June 9-10, 2002, Pooks Hill Marriott, Bethesda, Maryland
Introduction
To promote interactions and discussions between researchers studying embryonic and adult mammalian stem cells and basic researchers working in other areas such as chromatin, gene expression, cell cycle, cell signaling, and development, the National Institute of General Medical Sciences (NIGMS) hosted a workshop on the basic biology of mammalian stem cells on June 9-10, 2002 at the Pooks Hill Marriott in Bethesda, Maryland. In hosting the workshop, NIGMS aimed to learn how best to stimulate research that will advance the basic biology of mammalian stem cells, a field with the potential for rapid growth following the recent successful isolation of human embryonic stem cells and human embryonic germ cells in 1998 and the establishment of policies governing the use of human embryonic stem cells in Federally funded research. Dr. Judith H. Greenberg, acting director of NIGMS, who participated in planning the meeting along with co-chairs Dr. James Thomson of the Wisconsin Regional Primate Research Center of the University of Wisconsin, Madison, and Dr. Kenneth Zaret of Fox Chase Cancer Center, recognized the pressing need to foster communication between investigators. In particular, one goal was to develop a better understanding of the molecular and cellular properties that make stem cells unique, so they might be manipulated rationally for therapeutic purposes. Another goal was to explore the notion of employing stem cells as a model system for addressing fundamental problems in biology.
Background
On August 9, 2001, President Bush announced his decision to allow Federal funds to be used for research on existing human embryonic stem cell lines as long as, prior to his announcement, (1) the derivation process (which commences with the removal of the inner cell mass from the blastocyst) had already been initiated and (2) the embryo from which the stem cell line was derived no longer had the possibility of development as a human being. In addition, the President established the following criteria that must be met: the stem cells must have been derived from an embryo that was created for reproductive purposes; the embryo was no longer needed for these purposes; informed consent must have been obtained for the donation of the embryo; and no financial inducements were provided for donation of the embryo. On November 7, 2001, NIH posted the Human Embryonic Stem Cell Registry (http://escr.nih.gov/), a list of human embryonic stem cell lines at varying stages of characterization that meet the President's eligibility criteria. Since that time, more than 70 embryonic cell lines have been listed in the registry. At this junction in time, NIH realized the importance of looking beyond merely listing the availability of the stem cell lines to encouraging the conduct of research that will help unite the fields of basic molecular and cell biological research with more biomedically applied stem cell biological pursuits.
Keynote Addresses: Stem Cells and Basic Biology
The workshop began with keynote speaker Dr. James Thomson, who gave an overview of the stem cell research field. Dr. Thomson, who led one of the first groups to isolate embryonic stem cells from humans, described the properties of pluripotent embryonic stem cells and recounted the extreme difficulty in culturing these cells in the laboratory. Under the right set of conditions, Dr. Thomson and other groups have succeeded in growing embryonic stem cells for long periods of time without observing karyotype alterations or senescence. The cells retain an organizational ability to segregate into the three developmental compartments--endoderm, mesoderm, and ectoderm. Dr. Thomson stressed that significant differences exist between embryonic stem cells derived from mice and humans, cautioning that the use of murine embryonic stem cell models may be misleading in understanding the earliest events in human embryonic development and urging the need for continued study using non-human primate model systems. Dr. Thomson predicted that human embryonic stem cells will be important for three main reasons: to provide a tissue culture model system for understanding the differentiation and function of human tissues; to provide a testable cell population for drug discovery efforts; and as a possible transplantation source. Dr. Thomson expressed serious doubt that therapeutic cloning using human oocytes would be used widely in the foreseeable future to produce human embryonic stem cells for transplantation, given the likely inefficiencies of the process and the high costs that would inevitably coincide with patient-specific cell therapy. However, Dr. Thomson stated that understanding the mechanisms that mediate nuclear reprogramming will be essential, as it should be possible to one day replace the oocyte in the process. Dr. Thomson also predicted that in the next decade, relatively few clinical trials in "cell therapy" will be initiated with human embryonic stem cells, due to safety concerns and possible immune interference; however, use of the cells in a co-transplantation setting may help to alleviate allogeneic barriers leading to rejection. Forecasting that stem cells would have a greater impact on understanding the roots of disease rather than on treatment, Dr. Thomson encouraged interdisciplinary approaches to move the field forward as quickly as possible.
