RFA #: 0804221050
Targeted RFA for Investigation of iPS and Other Derivation Approaches Scientific Abstracts
Characterization of IPS Using a Novel High Throughput Replication Timing Assay (IIRP)
Eric Bouhassira, PhD
Albert Einstein College of Medicine
Embryonic stem cells give rise to all tissues of the body and can in theory be used to create replacement cells to cure many diseases. Before therapies can be developed from these cells, a number of problems must be resolved. The first problem is that the cells could be rejected by the body of the patient in the same way as other grafts are rejected. One elegant solution to the problem is to produce embryonic stem cells from every patient since that would completely eliminate the immune rejection problem. The second problem is that embryonic stem cells are isolated from very young embryos which are in short supply. In addition the embryos are destroyed in the process of extracting the stem cells and this is objectionable on ethical ground for part of the population. Recently, a research team in Japan has developed an extraordinary technique to reprogram skin cells into induced pluripotent stem cells (iPS), a type of cell that is very similar to embryonic stem cells and could be used instead of embryonic stem cells, if a number of technical issues were solved. Before iPS can be used to develop therapies, methods must be developed to determine whether the reprogramming has worked, or whether the cells have become dangerous and would cause cancer or other diseases if they were used to treat patients. Cells must duplicate their DNA before every cell division. This is a highly regulated process that is different in every kind of cell; brain cells replicate DNA that is active in the brain first; blood cells replicate DNA that is active in the blood first and embryonic stem cell replicate the DNA that is active in early embryos first. Some researchers, including us, believe that the order in which the DNA is replicated is very important and that if the DNA is not replicated in the proper order, the stem cells cannot function normally. We have developed a technique that measures very precisely the order in which the DNA replicates. We propose here to determine if we can use this technique to predict if iPS are properly reprogrammed and are identical to embryonic stem cells or not. Developing techniques to determine if iPS cells are properly reprogrammed could have a very high impact because iPS cells have the potential to be used to treat many diseases.
Intracellular Signaling Cascades in IPS Reprogramming (IIRP)
Asa Abeliovich, MD, PhD
Columbia University Medical Center
Intriguingly, ectopic expression of only 4 transcription factors (Oct4, Sox2, Klf4, and c-myc) reprogrammed fibroblasts into induced pluripotent stem (iPS) cells. Having this small set of genes provides the opportunity to dissect and understand mechanisms and kinetics of iPS cell generation, which is the key in developing new reprogramming strategies. iPS cells hold great promise for regenerative medicine. Yet, human clinical applications of iPS cells are still hindered by several obstacles, such as low efficiency of reprogramming and the danger of tumor formation. If we understand the reprogramming process better, then we can develop new approaches to improve iPS cell generation. As demonstrated by gene expression profiling, reprogramming is a multi-step process. But, which intracellular signaling cascades are downstream targets of reprogramming factors at the various stages, has yet to be determined. Rapid kinetics via phosphorylation and other protein modifications, switch-like regulation, and the availability of well established inhibitors (chemical compounds) make signaling cascades an exciting target for manipulations to improve iPS cell generation. Here, we explore an area of reprogramming which has not been considered yet, but has great potential to enhance iPS cell generation. We seek to illuminate intracellular signaling molecules which might become valuable drug targets to achieve better reprogramming. These studies will further our understanding of mechanisms and kinetics of reprogramming which is critical in developing new strategies for enhanced efficiency and safety of iPS cell generation, may lead to replacement of reprogramming factors with pharmacological tools or chemical compounds, and to iPS cells less prone to tumor formation.
Human iPS Cell-Based Models for Neurodegeneration (IIRP)
Asa Abeliovich, MD, PhD
Columbia University Medical Center
No treatments are currently available that slow the course of neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. To generate novel therapies and gain further insight into these disorders, it is essential to generate accurate disease models. Existing animal models are not good enough: they do not display basic features of these disorders, they only represent rare familial inherited forms, and they may not reflect potential treatments in human patients. Accurate models could provide mechanistic insights and allow for therapeutic approaches such as drug screens, and would be informative for the development of cell therapy. Our preliminary data and published work strongly support the utility of stem cell-based models of Parkinson’s, including ES-derived neurons. In this application, we propose to validate the accuracy of iPS cell-based models in the context of genetic Mendelian inherited forms of Alzheimer’s and Parkinson’s. We focus initially on the inherited genetic forms of Alzheimer’s (Presenilin 1 and 2 mutations) and Parkinson’s (LRRK2 mutations). The ‘stemness’ properties of iPS cell lines will be validated using established marker criteria. iPS cell differentiation to neuronal phenotypes will be evaluated in normal and disease associated fibroblasts (human ES cells will serve as a control). Finally, cells will be transplanted into rodent CNS and evaluated for integration and pathology. Subsequently we will demonstrate the utility of the models to test fundamental common hypotheses about ‘sporadic’ forms of Alzheimer’s and Parkinson’s. These studies will generate, validate, and characterize iPS cells from neurodegenerative models for the community. They will further explore mechanisms of disease, particularly with an interest in the unique opportunity to study ‘sporadic’ forms of neurodegeneration.
