Draft version 1.0
NIDDK should catalyze a nation-wide effort to characterize the molecular and cellular features of stem cells during and following development of the pancreas, liver, stomach and intestine, kidney and GU tract, bone and hematopoietic tissues. Such an analysis should provide entirely new strategies for repairing or replacing damaged organs in individuals of all ages, and new insights about pathologic processes underlying disordered development, disordered maintenance, and neoplastic transformation of these organs. The scope of this scientific problem is not only great but requires innovation at every level of planning and execution. It will necessitate development of new in vivo models for studying stem cell function, new methods for recovering stem cells as well as other cell populations necessary to maintain 'stemness' ex vivo, new ways of assaying stem cell functions in vivo and ex vivo, plus application of existing and new methods in genomics, proteomics, and bioinformatics that will allow the molecular features of stem cells and their committed daughters to be characterized. This scientific problem will necessitate NIDDK-sponsored melding of individuals and technologies from diverse disciplines, ranging from developmental biology, genomics/genetics, computational biology, to the material sciences (including bio-engineering). Such a melding, in turn, requires a trans-NIH planning effort that embraces a spectrum of activities including but not limited to:
- planning and adequately funding basic and translational clinical research initiatives
- promoting distribution of relevant technologies to stakeholders
- providing a means for making biological reagents from model organisms and humans available to the research community
- adequate training of scientists and physician-scientists in areas supportive of stem cell research
- adequate education of the public concerning the importance of this area of investigation.
- Development of reliable and convenient clonogenic assays for stem cell populations in developing and adult pancreas, liver, stomach/intestine, kidney/bladder, bone and hematopoietic tissues so that these cells can be purified, characterized, and the factors that influence their biological properties assayed ex vivo. These populations should be obtained from model organisms as well as humans.
- Sponsorship of 'genome anatomy projects' (GAPs) designed to profile expression of mRNA and proteins in recovered progenitor cell populations in developing and adult normal tissues. GAP projects should generate searchable, annotated, Internet-accessible databases of expressed genes that can be compared to databases obtained from diseased tissues from model organisms and humans.
- An additional GAP agenda should be to support efforts to develop broadly applicable methods for amplifying mRNAs from single or small numbers of recovered progenitor cells so that gene expression profiling can be performed. GAPs should help develop sensitive methods for rapid follow-up confirmation of cellular patterns of gene expression in tissues from model organisms and humans: the results should be incorporated into easily searched and clearly illustrated databases.
- NIDDK should create economical ways to distribute to the research community well characterized progenitor cells, cell lines that mimic progenitor cells, plus cDNA clones, microarrays, proteins and antibodies relevant to genes expressed in progenitor cell populations.
- Sponsored research that assesses the contributions of various cell populations to creating and maintaining stem cell niches in tissues.
- Sponsored research that develops methods for manipulating gene expression in stem cells and their immediate descendants.
- Promotion of translational research programs that allow application of lessons learned from basic stem cell research to humans.
- Sponsor building of interdisciplinary communities of individuals who study stem cell populations. This community building can be achieved, in part, through:
- NIDDK-sponsored meetings
- Crafting a diversified portfolio of research funding initiatives including RFAs, expanded financial support for program projects, RO1 supplements, and use of SBA money to create 'shadow RFAs' that sponsor technology development and enhancement of resources available to NIDDK scientists
- Development of core facilities for functional genomics/proteomics (local and national)
- Sponsorship of bioinformatics resources
- Training grants in areas needed to support stem cell research
- Sponsorship of activities that promote public education in stem cell research; support of broad-based society discussions of ethical issues arises from stem cell research
- Crafting of appropriate technology transfer policy that ensures the fullest possible public access to government-sponsored research findings.
Endoderm Organ Development and Stem Cell Biology
Overview of current knowledge
One of the goals of basic research is to understand how the different endodermal organs, for example, the liver, lungs, pancreas, and intestines, develop from small founder populations of stem cells. We also need to know how the organs continue to be maintained and even regenerate during adult life and why this capacity declines with age. Until recently, far more was known about the development of ectodermal and mesodermal tissues, including skin, nerves, muscle, and blood than about endoderm. However, in the past few years, advances in model experimental systems, from worms and flies to frogs, fish, and mice, have resulted in major breakthroughs in our understanding of all organ systems. Genes discovered to control endoderm development in readily manipulable, non-mammalian organisms often possess analogous functions in mammals. The evolutionary conservation of critical regulatory molecules, along with advances in the isolation, culture, and transplantation of endoderm tissue and other stem cells, indicates that the time is ripe for an aggressive investment in the field of developmental biology and its application to human disease and aging.
