human cortex. Finally, we will examine the differentiation and maturation of multiple lines of human Rett syndrome induced pluripotent stem cells towards cortical excitatory neurons at population and single progenitor levels. Our study will provide an expanding set of tools to decipher the mechanisms underlying human cortex development under physiological and pathological conditions, and in the long run, to open the door for human cortical disease modeling for therapeutic purposes.


Role of Nfi Genes in Neural Stem Cell Homeostasis

Richard Gronostajski, PhD
University at Buffalo – SUNY

Neurodegenerative diseases, spinal cord injury and traumatic brain injury represent a huge loss of quality of life and economic productivity for those who suffer from them; the economic costs of treating these conditions is high (estimated at over $100 million/year in the US for Alzheimer's disease alone) and to date therapies have been of limited effectiveness. Gaining an understanding of how we can regulate the proliferation and differentiation of Neural Stem Cells (NSCs) represents an early step in developing new methods to treat these conditions and create better therapeutic strategies. Lastly, understanding the mechanisms regulating NSC function also holds out the promise of preventing or ameliorating the cognitive loss that may accompany advanced aging and could even provide means to improve brain function in younger adults. The goal of the proposed studies is to understand how NSCs are regulated by a specific family of proteins, the Nuclear Factor I (NFI) family of transcription factors. We have good evidence that three members of this family control NSC proliferation and differentiation and this proposal is designed to discover the biochemical and genetic mechanisms by which these proteins regulate NSC function. We will investigate how loss of NFIX affects NSC growth and differentiation, and how NFIA and NFIB function with NFIX to regulate these processes. We will use state-of-the-art Genomics and Bioinformatic analyses to discover genes regulated by NFIX, NFIA and NFIB that control NSC proliferation and differentiation. These studies are designed to help us better understand how NSCs proliferate and differentiate with the ultimate goal of being able to stimulate NSC function as a therapy for a variety of brain insults and injuries.


Understand the Pathophysiology of Parkinson's Disease Using Genetically Modified iPS Cells

Jian Feng, PhD
University at Buffalo – SUNY

Parkinson’s disease (PD) is a movement disorder clinically diagnosed by locomotor problems including rest tremor, rigidity, bradykinesia and postural instability. These symptoms are caused by dysfunctional basal ganglia circuits in response to severe loss of dopaminergic (DA) input from nigral DA neurons. Rhythmic bursting of neuronal activities are seen in the basal ganglia of PD patients but not in normal brains. We found that neurons differentiated from induced pluripotent stem cells (iPSCs) of PD patients with parkin mutations exhibit oscillatory neuronal activities, which were not observed in normal human neurons. To test the idea that mutations of parkin cause the oscillatory neuronal activities associated with PD, we will repair parkin mutations in patient iPSCs and introduce parkin mutations to normal iPSCs to generate pairs of iPSCs with the same genetic background but that differ only in the presence or absence of parkin mutations. Using midbrain neurons derived from these isogenic iPSCs, we will establish the causal relationship between parkin mutations and oscillatory neuronal activities in PD. We will aslo genetically label dopaminergic (DA) neurons with GFP to examine the oscillatory neuronal activities in DA neurons and non-DA neurons derived from iPSCs with or without parkin mutations. Results of the study will be very useful for the development of predictive diagnostic tests for PD based on the inherent pathophysiology of the disease, as well as disease-modifying therapies that mimic the beneficial function of parkin in preventing these pathogenic oscillatory neuronal activities.