Human iPS Cells as a Model to Study ALS Pathogenesis (IIRP)
Hynek Wichterle, PhD
Columbia University Medical Center
Generation of pluripotent stem cells directly from individual patients is a prerequisite for production of relevant cell types needed to study human diseases and for development of new therapies. Although recently generated iPS cells have been shown to differentiate into many different cell types including nerve cells, it is not known whether these cells are identical to cells found in our body. Careful and detailed characterization of clinically relevant cell types derived from iPS cells is therefore an important milestone. Spinal motor neurons (MNs) are one of the best-characterized cell types in the nervous system and one of few cell types that have been successfully generated from human embryonic stem cells in cultures. Examining motor neuron specific attributes in iPS derived MNs will serve as a robust bench-mark to determine how faithfully iPS cells can acquire correct cellular identity. The ultimate goal of stem cell research is to model human disease and develop new stem cell based therapies. Towards that end, we have derived iPS cells from a patient with amyotrophic lateral sclerosis (ALS), a devastating motor neuron disease. We will determine whether defects observed in mouse models of this disease can be reproduced in human iPS cells. The proposed set of studies will provide foundation for future use of iPS cells derived from both hereditary and sporadic cases of ALS to study defects leading to motor neuron degeneration and to develop much needed new therapies for patients afflicted with this devastating disease.
Exploring Human iPS Cells for the Hematopoietic and Genetic Correction of β-Thalassemia (IIRP)
Michel Sadelain, MD, PhD
Memorial Sloan-Kettering Cancer Center
The reprogramming of somatic cells to a pluripotent stem cell state holds great promise for disease modeling and therapy. In a recent breakthrough study, researchers in Japan achieved direct reprogramming of mouse embryonic and adult fibroblasts to an embryonic stem (ES) cell-like state through retroviral-mediated gene transfer of the four transcription factors Oct-4, Sox-2, Klf-4 and c-myc. Several groups recently reported that induced pluripotent (iPS) cells could also be generated from human fibroblasts, a finding we have reproduced with our own lentiviral vectors. There are however numerous challenges to address before iPS cells might be used therapeutically. We propose to address three aspects of iPS cells that have great relevance to their potential therapeutic utility. The first is whether we can generate human iPS cells utilizing non-integrating lentiviral vectors, i.e., vectors that do not result in the permanent insertion of foreign DNA into the iPS cell’s chromosomes (an important safety feature). The second is whether we can augment the engraftment potential of hematopoietic cells derived from iPS cells. The third is whether we can genetically modify human iPS cells generated from thalassemic subjects and restore hemoglobin synthesis in the red cells that derive from these iPS cells. The studies will help the scientific community gain important insights into the therapeutic potential of iPS cells for the treatment of hematological disorders. In addition to establishing biological proof-of-principles, our approach remains focused on developing and utilizing methodologies that could eventually be employed in the clinical setting.
In Vivo Function of Human iPS Derived Neural Precursors (IIRP)
Viviane Tabar, MD
Memorial Sloan-Kettering Cancer Center
The derivation of human iPS cells has resulted in significant interest in their potential therapeutic applications. Our team and others have reported the successful derivation of neural progeny from several iPS lines. The cells obtained exhibit appropriate marker expression but more extensive analyses have not been performed to date. While current iPS cell line derivation strategies are likely to evolve rapidly towards a safer paradigm, it is crucial to initiate studies that address the phenotypic stability of iPS derivatives as well as their ability to integrate and function upon grafting. In this study, we propose to differentiate hES and hiPS cells into neural precursors and dopamine neurons, establish comparative molecular profiles at specific differentiation stages and transplant cells into two different rat models: the neurogenic regions of the normal adult rat brain and the 6-hydroxydopamine (6-OHDA) rat model of Parkinson’s disease. We hypothesize that hiPS derived neural progeny can survive, integrate and function in a host brain upon transplantation in the neurogenic zones of the normal rat brain or the Parkinson rat model where they lead to behavioral recovery. In the first aim, we will induce neural induction in hiPS derived ES like cells and analyze their ability to undergo anterior and posterior patterning. We will also direct hiPS differentiation into specific neuronal subtypes such as dopamine neurons. In the second aim, hiPS derived neural precursors will be grafted in the subventricular zone (SVZ) of adult rats. In the third aim, hiPS derived dopamine neurons will be grafted into the 6OHDA Parkinson rat model. Survival, phenotypic stability and behavioral recovery will be assessed. The proposal will have a significant impact on our understanding of the biology of iPS cells and more importantly, on their potential role as a source of stem or other cell types for transplantation.