Dr. Marc Kirschner of Harvard University delivered the second keynote address, on how basic biology intersects with the study of stem cell biology. Dr. Kirschner described stem cell behavior as an evolutionarily ancient process used not only to form adult tissues, but also to establish a body plan. Dr. Kirschner suggested focusing attention on the attributes of multicellularity in an effort to use stem cell biology to elaborate developmental processes in metazoans. Dr. Kirschner stated that in a metazoan organism, a stem cell's ability to generate all lineages would be useless without the capability to differentiate according to spatial and temporal constraints. These constraints give rise to complex cell arrangements, cell junctions, intercellular signaling, a defined body plan, and general overall complexity between cell types. In particular, Dr. Kirschner suggested considering compartmentation in addressing fundamental issues underlying the differentiation properties of adult stem cells. Within embryological compartments, cells are identical in expression and behavior, and cells within a compartment are renewable. Dr. Kirschner suggested that compartmentation promotes utilitarian evolutionary change by insulating changes in one field from those in another. Adult stem cells can be considered intermediates in embryonic development, with varying limitations in differentiation potential. Dr. Kirschner described regeneration as an obligate property of embryonic compartmentation, specifically selected in the case of some adult tissues (e.g., skin, blood, skeletal muscle) but not others (e.g., cardiac muscle, brain). Basic questions to divulge secrets of "stemness" may include addressing the limitations placed on cell proliferation within compartments as well as understanding which steps are reversible in compartmentation. Other critical issues open to inquiry through the use of stem cell models include characterizing the particular circuitry within a compartment that makes cells in that compartment independent of earlier progenitors, and asking why some organisms maintain a large competence for regeneration whereas others do not.
Adult Stem Cells
Dr. Pamela Gehron Robey of NIH's National Institute of Dental and Craniofacial Research spoke about her research on the biology of bone marrow stromal stem cells in health and disease, describing the existence and properties of postnatal multipotential skeletal stem cells in bone marrow, soft tissue, and blood that may provide a new reagent for regenerative medicine. Dr. Robey described the postnatal stromal cell network (consisting of cartilage, bone, fat, and hematopoietic stroma) as being in an equilibrium designed to suit the metabolic needs of an organism, leading to both the formation and resorption of bone. Dr. Robey described recent results reporting the identification in blood of circulating skeletal stem cells, which have a mostly fibroblastic appearance, but some of which have the ability to make bone. She described the extreme plasticity of these adult stem cells with regard to their ability to transform from one phenotype to another both in vitro and in vivo.
Dr. Margaret Goodell of Baylor College of Medicine provided an update on the biology and potential plasticity of marrow and muscle adult stem cells. Dr. Goodell stressed the importance of broadening the field of stem cell research to incorporate basic biological pursuits. Adult stem cells are present in nearly every human tissue, but with the exception of the hematopoietic system, most of these cell types have not been well studied. Dr. Goodell described recent experiments with satellite cells, stem cells previously defined electron microscopically as lining muscle fibers. Through conducting competitive repopulation assays, Dr. Goodell has found that in addition to the satellite stem cell population, hematopoietic stem cells also exist in muscle tissue, revealing that hematopoietic progenitors can reside outside of the bone marrow. Dr. Goodell urged the application of emerging technologies to the investigation of stem cell biology and underlying mechanisms.
Dr. Ron McKay of NIH's National Institute of Neurological Disorders and Stroke spoke about adult stem cells in the nervous system, stating that recent reports have shown cells to adopt "radical new fates." Dr. McKay described data showing that midbrain stem cells can generate functional, dopamine-producing neurons. At the right time in development, neuronal progenitors are plentiful, and stem cells can be cultured from central nervous system tissue under specified conditions. Dr. McKay discussed humoral determinants, such as bone morphogenetic proteins and other growth factors, which promote differentiation of neuronal stem cells along distinct lineages.
Basic Mechanisms (First Session)
Dr. Elizabeth Blackburn of the University of California, San Francisco, opened the session on basic mechanisms by giving an update on telomeres, telomerase, and growth effects. The precise functions of telomerase in cell senescence have not been clearly identified, although the enzyme performs many key roles in cell behavior and maintenance. Dr. Blackburn reported that the traditional role for telomerase in capping and replicating chromosome ends is being joined by new, unexpected functions. Dr. Blackburn reported recent data in which telomerase increases the stringency of responses to cellular insult, for example selectively stimulating cell death in response to DNA damage caused by ultraviolet radiation. Recent data also investigate the capacity for telomerase to prevent telomeric fusions in the absence of the action of a DNA damage checkpoint kinase.