Self-Renewing Erythroblasts from Human ES Cells as a Source of Blood

Michael Bulger, PhD
University of Rochester

The large and growing need for blood for transfusions, coupled with persistent bottlenecks in donated blood supplies, have created an intense interest in the artificial production of red blood cells (RBCs). The discovery of extensively self-renewing erythroblasts (ESREs) - precursor cells that can be grown and expanded indefinitely while maintaining the ability to produce RBCs - provides the foundation for a system to generate human blood. To this end, we propose to improve conditions for the production and efficient large-scale culture of ESREs from human embryonic stem cells. This will include the development of “bioreactors” in which large quantities of these cells can be grown and induced to form RBCs. In addition, we will engineer ESREs to make enzymes that effectively transform RBCs into long-lived drug carriers. The success of this approach would establish the first viable non-donor source of human red blood cells. In addition, our work would open up an entirely new avenue for the delivery of blood-borne therapies, making possible not only the artificial production of human blood, but of “designer” blood capable of ameliorating illness and disease.


Stem Cell-Mediated Craniofacial Skeletogenesis in Health and Disease

Wei Hsu, PhD
University of Rochester

The objective of this proposal is to identify and characterize the stem cells responsible for healthy development of the craniofacial skeleton. A growth center, separating two bone plates, also known as a suture, needs to be present for the continuous growth of a healthy skull in neonates. Craniosynostosis, affecting one in ~2,500 individuals, is one of the most common congenital birth defects of the craniofacial skeleton. In craniosynostosis, abnormalities in the suture cause it to close and disappear prematurely, resulting in skeletal defects. Surgical repairs to reestablish a suture are currently used for the clinical treatment of this disease. However, multiple rounds of surgery are often required for the patients because closure reoccurrence in the repaired suture is highly frequent. Therefore, it is critical to reconstruct a suture which does not close and disappear prematurely. We hypothesize that the presence of “suture stem cells” is necessary for suture existence. Using state-of-the-art genetic models in mice, we have identified putative suture stem cells residing in a healthy skull. These cells possess several properties which are classically defined for the characteristic behaviors of stem cells. In this proposal we will first demonstrate the existence of suture stem cells and their requirement in maintenance of the suture structure. Next, we will elucidate the mechanism underlying skeletal healing mediated by these naïve cells. Finally, to definitively assess the functionality of suture stem cells we will investigate their potential in bone regeneration and repair. Better understanding of suture stem cells is essential to achieve our goal of improving therapeutic strategies for future molecular and regenerative medicine.


Lysosomal Storage Disorder-Induced Precursor Cell Dysfunction in the CNS

Mark Noble, PhD
University of Rochester

We have developed new approaches to the understanding and treatment of Krabbe disease and other lysosomal storage disorders (LySDs). These disorders are characterized by severe neurological damage, particularly to myelinated regions of the central nervous system (CNS). We found that precursor cells that generate the myelin-forming oligodendrocytes of the CNS are the cells most vulnerable to the toxic lipids accumulated in LySDs, and found multiple new ways in which these agents disrupt cell function. Analysis of drugs approved by the FDA for other purposes reveals several compounds protective against all these toxicities, and our first agent taken in vivo demonstrates improved quality of life in a mouse model of Krabbe disease. We also found that >15% of all FDA-approved drugs make psychosine toxicity worse in vitro. All of these discoveries have converged on identification of new mechanisms of action of toxic lipids found in Krabbe disease, Gaucher disease and metachromatic leukodystrophy. Our goals are to improve treatment of Krabbe disease and other LySDs by application of compounds suitable for rapid transition to clinical use, understanding mechanisms of action of these drugs so as to facilitate rational design of combinatorial pharmacological interventions. In addition, our goal is to identify (and understand mechanisms of action) of compounds that may accelerate decline in children with LySDs so that their use can be better controlled. LySDs represent some of the most devastating pediatric diseases and current treatment approaches are severely limited. Identification of utility of substances already approved for other purposes will enable rapid clinical evaluation, while identification of compounds that enhance toxicity may provide benefit simply by switching to different drugs in the clinic. Discovery of convergent mechanisms of action will further provide new insights into even more effective threapeutic approaches.