Congenital Erythropoietic Porphyria: Evaluation of iPS Cells for Murine and Human Therapy (IIRP)
David Bishop, PhD
Mount Sinai School of Medicine
Congenital Erythropoietic Porphyria (CEP) is a rare inherited disorder caused by mutations in a gene necessary for the production of heme, the organic molecule that is essential for hemoglobin and other heme-containing proteins required for life. CEP is a metabolic disorder of red blood cells that results in their demise, causing severe anemia that requires repeated blood transfusions. In addition, toxic, photo-sensitive porphyrin molecules are released from the red cells which damage sun-exposed skin, leading to severe blistering, infections, cartilage/bone loss, and disfigurement. To avoid these consequences, patients with CEP must completely avoid sunlight and even some types of artificial lighting, use tinted window glass with shades, sunscreen and protective clothing. Many patients are so anemic, that they must get frequent blood transfusions to survive, but then suffer iron overload that injures the heart. If the patients have a relative who has a very good tissue match, bone marrow transplant/hematopoietic stem cells transplant can cure them, but it can also result in severe illness (rejection, etc.) and death. To make marrow replacement safer for this and other diseases, we have been studying other therapeutic strategies including gene and stem cell replacement. To evaluate the safety and effectiveness of new therapies, we generated a mouse model with CEP that mimics the human disorder. Our research will determine if we can correct a genetic disease of the bone marrow by transplanting the patient's own blood-forming cells that have been gene-corrected using new gene and stem cell techniques. The results of these studies could provide a better understanding of changes caused in cells due to this genetic disorder. The success of these murine and human "proof-of-concept" studies would provide the rationale for a safe and effective cure of CEP and other metabolic blood diseases.
Using a Novel Regulator of hES Cell Pluripotency in Generating iPS Cells (IIRP)
Saghi Ghaffari, MD, PhD
Mount Sinai School of Medicine
Recent ground-breaking discoveries have brought us closer to the promise of cell therapy in which cells from a host of debilitating or lethal conditions such as Parkinson’s disease, diabetes, spinal cord injuries and leukemias can be replaced by normal cells generated in Petri dish in the laboratory. This novel approach is based on using four nuclear factors to revert the nature of a mature cell (for instance skin cell) to a primitive embryonic stage. However, there are several hurdles in the currently used approaches that prevent this methodology to be used on a large scale for therapy. One major problem is the very low efficiency of the current methodology; another problem is the safety of this approach which may increase the tumor formation once cells derived from this strategy are transplanted. Therefore finding methods that increase the efficiency and/or safety of this approach will be extremely valuable. We have identified a nuclear factor (FoxO1) that may increase both the efficiency and the safety of the current methods. We hypothesize that the nuclear factor (FoxO1) we have identified has similar properties, as some of the factors currently used, in reverting mature cells back into embryonic stage. In this proposal we will test whether this factor (FoxO1) can increase the efficiency and/or the safety of current methods. We will also use human embryonic stem cells to further study the function of FoxO1 nuclear factor in maintaining the embryonic stage of the cell in which it can produce all cell types in the body. This study is likely to have a significant impact on the efficiency of the current methodology, and it may offer some safety improvement over the current methodology.
