Symposium Speakers List
- James F. Battey, Jr., M.D., Ph.D.
- Nancy E. Block, Ph.D.
- George Q. Daley, M.D., Ph.D.
- Meri T. Firpo, Ph.D.
- Gemma Flamberg, J.D.
- Daniela S. Gerhard, Ph.D.
- Steven A. Goldman, M.D., Ph.D.
- Thaddeus G. Golos, Ph.D.
- Della M. Hann, Ph.D.
- James Huettner, Ph.D.
- Timothy J. Kamp, M.D., Ph.D.
- Dan S. Kaufman, M.D., Ph.D.
- Gordon Keller, Ph.D.
- Barbara Knowles, Ph.D.
- Tenneille Ludwig, Ph.D.
- John W. McDonald III, M.D., Ph.D.
- Ronald McKay, Ph.D.
- Kevin E. Noonan, Ph.D., J.D.
- Martin D. Pera, Ph.D.
- Mahendra S. Rao, M.D., Ph.D.
- Mark L. Rohrbaugh, Ph.D., J.D.
- John W. Thomas, Ph.D.
- Esmail D. Zanjani, Ph.D.
- Su-Chun Zhang, M.D., Ph.D.
James F. Battey, Jr., M.D., Ph.D., earned a B.S. from the California Institute of Technology and M.D. and Ph.D. degrees from Stanford University. Following residency training in pediatrics at Stanford he received postdoctoral training at Harvard Medical School under the direction of Philip Leder. Dr. Battey came to NIH in 1983 as a Senior Staff Fellow and then Senior Investigator with NCI. In 1988 he moved to the NINDS as Chief of the Molecular Neuroscience Section and in 1992 returned to NCI to head the Molecular Structure Section of the Laboratory of Biological Chemistry. Dr. Battey was appointed Director of Intramural Research for NIDCD in 1995 and Chief of the Laboratory of Molecular Biology in 1996. He was appointed Director of NIDCD in 1998. Dr. Battey’s laboratory focuses on the molecular genetic analysis of biologic responses mediated by mammalian bombesin-like peptides and their receptors.
Dr. Battey is also the Chairman of the NIH Stem Cell Task Force, which is a team of leading scientists at NIH whose objective is to meet with and seek advice from scientific leaders in stem cell research to further advance this field.
Up to Top
Nancy E. Block, Ph.D., is a Licensing Manager, Wisconsin Alumni Research Foundation (WARF), and General Manager, WiCell Research Institute. Dr. Block joined WARF in 2000, where she manages the licensing of human embryonic stem cell technologies and directs the overall operations of WiCell. Prior to joining WARF and WiCell, Dr. Block served as a research scientist at the University of Wisconsin-Madison. She received her B.S. from the University of Wisconsin-Madison and her Ph.D. in molecular and cellular biology from the Medical University of South Carolina in Charleston, South Carolina. She has served as a research fellow at the Medical University of South Carolina and at Massachusetts General Hospital/Harvard Medical School in Boston. Dr. Block’s research career included studies on insulin action/diabetes and on the molecular, cellular, and developmental biology of skeletal muscle stem cells. She is the Program Director on an NIH Infrastructure Grant awarded to the WiCell Research Institute titled, “Expansion and Distribution of Human Embryonic Stem Cells.”
Presentation: This presentation will discuss the missions of the Wisconsin Alumni Research Foundation and the WiCell Research Institute (WiCell). The presentation will focus on the history behind the establishment of WiCell, the licensing of human embryonic stem cell technologies developed at the University of Wisconsin-Madison and WiCell, and WiCell’s efforts to promote the dissemination of human embryonic stem cell lines to the research community worldwide.
Up to Top
George Q. Daley, M.D., Ph.D., is a Research Scientist at the MIT-affiliated Whitehead Institute for Biomedical Research and is an Associate Professor of Medicine with appointments in the Department of Biological Chemistry and Molecular Pharmacology at Harvard Medical School and the Division of Pediatric Hematology/Oncology at The Children’s Hospital and Dana Farber Cancer Institute (transfer of appointments is in process). He received a bachelor’s degree magna cum laude from Harvard University, a Ph.D. in biology from MIT, and an M.D. summa cum laude from Harvard Medical School. Dr. Daley trained in internal medicine, hematology, and oncology and served as Chief Resident in Internal Medicine at the Massachusetts General Hospital. His research into the molecular basis of human chronic myeloid leukemia validated BCR/ABL as a target for chemotherapy and helped stimulate the development of Gleevec.
Dr. Daley’s current research interests include studies of the genetic regulation of blood stem cell development from embryonic stem cells. He is the Birnbaum Scholar of the Leukemia and Lymphoma Society of America and is the recipient of the Leon Reznick Memorial Research Prize from Harvard Medical School and the Burroughs Wellcome Fund Career Award in Biomedical Science.
Presentation: Self-Renewal Pathways in Human ES Cells
Whereas mouse ES cells can be readily maintained in a pristine pluripotent state by incubation with the antidifferentiation factor LIF (Leukemia Inhibitory Factor), no such factor has yet been identified to facilitate culture of human ES cells. We have investigated whether the LIF signaling pathways are functional in human ES cells. Despite expression of the LIF receptor and the gp130 signaling subunit, and demonstration of the activation of the STAT3 signaling intermediate by treatment of human ES cells with human LIF, the cells nonetheless fail to retain pluripotency. This argues that mechanisms of self-renewal may be distinct between murine and human ES cells. We are currently characterizing protein signaling pathways that mediate self-renewal and are employing expression cloning to identify factors that facilitate human ES cell culture. This work has been aided significantly by methods for viral gene transduction of human ES cells, which will be described.
Up to Top
Meri T. Firpo, Ph.D., is an Assistant Research Professor in the Center for Reproductive Sciences at the University of California, San Francisco, where she directs the Human Embryonic Stem Cell Laboratory. She received her Ph.D. in 1992 from the Cornell University Medical College Graduate School of Medical Sciences after completing a research project in the Developmental Hematopoiesis Laboratory at the Sloan-Kettering Institute. Dr. Firpo’s research at the Sloan-Kettering Institute focused on adult bone marrow stem cells. She then did a postdoctoral fellowship at the National Jewish Institute for Immunology and Respiratory Medicine in Denver, Colorado, where she completed a research project on generating hematopoietic stem cells from mouse embryonic stem (ES) cells in culture. After returning to the Bay Area in 1995, Dr. Firpo undertook a second postdoctoral fellowship at the DNAX Research Institute for Molecular and Cellular Biology in Palo Alto, California, where she studied the development of the human hematopoietic system and human models of leukemia.
In 1997 Dr. Firpo came to the University of California, San Francisco, where she directed the derivation of two of the human ES cell lines included in the National Institutes of Health Registry of Human Embryonic Stem Cells. She is currently working on using human ES cells as a model of human development, differentiating human ES cells into functional tissues for transplantation and deriving new lines suitable for transplantation therapies.