Dr. Robert Kingston of Massachusetts General Hospital discussed biochemical approaches to understanding body pattern specification through transcriptional memory. Transcriptional levels of developmentally important genes are established through one set of mechanisms but maintained by another set of mechanisms. Dr. Kingston described recent experiments analyzing the relationship between nucleosome structure and the mammalian homologs of the Drosophila polycomb-group and trithorax-group genes. Dr. Kingston predicted that using stem cell models to gain a better understanding of the mechanisms underlying chromatin remodeling may clarify mechanisms central to nuclear reprogramming.
Meeting co-chair Dr. Kenneth Zaret posed the question, "How do progenitor cells gain competence to differentiate into different cell types?" Developmental signals control tissue differentiation, and probing the mechanisms underlying the earliest signals that endow competence may shed light on these processes. Dr. Zaret described his approach to investigating the differentiation of mammalian endoderm, which uniquely gives rise to gut-derived tissues. Dr. Zaret described results revealing the importance of mesoderm-derived FGF signaling in the specification of pancreatic and hepatic tissues. Dr. Zaret also presented data using in vivo footprinting of embryonic tissues, in which he has identified both general and specific chromatin "openers." Understanding how transcription factors permit or deny subsequent events in chromatin opening could help guide future approaches to manipulating stem cell differentiation states.
Dr. Richard Young of the Whitehead Institute for Biomedical Research concluded the workshop's first basic mechanisms session by speaking about transcriptional regulatory networks in living cells. Dr. Young discussed recent efforts to define transcriptional regulatory potential that interdigitates distinct organismal processes such as metabolism, cell cycling, development, and environmental response. He noted that biological and microarray data are extremely noisy, requiring overlaid computational approaches for meaningful analysis. Dr. Young described a new technique that can identify chromosomal locations of transcriptional regulatory proteins in vivo. Insights gleaned include the identification of several transcriptional regulatory motifs, including autoregulation, feed-forward loops, regulatory cascades, multi-component loops, single-input modules, and multi-input modules.
Embryonic Stem Cells
Dr. Benjamin Reubinoff of Hadassah University Hospital began the session on embryonic stem cells by discussing neural differentiation of human embryonic stem cells. Dr. Reubinoff emphasized the need for the development of effective ways to obtain and develop lineage-specific progenitor cells. Dr. Reubinoff presented recent results of efforts to generate highly enriched preparations of neural progenitors from human embryonic stem cells and to induce their differentiation both in vitro and in vivo. He reported that following transplantation, the neural progenitors differentiated into progeny of the three fundamental neural lineages in a region-specific manner, indicating their capability to respond to local cues and participate in the processes of host brain development. Dr. Reubinoff described plans to use the experimental system to investigate issues related to early human neural development, and potentially, for the discovery of key genes regulating this process.
Dr. Martin Pera of Monash Medical Center discussed existing challenges in defining ideal culture conditions for perpetuating pluripotent stem cells in the laboratory. Unlike the case for the adult stem cell populations that constitute the hematopoietic system, pluripotent embryonic stem cell populations have not been systematically characterized. Although most current methods to culture embryonic stem cells depend upon a feeder cell layer and the addition of serum, Dr. Pera's laboratory and other groups are seeking ways to culture these cells in a serum-free environment, to wield more control over understanding their properties and behavior. In so doing, Dr. Pera reported early data on the ability of lysophospholipid ligands, which are major serum components in platelets, to regulate embryonic stem cell function, possibly through ERK signaling pathways. Dr. Pera also presented data describing the recent discovery of a putative, hepatoblast-resembling endodermal progenitor cell that undergoes budding and expresses gut and liver markers such as AFP and GCTM-5.
Dr. Hans Schöler of the University of Pennsylvania discussed gene regulation and function in embryonic stem cells and in the germ cell lineage, which together comprise the germline. Dr. Schöler spoke about the transcription factor Oct4 as a model system for studying specific questions about the germline, since Oct4 is expressed exclusively in the germline. Dr. Schöler reported results suggesting that Oct4 is stringently controlled such that too little or too much of this protein causes cells to exit to the trophoblast or hypoblast lineages. Dr. Schöler speculated that Oct4 may serve a useful purpose as a monitor of the efficiency of animal cloning, since clones with aberrant Oct4 expression and localization do not fare as well as those where levels and localization more closely mimic controls.
Basic Mechanisms (Second Session)
Dr. Stuart Kim of Stanford University began the second session on basic mechanisms by discussing global analysis of gene expression in the roundworm C. elegans, the first animal model for which all the genes were sequenced. Dr. Kim reported that only approximately 1,500 genes (6 percent of the C. elegans genome) have been studied using classical genetic or biochemical approaches, and more than 200 transcription factors have been identified through genome analysis. Dr. Kim reported results of a recent genome-wide analysis of gene regulation under various experimental conditions, representing 553 separate microarray experiments, many of which were obtained from other laboratories working with C. elegans. Dr. Kim presented the results as a three-dimensional topographical map developed in collaboration with Sandia National Laboratories, in which different mountains on the "topomap" represented different gene expression clusters.