Analyses of Pluripotency in Reprogrammed Induced Pluripotent Stem Cells (IIRP)
Ihor Lemischka, PhD
Mount Sinai School of Medicine
Embryonic stem cells (ESC) are pluripotent and can produce all cell types present in adult mammals. Because of this property human (h) ESC research promises to revolutionize medicine by providing cell populations for future transplantation therapies as well as for developing models to unravel the mechanisms of complex diseases. Realization of such promises is hampered by the relatively few available hESC lines and the genetic complexity of the human population. Somatic Cell Nuclear Transfer (SCNT) into oocytes is a potential avenue to produce genetically matched and patient/disease-specific hESC. However, this has not yet been possible with human material and even when accomplished will involve serious societal and ethical issues. Recent efforts have shown that human skin cells can be directly reprogrammed to a pluripotent state using defined molecules. The exact similarity of these human induced pluripotent (hiPS) cells and bona fide hESC is unclear. Moreover, the genetic manipulation-based methods to produce hiPS do not allow their clinical utilization. We propose that the properties of hESC and hiPS are very similar. We will rigorously compare the functional, molecular and biochemical properties of these two cell populations and establish how pluripotency is established and maintained. We further propose that there will be numerous alternative avenues to reprogram adult human cells into hiPS cells. We will explore this possibility using systems that will be developed where introduced reprogramming factors can be turned-on or off with small drug molecules. An understanding of how pluripotency is controlled is necessary in order to manipulate cells in ways that impact on medicine. Evaluating the pluripotent status of hiPS is of paramount importance if these cells are to be viable alternatives to hESC. In addition, it is likely that other, more effective ways of generating hiPS cells will be possible. Collectively, our proposed studies will impact on questions and issues of stem cell technology.
Noonan Syndrome and Related Disorders: Stem Cells and RAS Pathway Signaling (IIRP)
Bruce Gelb, MD
Mount Sinai School of Medicine
Noonan syndrome and related disorders are genetic diseases caused by mutations in several genes in a single pathway used in cells to signal from the outer membrane to the nucleus (the relevant one being called the RAS pathway). These disorders overlap in the problems they cause with the common issues being short stature, heart problems, mental retardation, and altered facial appearance. By and large, the RAS pathway mutations in these diseases result in an increase in signaling, termed gain of function. Until now, the fact that these disorders disrupt human embryonic development in a wide range of tissues and organs has made them impossible to study in humans. Efforts to date have relied on biochemical studies with artificially expressed mutant proteins and modeling in animals such as fruit flies and mice. We propose to examine several questions using the newly available technology for developing stem cells from cultured skin cells. Specifically, we want to determine how altered signaling from mutations in various RAS pathway genes results in specific disorders, affecting organ development and homeostasis in reproducible but different ways. We expect to find specific signatures of each disorder at the gene expression and protein levels. We have obtained skin cell lines from several patients with known mutations in RAS pathway genes. We will use the new technology to reprogram these cultured skin cells into stem cells. The stem cells will then be studied as such and differentiated along various developmental programs (for instance, developed into heart muscle cells). Stem cells and differentiated cells will be studied for their cellular properties such as the genes that they express and the status of RAS signaling in them. The results could provide new and important information about the pathogeneses of Noonan syndrome and related disorders. This might enable better prognostication for affected individuals as well as empower future efforts to development novel therapeutics.
Human RPE and Induced Pluripotent Stem Cells for Parkinson's Disease (IIRP)
Sally Temple, PhD
Regenerative Research Foundation
Parkinsons Disease (PD) affects approximately one million patients world-wide. PD is caused by loss of Dopamine-containing excitable cells (neurons) in the brain. Stem cell research offers the opportunity to create human Dopamine-containing neurons in tissue culture. These cells can be used to replace the lost cells in patients and to study how the disease forms and how it could be combated with novel medications. The cutting edge approach to making stem cells for PD is to generate ‘human induced pluripotent stem cells’ (hiPSCs), which can be done with a patient skin biopsy. We propose to generate over 20 hiPSC cell lines from PD patients in our collaborating clinical core. In addition, we will explore an entirely new way to generate Dopamine containing cells for PD, starting from an adult stem cell present in the human retina. From organ-donated eye tissue, we can derive human retinal pigment epithelial stem cells (hRPESCs), and we have evidence that these can produce Dopamine-containing cells relevant to PD. These hRPESCs cells may be less tumorigenic than hiPSCs, which can readily form tumors after transplantation. To our knowledge, we are the first group to isolate a stem cell from the adult human RPE. This is a highly novel approach to derive Dopamine-containing cells for PD applications. Unlike hiPSC, production of hRPESC does not require exposing the cells to genes that can cause tumors. Human iPSC cells, while tumorigenic after transplantation in vivo, could still offer an important platform for drug testing studies and studying Parkinsons Disease cause and progression. Consequently the hiPSC lines we will generate from PD patients with known genotypes will be highly useful to the research community, and in our future studies.