Presentation: Culture and Genetic Modification of Human Embryonic Stem Cells
The focus of the Firpo lab is the regulation of stem cell growth, both from the perspective of the maintenance of stem cell potential and the regulated differentiation to functional tissues. We are interested in embryonic, fetal, and adult stem cells. Our lab has two areas of active research. First, we have developed a model of in utero hematopoietic transplantation in mice. For this model, we isolate populations of cells containing hematopoietic stem cells from adult and fetal mice and embryonic stem cells using specific antibodies. The cells are then injected into fetal mice through the uterine wall. At various times after birth, the injected mice are analyzed to determine whether engraftment has occurred, and the degree of contribution of donor cells in hematopoietic tissues, including peripheral blood, bone marrow, and spleen.
The remaining two projects are part of our human embryonic stem (hES) program. This project covers the distribution of hES cells to other labs, both at UCSF and to academic institutions throughout the world. This project requires the expansion and characterization of the two hES lines that we have derived at UCSF and that are included in the NIH Human Embryonic Stem Cell Registry. As part of the characterization of the cell lines, we will determine what is required to maintain hES cells in an undifferentiated state. We are also characterizing the differentiation capacity of these cells. Finally, we have established a training program to help establish hES culture techniques in other labs through hands-on training of individuals visiting our lab.
The second project involving hES cells explores the regulation of differentiation of hES cells into mature cells of several tissues. Currently, we are actively differentiating hematopoietic precursors and mature populations, hepatocytes, pancreatic islet cells, neural progenitors, and neural cells. We have constructed plasmid expression vectors containing reporter genes under the control of both tissue-specific and ubiquitously expressed promoters. Ultimately, development of methods of genetic alteration will allow us to regulate differentiation, isolate populations that may be suitable for transplantation therapies, and follow the engraftment of embryonic stem cell- derived tissues. In addition to monitoring the differentiation and genetic status of subclones, we assay the differentiation to the various cell lineages through an assessment of the expression of tissue-specific cell-surface markers.
In summary, this lab is studying two questions related to stem cell biology. First, what is required to keep a stem cell a stem cell, while allowing it to proliferate? Second, what signals regulate the steps that stem cells take after they become committed to differentiate?
Up to Top
Gemma Flamberg, J.D., has been a Senior Legislative Analyst in the NIH Office of Legislative Policy and Analysis (OLPA) since 1998, where she follows congressional activities related to bioethics (e.g., stem cells, cloning, fetal tissue research, protection of human subjects in research) and technology transfer. Prior to joining the OLPA staff, she worked for 9 years for the Public Health Division of the Office of the General Counsel (OGC), HHS, where she provided legal advice to the Health Resources and Services Administration and the Substance Abuse and Mental Health Services Administration. While at OGC, she played a central role in setting up the National Childhood Vaccine Injury Compensation Program and the Ryan White AIDS programs.
Presentation (with Della M. Hann, Ph.D.): Federal Guidelines on Stem Cell Research: NIH Implementation
This session will provide an overview of existing Federal policy on support of human embryonic stem cell research and the process of how NIH is addressing many of the research challenges in this area of science. Some of the research challenges that will be addressed include access to cell lines, grant administrative issues, the acquisition of skills and experience, and scientific opportunities and funding.
Up to Top
Daniela S. Gerhard, Ph.D., is currently the Acting Director of Office of Cancer Genomics at the National Cancer Institute. She received her doctorate in molecular biology and genetics from Cornell College of Medicine and did her postdoctoral training at MIT in human genetics.
Dr. Gerhard was on the faculty of Washington University School of Medicine, where she did research in human genetics. She has published extensively in the areas of human genetics, physical mapping, and positional cloning of complex disorders, including cancer. She is a member of the American Society of Human Genetics, Human Genomics Organization, and International Society of Psychiatric Genetics.
Presentation: Elucidation of the Transcriptome of ES Cells: A Progress Report
Embryonic stem (ES) cells are derived from an inner cell mass of a blastocyst, 3 to 5 days postfertilization. These cells are totipotent and can proliferate without differentiation in vitro for an extended period of time. These cells can develop to derivatives from all 3 embryonic germ layers and are generating tremendous excitement. A limited number of the ES cells have been approved for further study by federally funded researchers.
Laboratories around the world are engaged in the study of regulation of specific genes transcribed in the ES cells; however, the full composition of the transcriptome is yet unknown. We have undertaken to fill in this gap by investigating the transcriptome of a number of the currently available cell lines by 5’ expressed sequence tag sequencing and serial analysis of gene expression (SAGE). In this presentation, I will report on the results of the first library from the WA01 cells and the corresponding trophoblasts, describe the other ongoing research, and provide Web addresses from which the data can be obtained.
Up to Top
Steven A. Goldman, M.D., Ph.D., is the Nathan Cummings Professor of Neurology and Neuroscience at Weill Medical College of Cornell University and is Attending Neurologist at New York Presbyterian Hospital. A summa cum laude graduate of the University of Pennsylvania, he obtained his Ph.D. from Rockefeller University in 1983 and his M.D. from Cornell in 1984. His thesis work, with Fernando Nottebohm at Rockefeller, included the discovery of neurogenesis in the adult songbird brain, one of the first reports of neuronal production in the adult CNS.
Dr. Goldman then interned in medicine and completed his residency in neurology at New York Hospital and Memorial Sloan-Kettering. In 1988, after serving a year as Chief Resident in Neurology, he joined the faculty and attending staff at Cornell-New York Hospital. In 1997 he was tenured as Professor of Neurology at Cornell, becoming at that time its youngest full professor of a clinical specialty. Dr. Goldman’s laboratory at Cornell is interested in neural regeneration and brain repair, with a special focus on neural stem and progenitor cells. His lab was the first to extract these cells from the adult human brain, and he has since focused on their potential therapeutic utility. Dr. Goldman has been elected to the American Neurological Association and American Society for Clinical Investigation and is a recipient of the Jacob Javits Neuroscience Investigator Award from NIH.