 

 

IDEA Awards

Stem Cell-Mediated Integrative Cartilage Repair
Helen Lu, PhD
Columbia University

The goal of this project is to evaluate the potential of stem cells for functional and integrative cartilage repair. Osteoarthritis, which is characterized by cartilage degeneration, is one of the most prevalent joint diseases, posing a significant societal burden, as it is the most common cause of work-related disability in this country. Current osteoarthritis treatment options often fail due to the lack of integration between the cartilage graft and the host tissues, including both the surrounding cartilage and underlying bone. Therefore, there exists an unmet clinical need for integrative solutions towards cartilage repair. Our primary goal is to maximize the repair potential of stem cells for cartilage integration. We propose that a cup-like, nanofiber-based scaffold, which releases homing factors from its walls, will attract cartilage cells, and encourage them to work with stem cells to connect the native and engineered cartilage. Furthermore, ceramic nanoparticles embedded in the base of the cup will promote integration with bone, effectively anchoring the graft in the body. The studies planned here will test the efficacy of this novel approach to both cartilage and bone integration. The cup design proposed here is highly innovative, with the ability to simulataneously integrate with both cartilage and bone. It is versatile and can be used with a variety of cartilage grafts. Moreover, it is biodegradable and will be replaced with living tissue over time. It is anticiapted that the successful completion of our studies outlined in this proposal will harness the regenerative potential of stem cells for cartilage integration, resulting in the development of a new generation of nanotechnology-driven graft fixation devices for orthopedic applications.

Stem Cell and iPSC Therapy for Epidermolysis Bullosa
Angela Christiano, PhD
Columbia University Medical Center

Human skin is the main barrier between the body and the environment. The epidermis is the outermost layer of the skin and is normally securely anchored to the skin. However, in the genetic disorder known as Epidermolysis Bullosa (EB), genetic mutations disrupt the functions of the protein molecules that anchor the epidermis in the skin, resulting in a fragile epidermis that can blister easily and cause pain and scarring. Easy detachment of the epidermis impairs the patients’ wound-healing capability, and the chronically wounded areas make EB patients prone to infections and to the development of lethal squamous cell carcinomas (SCC) in the skin. Thus, EB is among the most severe of skin disorders, which profoundly compromises the quality of life and significantly reduces the lifespan of affected patients. In the skin of EB patients, we sometimes observe normal patches surrounded by diseased skin, a phenomenon termed “revertant mosaicism” (RM), in which secondary mutations occur in these normal areas of skin and overwrite the deleterious effects imposed by the primary disease-causing gene mutations. Because spontaneous gene correction in RM-keratinocytes can give rise to clinically normal patches of skin, we postulate that skin grafting with RM-derived cells would be a valid approach to treat EB. However, because somatic RM keratinocytes have very limited growth potential, it is difficult to directly expand these cells. Thus, in this application, we will reprogram normal revertant cells into iPSCs that will then be differentiated into fibroblasts and keratinocytes for the construction of 3D skin. Alternatively, if the revertant cells are not available, gene correction will be performed on iPSCs derived from EB patients. Our experimental system is both innovative and feasible, and we have generated extensive preliminary data to develop iPSCs from patients and obtain large numbers of patient-specific cells for 3D skin construction using exclusively iPSCs. The techniques and knowledge gained from this work will be valuable for the development of treatment for EB patients, for both inside-out and outside-in treatment of diseases of the skin.

The Role of Endoplasmic Reticulum Stress in Diabetic Beta Cell Failure
Rudolph Leibel, MD
Co-PI: Dieter Egli, PhD
Columbia University Medical Center