The issues discussed here include: A brief review of the embryonic origin of the mammalian endoderm and the subsequent patterning that generates different tissues; suggestions for ways that far more can be learned about endodermal patterning in mammals; and suggestions for ways that the principles and practice of endoderm differentiation can be applied to the controlled differentiation of human embryonic stem (ES) cells and stem cells in adult tissues for biomedical purposes.
Embryonic endoderm cells are relatively restricted in the kinds of cell types they can generate, but they are derived from earlier endoderm cells that have more developmental options. Still, a small population of multipotent cells known as the definitive endoderm (DE) gives rise to all of the endoderm-derived organs in the adult. The DE is segregated from the pluripotent inner cell mass (ICM) of the blastocyst during the process of gastrulation that takes place soon after implantation. It is the ICM that is the source of pluripotent embryonic stem (ES) cells in the mouse and probably also in the human. The DE initially constitutes an epithelial sheet of about 500 cells covering the ventral surface of the embryo. As development proceeds, the sheet undergoes morphogenesis to form first the foregut and hindgut pockets and then a continuous tube running from the anterior to the posterior. Lineage marking experiments carried out when the DE is still a simple epithelial layer have shown that cells at the anterior normally gives rise to the foregut and to organs such as the lung, liver, stomach and pancreas. By contrast, the more posterior DE is fated to give rise to the midgut (intestines) and cloaca. The lineage of the hindgut, giving rise to the rectum and posterior large intestine, is less well studied. In addition to anterior-posterior regionalization of cell fate within the DE there is also dorsal-ventral (D-V) patterning of the endoderm, most clearly studied in the foregut pocket. The ventral foregut endoderm generates the precursors of the thyroid, lung, liver, and pancreas, while the dorsal foregut endoderm gives rise to part of pancreas and the intestine in its entirety.
Currently little is known about the mechanisms by which the DE layer is initially generated. Both intercellular signaling and cell-cell communication pathways are likely to be involved. There is evidence in the mouse from gene targeting studies that activation of FGF and TGF-related (possible nodal) signaling pathways are required for DE formation, as well as the forkhead transcription factor, HNF3ß. Studies with Xenopus, C. elegans, and zebrafish have also established roles for TGFß signaling and for transcription factors belonging to the paired type homeodomain, homeodomain, GATA, HMG, and T-box families. As mentioned above, most of these proteins are being found to have analogous roles in endoderm development in mammals. Future research should be directed towards using a variety of model organisms and methods to work out the entire interactive network of factors regulating normal DE development.
The rationale for understanding precisely how the DE is formed, for future therapeutic use, is that this information should allow the logical design of strategies for driving the in vitro differentiation of pluripotent embryonic stem (ES) cells and adult stem cell types into multipotent DE stem cells. For example, currently only a fraction of ES cells in culture can be induced to differentiate into DE cell type derivatives, and similarly a very small minority of hematopoietic stem cells, upon transplantation, can give rise to liver cells. Since we do not know the regulatory code that generates the DE during mammalian gastrulation, it is not yet possible to rationally generate the DE from other lineages.
Research Opportunities and Infrastructure Needs
Once the primitive gut tube has formed, several different organs develop from specific regions. For example, the lungs, liver and pancreas develop from the foregut as simple buds surrounded by a layer of splanchnic mesoderm. A great deal more needs to be learned about the factors controlling the position along the anterior-posterior A-P axis at which these buds form, as well as the factors and pathways involved in the specification of cell types particular to each endoderm-derived organ. In the foregut, homeodomain and Hox gene expression in the endoderm is important, as well as mesodermal cell signals such as fibroblast growth factor. In the hindgut, studies in both the chick embryo and mouse mutants suggest that Hox gene expression in the mesoderm, possibly regulated by Bmp signaling, regulates the specification of different cell types. Finally, there is evidence in the mouse that Parahox (Cdx) genes control differentiation and proliferation of the midgut. However, much more needs to be known about the mechanisms controlling such regional specification.
Interestingly, there are examples in the pathology literature showing that the developmental potency exhibited by the endoderm may be preserved, in part, in adult endoderm-derived tissues. Chronic pancreatic cell damage can lead to the formation of hepatocytes in the pancreas; chronic liver damage can lead to the formation of intestine glandular cells in the liver; and so on. These findings suggest that multipotent cells, possibly stem cells, can be activated in these adult tissues. More controlled studies of neural and hematopoietic stem cells have shown that these can also give rise to liver cells, demonstrating a reprogramming from apparent ectodermal and mesodermal lineages, respectively. A better understanding of these adult stem cell models seems highly likely to provide insight into new cell-based therapeutic approaches and the controlled differentiation of existing normal cells within a diseased individual. A major asset of these approaches for therapy is that the newly differentiated cells would have the same immunological characteristics as the host.