Presentation: Induction and Isolation of Spinal Motor Neurons from Human ES Cells: A Prototype for the Targeted Acquisition of Desired Neuronal Phenotypes (Additional Authors: Neeta Singh Roy and Takahiro Nakano, Cornell University Medical College)
Both the experimental and clinical use of human ES (hES)-derived neurons has been limited by the phenotypic heterogeneity of these cells and by the persistence of undifferentiated cells and undesired phenotypes in nominally enriched preparations of hES-derived neurons. To address the need for pure subpopulations of hES-derived neurons, of a defined and preselected phenotype and devoid of undifferentiated contaminants, we have established a promoter-based strategy for serially inducing and purifying specific neuronal subtypes from hES cells. As one of several initial target phenotypes, we have generated and purified spinal motor neurons, a cell type lost in the motor neuron degenerative diseases as well as in both spinal cord injury and peri-infectious motor neuronopathies. To select spinal motor neurons from hES cultures (WiCell, H1), we used retinoic acid (RA) and sonic hedgehog (SHH) to first induce motor neuron phenotype, as previously described in mouse ES cells (Wichterle, Cell 110:385, 2002). We then isolated the newly generated motor neurons from these cultures, using fluorescence-activated cell sorting (FACS) on the basis of GFP driven by Hb9, a gene that encodes a motor neuron-selective transcription factor. Specifically, we had found that a highly conserved 3.6 kb segment of the 5’ regulatory region of the Hb9 gene was sufficient to specify gene expression to motor neurons. On that basis, we established a motor neuron-selection vector, in which this 3.6 kb Hb9 enhancer was placed 5’ to an hsp68 basal promoter driving EGFP. The resultant plasmid E/Hb9:EGFP was transfected into hES cultures, which were treated with SHH/RA. This treatment resulted in the induction of motor neurons within the hES cultures, which could be identified on the basis of E/Hb9-driven GFP expression. Flow cytometry showed that the EGFP+ cells constituted 1.1 ± 0.3% (n=3, ± SE) of the total cell population, or 4.6% after correction, for our net transfection efficiency of 24%. FACS allowed the near-purification of these E/Hb9:GFP+ cells, virtually all of which co-expressed Islet1 protein. When raised in GDNF/BDNF, with and without human muscle cell co-culture, the E/Hb9:P/hsp68:EGFP+ cells also expressed choline acetyltransferase, indicating their cholinergic transmitter phenotype; all co-expressed the generic neuronal markers bIII-tubulin and MAP-2. Importantly, no detectable expression was noted of any markers of undifferentiated ES cells, which included SSEA-4 and Tra-1-81. The sorted motor neurons achieved functional maturation within 2 to 3 weeks, as indicated by both their glutamate-induced calcium responses upon confocal calcium imaging and by their fast sodium currents and action potentials on whole-cell patch-clamp analysis. Thus, the serial induction of motor neuron phenotype by RA and SHH, followed by E/Hb9(3.6):EGFP-based FACS, permitted the high-yield generation and purification of functional motor neurons from human embryonic stem cells. This approach may be used as a prototype for the specific selection and purification of defined neuronal subpopulations from otherwise mixed cultures of human embryonic stem cells.
This work was supported by Project ALS and the National Institute of Neurological Disorders and Stroke, NIH.
Up to Top
Thaddeus G. Golos, Ph.D., did his graduate training in reproductive biology in the Department of Physiology and Biophysics at the University of Illinois at Urbana-Champaign, followed by postdoctoral research at the University of Pennsylvania in the Department of Obstetrics and Gynecology. Dr. Golos has been at the University of Wisconsin since 1987, directing a research program in placental biology in nonhuman primates, including transcriptional control of hormone gene expression and the role of placental MHC class I molecules in maternal-fetal immune interactions. He is currently an Associate Professor of Obstetrics and Gynecology and an Associate Professor at the Wisconsin National Primate Research Center.
Presentation: Differentiation Into Placental Trophoblast Cells
Trophoblast differentiation and placental morphogenesis are the earliest events in mammalian development and yet are virtually impossible to study in humans. Human embryonic stem (hES) cells have captured the public imagination as a source of cells for regenerative medicine and reconstitution of diseased organs. Human ES cells also provide an unprecedented opportunity to improve our understanding of the basic processes of early human development. We have evaluated the expression of trophoblast markers in differentiating ES cells. Trophoblast differentiation is initiated during embryoid body formation, as demonstrated by secretion of chorionic gonadotropin, progesterone and estrogen, and the expression of the nonpolymorphic MHC class I trophoblast marker HLA-G. We propose that hES in vitro differentiation systems, such as the formation of embryoid bodies, will allow for an investigation of factors and regulatory pathways that direct both trophoblast differentiation and placental morphogenesis.
Up to Top
Della M. Hann, Ph.D., is Acting Director, Office for Reports and Analysis, and Senior Policy Advisor in the Office of Extramural Research, NIH. The Office for Reports and Analysis is responsible for a number of grant reporting and outreach activities including the CRISP database, Grants Info, and statistical analysis and reporting of NIH extramural funding data. As Senior Policy Advisor, she provides oversight of the extramural policies and issues related to the inclusion of women and minorities, human embryonic stem cell research, and the HIPAA privacy rules.
Previously, Dr. Hann was the Associate Director for Research Training and Scientific Collaborations within the Division of Mental Disorders, Behavioral Research, and AIDS at the National Institute of Mental Health. She provided administrative oversight of research centers, training grants, and individual fellowship awards for the Division, and served as Project Officer for several divisional special projects, including an international multisite cooperative agreement in AIDS prevention and several cooperative agreements for collecting national mental health epidemiological information. Before joining the Government in 1991, Dr. Hann was a research associate at Louisiana State University Medical Center in New Orleans, where she completed a postdoctoral fellowship from the John D. and Catherine T. MacArthur Foundation on adolescent mother-infant social and emotional development and served as an Instructor for the Department of Psychology at the University of New Orleans. Dr. Hann received her Ph.D. in psychology from the University of Tennessee in 1986 and a B.A., summa cum laude and with honors, from Catawba College in 1981.
Presentation (with Gemma Flamberg, J.D.): Federal Guidelines on Stem Cell Research:
This session will provide an overview of existing Federal policy on support of human embryonic stem cell research and the process of how NIH is addressing many of the research challenges in this area of science. Some of the research challenges that will be addressed include access to cell lines, grant administrative issues, the acquisition of skills and experience, and scientific opportunities and funding.
Up to Top
James Huettner, Ph.D., received his Ph.D. in neurobiology from Harvard Medical School in 1987. His graduate research with Robert Baughman focused on the physiology of synaptic transmission in cultures of rat cerebral cortex. From 1986 to 1988, he was a Junior Fellow in the Harvard University Society of Fellows. He performed postdoctoral research at Harvard with Bruce Bean on whole-cell and single channel currents mediated by glutamate receptors and was appointed Instructor in Neurobiology. In 1991 Dr. Huettner joined the faculty at Washington University Medical School in St. Louis, where he is currently Associate Professor of Cell Biology and Physiology. He began studying the physiology of pluripotent stem cells in 1992 as part of an ongoing collaboration with David Gottlieb and John McDonald to analyze the neural differentiation of P19 embryonic carcinoma cells, mouse embryonic stem (ES) cells and, more recently, human ES cells.