Dr. Haifan Lin of Duke University Medical School discussed D. melanogaster as a model system for investigating stem cell division. Dr. Lin has used the fruit fly model to identify mechanisms and structures involved in asymmetric division of stem cells in the germline, reproductive stem cells that proliferate and differentiate to become adult sperm cells. Dr. Lin reported results of studies characterizing the fruit fly piwi gene, its homologs in other organisms, and a cytoskeletal structure called the spectrosome in his group's efforts to further characterize the role of somatic cell signaling and the stem cell "niche theory" in D. melanogaster. Dr. Lin reported that homologs of piwi play essential roles in mammalian spermatogenesis, and that the overexpression of one of piwi's human homologs is highly correlated to seminoma, a form of testicular cancer caused by malignant proliferation of germline stem cells.
Dr. Sharon Dent of the University of Texas M.D. Anderson Cancer Center discussed the role of histone acetyltransferases (HATs) in mouse development. Dr. Dent provided an overview of studies by a number of researchers addressing how gene regulation is affected by changes in chromatin structure, culminating in a recently defined "histone code" that reflects covalent modifications of histone tail residues that appear to provide specific binding sites for regulatory factors. Dr. Dent presented results of her studies using knockout mice that lack certain HATs, such as GCN5. Dr. Dent also described the synergistic effects of combined mutations in GCN5 and p300 on cell survival during mouse development, stressing the importance of specific histone-modifying enzymes in controlling the process of embryogenesis.
Dr. Timothy Bestor concluded the workshop with a talk on mechanisms of imprint disruption in mammalian cloning. Previous studies on gene silencing in mammals have demonstrated that imprint establishment occurs in the germline of the previous generation. Dr. Bestor stated that DNA methylation has a crucial role in genomic imprinting and X chromosome inactivation in females, but that little is known about the mechanistic details of genomic imprinting mediated through DNA methylation. Dr. Bestor has investigated the role of Dnmt3L and Dnmt1, the only genes that have been demonstrated to be required for the establishment and maintenance of genomic imprinting. Dr. Bestor put forward the hypothesis that poor success rates and severe phenotypic abnormalities in cloned offspring may be caused by artificial and abnormal DNA methyltransferase constitutions at early stages.
Discussion and Conclusions
A major goal of the workshop was to determine how basic science experimental systems may be enhanced by employing stem cell model systems, as well as to clarify areas of need and potential collaboration. Following the meeting, the speakers convened for a half-day session and discussed several common themes.
Importance of stem cell models:
1. Stem cells will be an increasingly important model system to investigate fundamental biological problems. Significant, fundamental, molecular differences distinguish development among mammals, and studying human cell systems may be the best way to clarify seminal events in early human development and human tissue specification. Stem cell cultures, which are primary culture systems, may offer significant advantages over cell lines, which often possess karyotypic abnormalities and other idiosyncrasies in growth characteristics. Currently, access to primary somatic tissue is suboptimal, with cultures often being derived from diseased or damaged human tissues.
2. Stem cell model systems may provide an important tissue culture model system for drug discovery/testing, which may be superior to current preclinical animal model systems that often do not accurately reflect human cell physiology. Such stem cell models may also partially obviate the need to use research animals in drug toxicity studies.
3. A better understanding of stem cell biology may lead to the improvement of the therapeutic potential of stem cells. Answers to underlying questions such as the molecular logic that permits stem cells to be unique in their ability to self-renew and to retain plasticity to differentiate into any cell type in the body may lead to the ability to experimentally manipulate these properties. Additional basic research is required to provide molecular characterization of basic processes that contribute to "stemness," such as cell cycle control/arrest, chromatin structure and remodeling, transcriptional regulation, and genomic imprinting. A better understanding of the roles such basic processes play in nuclear reprogramming may also improve scientists' ability to condition stem cells for therapeutic uses.
4. Over time, stem cells may be useful in transplantation and/or co-transplantation settings, although the safety of such applications must be carefully evaluated and the cost-benefit ratio for individual disease applications may vary widely.
Areas of need:
1. Interdisciplinary research will be an essential ingredient for the success of stem cell research. Novel granting mechanisms, training programs, and cross-disciplinary meetings may facilitate healthy growth of the field and communication among researchers in this area. Establishing new centers with stem cell production core facilities could enable research projects to make use of the cells. Conference grants to fund interdisciplinary research meetings may foster better communication between stem cell biologists and researchers working in other areas of biomedicine. Mechanisms for training investigators in the use and maintenance of stem cells are needed. The availability of existing funding mechanisms, such as administrative supplements for stem cell research, could be better communicated to the scientific community.