Using Improved iPS Derivation and Differentiation Methods to study Parkinson's Disease (IIRP)
Jian Feng, PhD
SUNY - University at Buffalo
Parkinson’s disease (PD) is marked by the selective death of dopamine neurons in a brain region called substantia nigra. Interactions between environmental and genetic factors are thought to cause the disease. We have been studying how parkin, one of the most frequently mutated genes in Parkinson’s disease, interacts with environmental PD toxins such as rotenone in rat dopamine neurons. One of the most significant obstacles for PD research is the lack of live dopamine neurons from PD patients for long-term studies and drug development. Our previous studies have shown that rat dopamine neurons are particularly vulnerable to the disruption of microtubules – intracellular highway for the transport of dopamine vesicles from the cell body to nerve terminals over long distance. Our central hypothesis is that parkin stabilizes microtubules to protect against environmental PD toxins such as rotenone, which destroys microtubules and selectively kills dopamine neurons. Mutations of parkin disrupt this protective function and thus leave dopamine neurons unprotected against environmental toxins, whose effects over the years kill these neurons and produce the core clinical symptoms of Parkinson’s disease. A recent landmark breakthrough makes it possible for the first time to change human skin cells to induced pluripotent stem (iPS) cells. These iPS cells are like embryonic stem cells and can be converted to any types of cells including dopamine neurons. Thus, we would like to reprogram skin cells and blood cells from normal people and PD patients with parkin mutations to stem cells through this method. The resulting stem cells will be converted to dopamine neurons so we can study how mutations of parkin disrupt its ability to protect against environmental PD toxins. This study will allow us to better understand how parkin protects against environmental PD toxins in human dopamine neurons. It will also tell us how mutations of parkin disrupt this protective function and cause selectively death of human dopamine neurons and Parkinson’s disease. Knowledge gained from the study may enable us to design preventative measures and therapies for Parkinson’s disease.
In Vivo Assessment of the Tumorigenic Potential of iPS-Inducing Transcription Factors in Mouse Models (IIRP)
Ute Moll, MD
SUNY - Stony Brook University
Recent studies from several laboratories have shown that a defined set of 3-4 transcription factors can reprogram adult differentiated mouse and human cells back into pluripotent cells that exhibit properties of embryonic stem cells. They are called induced pluripotent stem cells, iPS. When engrafted into adult recipients, these iPS cells have already been shown to repair and restore some defective physiological functions in vivo. This cell culture reprogramming approach has the potential to provide a patient-specific source of cells for a broad array of therapeutic applications. However, the production of iPS cells is based on gene transfer via the use of retroviruses that express these factors, some of which are known cancer-causing genes. This requirement significantly hinders the potential application of in vitro reprogramming for therapeutic use in humans because such cells pose an increased risk of cancer. So far two cocktails of reprogramming factors are successful. While the c-Myc factor, a proven cancer gene, appears dispensable, both cocktails require the Sox2 factor and one requires the Nanog factor. However, the intrinsic cancer risk of these essential stemness factors is currently unknown. To assess the long-term in vivo cancer risk of Sox2 and Nanog, we will make classic transgenic and iPS-derived mouse models. Deliberate reactivation of Sox2 and Nanog in postnatal mice will reveal if unintended reactivation of these factors in iPS-derived adult tissues poses cancer risks. Long term in vivo assessment of cancer risks of iPS-inducing factors, individually and combined, is essential and represent one of the most imperative lines of investigation in cellular reprogramming at this time. Knowledge gained might reduce the risk of malignancy of iPS cell-derived tissues by allowing us to choose the optimal combination of reprogramming factors. This proposal evaluates the long-term safety risks posed by unchecked expression of the pluripotency-inducing factors Sox2 and Nanog in adult organisms.