Presentation: Physiology of Human ES Cells
Ion channels and electrical signaling play important roles in neurons, muscle, and secretory cells. The normal operation of these cells requires the coordinated interaction of currents through a variety of different channels that have distinct gating and permeation properties. Aberrant channel expression, or production of channels with abnormal properties, can have pathological consequences. Thus, the development of successful therapies involving human ES cells will require the functional evaluation of their electrical signaling capabilities. Physiological recordings from differentiating ES cells allow for the direct evaluation of ion channel characteristics and for cellular electrical signaling. Studies of mouse ES cells differentiating along the neural lineage have revealed physiological characteristics that parallel the properties of primary neurons and glia. Mouse ES-derived neurons express voltage-gated sodium and potassium channels that underlie the action potential. They also express a variety of neurotransmitter-activated ion channels as well as voltage-gated calcium channels, which are required for calcium-dependent transmitter release. Within 2 to 3 weeks after induction, mouse ES-derived neurons form functional excitatory and inhibitory synapses. Recordings from undifferentiated mouse and human ES cells reveal a much simpler physiological phenotype. Undifferentiated ES cells express voltage-dependent currents and intercellular junctions that may be important for cells in the undifferentiated state, but they lack most of the voltage- and ligand-gated currents that are observed in differentiated neurons and glia.
Up to Top
Timothy J. Kamp, M.D., Ph.D., is an Associate Professor in the Departments of Medicine and Physiology at the University of Wisconsin. Dr. Kamp received his M.D. and Ph.D. degrees from the University of Chicago. He has been at the University of Wisconsin since 1996 after completing his Internal Medicine residency and fellowship in cardiovascular diseases at Johns Hopkins University. Dr. Kamp’s research focuses on understanding the normal function of ion channel proteins in the heart and the dysfunction of these channels in disease states such as heart failure and arrhythmias. Recently, human embryonic stem cells have been utilized in this research to produce human cardiac myocytes for basic cellular electrophysiology studies.
Presentation: Human Embryonic Stem Cells Develop into Multiple Types of Cardiac Myocytes: Action Potential Characterization
Human embryonic stem (hES) cells can differentiate in vitro, forming embryoid bodies (EBs) composed of derivatives of all three embryonic germ layers. Spontaneously contracting outgrowths from these EBs contain cardiomyocytes (CMs); however, the types of human CMs and their functional properties are unknown. This study characterizes the contractions and action potentials (APs) from beating EB outgrowths cultured for 40 to 95 days. Spontaneous and electrical field-stimulated contractions were measured with video edge-detection microscopy. Beta-adrenergic stimulation with 1.0 µmol/L isoproterenol resulted in a significant increase in contraction magnitude. Intracellular electrical recordings using sharp KCl microelectrodes in beating EB outgrowths revealed three distinct classes of APs: nodal-like, embryonic atrial-like, and embryonic ventricular-like. The APs were described as embryonic based on the relatively depolarized resting membrane potential and slow AP upstroke. Repeated impalements of an individual beating outgrowth revealed a reproducible AP morphology from different cells, indicating that each outgrowth is composed of a predominant cell type. Complex functional properties typical of cardiac muscle were observed in the hES cell-derived CMs, including rate adaptation of AP duration and provoked early and delayed afterdepolarizations. Repolarization of the AP showed a significant role for- IKr based on E4031-induced prolongation of AP duration, as anticipated for human CMs. In conclusion, hES cells can differentiate into multiple types of CMs displaying functional properties characteristic of embryonic human cardiac muscle. Thus, hES cells provide a renewable source of distinct types of human cardiac myocytes for basic research, pharmacological testing, and potentially, therapeutic applications.
Up to Top
Dan S. Kaufman, M.D., Ph.D., received a bachelor of science degree from Stanford University. Subsequently, he completed a combined M.D./Ph.D. program at the Mayo Medical School and Mayo Graduate School in Rochester, Minnesota. His graduate work was in immunology and focused on natural killer cell interactions with MHC class I molecules. Dr. Kaufman then went to the University of Wisconsin-Madison for a residency in internal medicine and fellowship in hematology. While at the University of Wisconsin, he began studies of hematopoietic development of human embryonic stem (ES) cells as a postdoctoral fellow in the lab of Dr. James Thomson.
In 2002 Dr. Kaufman took a faculty position at the University of Minnesota as an assistant professor in the Stem Cell Institute and Department of Medicine, Division of Hematology, Oncology, and Transplantation, where he continues laboratory-based research on human ES cells and does clinical work in hematology and hematopoietic cell transplantation.
Presentation: Stromal and Cytokine Requirements To Support Hematopoietic Differentiation of Human Embryonic Stem Cells
Human embryonic stem (ES) cells offer an extraordinary resource to define the proteins and genetic pathways that regulate the earliest stages of human blood development. We have compared two different methods to support derivation of hematopoietic cells from human ES cells: stromal cell co-culture and embryoid body (EB) formation. Initial studies demonstrated co-culture of human ES cells with stromal cells derived from hematopoietic microenvironments in media containing FBS, but no other exogenous cytokines promoted blood development. Under these conditions, we could readily identify CD34+ precursor cells and hematopoietic colony-forming cells (CFCs) that produce characteristic erythroid, myeloid, and megakaryocytic colonies when placed in a standard methylcellulose assay. Mature CD45+ cells are also demonstrated in this system. Subsequent studies have been done to better characterize the contribution of cell-bound and soluble factors to support this hematopoietic development. Coculture of ES cells and S17 cells in serum-free media leads to few CFCs. However, the addition of cytokines known to support hematopoietic expansion, stem cell factor (SCF), thrombopoietin (TPO) and flt3-ligand (flt3L) to the serum-free media results in increased numbers of CFCs. Transwell culture experiments demonstrate that direct contact between ES cells and stromal cells is not required. This led us to examine hematopoietic differentiation by EB formation. Here, we observe derivation of CD34+, CD45+ cells, and CFCs when EBs are allowed to differentiate in media-containing serum, but not under serum-free conditions. However, unlike the stromal cell co-culture method, the addition of SCF, flt3L, and TPO to EBs grown in serum-free media did not result in significant CFC development. These results suggest that cytokines may act on stromal cells to produce additional factors that support survival and growth of hematopoietic cells.
Up to Top
Gordon Keller, Ph.D., is Professor of Gene Therapy and Molecular Medicine at the Mount Sinai School of Medicine in New York. His research focuses on defining and characterizing the early events involved with the establishment, growth, and maturation of the embryonic hematopoietic and vascular systems. Using the mouse ES differentiation system, he has identified a novel progenitor with the potential to generate both hematopoietic and endothelial progeny, a cell with characteristics of the hemangioblast. Through the identification of this progenitor, Dr. Keller has been able to define the role of a number of different genes in the earliest stages of hematopoietic and endothelial commitment. Dr. Keller serves on the Board of Directors for the International Society for Stem Cell Research.
Presentation: Differentiation of ES Cells to the Hematopoietic Lineage
This presentation will cover important aspects of the commitment of mouse ES cells to hematopoiesis and the process of how we plan to use this information for the differentiation of human ES cells.
