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Investigator Initiated Research Projects and Innovative, Developmental or Exploratory Activities (IDEA) in Stem Cell Research - 2011

RFA #: 0912180242

RFA #: 0812220315

IIRP Awards

Institution PI Amount Project Title

Albert Einstein College of Medicine

Rogler, Leslie

$1,080,000

The Role of the Long Non-Coding RNA, RMRP in Stem Cell Biology

Albert Einstein College of Medicine

Steidl, Ulrich

$1,080,000

Regulation of Hematopoietic Stem Cells by the Chromatin-Remodeling Protein SATB1

Columbia University

Vunjak-Novakovic, Gordana

$1,077,792

Spatial-Temporal Studies of Stem Cells Using a Microbioreactor Platform

Columbia University Medical Center

Doetsch, Fiona

$1,023,290

MicroRNA Regulation of Adult Neural Stem Cells

Cornell University

Nikitin, Alexander

$1,042,227

Roles of Ovarian Surface Epithelium Stem Cells

Mount Sinai School of Medicine

Soriano, Philippe

$1,080,000

FGF Signaling in Pluripotent Stem Cells

Mount Sinai School of Medicine

Wang, Jianlong

$1,079,000

Connecting the Tet Proteins to the Pluripotency Network

New York University School of Medicine

Aifantis, Iannis

$1,080,000

Regulation of Pluripotency and Stem Cell Differentiation by the Ubiquitin-Proteasome System

New York University School of Medicine

Bach, Erika

$1,067,116

Genetic Circuits that Regulate Self-Renewal and Niche Competition in the Testis

New York University School of Medicine

Fishman, Glenn

$986,819

Directed Differentiation of Cardiac Purkinje Cells from ESCs

New York University School of Medicine

Schneider, Robert

$1,080,000

Translational Regulation for Treatment of the Breast Cancer Stem Cell

New York University School of Medicine

Smith, Susan

$360,000

The Molecular Mechanism of Stem Cell Dysfunction in TIN2-Associated Dyskeratosis Congenita

The Rockefeller University

Brivanlou, Ali

$1,080,000

Directed Differentiation of Human Embryonic Stem Cells to Defined Neocortical Subtypes

Sloan-Kettering Institute

Lai, Eric

$1,058,640

Control of Neural Stem Cell Identity by the Zinc Finger Protein Ars2

Sloan-Kettering Institute

Ptashne , Mark

$1,044,317

Chromatin Structure and the Role of Myc and Variants Thereof in Pluripotency, Differentiation, and Cancer

Stony Brook University - SUNY

Ma, Yupo

$1,077,486

Transformation of Stem Cell Based Transplantation Therapy

University at Buffalo - SUNY

Feng, Jian

$1,080,000

Redefining Idiopathic Parkinson's Disease through Induced Pluripotent Stem Cells

University of Rochester

Goldman, Steven

$1,077,552

Human Induced Pluripotential Cell (iPSC)-Derived Oligodendrocyte Progenitor Cells for the Treatment of Myelin Disorders

Weill Medical College of Cornell University

Rafii, Shahin

$1,079,993

Contribution of the Vascular Niche to Liver Regeneration and Repair

IDEA awards

Institution

PI

Amount

Project Title

Albert Einstein College of Medicine

Bouhassira, Eric

$330,000

Clinical Grade iPS

Albert Einstein College of Medicine

Guo, Wenjun

$330,000

Understanding the Role of Normal Mammary Stem Cell Program in Breast Cancer Stem Cells

Columbia University Medical Center

Christiano, Angela

$330,000

Skin Regeneration in the Setting of Epidermal Stem Cell Ablation

Columbia University Medical Center

Doetsch, Fiona

$328,359

Three-Dimensional High Resolution Reconstruction of the In Vivo Adult Neural Stem Cell Niche

Columbia University Medical Center

Laufer, Edward

$330,000

Canonical Wnt Signaling Regulation of Adrenocortical Stem Cells

Columbia University Medical Center

Laufer, Edward

$328,166

Identification of Novel Adrenocortical Stem Cell Markers

Columbia University Medical Center

Sussel, Lori

$325,778

Regulating the Directed Differentiation of CNS and Pancreatic Islet Cell Populations

Cornell University

Liu, Jun

$320,966

Induced Cellular Reprogramming in C. Elegans

Mount Sinai School of Medicine

Ezhkova, Elena

$330,000

Characterization of Multipotent Embryonic Skin Stem Cells

Mount Sinai School of Medicine

Wang, Jianlong

$329,800

A "BTB-POZ" Key to Ground-State Pluripotency During Somatic Cell Reprogramming

New York University School of Medicine

Reinberg, Danny

$329,946

Understanding How Erk Signaling Promotes Lineage Competence through Chromatin Regulation

Stony Brook University - SUNY

Thomsen, Gerald

$325,000

The Function of Stem Cells in Sea Anemone Regeneration

University at Buffalo - SUNY

Sim, Fraser

$329,984

Directed Reprogramming of Human Fibroblasts to Oligodendrocyte Progenitors

University of Rochester

Benoit, Danielle

$314,494

Promoting MSC-Mediated Musculoskeletal Tissue Regeneration Using Sustained, Localized siRNA Delivery

Weill Medical College of Cornell University

Evans, Todd

$330,000

Discovery of Novel Retinoids for Stem Cell Biology

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IIRP awards

The Role of the Long Non-coding RNA, RMRP in Stem Cell Biology

Leslie Rogler, PhD
Co-PI: Charles Rogler, PhD
Albert Einstein College of Medicine

A major frontier in biology is the identification and functional characterization of a set of genetic elements called long non-coding RNAs (lncRNAs). One such lncRNA is a ribozyme called RMRP. It has long been known that point mutations in RMRP cause a spectrum of devastating human disease syndromes, such as Cartilage Hair Hypoplasia (CHH). The deficits exhibited by patients with CHH suggest that that mutations in RMRP may lead to a paucity of stem cell function. However there has been no understanding of how these mutations lead to disease and thus no effective therapies. We recently discovered a mechanism by which RMRP is the source of at least two small silencing RNAs that can regulate genes that appear to be responsible for CHH disease characteristics. In this proposal, we will investigate the molecular mechanism of action of these new gene-silencing RNAs, with the goal of understanding how mutations in them cause impairment of stem cell function. Specifically we will determine the impact of RMRP mutations on differentiation capacities of hES and hiPS cells. This work stands to establish a new paradigm for the functions of lncRNA in stem cell biology and to strongly impact future therapeutic applications.