2. Significant improvements in the development and standardization of culture conditions for embryonic stem cells and various types of adult stem cells are required to move stem cell research away from phenomenological characterizations that have plagued the field. Systematic analysis of culture conditions, performed by senior scientists, is needed to refine conditions to enhance basic biological studies and also to pave the way for potential future use in therapeutic cell therapy applications. Mechanisms should be put into place to encourage these types of non-hypothesis-driven studies that are inherently risk-prone. Developing and implementing protective mechanisms for faculty who invest time in characterizing culture conditions may attract talent in this area.
3. Increased application of emerging technologies to the study of stem cell biology may enhance understanding of genetic factors that contribute to self-renewal and differentiation potential. Some of these technologies include microarray analysis (including "chip-chip" analysis), and both temporary (e.g., RNAi and antisense) and global gene inactivation strategies to investigate human genetics in an unbiased manner, in an "open system." (Mouse knockout technology, in contrast, may be considered one example of a closed system, with less flexibility for ongoing manipulation). Improved imaging technologies may improve the identification of reliable cell surface markers to identify and/or isolate stem cells and progenitor cells.
4. Specialized study sections may be necessary to evaluate research applications for stem cell research, specifically for proposed projects that are high-risk and non-hypothesis driven in nature.
5. The creation of national, centralized facilities to develop, culture, and distribute stem cells may be a useful strategy for providing biomedical scientists' access to stem cells. Alternatives include the expanded use of infrastructure grants to the stem cell derivers to enable them to supply larger quantities of stem cells or cell-derived products to investigators who do not want to develop expertise themselves in stem cell culture.
6. Completion of the Mammalian Gene Collection, in which cDNAs of murine, human, and other mammalian genes are packaged into vectors appropriate for use in stem cell studies, will catalyze basic studies of stem cell biology.
As a result of the changing landscape in the area of stem cell research, NIH has been trying to facilitate stem cell research. To date, relatively few international policies have been implemented and several are still under development; however, NIH has been working to resolve issues dealing with intellectual property and the importation of cell lines. The first applications submitted to NIH involving the use of embryonic stem cells were funded in May 2002. Many NIH components have established administrative supplement programs to fund existing investigators who wish to extend their studies using embryonic stem cells. Visit the NIH Web site for more information about stem cell funding opportunities and their individual requirements (http://grants1.nih.gov/grants/stem_cell_faqs_topic.htm#assistance).
Roster
Dr. Timothy H. Bestor
Department of Genetics and Development
College of Physicians and Surgeons of Columbia University
Dr. Elizabeth Blackburn
Department of Biochemistry and Biophysics
University of California, San Francisco
Dr. Sharon Y.R. Dent
Department of Biochemistry and Molecular Biology
University of Texas M.D. Anderson Cancer Center
Dr. Gregory Downing
Office of Science Policy and Planning
National Institutes of Health
Dr. Margaret A. Goodell
Center for Cell and Gene Therapy
Baylor College of Medicine
Dr. Judith H. Greenberg
National Institute of General Medical Sciences
National Institutes of Health
Dr. Stuart Kim
Department of Developmental Biology
Stanford University
Dr. Robert Kingston
Department of Molecular Biology
Massachusetts General Hospital
Dr. Marc W. Kirschner
Department of Cell Biology
Harvard University
Dr. Haifan Lin
Department of Cell Biology
Duke University Medical School
Dr. Ron McKay
Laboratory of Molecular Biology
National Institute of Neurological Diseases and Stroke
National Institutes of Health
Dr. Martin Pera
Centre for Early Human Development
Monash Institute of Reproduction and Development
Monash Medical Centre
Clayton, Victoria, Australia
Dr. Benjamin Reubinoff
Goldyne Savad Institute of Gene Therapy
Hadassah University Hospital
Jerusalem, Israel
Dr. Pamela Gehron Robey
Bone Research Branch
National Institute of Dental and Craniofacial Research
National Institutes of Health
Dr. Hans Schöler
Center for Animal Transgenesis and Germ Cell Research
University of Pennsylvania
Dr. James Thomson
Wisconsin Regional Primate Research Center
University of Wisconsin, Madison
Dr. Richard A. Young
Whitehead Institute for Biomedical Research
Dr. Kenneth Zaret
Cell and Developmental Biology Program
Fox Chase Cancer Center