Up to Top
Barbara Knowles, Ph.D., received her Ph.D. in zoology from Arizona State University. After a postdoctoral fellowship in the Department of Genetics at the University of California, Berkeley, she had a distinguished career at the Wistar Institute of Anatomy and Biology in Philadelphia. Additionally, Dr. Knowles was a Professor of Microbiology and a Professor in Pathology and Laboratory Medicine at the University of Pennsylvania School of Medicine. She is currently the Associate Director of The Jackson Laboratory, where she serves as the Director of Research and Training and as a Senior Staff Scientist.
Her graduate and professional training was in Drosophila genetics, and her subsequent research career is marked by groundbreaking publications in three main areas of mammalian genetics: the immune control of tumor growth, hepatocellular carcinogeneis, and the molecular control at the initiation of embryonic development. Dr. Knowles serves on many advisory and editorial boards, and she is a Presidential Professor of the University of Maine. Since 1995, she has served as the Consulting Director for the European Mouse Mutant Archive in Monterotondo, Italy, with nodes in France, Sweden, Portugal, and England.
Dr. Knowles, together with Dr. Davor Solter, developed a technique to immunosurgically isolate the embryonic stem cell-enriched inner cell mass of the mouse blastocyst and made the monoclonal antibodies that characterize the stage-specific embryonic antigens (SSEAs1, 3, and 4), which variously marked stem cells and differentiated cells of mouse embryos, mouse and human embryonal carcinoma cells, and human embryonic stem cells.
Presentation: Human Embryonic Stem Cells, a Practicum (and Stem Cells on Land and on Sea)
Keeping current with cutting-edge techniques requires flexible, expedient approaches to effective practical training. In response to the rapid rate of technological advancement in biomedical sciences, The Jackson Laboratory (TJL) has instituted a series of intensive, hands-on courses. Our premise is that a quick response to the immediate needs of the everchanging scientific and technological landscape is crucial. Efficient transfer of techniques and methodologies to researchers who will use and disperse them brings rigor to a rapidly emerging field. Moreover, gathering a small group of innovators from around the world to teach the students promotes learning and interactions among them, further advancing nascent research areas. The formula we have adopted focuses faculty attention on training a cadre of students through short morning lectures to illustrate hands-on principles and through extended long sessions in the laboratory. A separate meeting immediately following the practicum supplies perspective and further emersion in the field of study.
The 2002 Human Embryonic Stem Cell Course at TJL, and the accompanying meeting (a joint venture with the Mount Desert Island Biological Laboratory), Stem Cells on Land and on Sea, were conceived as a response to the change in the political climate that made Government funding of this research area a possibility. Informal interactions led to a course and a conference outline and NIH grant applications for funding. We will discuss both meetings, which were held in 2002 and are planned for July or August 2003.
Up to Top
Tenneille Ludwig, Ph.D., received both her B.S. and M.S. degrees in reproductive endocrinology from Washington State University’s Department of Animal Sciences. She completed a Ph.D. in embryology and developmental biology with a minor in bioethics, in 2001 under the direction of Dr. Barry Bavister at the University of Wisconsin-Madison. Her Ph.D. thesis focused on the effect of the culture milieu on the physiology and developmental competence of embryos in vitro. Dr. Ludwig currently leads the culture optimization program for both human and nonhuman ES cells in the laboratory of Dr. James Thomson at the University of Wisconsin-Madison.
Presentation: Human Embryonic Stem Cell Culture: Strategies To Improve Success
Techniques used to culture human ES cells successfully differ from those used with other cell types—even other stem cell types. This workshop will highlight the areas of human ES cell culture protocols that are the most critical to success and those that have traditionally been the most troublesome. A panel discussion will follow, with the opportunity to have specific technique questions answered.
Up to Top
John W. McDonald III, M.D., Ph.D., is Assistant Professor of Neurology and Neurological Surgery at Washington University School of Medicine and is Director of the Spinal Cord Injury Program and the Restorative Treatment and Research Center. His career goals have been to bridge basic science, translational, and clinical research through clinical trials in the realm of the regeneration and recovery of function in spinal cord injury. Dr. McDonald’s clinical focus is spinal cord injury from all causes. The emphasis of his research is regeneration, with a focus on remyelination and optimization of spontaneous regeneration and recovery of function. One active strategy is utilizing embryonic stem (ES) cells for neural transplantation. His group is developing remyelination as an approachable strategy for the restoration of function in the injured nervous system. The second strategy is maximizing spontaneous regeneration in part by optimizing patterned neural activity. Functional electrical stimulation and partial weight-supported robotic ambulation are two active approaches being studied at the level of regeneration in animals and at the level of fMRI and recovery of function at the human level. Dr. McDonald has received numerous awards for his research, including the Research for Freedom Award, Gateway to a Cure; L.W. Freeman Award for significant contributions to regenerative spinal cord research, National SCI Association; Keck Foundation Award for ES cell transplantation in the injured spinal cord; Murray Goldstein Award, Neurotrauma Society; and S. Weir Mitchell Award for research excellence from the American Academy of Neurology. Dr. McDonald’s ES cell transplantation work was cited as one of the top 10 scientific discoveries of 1999 by Science magazine.
Presentation: Human ES Cells: Immunological and Ultrastructural Features; Towards Novel Strategies of CNS Repair (Additional authors: Aileen Lu and Jim Huettner, Restorative Treatment and Research Center, Washington University in St. Louis)
We have evaluated multiple human ES cell lines that have been previously approved for use with Federal research (WA01:NIH code, H1:Provider’s code from WiCell Research Institute and BG01, hESBGN.01 from BresaGen, Inc.). We have compared multiple strategies for neural differentiation toward oligodendrocytes. A particular early focus has been characterizing undifferentiated human ES cells and later staged cells in the process toward neural differentiation using immunological markers, ultrastructural examination, and physiological properties. (See the presentation by our collaborator, Dr. Huettner, for physiological characteristics.) We are in the process of characterizing the expression of connexin hemichannels using the above approaches. We have compared and contrasted these characteristics of human ES cells with our previous work on mouse ES cells and will present a summary of these results. Multiple novel features of human and mouse ES cells have been uncovered that offer potential toward strategies to repair the damaged nervous system. The human ES cell system will be discussed within this therapeutic target domain.
Up to Top
Ronald McKay, Ph.D., is the Chief of the Laboratory of Molecular Biology in the Basic Neuroscience Program of the National Institute of Neurological Disorders and Stroke. He has held this position since April 1993 when he moved from MIT. Dr. McKay has also worked at Oxford University and at Cold Spring Harbor Laboratory. He received his doctorate for work in nucleic acid chemistry with Dr. Ed Southern at the University of Edinburgh. His recent work focuses on the stem cells of the central nervous system but also influences thinking in other areas of medicine, including diabetes and cancer.