Regulation of Hematopoietic Stem Cells by the Chromatin-Remodeling Protein SATB1

Ulrich Steidl, MD, PhD
Albert Einstein College of Medicine

Blood stem cells (hematopoietic stem cells; HSCs) rely on a tightly regulated balance between quiescence and activation, as well as self-maintenance and specialization, to ensure the appropriate production and function of the entire spectrum of mature blood cells. Dysregulation of HSC function also plays a key role in many hematological disorders, including myelodysplasias and leukemias. In preliminary studies we have discovered that the chromatin-remodeling special AT-rich sequence-binding protein 1 (SATB1) is highly expressed in HSC, that loss of SATB1 leads to multiple defects in HSC function, and that reduced levels of SATB1 are found in aged HSC as well as in patients with acute myeloid leukemia (AML), where low SATB1 levels correlate strongly with poor overall survival. Based on these observations we hypothesize that SATB1 is a key epigenetic regulator of critical HSC functions, and that low levels of SATB1 contribute to stem cell aging, and to the development of leukemia. Our specific research aims are: 1) To characterize SATB1-dependent regulation of HSC functions; 2) To examine the molecular mechanisms of SATB1-dependent regulation of HSC; 3) To investigate the function of SATB1 as an epigenetic regulator in stem cells in acute myeloid leukemia. In summary, our study will demonstrate that SATB1 is a critical epigenetic regulator of pivotal functions of HSC, that disturbed SATB1 function contributes to stem cell aging, and that aberrantly low levels of SATB1 are crucially involved in the pathogenesis of AML. Our study will provide novel functional and mechanistic insights into the regulation of hematopoietic stem cells by chromatin-remodeling proteins, and may open new avenues for targeted therapeutic intervention in hematological diseases including leukemia, as well as in blood cell engineering using HSCs.

Spatial-Temporal Studies of Stem Cells Using a Microbioreactor Platform

Gordana Vunjak-Novakovic, PhD
Co-PI: Elisa Cimetti, PhD
Columbia University

The use of stem cells offers tremendous opportunities for study of development, regeneration and disease, with the ultimate goal to provide new treatment modalities for some of the most devastating diseases. In the human body, cells are regulated by the context of their entire environment, with its dynamic sequences of molecular and physical signals and complex interactions with the other cells and extracellular matrix. As the importance of replicating the in vivo milieu in cell culture is increasingly recognized, efforts are being invested into the design of advanced culture systems. Still, major limitations remain and we propose to develop a new generation of culture systems, Microbioreactor Platforms, enabling dynamic changes in environmental conditions, experimentation in a large parameter space, and real-time insights into cellular responses. Studying stem cells within our Microbioreactor Platforms would radically advance biological and medical studies of human cells, by implementing complex patterns of molecular and physical signals controllable in space and time. Using our technological solution, we will be able to study stem cell behavior in conditions closely resembling those established in vivo, inside our bodies. Fulfilling the proposed aims, we will optimize the protocols for obtaining ‘better’ differentiated cells from embryonic stem cells (i.e. cardiomyocytes) and will establish a disease model for human ischemia that will help researchers find novel therapeutic strategies and test new drugs.

MicroRNA Regulation of Adult Neural Stem Cells

Fiona Doetsch, PhD
Columbia University Medical Center

In the adult brain, neural stem cells continuously generate new neurons. The stem cells are a subset of glial cells, classically associated with support functions in the brain. This raises the possibility that glial cells elsewhere in the brain are also latent stem cells. Neural stem cells are largely found in a quiescent state, and occasionally become activated to divide. However the molecular mechanisms that control the dormant state are largely unknown. microRNAs pose an elegant solution to this puzzle; these small, non-coding RNAs are capable of rapidly suppressing translation of hundreds of transcripts, making them strong candidate mediators of rapid changes in cell state. Here, we have identified microRNAs as important mediators of the transition of stem cells from quiescence to activation. We identify a family of microRNAs that are highly enriched in activated stem cells, while their targets are enriched in quiescent stem cells. This project will define the functional role of microRNAs in the regulation of adult neural stem cell quiescence and activation. By elucidating the regulatory networks that control stem cells in the adult mammalian brain it will provide insight into the possibility of harnessing endogenous stem cells, as well as activating other cells in non-neurogenic regions, for brain repair.

Roles of Ovarian Surface Epithelium Stem Cells

Alexander Nikitin, MD, PhD
Cornell University

Epithelial ovarian cancer (EOC) is the most deadly gynecological malignancy. Due to its latent progression, the majority of women are diagnosed at advanced stages of disease and the 5-year survival of patients with EOC is below 30%. No significant progress has been made in treatment of EOC during the past 30 years, mainly due to poor understanding of the pathogenesis of this disease. Recent studies have shown that ovarian cancer cells frequently have stem cell-related properties. Such cells are responsible for the aggressive course of the disease and development of drug resistance, thereby providing a rationale for development of diagnostic and therapeutic approaches aimed towards detection and elimination of malignant cells with stem cell properties. The success of such work greatly depends on better understanding of mechanisms regulating the normal stem cell compartment and unraveling how alterations in such mechanisms may result in cancer. Unfortunately, the existence of a stem cell compartment for the ovarian surface epithelium (OSE), which is believed to be the main cell of origin of EOC, remains insufficiently confirmed and its roles in physiological regeneration and malignant transformation are unknown. We have identified OSE stem cells in a specific anatomical location of the ovary and propose to determine their biological functions and to determine their role in carcinogenesis. This work is based on advanced cell biology of somatic stem cells, genetic modeling, genomics and cancer research approaches. In addition to characterization of the novel stem cell compartment for OSE stem cells, it is expected that proposed research will determine whether distortion of mechanisms regulating normal physiological functions of OSE stem cells leads to cancer. It is expected that proposed studies will significantly advance our understanding of mechanisms of EOC formation, thereby leading to development of more effective approaches for detecting and targeting EOC cells.

FGF Signaling in Pluripotent Stem Cells

Philippe Soriano, PhD
Mount Sinai School of Medicine

Fibroblast growth factors (FGFs) play pivotal roles in pluripotent stem cells of the early mouse embryo and in regenerative biology. FGF induced signaling pathways have been extensively analyzed in established cells lines, but remarkably little is known about their roles in stem cells. We will first define FGF signaling pathways that play critical roles in trophoblast stem (TS) and extraembryonic endoderm (XEN) cells. Fgf4 and Fgfr2 are expressed reciprocally in the epiblast and extraembryonic structures of the preimplantation embryo and are required for implantation. Consistent with these observations, TS cells require FGF signaling for their establishment and maintenance, and the primitive endoderm is regulated by FGF signaling in vivo. To identify critical FGF signaling pathways, we will generate an allelic series at the Fgfr2 locus, carrying point mutations that prevent binding of multiple effector binding sites, and investigate their impact on TS and XEN cell derivation and maintenance. We will next define FGF signaling pathways that play critical roles in ES cell differentiation and in epiblast stem cells (EpiSCs). Autocrine FGF signaling in ES cells through Fgfr1 is required to induce their differentiation and EpiSCs depend on FGF signaling. We will generate an identical allelic series at the Fgfr1 locus as for Fgfr2, preventing binding of multiple binding sites, alone or in combination. We will assay the ability of Fgfr1 mutant ES cells to differentiate along the neural pathway and the ability to isolate and maintain mutant EpiSCs. As Fgfr2 is subsequently coordinately expressed in the epiblast, we will assay EpiSC functions in Fgfr1/Fgfr2 double mutants carrying point mutations for identical effectors. This will establish the coordinate roles of FGF signaling pathways in stem cells of the early embryo. By using a combination of mouse molecular genetic approaches, our studies will help define the roles of signaling processes engaged by FGF signaling in stem cell isolation and maintenance.