Pluripotent embryonic stem (ES) cells derived from mouse or human embryos can give rise to many different cell types in culture, including cells specific to endodermal tissues. If ES cells are to be used therapeutically to generate tissue for transplantation, then we need to know much more about how to drive the undifferentiated cells efficiently down different developmental pathways. The discoveries made both with normal embryos and with adult stem cells should facilitate this quest. In turn, advances in our understanding of how ES cells differentiate should provide clues for the reprogramming of stem cells from adult tissues. For this research to go forward expeditiously we need more human ES cell lines, and both human and mouse ES lines engineered to express marker genes specific to different endoderm-derived cell types and lineages.
The elucidation of the molecular circuitry driving multipotent endodermal stem cells down different lineage pathways and maintaining specialized phenotypes will be important for controlling the development of endodermal cell types from various stem cells. For these studies, it will be important to have banks of marker genes, mice expressing reporter genes (lacZ and GFP, for example) in specific cell types, and mice expressing cre recombinase (either constitutively or after induction) in specific regions of the embryonic gut. These tools can be used in conjunction with high-throughput screening assays to identify factors regulating differentiation and with microarray technology to identify large numbers of genes expressed in specific cell populations. Other tools are normalized cDNA libraries for subtractive hybridization and two-hybrid screening, and advances in cell and embryo tissue explant culture methodology to screen new molecules for biological activity.
In summary, the continued maintenance and regeneration of adult tissues depends on the same mechanisms and cell types that are used to make the animal in the first place. This provides a compelling rationale for investigating the mechanisms of endodermal tissue development and the application of such mechanisms to issues in human disease.
Hematopoietic Stem Cells
Overview of current knowledge
Hematopoiesis involves the production and maintenance of blood stem cells, and their proliferation and differentiation into the lineages of the peripheral blood. The hematopoietic stem cell is derived early during embryogenesis from ventral mesoderm and becomes deposited in unique hematopoietic sites within the embryo. In the human, these sites include the yolk sac, the paraaortic splanchnopleura or aorta-gonado-mesonephros (AGM) region, the fetal liver, and the bone marrow. Hematopoiesis is an evolutionarily conserved process that can be studied more easily than other organs based on the accessibility of the cells. Much of the basic science of stem cell biology has been obtained by the study of hematopoietic cells. The hematopoietic stem cell is capable of both short-term and long-term repopulation of a lethally irradiated animal. This is the gold standard for the measure of stem cell activity. Other surrogate assays such as long-term colony initiating cell (LTCIC) and cobblestone assays have been developed. Hematopoietic stem cells have been purified through the use of monoclonal antibodies such as CD34. Recently, lineage-specific progenitors have been purified. For instance the common lymphoid progenitor and myeloid-erythroid progenitor have been isolated and characterized.
The signals that regulate the number of hematopoietic stem cells would be extremely useful clinically. The numbers of bone marrow and core blood transplants have been increasing in the 21st century.
In addition, recent studies have demonstrated the plasticity of stem cell populations within individual organs. The marrow contains stem cells that are committed to hematopoiesis as well as stem cells that are mesenchymal in origin. These stem cells can be driven to a large number of tissues with exogenous factors. Although the process is relatively inefficient in vitro, contribution of marrow cells to non-hematopoietic lineages have been documented in vivo. The marrow thus may represent an undifferentiated tissue derived during embryogenesis that can maintain highly plastic stem cell populations from diverse tissues. The plasticity of the hematopoietic marrow possibly may be used to derive any organ.
Patients undergoing chemotherapy have decreased peripheral blood lineage counts. It is critical to have an ample number of hematopoietic stem cells for both transplantation experiments as well as for gene therapy.Identification of Major Research Issues: Opportunities and Obstacles
- Understanding the plasticity of hematopoietic stem cells and marrow. It would be of great advantage to understand the exogenous factors that could be added to marrow cells to derive individual hematopoietic lineages, amplify stem cells or progenitors and/or convert the marrow to other organ tissue.
- The derivation of hematopoietic stem cells during embryogenesis should be examined to provide clues to the normal pathways utilized by stem cell populations. Cell migration and homing of hematopoietic stem cells to their environment should be investigated.
- Gene Expression. The individual populations of hematopoietic cells must express lineage-specific gene programs. These could be defined using large-scale approaches for studying gene expression such as microarrays. Proteomics also may be useful.
- Clinical use of marrow stem cells. An extensive literature is evident for the hematopoietic stem cells, both purified and unpurified, in the clinical world of transplantation and gene therapy. A better understanding of the use of these stem cell populations after being treated with particular factors, or transduced by certain vectors, would be helpful.