Presentation: Functional Somatic Cells From ES Cells
Work from our group played an important role in the early period when stem cells of the central nervous system were first defined (McKay R. Science. 276:66-71, 1997). Our more recent work contributes to our fundamental understanding of three major processes in the developing nervous system: (1) cell cycle control (Tsai, R.Y. and R. McKay Genes & Dev. 16:2991-3003, 2002), (2) the control of cell fate (Panchision, D.M. et al. Genes Dev. 15: 2094-2110, 2001) and (3) the early steps in neuronal differentiation (Vicario-Abejon, C. et al. Eur.J.Neurosci. 12: 677-688, 2000). Recent advances in the application of stem cell biology to Parkinson’s disease clearly demonstrates the potential importance of stem cells in models of neuronal loss or injury (Studer, L. et al. Nat.Neurosci. 1: 290-295, 1998; Lee, S-H. et al. Nat.Biotechnol. 18: 675-679, 2000; Kim, J-H. et al. Nature 418: 50-6, 2002). We have also made contributions in other areas, for example, in glial transplantation (Brustle, O. et al. Science 285: 754-756, 1999), adult neurogenesis (Cameron, H.A. and R.D. McKay Nat.Neurosci. 2: 894-897, 1999; Cameron, H.A. and R.D. McKay J.Comp.Neurol. 435: 406-17, 2001) and endocrine pancreatic differentiation (Lumelsky, N. et al. Science 292: 1389-1394, 2001). These results suggest that this field will generate a powerful technology to analyze fate and function in many cell types.
Up to Top
Kevin E. Noonan, Ph.D., J.D., a Partner with McDonnell Boehnen Hulbert & Berghoff, Chicago, IL, has extensive experience in biotechnology and the chemical arts. A registered patent agent since 1991, Dr. Noonan brings over 10 years’ experience as a molecular biologist working on high-technology problems to his legal work. Dr. Noonan has wide experience in all aspects of patent prosecution and client counseling on validity, infringement, and patenting strategy matters. He represents pharmaceutical and biotechnology companies both large and small, and he is particularly experienced in representing university clients in both patent prosecution and licensing to outside investors.
Dr. Noonan received a Ph.D. in molecular biology from Princeton University, where his thesis work involved genetic analysis of oncogenesis in mammalian cells. Dr. Noonan was also a postdoctoral fellow supported by the National Cancer Institute at the University of Illinois at Chicago, where he studied multidrug resistance in mammalian tumor cells. During his fellowship Dr. Noonan developed a variety of novel methods based on the polymerase chain reaction, including quantitative analysis of mammalian gene expression. Dr. Noonan is the author of several articles and has lectured extensively on his scientific work. Dr. Noonan graduated, cum laude, from the John Marshall Law School. He is admitted to practice in Illinois and Massachusetts and is currently an adjunct professor of law at the De Paul University Law School, where he teaches biotechnology patent law.
Presentation: This presentation will provide an overview of the patent and intellectual property landscape for human embryonic stem cells. In addition, it will address the potential impact of this intellectual property on companies and product development.
Up to Top
Martin D. Pera, Ph.D., is Associate Professor and Co-Director of the Centre for Early Human Development at the Monash Institute of Reproduction and Development. He obtained his B.A. degree in English language and literature from the College of William and Mary in Virginia and his Ph.D. in pharmacology from George Washington University. Dr. Pera was awarded an NIH National Research Service Award, and conducted postdoctoral research at the Institute of Cancer Research and the Imperial Cancer Research Fund, both in London. Thereafter, he led research teams at the Institute of Cancer Research and at Oxford University before coming to Monash University.
Dr. Pera is an expert in the cell biology of human pluripotent stem cells, having pioneered the development and characterization of human embryonal carcinoma cell lines, work which led directly to the isolation of human embryonic stem (ES) cells from blastocysts by his laboratory. The Monash group was the second in the world to isolate human ES cells and the first to demonstrate somatic differentiation of these cells in vitro. His recent invited lecture presentations include those at the International Alpha Congress on Assisted Reproduction (Copenhagen, 1999), Ontogeny Corporation (Boston, 2000), Australian Society for Biomaterials (Melbourne, 2000), Serono Symposium on Embryos Stem Cells and Transplantation (Canberra, 2000), IMSUT Symposium for Stem Cell Biology (Tokyo, 2000), Royal Society of Edinburgh Meeting on Cloning Stem Cells and Cell Therapy (Edinburgh, 2000), ICDB (Gold Coast, 2000), Genomics Institute of the Novartis Foundation (La Jolla, 2000), Japanese Society for Hematological Transplantation (Kyoto, 2000), Cold Spring Harbor Stem Cell and Progenitor Cell Conference (2001), Royal Society Discussion Meeting on Stem Cells (2001), Sheffield University Symposium on Stem Cells (2001), the Seoul Symposium on Stem Cells and Therapeutic Cloning (2001), and the International Society for Developmental Neuroscience (2002). Dr. Pera’s recent peer review responsibilities have included grant reviews for NHMRC, ARC, UK BBSRC, Wellcome Trust, and Yorkshire Cancer Research Campaign; manuscript referee for PNAS, International Journal of Cancer, Experimental Cell Research, Science, International Journal of Radiation Biology, Reproduction Fertility and Development (Editorial Board), Nature, Stem Cells, Molecular Reproduction and Development, Developmental Biology, and Journal of Cell Science. He has advised the Australian federal government and the Victorian State government, the U.S. NIH, and the UK Department of Health on scientific matters relating to the ethics of the use of ES cells in research and medicine. Dr. Pera is co-inventor on four provisional patents relating to human ES cells and is a founding scientist of ESI, an Australian-Singapore biotechnology company founded to develop and exploit human ES cell technology.
The research in Dr. Pera’s laboratory focuses on the control of growth and the differentiation of human ES cells. His team members are trying to identify the signals that determine whether a stem cell will multiply to produce more stem cells, or instead turn into a precursor of a specialized body cell, such as nerve or muscle. Dr. Pera’s laboratory has a special interest in the differentiation of stem cells into endodermal progenitors, cells in the embryo committed to forming the liver, gut, lung, and pancreas. By understanding the early events in this pathway, the team can direct stem cells to form pancreatic cells more efficiently. These pancreatic cells will in turn have many uses in research into diabetes and may eventually be used in transplantation therapy to treat these diseases.
Presentation: Human Embryonic Stem Cells: Technical Aspects of Growth Characterization and Manipulation
Our laboratory studies the basic biology of human embryonic stem cells and the early phases of their differentiation. I will discuss systems used currently in our laboratory for the derivation and culture of embryonic stem cells, approaches to the characterization of stem cells, and some methodologies used in studying embryonic stem cell differentiation.