Diabetes mellitus is characterized by elevated concentrations of blood glucose due to insufficient production of insulin relative to metabolic need. Diabetes affects all ages, is increasing rapidly in prevalence, and takes a terrible physical, psychological and financial toll on the individuals and families in which the disease occurs. Over 8% of the U.S. population (over 25 million adults and children) is now diabetic, with NY State proportionately affected. Over 3 times this number of Americans are pre-diabetic. Medical costs are over $250 billion per year, and growing rapidly. Stem cells are an increasingly important tool with which to interrogate the molecular and cellular mechanisms of a disease. Unlike primary tissues, which can often only be obtained post mortem, stem cells can allow studying insulin-producing beta cell demise in diabetes as it unfolds, and test strategies to prevent or reverse the damage. While there are several molecular and clinical forms of diabetes, the mechanisms of ultimate beta cell failure are not unique to a single form. Protein folding stress appears to play an important role in beta cell failure in most if not all forms of diabetes, including type 1 (T1D) and type 2 (T2D). This stress is created in the insulin producing beta cells by immunologic and metabolic “pressure” brought to bear by the underlying mechanisms for the respective types of diabetes. In the proposed studies we will establish stem cell models of diabetes and investigate the role of protein folding stress in beta cell failure. We will use stem cells with known hypersensitivity to protein folding stress, as well as stem cells from T1D, T2D and controls. This will allow us to test safe compounds able to relieve protein folding stress and improve beta cell function. An extension of the proposed research will be the clinical translation of these compounds.

Impact of Microbes on Intestinal Stem Cells in Drosophila
Nicolas Buchon, PhD
Cornell University

Diseases of intestinal origin are a major health problem worldwide, including inflammatory bowel diseases (IBD) and colorectal cancer. Epidemiologic studies have shown that inflammatory states of the gut increase the risk of cancer development, suggesting that distinct intestinal pathologies are actually connected. However, we do not know precisely how the different facets of intestinal physiology are linked, and what mechanisms are at the basis of these pathologies. The intestine is routinely renewed by intestinal stem cells (ISCs) and it is likely that those cells have an important role both to maintain the organ’s integrity and in initiation of cancer development. In addition, an increasing number of studies suggest that intestinal microbes, either non-invasive (microbiota), or pathogenic, are etiological factors for disease initiation and progression. However, the high complexity of the microbiome of humans impedes our ability to understand the mechanisms by which microbes affect intestinal stem cell activity. We previously demonstrated that the basic mechanisms underlying the control of intestinal stem cell activity appear well conserved between Drosophila and mammals. We will use the simplicity of the Drosophila model to decipher the mechanisms that regulate intestinal stem cell activity and characterize how gut microbes affect their maintenance and proliferation. We will focus on the precise links between the gut immune response to bacteria and the control of intestinal stem cell function. This should improve our general understanding of how stem cells respond to their environment and how microbes impact intestinal health. Our work will not only shed light on how intestinal stem cell functions are maintained in healthy conditions, but also how they are affected in diseases of immune or microbial origin.

Using C. elegans Germline as a Model to Study the Functions of HCF-1, a Critical Factor in Stem Cell Pluoripotency
Siu Sylvia Lee, PhD
Cornell University

The proper functioning of stem cells is key to many biological processes, and a gradual decline of stem cell function is thought to account for some of the tissue/organ degeneration that accompanies aging. However, exactly how stem cell function and aging are interconnected, and whether rejuvenation of stem cell function can really slow down aging are still open questions. A clear understanding of the relationship between stem cell function and aging will be essential for developing regenerative therapies that can slow down age-dependent degeneration and alleviate age-dependent diseases. In this project, we will attempt to resolve the complex relationship of stem cells and aging by focusing on the protein HCF-1, which is known to have an essential role in preserving proper stem cell function, and also in determining the overall lifespan of an animal. We reason that if we can uncover in detail how HCF-1 regulates stem cells and modulates aging, we wil gain important insights about the interconnection of stem cell function and aging. We propose to use the free-living worm C. elegans as a model and employ different genetic, genomic, cell and molecular techniques to address the important question of how HCF-1 ensures proper functioning of stem cells and determines overall lifespan. C. elegans is an ideal model for our study because it has a short lifespan and many tools available to probe how different players work together to regulate biological processes. It is well known that many of the findings uncovered in C. elegans are directly relevant to higher organisms like humans, so we expect our proposed research will reveal important insights about stem cell and aging biology applicable to humans. Our study will facilitate future regenerative therapies aiming to slow down age-dependent degeneration and alleviate age-dependent diseases.