Connecting the Tet Proteins to the Pluripotency Network

Jianlong Wang, PhD
Mount Sinai School of Medicine

Pluripotent stem cells can be derived from developing early embryos as embryonic stem cells (ESCs), and also from readily available cell sources such as skin after introduction of a gene cocktail (Oct4, Sox2, Klf4 and cMyc) as induced pluripotent stem cells (iPSCs). Both ESCs and iPSCs can propagate in a petri dish forever and are also able to turn into other cell types under proper cues for therapeutic application. These unique properties are regulated by a process that does not directly change the DNA coding sequence but rather involves adding or removing a small molecular attachment known as a methyl group to one of the four building blocks of DNA (cytosine), a process known as epigenetic regulation. The addition (methylation) and removal (demethylation) of the methyl group control the gene expression and consequently the cell behavior. In order to exploit therapeutic benefits of ESCs/iPSCs in regenerative medicine, we need to have a full understanding of how such epigenetic regulation is controlled during ESC maintenance and iPSC generation. DNA methylation is considered a major roadblock to efficient generation of iPSCs and molecular tools to reverse the process (i.e., demethylation) are much needed. The recent discovery of a family of proteins known as Tet1, Tet2 and Tet3 as DNA demethylation enzymes offers a great opportunity to move the field forward. Our goals are to pursue a detailed characterization of Tet1/2 for their function in stem cell maintenance and iPSC generation, dissect their molecular and biochemical relationship with the famous stem cell regulators Nanog and Oct4, and investigate the underlying mechanisms by which Tet1/2 regulate pluripotency and reprogramming. The innovative elements of the project include the proteomic approaches for discovering the connection of Tet proteins with the core pluripotency network and functional dissection of these novel DNA demethylation factors for both ESC pluripotency and iPSC generation.

Regulation of Pluripotency and Stem Cell Differentiation by the Ubiquitin-Proteasome System

Iannis Aifantis, PhD
New York University School of Medicine

Embryonic stem (ES) and induced pluripotent stem (iPS) cells can generate all tissues of the embryo (pluripotency) and are capable of indefinite cell growth in vitro. The ubiquitin- proteasome system (UPS) is complex cellular machinery, which controls protein stability, localization and function. Strong preliminary data suggest that the UPS may be a novel important regulator of stem cell pluripotency and differentiation. To investigate the role of the UPS in stem cells, we have previously: a) identified proteins ubiquitinated in ES, and iPS cells using protein analysis, and b) identified novel substrate identifying proteins (E3 ligases) of the UPS for ES cell pluripotency and differentiation using RNA interference (RNAi)-based screens. These studies suggested that key elements of the ES cell pluripotency network are controlled by ubiquitination, and that a significant number of UPS members regulate pluripotency and influence ES cell differentiation. Our preliminary data also indicate that the expression of the UPS member Fbxw7, an enzyme regulating the half-life of target proteins, is dynamically regulated during ES cell differentiation and that Fbxw7 expression silencing inhibits ES cell differentiation, promoting pluripotency. The recent advances in cellular reprogramming (generating inducible pluripotent stem cells from somatic tissue) has enhanced the understanding of the molecular mechanisms underlying pluripotency, however, very low efficiency of cellular reprogramming suggest unidentified molecular roadblocks. The identified UPS members (like Fbxw7) may improve iPS efficiency and provide means of identifying small molecule inhibitors that enhance reprogramming. These findings lead to the hypothesis that the ubiquitinatin-proteasome system, a key modulator of protein stability and function, can control pluripotency, cellular reprogramming and stem cell function.

Genetic Circuits that Regulate Self-Renewal and Niche Competition in the Testis

Erika Bach, PhD
New York University School of Medicine

Stem cells are uniquely endowed with the property of producing multiple specialized cells. They can regenerate damaged tissues throughout the lifetime of an individual. After a stem cell divides, one descendent remains a stem cell, while the other one becomes a specialized cell type, like skin or hair. Understanding how a stem cell’s offspring chooses between these paths holds great potential for breakthroughs in the treatment of cancer, diabetes and spinal cord injuries. We are focused on finding new factors that let a stem cell remain in a pluripotent state and studying how new factors work together with known ones, particularly with the transcription factor STAT. We discovered proteins that allows some stem cells survive in the niche (the stem cells’ home) at the expense of others, indicating that some stem cells are more “fit” and competitve than others. This study is designed to identify factors that control self-renewal and survival of germ-line stem cells (GSCs) and adult stem cells in the testis of the fruit fly. Aim 1 is focused on the relationship between STAT and other transcription factors in stem cells. Aim 2 will identify how descendents of a “fitter”, more competitive GSC displace normal GSCs from the niche. Aim 3 will examine if colonizing stem cells have increased cell division or adhesiveness. We use Drosophila to uncover new stem cell self-renewal pathways. Flies are a highly effective tool for analyzing the function of human disease genes. 60% of such genes have counterparts in Drosophila, highlighting the relevance of Drosophila to human health. We found that stem cells that proliferate the fastest are the ones that occupy the tissue, which is similar to what has been observed in mammals.

Directed Differentiation of Cardiac Purkinje Cells from ESCs

Glenn Fishman, MD
New York University School of Medicine

During a normal lifespan, our heart beats several billion times. The heart cells that regulate the heartbeat form a network known as the specialized conduction system. These cells are susceptible to many diseases, leading to cardiac arrhythmias, which when severe can result in death. From a societal point of view, the government pays out more $3 billion dollars each year to Medicare beneficiaries for cardiac arrhythmia-related diseases. Despite this enormous expense, the treatments we have available are inadequate. Our major goal is to better understand how the cells of the cardiac conduction system normally develop and the mechanisms that lead to disease. With this knowledge, we can design more effective treatments. Embryonic stem (ES) cells have the capacity to differentiate into virtually any cell type in the body, including these conduction system cells, however the efficiency of creating these specialized cells from ES cells is currently quite poor. Our team proposes to address this problem and develop much more efficient methods. If we can generate conduction system cells more easily, we can use such cells to study heart rhythm disorders and potential new therapies. Our team has created embryonic stem cells that are especially good at turning into heart cells. In addition, we have created embryonic stem cells that give off a special signal when they are coaxed into becoming the specialized conduction system cells we wish to study. In addition, our team is comprised of researchers with complementary expertise in the areas of stem cell biology, heart development and heart rhythm disorders. Together, these elements of our project increase the likelihood that we will be successful in completing our aims and making important advances to diminish the morbidity and mortality associated with heart rhythm diseases.