Up to Top
Mahendra S. Rao, M.D., Ph.D., currently serves as Stem Cell Section Chief, Laboratory of Neurosciences, Gerontology Research Center, National Institute on Aging, in Baltimore, Maryland. Dr. Rao earned his MBBS (M.D.) from Bombay University, India, and his Ph.D. from the California Institute of Technology (Caltech), Pasadena, California. He served as a Postdoctoral Fellow at the California Institute of Technology from 1992 to 1994 and at Case Western Reserve University, Cleveland, Ohio, from 1991 to 1992. Dr. Rao was a Research Associate at the Howard Hughes Medical Institute at Caltech from 1992 to 1994, and from 1994 to 2000, he was an Assistant Professor, then an Associate Professor of Neurobiology and Anatomy at the University of Utah School of Medicine, where he taught neuroanatomy. Dr. Rao served on the Food and Drug Administration (FDA) Stem Cell Advisory Committee in 2000 and on the NIH Stem Cell Committee in 1999. Since 1997, he has served on the MDCN6 study section. Since 1988, Dr. Rao has been a reviewer for the National Science Foundation and for three journals—Journal of Neuroscience, Developmental Biology, and Glia. During the same period, he served as a consultant to Geron, Inc., in Menlo Park, California, and Neuronyx in Malvern, Pennsylvania. Since 1998, Dr. Rao has been an Associate Professor at the National Center for the Biological Sciences in Bangalore, India. He currently is an Adjunct Associate Professor at Johns Hopkins University in Baltimore, Maryland, and his research interests include stem cells and the developing nervous system.
Presentation: Stem Cell Biology
The Stem Cell Biology section of the Laboratory of Neurosciences at the NIA focuses on the cellular and molecular mechanisms that regulate the proliferation, differentiation, and survival of stem and progenitor cells during development and in the adult. This research is based firmly on the concept that the same signaling mechanisms that regulate the development and plasticity of the nervous system are altered during aging and in age-related neurodegenerative disorders. Accordingly, an understanding of developmental mechanisms is likely to lead to novel approaches to preventing and treating neurological disorders of aging. Ongoing research is divided into four interrelated areas: (1) signal transduction mechanisms regulating the proliferation, differentiation, and survival of embryonic stem cells and pluripotent neural stem cells; (2) cellular and molecular alterations that occur in neural stem cells during aging and in age-related neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases; (3) elucidation of the mechanisms whereby environmental factors, such as diet and intellectual and physical activity, can alter stem cell biology; and (4) development of novel stem cell therapy-based approaches for repairing the aging and diseased nervous system.
Up to Top
Mark L. Rohrbaugh, Ph.D., J.D., is the Director of the Office of Technology Transfer (OTT), National Institutes of Health (NIH), where he oversees the patenting and licensing of NIH inventions and contributes to intramural and extramural technology transfer policy at NIH and in the U.S. Department of Health and Human Services (HHS). Dr. Rohrbaugh is Vice-Chair of the Public Health Service Technology Transfer Policy Board and represents HHS on the National Science and Technology Council Technology Committee.
Dr. Rohrbaugh began his NIH career in 1991, administering the review of grants and contracts as the Scientific Review Administrator for the National Institute of Allergy and Infectious Diseases (NIAID) Allergy, Immunology, and Transplantation Research Committee. He served for 5 years as Director of the Office of Technology Development for NIAID, managing a staff responsible for negotiating technology transfer agreements with industry and academic institutions for the conduct of both intramural basic and clinical research and extramural cooperative networks funded by NIAID. He joined the OTT in 2001 as Deputy Director.
Before beginning his work at the NIH, Dr. Rohrbaugh conducted molecular and cell biology research in academic and industrial laboratories. He received his Ph.D. in biochemistry from The Pennsylvania State University (1984) and a J.D. with honors from George Washington University Law School (1997), where he served as an Articles Editor for the American Intellectual Property Law Association Quarterly Journal. Dr. Rohrbaugh is licensed to practice law in the State of Maryland and is registered to practice before the U.S. Patent and Trademark Office.
Presentation: NIH Technology Transfer: Facilitating Access to hESCs
After President Bush announcement his human embryonic stem cell (hESC) policy on August 9, 2001, it became clear that one of the many hurdles to the distribution of these cells would be the negotiation of agreements governing their transfer. NIH decided that its Office of Technology Transfer (OTT) would negotiate agreements with the hESC providers to provide cells to Public Health Service intramural researchers, including researchers working at the NIH, Food and Drug Administration, and Centers for Disease Control and Prevention (CDC). The agreements would also require that the hESC providers offer no more restrictive terms to NIH-funded nonprofit institutions. Extramural institutions could then choose to accept the same reasonable terms or attempt to negotiate alternative agreements for themselves to suit their particular needs, policies, or local laws.
The OTT began negotiations first with WARF/WiCell, which owns the dominant patents that likely govern most, if not all, the approved hESC lines. WARF/WiCell entered into a Memorandum of Understanding in September 2001. By spring 2002, NIH had entered into similar agreements with BresaGen, ES Cell International, and the University of California, San Francisco. In addition, NIH has required recipients of infrastructure grants for the scale up and distribution of hESCs to provide an acceptable plan for the distribution of cells to researchers. This talk will survey the intellectual property and policy issues that surround these agreements and the sharing of research materials that will ultimately be derived from hESC lines. Such policies seek to balance the needs of the research community to conduct research in an unencumbered manner with the intellectual property incentives companies need to invest in the development of new therapeutics that ultimately better public health.
Up to Top
John W. Thomas, Ph.D., is a Health Scientist Administrator in the Division of Blood Diseases and Resources at the National Heart, Lung, and Blood Institute (NHLBI) at the National Institutes of Health (NIH) in Bethesda, Maryland. At the Blood Division, he administers a grant portfolio focusing on hematopoiesis, blood and stem cell biology, transplantation, and cellular therapies.
In addition, Dr. Thomas leads the NHLBI Cell-Based Therapy Group formed to coordinate stem cell and cellular therapy research planning and to implement new programs, including the Institute’s human embryonic stem cell line research program. He also serves as the NHLBI representative to the trans-NIH Stem Cell Implementation Group and is a member of the NIH Stem Cell Task Force formed in August 2002 by the Secretary of HHS and the NIH Director.
Presentation: Human Embryonic Stem Cell Research Training
This workshop on NIH stem cell training programs will focus on the new T15 training course program titled “Short-Term Courses in Human Embryonic Stem Cell Culture Techniques” (PA-02-054). These course offerings will include hands-on experience to improve the knowledge and skills of biomedical researchers to maintain, characterize, and utilize human embryonic stem cells in basic research. Drs. Barbara Knowles (The Jackson Laboratory) and Martin Pera (Monash Institute) will provide information based on last year’s human embryonic stem cell course at The Jackson Laboratory and course representatives from upcoming T15 training programs will be on hand for a panel discussion featuring a question-and-answer session. In addition, other NIH training opportunities will be discussed, in particular, the K18 sabbatical program (PAR-02-069), specifically designated for stem cell research. Information will be available at the session on NIH stem cell training programs, including all the upcoming T15 course offerings and the K18 program.