Versatile microRNA Regulation for Cell Fate Decision
Xiling Shen, PhD
Cornell University

Stem cells need to make decisions to self-renew (remain as stem cells) or differentiate into other cell types. These cell fate decisions are often regulated by microRNAs, which are small RNAs that do not code for protein. microRNAs are often thought to have only subtle, fine-tuning influence on their many target genes. How does this "subtle" regulation control the drastic, on-off cell fate decision? A recent discovery from our lab provides a counter example. The microRNA miR-34a, which controls normal and cancer stem cell fate, forms a strong binary switch to determine cell fate asymmetry. This study suggests that microRNA regulation is more versatile than it was previously given credit for. How does this versatility contribute to cell fate determination in stem cells? Do microRNAs provide any benefits such as robustness, programmability and speed? This multidisciplinary proposed study will combine engineering, computational modeling and experiments to approach these questions from three angles: (1) Do microRNAs form network motifs with protein regulators to enhance cell fate decision? (2) Do microRNAs switch from fine-tuning regulation to binary regulation for genes that regulate cell fate? (3) Are microRNAs capable of filtering perturbations from upstream regulators to protect the robustness of cell fate decisions? microRNAs add an extra layer of complexity to the regulatory network that controls cell fate decision in stem cells. The proposed study will reveal the versatility of microRNAs and help explain why certain microRNAs serve as regulatory hubs for stem cell programming. The system biology insights may help improve stem cell engineering and iPSC reprogramming as well as microRNA-dependent diagnostics and therapies (e.g., against cancer stem cells).

Exploring Pathophysiology and Therapy Strategies of Duchenne Muscular Dystrophy Cardiac Dysfunction in Human iPSC-Derived Engineered Heart Tissue
Nicole Dubois, PhD
Icahn School of Medicine at Mount Sinai

Duchenne Muscular Dystrophy (DMD) is a genetic disease, affecting approximately 1 out of 2400 boys worldwide. For a long time, DMD was considered predominantly a skeletal muscle disease, with progressive muscle degeneration and breathing problems. Cardiac complications in DMD became more prominent only recently as the life of DMD patients could be prolonged with improved therapy for the skeletal and respiratory systems. The most common heart problems in DMD are arrhythmias and dilated ventricular cardiomyopathy. About 95% of patients with DMD develop cardiac problems by 20 years of age and for about 20% of these patients this is fatal. However, due to the previous focus on skeletal and respiratory defects, the specific problems in DMD heart disease are still substantially understudied. Several mouse models with mutations in the same gene as in humans, dystrophin, have previously been used to study DMD skeletal and heart defects. However, these mice fail to accurately recapitulate the human disease, displaying only a mild skeletal and cardiac phenotype with near to normal life span. While significant insights have been obtained from these studies, the field is currently missing a robust model to investigate the precise cardiac defect of DMD in human cells. Here we propose to use human induced pluripotent stem cells (hiPSCs) to address this problem. hiPSCs can be derived from any patient of interest and can be directed to generate heart cells that will be identical to the heart cell population of the patient. The objective of this proposal is to establish a human disease model for DMD and characterize the specific cardiac defects in human cells. Findings from our study will lead to an understanding of how dystrophin gene mutations affect human heart tissue formation and function and will give insights into improving treatment options for DMD patients.