iPS Cell Therapy for Diseases of Adult Acquired Demyelination (IIRP)
Steven Goldman, MD, PhD
University of Rochester Medical Center
A number of strategies have been developed for the cell-based repair of demyelinated lesions of the brain and spinal cord. In particular, glial progenitor cells (GPCs) capable of oligodendrocytic maturation and myelination have been derived from human brain tissue, as well as from human embryonic stem cells, and have proven effective in myelinating both congenitally hypomyelinated and adult demyelinated brain and spinal cord. We have derived a set of techniques for the identification, isolation, transplantation and assessment of human GPCs, and have achieved the widespread myelination of congenitally dysmyelinated experimental brains; this work has included the functional and phenotypic rescue of a mouse model of congenital hypomyelination, the shiverer mouse. Yet these experiments were performed in immunodeficient mice; immune rejection has otherwise hindered the development of human cells for allogeneic (not-self) transplant. To address this issue, we propose to develop human induced pluripotent stem cells (iPS cells) as a source from which new oligodendrocytes may be generated. By doing so with recipient cells taken from a given patient, we may hope to generate central GPCs in sufficient numbers to provide myelinogenic autografts, largely free of rejection risk. The intended production of oligodendrocytes will challenge the ability of iPS cells to generate such a post-mitotic and late-appearing phenotype in significant and scalable numbers. The coupling of this feat with our extant ability to both purify and transplant glial progenitor cells, and to assess their myelination and functional competence, may provide an early and compelling opportunity for the clinical development of iPS cells, as autologous myelinogenic progenitors for the treatment of adult acquired demyelinating disorders. We are developing the means by which to generate and purify myelinogenic glial progenitor cells from human iPS cells, to establish their similarities in profile and performance to human ES and tissue-derived GPCs, and to establish their myelination and safety in vivo, after transplantation into the brains of neonatal, myelin-deficient shiverer mice. Our intent is to establish a conceptual and operational basis for producing myelinogenic autografts derived from somatic cells of patients with demyelinating disease, using a means largely free of rejection risk. This should prove an especially attractive strategy for restoring lost myelin in diseases such as multiple sclerosis, transverse myelitis, optic neuritis, and subcortical stroke, in which no genetic abnormalities prevent the use of a patient’s own cells as iPS source.
Stem Cell Activity and Human Longevity (IDEA)
Todd Evans, PhD
Weill Cornell Medical College
Why do humans age, and why is there a typical expected natural lifespan (barring accident, war, or other “un-natural” causes)? While healthy life (healthspan) has been extended in many societies by public health measures (clean water) and medical advances (antibiotics), it is clear that aging remains associated with the development of specific diseases, such as arthritis, diabetes, brittle bones, heart failure, and cancer. Is it possible for the human genome to support longer healthier lives, for example by delaying the propensity toward these debilitating aging diseases? Yes, healthspan can be extended. Indeed, there are rare families whose members (centenarians) live a healthspan of exceptional longevity (EL). Studies have clearly shown that this “trait” is inherited, and therefore is driven by genes. Thus, not only can you inherit genes that increase the risk of disease and death, but you can also inherit genes that increase the chance to live healthy for decades longer than average. If we could identify the genes responsible for EL, it should be possible to use this information to enhance the healthspan for everyone. We hypothesize that at least part of the reason the EL individuals have such a long healthspan is due to their stem cells, or genes that affect stem cell activities. In particular, tissue-specific or adult-stage stem cells are required to maintain healthy populations of cells, and are recruited upon stress or injury to help stimulate regeneration and recovery. We doubt that centenarians have more stem cells, but rather that EL individuals have stem or progenitor cells that remain more active or responsive at older age, or that respond better to signals that call out for a regenerative response (healing, restoring dying or defective tissue, fighting cancer). We are proposing to develop iPS lines from a remarkable “patient” group and these lines will provide novel basic research tools to study the stem cell biology. The findings of this specific research will confirm whether or not EL can be associated with a stem cell phenotype. It will provide reagents to evaluate differences in regenerative capacity and could identify genes that regulate stem and progenitor cell activity related to a long and healthy life. Ultimately, this approach may lead to a fundamental understanding of the molecular basis of important aging-related diseases, and to novel preventive and treatment strategies for these diseases. This would have a profound impact on morbidity and mortality.