Translational Regulation for Treatment of the Breast Cancer Stem Cell

Robert Schneider, PhD
New York University School of Medicine

Metastatic breast cancer, in which cancer recurs and spreads to distant parts of the body, is responsible for the majority of breast cancer deaths because it is difficult to treat. Consequently, there has been only a small increase in the survival of women with metastatic breast cancer in the last 20 years, and no woman is “cured” of her disease. Moreover, there are no drugs that have been specifically developed for metastatic breast cancer. Progress requires a shift in our way of thinking, focusing on the source of breast cancer late recurrence, resistance to treatment and metastasis. That shift involves recognition of the central role of breast cancer stem cells, cancer cells that possess stem-like qualities that permit them to survive harsh chemo- and radiation therapy, and spread throughout the body in the process of metastasis. Our research, and this grant application, have identified unique alterations to the protein synthesis machinery of the breast cancer stem cell as crucial for its survival, treatment resistance and ability to develop metastatic breast cancers. This proposal will investigate the molecular mechanisms by which breast cancer stem cells orchestrate and utilize unusual changes in their protein synthesis programs for their survival, and conduct development of exciting new experimental therapeutics that target the specific protein synthesis factors responsible as a means of treating metastatic breast cancer.

The Molecular Mechanism of Stem Cell Dysfunction in TIN2-Associated Dyskeratosis Congenita

Susan Smith, PhD
New York University School of Medicine

The goal of this proposal is to elucidate the mechanism of stem cell dysfunction in TIN2-associated dyskeratosis congenita (DC). Telomere length homeostasis is achieved by a balance of telomere shortening caused by DNA replication and nucleolytic attack and telomere lengthening by telomerase. The importance of telomere length maintenance to human health is best illustrated by dyskeratosis congenita (DC) a disease of telomere shortening caused by mutations in telomerase subunits. DC patients suffer stem cell depletion and die of bone marrow stem cell failure. Recently a new class of particularly severe DC patients was found to harbor mutations in the shelterin subunit of TIN2. The DC-TIN2 mutations were clustered in a small domain of unknown function. In a recently published study we showed that the DC mutation cluster in TIN2 harbored a binding site for heterochromatin protein 1 (HP1) and further, that HP1 binding to TIN2 was required to establish sister chromatid cohesion at telomeres and for telomere length maintenance by telomerase. In this grant we will determine how telomere cohesion influences telomere length maintenance by telomerase. In addition, we will determine if defective sister telomere cohesion impacts telomere length maintenance by recombination, an alternative mechanism used by embryonic stem cells to lengthen their telomeres. We will carry out this analysis initially in human model cell lines to elucidate the molecular mechanisms, but then we will turn to human patient cells and use induced pluripotent stem (iPS) cell reprogramming to determine if we can rescue or recapitulate features of the disease. The results of our work will have direct relevance to DC patients and will provide insight into the role of telomerase in stem cells and in human health.

Directed Differentiation of Human Embryonic Stem Cells to Defined Neocortical Subtypes

Ali Brivanlou, PhD
The Rockefeller University

This proposal seeks to elucidate the molecular mechanisms that determine the ultimate cell type of neural progenitor cells in the earliest stages of human brain development. The successful completion of this study will enhance our basic understanding of human brain development and will have direct relevance for clinical application. At the basic scientific level, knowledge of the molecular mechanism responsible for the formation of the neocortex will provide the first blueprint of human brain development - one that cannot be addressed with direct scrutiny during embryonic development. At the clinical level, this study will inevitably impact our knowledge of degenerative diseases, such as brain injury, stroke, autism spectrum disorders, and neuropsychiatric diseases.

Control of Neural Stem Cell Identity by the Zinc Finger Protein Ars2

Eric Lai, PhD
Sloan-Kettering Institute

Stem cells are endowed with the fundamental properties of self-renewal and multipotency. The former quality means that they can generate additional stem cells through division, and the latter means that they have the capacity to differentiate into diverse cell types. A general hope of stem cell research is that understanding the mechanisms that endow stem cells with these core properties will permit and inform their directed manipulation for regenerative medicine. Our studies of adult neural stem cells (NSCs) identified a novel factor termed Ars2 that mediates both self-renewal and multipotency; moreover, Ars2 is sufficient to drive self-renewal of adult NSCs. We determined how Ars2 works by showing that it binds DNA and directly activates another self-renewal gene called Sox2. This establishes a novel and vital regulatory connection between these key NSC genes. To determine the generality of Ars2 function across different NSCs, we propose to study the function of Ars2 in other classes of NSCs located in the adult hippocampus and embryonic neocortex. We also propose a series of tests to understand how Ars2 operates to bind DNA and regulate gene expression. We will use the latest whole-genome profiling methods to elucidate all of the regulatory targets of Ars2 across a variety of cell types, to obtain a comprehensive view of its regulatory action. Finally, we will explore whether Ars2 plays similar roles in non-neural stem cell settings, including during embryonic development as well as in the generation of induced pluripotent stem cells. We have generated substantial preliminary data on all these proposed directions, and our proposed studies of this completely novel stem cell factor have great potential to open doors for manipulating neural stem cells under trauma or neurodegenerative conditions. This work is also poised to yield general insights on the regulatory controls of other types of stem cells.

Chromatin Structure and the Role of Myc and Variants Thereof in Pluripotency, Differentiation, and Cancer

Mark Ptashne, PhD
Sloan-Kettering Institute

Pluripotent cells (e.g. ES, iPS cells) express “pluripotency” genes while not expressing, or expressing at very low levels, other genes that drive differentiation. What are the regulatory mechanisms that underlie this difference? Previous experiments have described in general terms an aberrant state of chromatin (DNA plus associated proteins) in ES cells, and have related that to cancer cells, in particular those expressing the oncogene Myc. ES cells and cancer share similarities in their ability to grow and differentiate and Myc, a powerful oncogene, is involved in promoting these effects in both cases. We have developed a new way to examine chromatin structure in mammalian cells, and to relate changes therein with gene expression. We will apply these methods and findings to examining possible aberrancies in chromatin structure in pluripotent cells, and determine whether such aberrancies might be caused, or exacerbated, by Myc. We will follow the state of these genes as pluripotent cells are caused to differentiate, experiments done in collaboration with Lorenz Studer at Sloan-kettering, an authority on pluripotency and differentiation. We will use another novel tool – “fusion” proteins – designed to convert Myc into a protein that works solely as a transcriptional activator (in one case) and a transcriptional repressor (in the other). We will determine which form, if either, powerfully affects chromatin structure, and in what way. These experiments, in addition to their hoped-for explanatory power, might yield practical results as well. Another collaborator at Sloan-kettering, Guiddo Wendel, has found, as have others, that Myc plays a crucial role in forming and maintaining many cancers. One of our Myc fusion proteins might turn out to be a powerful anti-oncogenic tool. With this possibility in mind, we will test the anti-oncogenic effect of expressing our Myc fusion proteins in animal models for cancer.