Up to Top
Esmail D. Zanjani, Ph.D., is a Professor in the Departments of Animal Biotechnology and Internal Medicine at the University of Nevada, Reno. His areas of interest include hematopoietic stem cell biology, in utero stem cell and gene therapy, and the assessment of the in vivo potential of human stem cells in the pre-immune human/sheep xenogeneic model. Dr. Zanjani was a member of the NIH Hematology I Study Section from 1998 to 2002, a member of the Nominating Committee for the HHS Assistant Secretary for Health (ASH) in 1998, and a member of the Subcommittee on Hematopoietic Growth Factors for the ASH from 1999 to 2002. In addition, he was Vice President (1999), President-Elect (2000), and President (2001) of the International Society for Experimental Hematology. Currently, Dr. Zanjani is a member of the Publications Committee for the ASH (2003 to 2006), a member of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Subcommittee D Study Section (2002 to 2006), and Editor-in-Chief of Experimental Hematology (2004 to 2009).
Presentation: In Vivo Engraftment and Multilineage Differentiation of Human Embryonic Stem Cell (hESC)-Derived Hematopoietic Cells in Primary Fetal Sheep Recipients (Additional authors: A.D. Narayan, J.A. Thomson, R.L. Lewis, D.S. Kaufman, and G.Almeida-Porada, Department of Animal Biotechnology, University of Nevada, Reno, NV; Wisconsin Regional Primate Center; and Departments of Anatomy and Internal Medicine, University of Wisconsin, Madison, WI)
We used transplantation into primary pre-immune fetal sheep recipients (55 to 62 days-old, term: 145 days) to evaluate the in vivo potential of hematopoietic elements derived from hESCs. The in utero human/sheep xenograft model has proven valuable in assessing the in vivo hematopoietic activity of stem cells from a variety of fetal and postnatal human sources. Three transplant groups with cells from differentiated hESCs (H1 and H1.1) were established. Human ESC was differentiated on the mouse S17 cell line for 17 days. The 17-day cultures were found to be positive for CD34, CD133, CD38, gly-A, CD33, CD15, CD10, CD56, and nestin and were negative for CD45, CD3, CD2, HLA-DR, CD19, CD8, CD4, CD14, and CD20. Each fetus (n=7) in group 1 was transplanted with 0.75-2.8 x 105 CD34+/CD38- cells isolated from the day 17 cultures by sorting. The fetuses in group 2 (n=13) were given 0.13 – 0.95 x 105 CD34+/Lin- cells/fetus obtained from the day 17 cultures by sorting, while the fetuses in group 3 (n=12) were transplanted with 1 – 1.5 x 105 whole-day 17 culture cells/fetus. The animals in groups 1 and 2 were allowed to complete gestation and be born. As can typically happen, 3 recipients (2 in group 1, 1 in group 2) were lost to study (fetus absorbed). Four animals in group 1 and 5 animals in group 2 were found to be chimeric with a variety of donor (human) cell types that in some cases have persisted for 13 months post-transplant. For example, the relative percentages of human cells expressing CD34, CD45, CD3, CD13, CD133, CD38, HLA-DR, and CD2 at 5 months post-transplant in a representative animal in group 1 were 0.05, 0.26, 0.20, 0.10, 0.15, 0.09, 4.4, and 0.04, while at 3 months post-transplant the values for cells expressing CD45, CD3, CD133, CD38, HLA-DR, and Gly-A for a representative animal in group 2 were: 0.5, 0.6, 0.4, 0.3, 0.6, and 0.5 respectively. The donor (human) cells appear to be responsive to human cytokines. The administration of human G-CSF to animals chimeric with CD34+/CD38- cells on two separate occasions, at 4 and 12 months post-transplant, resulted in increased donor cell activity. Increases in human cell activity were also noted in chimeric group 1 and group 2 animals treated with human GM-CSF. Two of the recipients in group 3 were sacrificed at 2 months post-transplant (i.e., 1 month before birth). Both were negative for human hematopoietic cell activity. The remaining group 3 animals will be evaluated at birth and at intervals thereafter. Careful examination of these two animals and of 4 additional animals from groups 1 and 2 sacrificed at intervals after birth failed to reveal any gross anatomical abnormalities; all live animals appear to be healthy. These findings indicate that hematopoietic cells derived from hESC can engraft and undergo multilineage differentiation in the human/sheep model. Serial transplantations into secondary and/or tertiary fetal sheep recipients will establish whether the hematopoietic activity in these primary hosts can be attributed to long-term engrafting cells.
Up to Top
Su-Chun Zhang, M.D., Ph.D., received his M.D. (1984) and M.Sc. degrees (1989) in China and his Ph.D. training (1996) in Canada in neural and cell biology, with a focus on the biology and pathology of oligodendroglia and microglia. In his subsequent career, he studied the differentiation of oligodendroglia from neural stem cells and the transplantation of neural stem/progenitors in animal models of myelin disorders. Dr. Zhang is currently an Assistant Professor of Anatomy and Neurology at the Waisman Center, University of Wisconsin-Madison. His research, funded by NIH and private research foundations, explores the potential of embryonic stem cells as a model for early human neural development, such as neural induction and neural patterning, and as a source for generating therapeutically active cells for neurodegenerative diseases.
Presentation: Neural Specification of Human Embryonic Stem Cells
Human embryonic stem (hES) cells not only provide a continuous cell source for potential cell therapy but also offer an otherwise inaccessible system to unveil events of early embryonic development in humans. We have established a stepwise, chemically defined culture system that directs the hES cells to neuroepithelia, the earliest neural cells. The process of neuroepithelial specification resembles in vivo neural induction in terms of timing, formation of neural tube-like structures, and response to FGF signaling. Using this in vitro model system, we are dissecting the molecular mechanisms underlying neural specification from hES cells. The in vitro-generated neuroepithelial cells can differentiate into neurons and glial cells in culture and after transplantation into rodent brain. To determine whether the hES cell-derived neuroepithelial cells can be preferentially fated to a particular neural lineage, we employ the principle of neural patterning and are establishing culture systems that persuade the neuroepithelial cells to choose a particular neuronal or glial fate, such as midbrain dopamine neurons, spinal cord motoneurons, or myelinating oligodendrocytes. The function of the in vitro-generated mature neural cells will be assessed using electrophysiological techniques to measure action potentials and synaptic communication in neurons in culture and after transplantation into rodent brain and spinal cord. We hope that an understanding of neural induction and patterning in humans will lead to an optimized procedure for generating enriched or purified neuroepithelia and specialized neural cells, which will lay groundwork for potential future use of human ES cells in the treatment of neurological injuries and diseases.
Return to top