Terminal Differentiation of Striatal Medium Spiny Neurons from H9 Cell Line
Michelle Ehrlich, MD
Icahn School of Medicine at Mount Sinai

Huntington's disease is an autosomal dominant, 100% penetrant, fatal disease. In patients with the mutation and an expanded polyglutamine tract >37, the disease develops around the 4th decade of life and the patients die within 10-20 years. Each child of a carrier has a 50% chance of carrying the mutant gene. Genetic testing is available, but there is no treatment to arrest or cure the disease. Major symptoms are an uncontrollable movement disorder, i.e. chorea, abnormal affect, and progressive dementia. The protein is expressed in all cells of the body, but major symptoms arise from degeneration in the deep brain nucleus known as the striatum, comprised of the caudate and putamen (CP). Within the CP, 95% of the nerve cells (neurons) are medium spiny neurons (MSNs). HD researchers hope to establish a source of MSNs for transplantation. Small studies using fetal-derived neurons for transplant have shown clinical improvement in some patients. There is a crucial need for a renewable source of cells. Attempts to induce MSNs from stem cells and pluripotent cells have yielded an increasing number of differentiated cells, but the neurons remain quite immature. New protocols are needed to specifically induce this type of neuron in its fully differentiated form. We have been identifying factors and signal transduction pathways required for MSN differentiation. We discovered several transcription factors and pathways that are required for differentiation. We also developed a unique reagent: a genomic DNA construct that expresses only in the MSNs in the mouse forebrain. We will employ a human embryonic stem cell line as our starting point in this project. We will use our MSN-specific construct as a marker of differentiation as we activate pathways that we have implicated in MSN differentiation. We will also employ this system to identify other MSN markers as well as other transcription factors required for MSN differentiation.

Control of the Intestinal Stem Cell/Progenitor Pool by IL-23-Dependent Lymphocyte Signaling During Homeostasis and Repair
Dan Littman, MD, PhD
New York University School of Medicine

In the intestine, a single layer of cells known as an epithelium is essential for absorbing nutrients from food and preventing pathogens from entering the body. Maintaining and repairing this barrier requires the activity of epithelial stem and progenitor cells that are constantly dividing to replace aging or dying epithelial cells. The commensal microbiota, “good bacteria,” which normally inhabit the gut, play an important role stimulating the ability of stem and progenitor cells to divide and repopulate the epithelium. Similarly, pathogens, or "bad bacteria," damage the epithelium, requiring repair by new cells descended from stem or progenitor populations. How the commensal microbiota or pathogens control stem and progenitor cells in the epithelium is poorly understood. Here we propose to test the role of a particular immune cell signaling pathway mediated by Interleukin-23 (IL-23) in controlling epithelial stem and progenitor cells. IL-23 signaling can be activated by the commensal microbiota as well as pathogens, and appears to promote epithelial maintenance and repair. Our experiments will determine how progenitor cells are regulated by IL-23 signaling. We also will identify novel pathways acting in epithelial progenitors that are regulated by IL-23 signaling. We expect that our studies will shed light on how the microbiota and pathogens regulate the epithelial progenitor pool. Further, by identifying new genes and pathways that promote the ability of stem and progenitor cells to renew the epithelium we may identify new targets that can be modulated to treat patients suffering from chronic epithelial damage as in Crohn's Disease or Ulcerative Colitis.

Identifying Telomerase Regulators in Pluripotent Cells
Agnel Sfeir, PhD
New York University School of Medicine

Normal human cells lose DNA from the ends of their chromosomes (telomeres) with each replication cycle. When telomeres reach a critically short length, cells cease dividing and undergo growth arrest or senescence. To achieve longterm proliferation, such as with stem cells and cancer cells, it is essential to counteract this persistent telomere shortening. This is accomplished primarily by activating the enzyme telomerase, which adds telomeres de novo. Being the obligate means for indefinite growth of cancer cells, telomerase has emerged as a promising target for cancer therapeutics, and telomerase inhibitors are now entering phase II clinical trials. However, the greatest concern behind telomere-targeting cancer therapies is the potential deleterious side effects to stem cells. Despite the importance of telomere maintenance in stem cell survival, the mechanisms by which these cells regulate telomerase remain largely unknown. We plan to decipher the mechanism of telomerase regulation by developing an assay that will allow us to screen for genes mediating telomerase activity in embryonic stem cells. Once we validate this assay, we will perform a genome-wide screen using an shRNA library and identify the full spectrum of telomerase regulators in pluripotent cells. Last, we will study the function of the identified gene(s) and determine the effect of inhibiting these telomerase regulators on the proliferative capacity of pluripotent cells.