Transformation of Stem Cell Based Transplantation Therapy

Yupo Ma, MD, PhD
Co-PI: Cecilia Avila, MD, MPH
Stony Brook University - SUNY

Although bone marrow transplantation has been applied clinically for more than three decades, the use of HSCs (hematopoietic stem cells) remains limited due to the inability to expand these cells ex vivo. An innovative approach is urgently needed if the research community is going to succeed in unraveling HSC expansion biology. A breakthrough in the ability to expand HSCs in vitro to clinically useful numbers will have tremendous impact on both bone marrow transplantation and gene therapy for hematologic disorders, and in doing so, will save many lives. We have recently developed an innovative, rapid and efficient method to expand HSCs using SALL4 which could profoundly improve bone marrow transplantation, potentially minimizing or even eliminating the need for matched donors. Our findings are very exciting and suggest that with further testing in vivo, an entirely new approach for HSC expansion could be developed. The goals of our proposal will focus on improving the effects of SALL4 on HSC expansion and elucidating the basic mechanisms necessary for achieving this objective.

Redefining Idiopathic Parkinson's Disease Through Induced Pluripotent Stem Cells

Jian Feng, PhD
University at Buffalo - SUNY

Parkinson’s disease (PD) is a complex movement disorder with many different features. A well-recognized categorization of Parkinson’s disease is based on whether rest tremor is present or not at disease onset. PD patients who have rest tremor generally have slower progression and better prognosis than PD patients without rest tremor at onset. This proposal aims to redefine Parkinson’s disease by finding out whether PD with tremor and PD without tremor have a different molecular signature in their midbrain dopaminergic neurons. Our preliminary studies using fibroblasts derived from 30 subjects (9 PD patients with rest tremor at onset, 14 PD patients without rest tremor at onset and 7 healthy spousal controls) showed that the expression levels of several key genes involved in dopamine functions are significantly different between the non-tremor group vs. the tremor and control groups. Since these genes are highly expressed in midbrain dopaminergic neurons, we would like to generate and analyze induced pluripotent stem cells (iPSC) derived from these fibroblasts. We will analyze iPSC-derived midbrain DA neurons to establish whether there is significant difference in the expression levels of these key genes involved in dopamine function. In addition, we will use mRNA profiling and proteomic methods to obtain a molecular signature of midbrain DA neurons that may distinguish PD with or without rest tremor at onset. The proposal will generate a very useful collection of cellular models of Parkinson’s disease; shed novel insights on the vulnerability of midbrain DA neurons in Parkinson’s disease as compared to normal spousal controls; offer new opportunities to identify neuroprotective strategies based on different subtypes of Parkinson’s disease; and provide improved design for clinical trials based on disease subtypes.

Human Induced Pluripotential Cell (iPSC)-derived Oligodendrocyte Progenitor Cells for the Treatment of Myelin Disorders

Steven Goldman, MD, PhD
University of Rochester

Many neurological diseases involve loss of the brain’s white matter, the myelin. In a previous NYSTEM grant, now ending, we proposed to develop the means by which to generate and purify myelin-producing oligodendrocyte progenitor cells (OPCs) from human iPS cells, and to assess their ability to myelinate neuronal axons in vivo, after transplantation into the brains of neonatal, myelin-deficient shiverer mice. We accomplished these goals, and now propose to continue this work with a set of animal studies intended to establish the clinical feasibility of transplanting human iPS cell-derived OPCs as a means of remyelinating the demyelinated brain. We intend to evaluate the safety and efficacy of human iPSC-derived OPCs in several models of both congenital and acquired myelin loss. These studies include trials intended to establish whether hiPSC OPCs are sufficient to rescue congenitally hypomyelinated mice, and to assess the risk of these grafts in terms of potential tumorigenesis. We will compare the relative yields, performance and risks of hiPSC cultures highly enriched in hOPCs, with those sorted to purity at different stages of maturation from these cultures, so as to define the optimal preparation for clinical use. We will then assess the ability of these cells to migrate and myelinate within the adult brain, using two distinct models of chemical demyelination in mice, chosen to model different categories of human demyelinating disease. Our intent is to establish a conceptual and operational basis for producing myelinogenic autografts from somatic cells of patients with demyelinating disease, in a manner largely free of rejection risk. This should prove an especially attractive strategy for restoring lost myelin in multiple sclerosis, transverse myelitis, optic neuritis, and subcortical stroke, in which no genetic abnormalities prevent the use of a patient’s own cells as iPSC source.

Contribution of the Vascular Niche to Liver Regeneration and Repair

Shahin Rafii, MD
Weill Cornell Medical College

Liver transplantation is the mainstay of treatment for patients with end-stage liver disease due to chemical injury (alcohol-induced, hepatotoxin ingestion), viral hepatitis, genetic disorders, hepatocellular tumors and tumor metastasis. However, the paucity of genetically matched donors results in increased morbidity and mortality of many patients, who could have otherwise been treated effectively with liver or hepatocyte transplantation. Indeed, every year more than 50,000 individuals succumb to the complications of liver disease with more than 15,000 patients awaiting a curative liver transplant. Hepatocyte transplantation provides for a clinically plausible approach to improve liver function. Therefore, identification of the molecular and cellular pathways that allow expansion and long-term engraftment of hepatocytes or augment liver regeneration and repair will have significant therapeutic impact. We have shown that liver blood vessels that are lined by specialized endothelial cells produce a specific set of unique growth factors that support expansion of hepatocytes and augment liver regeneration after injury. In this proposal, we have set forth experiments to identify the factors that stimulate liver blood vessels to support long-term hepatocyte engraftment into the liver as well as augmenting liver regeneration and repair. We hope our studies will lay the foundation for designing pre-clinical and clinical trials to exploit the potential of liver blood vessel endothelial cells to improve the outcome of hepatocyte and liver transplantation, as well as setting the stage for stimulating liver regeneration and repair in patients who are suffering from end stage chemical- and hepatitis- induced liver diseases.

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IDEA Awards

Clinical Grade iPS

Eric Bouhassira, PhD
Albert Einstein College of Medince

Hemoglobinopathies are among the most common single gene hereditary disorders in the US and worldwide. Currently available curative methods are not available for all patients because of lack of transplantable matching cells and because the treatments carry serious risks of secondary adverse events. Novel stem cell technologies have the potential to provide new therapeutic options. A novel method invented in Japan about five years ago allows researchers to create stem cells out of ordinary cells, to correct the genetic defect in the cells, and to finally transform these custom stem cells into blood stem cells that can then be reinjected in the patients to cure them. This revolutionary procedure, which eliminates all risk of rejection since the transplanted cells are derived from the patient’s own cells, is effective in mice but is currently too risky for use in humans. The goal of this proposal is to ameliorate these techniques in order to reduce the risk of adverse effects and therefore help bring stem cell technology to the patients. The techniques to transform ordinary cells into stem cells and to correct the genetic mutations in our approach are novel.