Induced Pluripotent Stem (iPS) Cells Derived from Mesenchymal Stem Cells (IDEA)
Timothy Wang, MD
Columbia University Medical Center
Stem cells are believed to hold much promise in the treatment of chronic diseases such as Parkinson’s, diabetes and spinal cord injuries. While embryonic stem (ES) cells have shown the greatest potential in the past, a new type of stem cell called induced pluripotent stem (iPS) cell, has recently been reported. These new iPS stem cells can be generated from almost any cell type and appear to be essentially equivalent to ES cells. The advantage then of iPS cells is that one could create “patient-specific” stem cells from an individual for treatment of that patient (so no immunosuppression would be required). However, the generation of iPS cells is technically challenging, and while reproducible, is far from routine use. The creation of iPS cells requires using viruses to introduce 4 genes simultaneously into cells, and only 1 in 10,000 cells in the end become an iPS cell. Our lab has been working with a different type of stem cell called a mesenchymal stem cell or MSC, which is present in the bone marrow of adults. These cells show some similarities to ES cells but they are not as good in that they can give rise to some but not all tissues. However, our MSCs express 3 of the 4 genes that are critical for iPS cells, but do not express one gene (Oct 3/4) that is most closely associated with embryonic development. Our central hypothesis is that bone marrow (MSC) stem cells are much closer to iPS stem cells, since they already express most of the genes needed except for one gene (Oct 4). In a sense, they are more than half way there. We believe that choosing the right starting cells will be critical for high yield production of iPS cells. We propose that MSCs can possibly be converted to iPS cells by overexpression of Oct4 in these cells. We are interested in answering the question as to whether putting the one missing gene (Oct3/4) into our adult mesenchymal stem cells is sufficient to increase the stem-like properties of our cells and can possibly change our stem cells into iPS cells, or cells that are equivalent to embryonic stem cells in being able to give rise to every tissue of the body. If iPS cells can be derived from bone marrow MSCs by introduction of a single gene, Oct4, the advantages of this approach will be significant. MSCs can easily be obtained and grown from any patient, and it is much easier to introduce genes into these cells. In theory, the approach would allow rapid generation of patient-specific iPS cells. The studies will also provide new insight into the nature and potential of bone marrow stem cells.
The Development of Patient-Specific Cardiomyocytes Differentiated from Induced Pluripotent Stem Cell (iPS Cells) as a Cellular Model for the Molecular, Cellular, and Electrophysiologic Characterization of Long QT Syndrome (IDEA)
Jonathan Lu, MD, PhD
Columbia University Medical Center
Recent development in human embryonic stem cell biology has generated a great deal of excitement regarding its potential role as a therapeutic agent in human disease. However, its use has been hampered by political and ethical controversies. The discovery that skin cells can be directly reprogrammed to become stem cells offers a way to not only circumvent the controversies, but a way to generate patient-specific tissues. While this process holds great potential for personalized regenerative medicine, many more years of research and development are required before it can be a reality. We believe these reprogrammed stem cells (iPS cells) can immediately begin to impact the way we diagnose and treat cardiovascular disease by its ability to potentially turn skin biopsy into “heart biopsy” without actually invading the heart: the skin cell can be reprogrammed into stem cells, which in turn can be directed to become beating heart cells in the research laboratory. In effect, this process offers us the ability to have a replica of individual patients heart cells to be kept in the laboratory for virtually unlimited cellular studies just by taking a piece of patient’s skin. We believe that the skin-turned-heart cells will be invaluable research tool to uncover new mechanisms of cardiovascular diseases. In order to explore the potential use of reprogrammed stem cells (iPS) as a research tool, we propose to begin by determining whether heart cells generated from iPS cells closely resemble heart cells that have been naturally developed. Long QT syndrome, a well-characterized inherited arrhythmia which results in sudden cardiac death, has been chosen as an initial model for these studies because past investigations have clearly established that the electrical abnormalities responsible for the disease can be detected at a single cell level, and that many genetic mutations have been cataloged in both mice and men. In this proposal, we will try to address the question of 1) how closely iPS cell derived cardiomyocyte resemble native cardiomyocytes. 2) If the iPS cell derived cardiomyocytes are derived from patients carrying a well characterized Long QT mutation, whether the abnormalities can be detected in the research laboratory. These studies will lay the foundation of recently described iPS cells as a way to derive patient- and disease- specific heart cells for studies in the laboratory. While the proposed experiments utilize long QT syndrome as a model disease, once the system is developed and validated, it can be utilized to not only other forms of inherited arrhythmias, but also has potential of affecting profound change in the way we personalize the treatment for more common forms of arrhythmia such as atrial fibrillation.