Molecular Roadblocks for In Vitro Hematopoietic Stem Cell Specification
Matthias Stadtfeld, PhD
New York University School of Medicine

Reprogramming technology enables the straightforward generation of patient-specific pluripotent cells, which hold tremendous potential for regenerative medicine. Harnessing this potential for the benefit of future patients will require developing ways to differentiate pluripotent cells in a safe and reproducible manner into adult stem cells that are able to replenish damaged or diseased tissues and organs such as blood, heart or brain. Unfortunately to date, all attempts to generate fully functional adult stem cells from pluripotent cells have failed, representing one of the most serious obstacles for the translation of reprogramming technology into clinical application. Our research aims at identifying the molecular reasons for the inability to derive transplantable blood stem cells from pluripotent cells in cell culture. To do so, we are developing a novel approach that allows us to directly compare blood cells generated in cell culture to “gold standard” blood stem cells that formed within the body. Analyzing these two cell populations side by side will point us towards specific genetic pathways that are abnormal in blood cells generated in cell culture. We will then test, using novel mouse and human pluripotent cell lines, whether the manipulation of these genes can indeed increase the quality of blood cells generated in vitro and ultimately yield cells of sufficient quality for transplantation purposes. In doing so, we hope that our work will bring us one step closer to the clinical use of patient-specific pluripotent cells.

Precursor Cells as Viral Targets: a New Approach to Understanding Failure of Repair in the Human CNS
Margot Mayer-Proschel, PhD
University of Rochester

This proposal focuses on three related questions. Our primary goal is to better understand the failure of human oligodendrocyte progenitor cells (hOPCs), the ancestors to the myelin producing cells of the CNS, to effectively repair lesions in diseases like multiple sclerosis (MS) despite the presence of many hOPCs in MS lesion sites. The second concern addresses the poor ability of animal models to predict clinical outcomes in MS treatment, raising the possibility that factors specific to human diseases play critical roles in preventing repair. The third question is why some promising treatments for MS help some patients but make others worse. We pursue the novel hypothesis that latent infection with human herpesvirus 6 (HHV-6), which is widespread in the human population and particularly found in MS lesions, is a human-specific factor that impairs repair functions of hOPCs. We have shown that HHV6 infection of hOPCs causes impaired proliferation and migration in vitro. We also found a surprising inhibition of cholesterol synthesis by HHV6, leading us to propose that HHV6 infection of hOPCs causes statins (which appear to help some MS patients but to make others worse) to become toxic rather than beneficial. In Aim 1 we will analyze the impact of different stages of HHV6 infection on hOPCs in a controlled in vitro setting in the absence or presence of statin treatment. Aim 2 then will define the in vivo capacity of infected hOPCs to contribute to myelin replacement in the absence or presence of inflammation and statin treatment. By studying human OPCs in vitro and in in vivo transplantation models, this work will open up new avenues for identifying the human-specific factors that impact repair in demyelinating diseases, and that may have still broader implications for understanding failures in repair by endogenous precursor cells.

Modulation of Intestinal Differentiation in Esophageal Basal Stem Cells
Jianwen Que, MD, PhD
Co-PI: Jeffrey Peters, MD
University of Rochester