Understanding the Role of Normal Mammary Stem Cell Program in Breast Cancer Stem Cells

Wenjun Guo, PhD
Albert Einstein College of Medince

In many cases, only a small fraction of cancer cells are truly capable of forming tumors, while the bulk of cancer cells have lost such ability. The tumor-forming cells and non-tumor-forming cells are organized in a hierarchical relationship, in which the tumor-forming cells generate the non-tumor-forming cells. Such a hierarchy resembles the relationship of stem cells and differentiated cells within normal tissues. Therefore, the tumor-forming cells are often referred to as cancer stem cells to reflect the fact that these cells are the driving force of tumor growth and progression. This implies that a successful cancer therapy has to destroy the cancer stem cells. In order to target cancer stem cells, we need to develop means to identify these cells. In addition, we need to understand how cancer stem cells survive and produce additional cancer stem cells (i.e. self-renewal). Because of the similarity of cancer stem cells and stem cells in normal tissues, it has been speculated that these two types of cells share many common genes that help maintain stem cells as stem cells and allow them to reproduce. We have found two novel genes that are necessary for maintaining normal breast stem cells. In this proposal, we will investigate whether these two genes also play a key role in maintaining breast cancer stem cells. In addition, we will develop a method to label cancer stem cells using these two genes. This work will use novel breast cancer models to identify novel identifiers and key regulators of cancer stem cells. The findings obtained here will pave the way for developing therapeutic approaches to target breast cancer stem cells.

Skin Regeneration in the Setting of Epidermal Stem Cell Ablation

Angela Christiano, PhD
Columbia University Medical Center

The skin is the largest and most regenerative tissue of the adult organism. It is the first line of defense against pathogens and is subjected to a continual barrage of physical, thermal and chemical assaults. As such, the skin requires continuous replenishment from skin-resident stem cells to ensure optimal function over a lifetime. With injury or wounding, normal skin regeneration is perturbed, and stem cell activity has to be adjusted to compensate for lost tissue. This process of repair and regeneration, specifically after chronic injury, often leads to abnormalities in skin formation and function. Therefore, the ability to regenerate fully functional skin after severe injury represents a major unmet medical need. In this proposal, we investigate the stem cell-dependent mechanism involved in the skin’s response to repeated injury. Using genetic means to kill individual cells in mouse epidermis, we examine the outcomes of perturbing normal skin regeneration, without injuring the skin and causing inflammation. Our preliminary data show that chronic cell death in the skin results in the hyperactivity of stem cells, yielding a thicker-than-normal epidermal layer. Our experimental system is novel in that it combines the ability to control cell death in a genetic and drug-dependent way with the ability to track and visualize how stem cells respond to that loss. Unraveling the regulatory machinery responsible for cellular repair in response to skin damage will allow us to translate this knowledge into improved therapies for treating a multitude of skin ailments.

Three-Dimensional High Resolution Reconstruction of the In Vivo Adult Neural Stem Cell Niche

Fiona Doetsch, PhD
Columbia University Medical Center

Adult neural stem cells continuously generate neurons in restricted parts of the brain and may represent an important source of endogenous cells that can be stimulated for brain repair. Stem cells reside in specialized niches, or microenvironments, that support their self-renewal and maturation into different cell types. The subventricular zone (SVZ) is the largest germinal area in the adult brain. Here we will visualize the entire SVZ niche using a novel serial reconstruciton approach which will illminate all cell interactions. This will provide unprecedented three-dimensional information about the stem cell niche.

Canonical Wnt Signaling Regulation of Adrenocortical Stem Cells

Edward Laufer, PhD
Columbia University Medical Center

The adrenal cortex is a critical endocrine gland that produces steroid hormones. The hormones regulate blood salts and total volume, and control stress responses. Defects in adrenocortical function are life-threatening without daily treatment, and also pose a significant hypertension risk. Adrenocortical cells constantly turn over, and steroid-producing populations expand or contract in response to changing diet or stress levels, and it is thought that stem cells provide the replacement steroidogenic cells. We identified a population of cells within the mouse adrenal that behave like stem cells, although their regulation is poorly understood. This project will explore the behavior of the adrenocortical stem cells using two approaches. The first experiments will ask if all of the stem cells behave similarly. This will be tested by genetically marking both daughter cells of a single stem cell and comparing their behavior, and also comparing the behavior of all daughters across a population of stem cells. The second set of experiments will address how the properties of the stem cells are maintained by external signals, focusing on a signal called Wnt, and a molecule that functions in this pathway called beta catenin. Daughter cells will be genetically marked similar to the first experiments, except now one daughter will lack a functional copy of the beta catenin gene, thus preventing activity of the Wnt signal. The function of the Wnt signal will be inferred by comparing the behavior of the normal and mutant cells in a variety of physiological contexts. This project will combine several state of the art technologies including genetic lineage tracing and the technique that allows us to follow the behavior of daughter cells, to investigate fundamental questions in molecular endocrinology and stem cell biology, namely how external signals regulate stem cells.

Identification of Novel Adrenocortical Stem Cell Markers

Edward Laufer, PhD
Columbia University Medical Center

The adrenal cortex is a critical endocrine gland that produces steroid hormones which regulate ionic balances and blood volume, as well as mediating responses to stress. Defects in adrenocortical function can be life-threatening without daily treatment, and can also contribute to high blood pressure. Adrenocortical cell populations are subject to constant turnover, and functional steroid-producing populations expand or contract in response to changing diet or stress levels. It is thought that adrenocortical stem cells are a fundamental source for replacement steroidogenic cells. We recently identified cells in the adrenal cortex that behave like stem cells, as they can generate all steroidogenic cell types, and persist throughout life. However progress in deciphering the molecular mechanisms that control these cells is hampered because we do not know which genes are critical for regulating their behavior. The goal of this project is to identify functionally important genes expressed specifically in the adrenocortical stem cells. We will use fluorescent labeling to isolate multiple adrenocortical cell populations, identify genes differentially expressed in the stem cell population, and investigate how and where they are expressed and how this expression is regulated in intact adrenal glands. We will also investigate whether cells that express one of the genes behave like stem cells, and also what the function of one is in regulating stem cell behavior. The outcome of these experiments will provide the foundation for future studies that will explore the regulatory logic of organ maintenance and remodeling, both in vivo and in vitro. This project will combine several state of the art technologies including fluorescently labeled transgenic cell populations, fluorescence activated cell sorting and genome-wide microarray transcriptional profiling to investigate fundamental questions in molecular endocrinology, namely how adrenal stem cells are regulated.