Genetic Background and Efficient Generation of Induced Pluripotent Stem (iPS) Cells (IDEA)
Lisa Fortier, DVM, PhD
Stem cells have potential to treat diseases such as juvenile diabetes, spinal cord injury, Parkinson’s Disease, myocardial infarction, and arthritis. Recent technology enables scientists to use a patient’s skin cells to create embryonic stem-like cells, called induced pluripotent stem (iPS) cells, without the generation or destruction of embryos. These iPS cells have the potential to become any type of cell in the adult body, and therefore can be used to regenerate diseased or damaged tissue. iPS cells will not be subject to immune rejection in the patient from which they are obtained. There are many questions left to answer before iPS cells can be applied therapeutically. For example, it is not clear if the qualities of iPS cells that can be generated are influenced by the genetic makeup of the patient. The concept that the genetic make-up of an individual affects their response to treatment is clear in drug-response studies and by the fact that generation of embryonic stem (ES) cells is efficient in some strains of mice, but not others. If iPS cells vary depending on the genetic background of the patient who donated the skin cells, then further investigations into generating optimal iPS cells from all patients will be necessary prior to clinical applications. In this proposal, we aim to determine if genetic background influences the generation of iPS cells in two species. We hypothesize that the ability/efficiency of generating iPS cells, and the pluripotent quality/stability of iPS cells, will be strongly influenced by the genetic background of an individual. We propose that by investigating the effect of genetic diversity in the many different strains of laboratory mice, and in widely differing breeds of dogs (an animal more relevant to human health), we will gain important insight into the genetic factors affecting iPS derivation. Furthermore, using the lessons of mouse ES cells, we propose that it will be possible to devise conditions that are efficacious for iPS cell generation for genetically diverse individuals. Successful completion of the studies outlined in this proposal will determine if genetic background influences the quality and/or quantity of iPS cells that can be generated. This is an important first step before consideration of therapeutic applications of iPS cells.
Replication Errors in Hematopoietic Stem Cells (IDEA)
Steven Pruitt, PhD
Roswell Park Cancer Institute
Genetic damage is thought to play a role in both the etiology of cancer and in age related dysfunction. Recent studies by both the Pruitt and Blow laboratories have lent support to the hypothesis that much of the damage that leads to cancer and other age related disorders is a consequence of replication related DNA damage. Specifically, these studies have shown that deficiency for components (termed Mcms) of a complex of proteins that bind to the DNA to both specify where replication will start (in normal cells replication starts at many thousands of different sites) and to prevent more than one replication event per cell division result in use of fewer start sites in cell culture studies and in both a deficiency in the number of adult stem cells and a highly elevated rate of cancer when tested in mice. Here we hypothesize that Mcm deficiency acts to accelerate the rate at which replication related errors occur normally during development and aging. In support of this hypothesis, sites in the genome which are prone to chromosome breakage under conditions of replication stress have been identified and correlated with the locations on the chromosomes of cancer causing genetic damage. A corollary to the hypothesis is that the frequency with which genomic rearrangements occur at a given chromosomal location may be dependent on differences between individuals in the sequence of the DNA at sites where replication starts. That such differences exist is suggested by the finding that some damage prone sites are present in most individuals (referred to as common fragile sites), while others are present in only a subset of the population (referred to as rare fragile sites). Hence the hypothesis we seek to test is that individuals carrying genetic differences resulting in inefficient replication origin usage near to disease causing genes will undergo genetic instability at these sites at higher rates than individuals with more favorable DNA sequences. The key innovative component of this project is in defining the link between mechanisms that control the replication of DNA and genetic damage that results in disease. These studies are anticipated to enhance our understanding of mechanisms responsible for the etiology of hematological malignancies and may be useful in assessing the risk of disease progression in the clinic.
Human Retinal Pigment Epithelial Multipotent Cells (IDEA)
Enrique Salero, PhD
Regenerative Research Foundation
The retinal pigment epithelium (RPE) forms a single layer of highly specialized cells that is required for maintenance of the retina. The RPE remains non-proliferative throughout life. In amphibians and in embryonic chick and mouse, RPE cells can produce other retinal and even lens tissues, suggesting an inherent plasticity. In humans, RPE cells can be activated and proliferate when they are removed from their normal environment. Retinal stem cells have been identified in the ciliary epithelium and iris pigmented epithelium of adult rodents and human that can self-renew in vitro and differentiate into retinal neurons and glia. Nevertheless, until now, there has been no evidence for a stem-like cell from the adult RPE. The ability of stem cells to self-renew, to form cell lines and differentiate into many cell types make them an ideal focus for treatment of diseases. We propose to isolate RPE-specific stem cells (RPESCs) for expansion in vitro and test their potential to become different types of cells in the body. It is particularly important to determine whether these cells can be obtained from fluid that is normally discarded when patients undergo retinal surgeries. Patient-matched stem cells lines derived from RPE biopsies could be a unique source of multipotent stem cells for the study of cell fate choice and could be used to generate specific cell types for cell replacement therapy. This represents a novel approach to develop therapeutics to treat human diseases, such as ocular diseases: retinitis pigmentosa, cone dystrophy, age-related macular degeneration or Parkinson disease, spinal cord injury. Patient-specific RPESC lines also have the potential to improve the efficiency of drug discovery. Furthermore, this reprogramming system could make the idea of customized patient-specific screening and therapy both possible and economically feasible.