Long-standing reflux of stomach contents into the esophagus (GERD), results in changes of the cells lining the esophagus, a disease known as Barrett’s esophagus (BE) that affects an estimated 3-4 million Americans. When this happens, there is a much higher (~40-fold) risk of developing cancer of the esophagus, one of the most rapidly increasing and deadly forms of cancer. In this proposal we aim to identify the underlying cellular mechanisms of how stomach contents reflux into the esophagus to initiate BE formation. Experiments by our groups and others have shown that bile acid, which is commonly present in reflux of patients, breaks down cell-cell junctions and creates inflammation in the esophageal layer where its stem cells are located. It also appears from these studies that cellular pathways important for gut development in the human embryo are active in BE biopsies, and can be turned on with bile exposure in cell culture. Additionally our mouse genetic studies showed that inducing the activity of one such cellular pathway [bone morphogenetic protein (BMP) pathway] caused BE-like pathological changes in the developing mouse esophagus. However, it remains unknown what triggers the abnormal signaling activity and how it exactly relates to the formation of human BE. For the experiments in this proposal, we will test the hypothesis that reflux contents, including bile acid, work in concert with tissue inflammation created by chronic GERD to activate cellular signaling pathways in human esophageal stem cells, leading them to differentiate towards cells that make up the esophageal lining and cause Barrett’s esophagus. We expect findings from these studies will shed new light on the fundamental mechanisms causing the abnormal esophageal lining of BE. We predict that manipulation of the relevant signaling pathways will block this abnormal differentiation process, and provide new treatments to prevent or reverse BE.

Proteasomal Regulation of Osteoblasts
Lianping Xing, PhD
University of Rochester

Bone fracture is a major public health problem and needs new therapies. Stem cells have been used to treat fractured patients with promising results. Discovering new ways to control stem cell function during fracture healing will improve the efficiency of stem cell treatment. We found that stem cells from mice, in which a protein called Itch has been removed, have better capacity to make bone, an important part of fracture healing. In stem cells, Itch causes the breakdown of proteins in the proteasome, a place where cells get rid of broken proteins. Interestingly, the drug Bortezomib, which blocks proteasome action, increases fracture healing and makes fractured bone stronger in mice. These findings suggest that Bortezomib could be used to treat fracture and that Itch may be a new drug target for stem cell therapy. Here, we hypothesize that Itch delays fracture healing by reducing the bone forming capacity of stem cells via increased protein breakdown in the proteasome. This can be prevented by Bortezomib or depletion of Itch in stem cells. Injections of stem cells without Itch enhance fracture healing. We will use a mouse fracture model to examine the location of stem cells, levels of Itch, and Itch target proteins in fracture sites at different time points post-fracture. We will treat fractured mice with Bortezomib to examine its effect on bone repair. We will generate mice with depleted Itch in stem cells to examine if these mice have better fracture repair. We will label stem cells lacking Itch and put them into fractured mice to examine the effect of Itch null stem cells on fracture healing. If our hypothesis is correct, our research will open a new area of investigation regarding a possible association among protein breakdown, stem cells, and fracture healing, and a new use for Bortezomib.

Role of MECOM in Leukemia Stem Cell Quiescene
Yi Zhang, PhD
University of Rochester

Stem cell research encompasses not only the application of stem cells for regenerative medicine, but also the study of cancer stem cells. It is believed that our inability to entirely eliminate certain cancers is due to the resistance of cancer stem cells to conventional chemotherapy. Our studies to date indicate that a protein called MECOM is critical after stress by chemotherapy drugs; we believe it confers resistance to chemotherapy by inducing queiscence. In this grant application, we plan to develop reagents that will block MECOM function and thereby augment the killing of leukemia stem cells by chemotherapy. We believe that by inhibiting MECOM, we will induce the cells to leave their dormant, quiescent state and begin dividing, thereby making them more susceptible to death by chemotherapy. While our preliminary studies will use powerful mouse models, we will also test our ideas on primary human leukemia patient samples. At present, no therapeutic agents are clinically available that specifically target cancer stem cells. If successful, the proposed experiments will provide critical groundwork for the development of effective new therapies for the elimination of cancer stem cells, and improve the response of leukemia stem cells to chemotherapy. The targeting of leukemia stem cells via their stress response is a creative and novel approach. The development of the technologies we are employing for stem cell research is also novel, and if successful, will provide novel approaches for use by other investigators. Furthermore, by developing and testing compounds on human patient samples, this study will hopefully provide the first compounds to specifically target the resistance of leukemia stem cells to chemotherapy; this study will thereby benefit

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