Regulating the Directed Differentiation of CNS and Pancreatic Islet Cell Populations

Lori Sussel, PhD
Co-PI: Hynek Wichterle, PhD
Columbia University Medical Center

The potential to use human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) for regenerative medicine relies on our ability to direct the differentiation of specific cell populations. Although protocols for the in vitro differentiation of many tissue-specific cell types have been established, the differentiation process remains inefficient, and pure populations of functional cell types have not been achieved. Since the most successful strategies for directed differentiation of specific cell populations have been modeled after normal developmental processes, it is likely that our failure to achieve pure populations of specialized cell types is due to an incomplete understanding of the regulatory events needed to specify unique cell type identities. Therefore greater understanding of the normal developmental pathways required for cell type diversification will contribute to the optimization of the current ESC directed differentiation protocols. We propose to take advantage of information we have gained from our studies on the development of pancreatic islet cells and spinal motor neurons during mouse fetal development and apply them to ESC differentiation protocols. We have demonstrated that the transcription factor Nkx2.2 is a key regulator of cell fate decisions within the pancreas and nervous system. We hypothesize that Nkx2.2 differentially regulates the induction of insulin-producing beta cells within the pancreas and motor neurons within the CNS through distinct protein domains that function to initiate unique cell type specific gene regulatory networks. In this IDEA grant, we will test whether Nkx2.2 can induce important cell differentiation programs to optimize directed differentiation protocols for CNS motor neurons and insulin-producing beta cells, cell types that are needed for the treatment of neurological disorders and diabetes, respectively. Importantly, this study combines established mouse embryonic and mESC systems, and brings together the complimentary expertise of the Wichterle and Sussel labs to ensure success of this IDEA project.

Induced Cellular Reprogramming in C. Elegans

Jun Liu, PhD
Cornell University

The reprogramming of cell identity has tremendous potential for cell replacement therapies of various kinds of human diseases. One of the most exciting possibilities for cellular therapy is to allow direct replacement of the diseased or damaged cells by other cells from the same patient. While a number of studies have successfully demonstrated direct reprogramming of one differentiated cell type to another via ectopic expression of certain tissue-specific transcription factors, how and when certain somatic cells are capable of changing their identify is still poorly understood. The powerful genetics and the availability of the entire lineage of C. elegans make it an attractive model to study the mechanistic basis of cellular plasticity and cell reprogramming in vivo at single cell resolution. Recently, Tursun and colleagues reported that ectopic expression of neuronal cell fate determinants together with the knockdown of a histone chaperon protein, LIN-53/RbAp, led to the reprogramming of germ cells to specific types of neurons in C. elegans. However, knocking down LIN-53 did not permit reprogramming of germ cells to myogenic fate upon ectopic expression of the muscle fate determinant. These findings suggest an exciting possibility that germ cells need to overcome different inhibitory chromatin environments in order to be converted to different cell types. We have found that two transcription factors, a forkhead transcription factor LET-381 and a homeodomain transcription factor CEH-34, function together to specify the non-muscle macrophage-like mesodermal cells, the coelomocytes (CCs). We further showed that LET-381 and CEH-34 are sufficient to re-program other cells to CCs in a context-dependent manner. We propose to use these non-muscle CCs as a model and investigate roles of cell- or lineage-specific chromatin-based mechanisms that restrict the plasticity of cell fates. Results from the proposed studies will have significant implications on in vivo cellular reprogramming.

Characterization of Multipotent Embryonic Skin Stem Cells

Elena Ezhkova, PhD
Mount Sinai School of Medicine

Stem cells can both self-renew and differentiate into particular cell types, making them an attractive target for potential clinical applications involving tissue regeneration following injury or genetic disease. During embryogenesis, stem cells become progressively more restricted in their fates as tissues and organs develop. In some adult tissues, however, niches of multipotent stem cells are set aside for homeostasis and injury repair. A fundamental question in stem cell biology is the degree to which adult stem cells resemble their embryonic counterparts within a developing tissue. Skin epithelium is an excellent model to address this question since its lineages are all derived from a single layer of embryonic skin stem cells. We have devised strategies to isolate and characterize embryonic skin stem cells and compare them to the multipotent and unipotent stem cells of adult skin. Through transcriptional profiling, we propose to determine how these populations differ and will select factors that might be involved in stem cell maintenance and fate specification. By performing in vivo functional studies, we will determine the functional significance of selected candidates in skin stem cell control. These studies will address the extent to which the transcriptional and signaling differences are relevant to stem cell regulation and cell fate determination. Here, we present strategies to isolate embryonic skin stem cells as they transition from a multipotent to a lineage committed state. We also propose to use novel in vivo genetic methods to dissect key regulatory proteins that control skin stem cells. In summary, upon completion of this study we will fill in multiple gaps in our current understanding of skin stem cell control and fate determination. Additionally, this study will expand our knowledge on the use of skin stem cells in tissue regreneration to treat chronic wounds and severe burns.

A "BTB-POZ" Key to Ground-State Pluripotency During Somatic Cell Reprogramming

Jianlong Wang, PhD
Mount Sinai School of Medicine

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can self propagate and make other cell types under proper cues, therefore, they provide unlimited material for cell-based therapies and offer great hope for personalized medicine using patient-specific iPSCs. The goal of this grant proposal is to identify and characterize a key regulator for efficient generation of iPSCs and to understand how this novel reprogramming factor promotes ground-state pluripotency during reprogramming. We hypothesize that the BTB-POZ transcription factor Nac1, while dispensable for early development and stem cell maintenance, is essential for establishing ground-state pluripotency during reprogramming. Indeed, by interacting with and sharing numerous target genes with many other important stem cell and reprogramming factors, namely Nanog and Oct4, and chromatin remodeling and modifying factors, such as SWI/SNF complexes, Nac1 may play a fundamental role in establishing ground state pluripotency. We will characterize the pluripotent state of iPSC clones reprogrammed in the absence of Nac1. We will also dissect the potential molecular mechanisms by which Nac1 promotes ground state pluripotency and connect the Nac1 reporgramming activity with function of Nanog, Oct4, and chromatic remodeling proteins during reprogramming. The innovative aspects of this proposal lie in the discovery of a novel transcriptional regulator that may control the well known pluripotency and reprogramming factor Nanog, and the proteomic and genetic approaches we will undertake to dissect its unique protein structure and function in organizing ESC-associated enhanceosomes encompassing pluripotency factors and epigenetic regulators on critical target genes that promote the ground state pluripotency of iPSCs during somatic cell reprogramming.

Understanding How Erk Signaling Promotes Lineage Competence through Chromatin Regulation

Danny Reinberg, PhD
New York University School of Medicine

Pluripotent embryonic stem cells have the unique ability to undergo indefinite self-renewal and yet possess the ability to differentiate into all cells of the body. Understanding this remarkable property offers opportunities for regenerative medicine. In mouse ES cells, the extracellular-signal regulated kinase (Erk) signaling pathway plays an important role in governing the switch between self-renewal and lineage commitment. Therefore, understanding how Erk impacts the gene regulatory circuitry is imperative. This proposal aims to dissect how lineage differentiation signals Erk such that the transcriptional and epigenetic machineries are modulated to confer lineage competence in mouse ES cells. We show that Erk converges on both RNA Poiymerase II as well as the Polycomb repression machinery. We plan to identify other novel regulators, downstream of Erk, to ascertain the mechanistic principles governing the interplay between these factors and the resultant contributions to the multi-lineage differentiation potential of mouse ES cells. The interdependency between these components likely underscores the ability of ES cells to respond quickly to differentiation cues. We combine a state of the art proteomic approach with powerful biochemical tools to examine the dynamic changes in chromatin structure as a function of Erk activity. Our proposed studies also open up new avenues to examine how signal-induced transcriptional changes may trigger Polycomb mediated repression. These changes point to a highly dynamic role for Polycomb repression that is attuned to environmental perturbations

The Function of Stem Cells in Sea Anemone Regeneration

Gerald Thomsen, PhD
Stony Brook University – SUNY

We propose an exploratory grant to study the stem cells of the sea anemone Nematostella vectensis. This animal can regenerate its entire body from just a small stump of amputated tissue, and we hypothesize that this happens through an extensive contribution of stem cells. We plan to identify the Nematostella genes that control the self-renewing capability of stem cells, based on their close evolutionary relationships to genes known to regulate stem cells of vertebrates like humans and mice. We will find out where those genes are expressed, and which cells express them, during regeneration. We plan to find out if the stem cells in the sea anemone are required for its ability to regenerate by removing the genes that maintain the stem cells. We expect that by removing these genes, the stem cells of the anemone will differentiate and thus lose their ability to self-renew, and the animal will no longer have the cells it needs to regenerate. We will also attempt to turn on expression of these genes permanently, and see what effects this has. Finally, we will use the genomic DNA controlling where these genes are expressed to control the expression of a gene we can easily see, such as a fluorescent protein, to visualize where the stem cells in the anemone are located, and determine whether they divide, migrate and contribute to new tissues during regeneration. By studying sea anemone regeneration as a new model for stem cell biology, our long-term goal and hope is to understand the nature of regeneration well enough to use discoveries made with the sea anemone to induce human cells to regenerate lost, diseased or defective human tissues and organs.

Directed Reprogramming of Human Fibroblasts to Oligodendrocyte Progenitors

Fraser Sim, PhD
University at Buffalo – SUNY

The development of cellular and molecular therapies to treat and diagnose developmental leukodystrophy and adult demyelinating diseases, such as multiple sclerosis, have been hampered by a lack of understanding of human glial development and regulation. In this project, we will help address these needs by defining the transcription factors necessary to reprogram human fibroblasts to oligodendrocyte progenitors. The primary goal of this application is to define transcription factors that are necessary and sufficient to induce oligodendrocyte fate from human fibroblasts. We believe that the direct reprogramming of oligodendrocyte progenitors from patient skin cells may provide a novel cellular source for stem cell repair in demyelinating disease. Importantly, rapid reprogramming will also permit tissue culture modeling of fatal leukodystrophies. This would be clinically important in Krabbe’s disease as it may allow accurate and early diagnosis of at-risk newborns. The direct induction or reprogramming of fibroblasts from human skin to oligodendrocytes has not been previously described. We believe that this project is highly innovative as it combines novel genomic data from primary sorted cells and new methods for the detection of human oligodendrocyte reprogramming. We have employed cell sorting techniques to purify and characterize human primary neural stem cells and progenitors to determine the transcription factors specifically expressed by oligodendrocyte progenitor cells in the normal human brain. We have developed a novel reporter virus to accurately and reliably identify oligodendrocyte progenitors by expression of green flourescent protein. This reporter will be used to determine which factors are important in defining the oligodendrocyte. Importantly, we will then utilize a mouse model to establish whether reprogrammed cells are bona fide oligodendrocyte capable of myelin formation and maintainance.

Promoting MSC-Mediated Musculoskeletal Tissue Regeneration Using Sustained, Localized siRNA Delivery

Danielle Benoit, PhD
University of Rochester

Although fractures generally heal, of the approximately 7.9 million skeletal fractures that occur each year in the U.S., 10-20% have impaired healing, including delayed healing or no healing at all. Reduced healing capacity is linked with dysfunctional mesenchymal stem cells (MSCs), cells recruited to fractures that orchestrate the bone regeneration/healing cascade. There is clear and immense potential of gene silencing techniques (e.g., through small interfering RNA, siRNA) to aid in fracture healing by improving the regenerative capacity of MSCs. However, there is no means to exploit siRNA as a therapeutic molecule for fracture healing due to significant delivery hurdles including the inability to locally and sustainably delivery siRNA to fracture sites. To address this, our goal with this project is to develop a delivery system for siRNA that will prolong siRNA treatment in vivo to affect MSCs over the time course of fracture healing (~2-4 weeks). Our objectives are: 1) further develop our proven nanoparticle (NP) delivery system for siRNA to ensure complete and potent siRNA delivery to MSCs through alterations in chemical compositions; 2) augment delivery characteristics of siRNA through introduction of controlled release chemistries that enable tethering and long-term release of siRNA NPs from hydrogel reservoirs; and 3) test that hydrogel reservoir delivery systems sustain and localize siRNA delivery to MSCs within fracture sites for 2-4 weeks. The innovation of this work is the development and optimization of the first siRNA delivery approach to achieve localized, sustained in vivo treatment to rescue MSC functions, resulting in enhanced healing of bone fractures.

Discovery of Novel Retinoids for Stem Cell Biology

Todd Evans, PhD
Co-PI: Bhaskar Das, PhD
Weill Cornell Medical College

Retinoic acid is the active component derived from Vitamin A. It is a small molecule that regulates gene expression through interaction with a family of different receptors that bind to DNA. Retinoic acid can regulate stem cell function, and its receptors are essential for normal function of many organ systems. Defects in the signaling pathway can cause diseases, including leukemia. Specific receptors are important for different functions of the retinoic signaling pathway. For this reason, much effort has gone into the development of retinoids that might activate or repress subsets of receptors. Unfortunately, the successful development of such compounds as drugs has not progressed and they are generally not available to the research community. This project is a collaboration between an organic chemist and a stem cell biologist to identify new active retinoids, define their specificity of action, and to test their ability to impact stem and progenitor cell biology. For this purpose we propose to exploit new chemical reactions to complete a library of novel compounds that have never previously been tested for activity. To evaluate biological function we will use our expertise in stem cell biology to direct differentiation of embryonic stem cells and to evaluate stem and progenitor activity in the zebrafish animal model. We have preliminary data suggesting that new compounds can be generated with receptor specificity. We expect our project to generate a set of new biologically active compounds ready to be used for research purposes and as leads for drug development.

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