- The major obstacle for understanding the plasticity of the hematopoietic stem cell is to identify the key regulatory factors in the environment that dictate particular cell fate and cell lineage decisions.
- There is a lack of availability of cDNA libraries or arrays representing distinct individual hematopoietic cell populations in the various model systems.
- A major effort should be placed on developing model systems. For instance, the mouse, chicken, and zebrafish have been previously utilized for hematopoietic studies. Building this infrastructure would be a major advantage for developing an understanding of the factors that regulate stem cell biology.
- Integration of genetics/genomics as well as the developmental biology of hematopoietic stem cells. There is a need to establish communication between investigators studying the genetics of hematopoiesis as well as cell biology.
Bioinformatics. The development of gene arrays to establish function as well as identity of hematopoietic cells would be extremely useful. Bioinformatics at a local level (adjacent to the bench scientist) is required to understand this process. National sources should attempt to derive programs that will evaluate gene expression arrays.
Recommended First Steps
The hematopoiesis community is relatively large and involves a diverse group of investigators. As a first step, it would be useful to have a meeting on stem cell biology to: (a) establish the needs of the community at large and to focus our efforts on hematopoiesis toward a better understanding of stem cell biology; and (b) establish grant proposals that will attack the aims listed in C and D.
Gut Stem Cell Biology
Overview of current knowledge
The gastrointestinal epithelium of humans undergoes continuous and rapid renewal throughout life. The differentiation programs of its component lineages exhibit considerable regional specificity, allowing regional diversification of GI function. Epithelial renewal in the stomach, small intestine, and colon is sustained by populations of multipotent stem cells that reside in distinct anatomic units. A major goal of current research is to define the properties of these stem cells, the factors that regulate their proliferative activity, and the molecular mechanisms that underlie the decisions of their daughters to differentiate along a particular pathway in a developmental and region-appropriate fashion.
Gastric stem cells - Lineage tracing studies using 3H-thymidine labeling followed by EM autoradiography have provided a detailed morphologic description of the presumptive multipotent mouse gastric stem cell, its immediate daughters, as well as the pathways their descendants follow during terminal differentiation. This description is more detailed than the descriptions of epithelial lineage progenitors in the small intestine or colon. Studies in transgenic mice have revealed that the fractional representation of normally rare gastric epithelial lineage progenitors can be markedly increased due to engineered ablation of acid-producing parietal cells. These types of mouse models provide a starting point for recovering these cells and defining their molecular features. Recent work has also suggested that gastric epithelial lineage progenitors may play an important role in determining whether some patients infected with Helicobacter pylori are at risk for developing gastric adenocarcinoma. ~50% of humans harbor H. pylori in their stomachs but only a subset go on to develop severe pathology, including ulcers or cancer. The host and microbial factors that determine the destiny of H. pylori infection are poorly defined, in part due to the genetic heterogeneity of both man and microbe. Chronic atrophic gastritis (CAG) in H. pylori-infected humans appears to be a risk factor for development of gastric adenocarcinoma. The amplified lineage progenitors in adult transgenic mice with genetically engineered ablation of parietal cells produce oncofetal carbohydrate epitopes that can serve as receptors for adhesins produced by clinical isolates of H. pylori. These sialylated glycans are also found in human gastric adenocarcinomas and its precursors - CAG, 'intestinal' metaplasia, and dysplasia. These findings have led to the hypothesis that when the relationship between host and microbe results in loss of parietal cells, as in CAG, bacterial tropism to lineage progenitors may occur if there is a matching of microbial adhesin and host receptor production. Bacterial binding to these progenitors with a high proliferative potential and a relatively long residence time in the stomach may have a large influence on their properties and could facilitate initiation and/or progression of tumorigenesis.Additional information about gastric stem cells comes from recent studies that implicate enhanced EGF receptor signaling in the pathogenesis of Ménétrier's disease, an acquired premalignant hypertrophic gastropathy. Expression of transforming growth factor a, a major EGF receptor ligand, is increased in the gastric mucosa of Ménétrier's disease patients. Transgenic mice overexpressing TGFa in their gastric epithelium exhibit histopathologic changes that phenocopy Ménétrier's disease, including loss of parietal cells. Observations obtained from these mouse models have been extended to the bedside. A Ménétrier's disease patient was successfully treated with an EGFR monoclonal antibody that blocks ligand binding. Clinical improvement was dramatic and parietal cells re-emerged after one month of therapy (Burdick et al., N. Engl. J. Med., 343: 1697 (2000)).
Intestinal stem cells - Epithelial renewal in the mouse intestine is sustained by a population of multipotent stem cells located in crypts of Lieberkühn. Studies in mice have shown that all active stem cells in a given crypt are derived from a common ancestor. These stem cells give rise to daughters that undergo several rounds of division, creating a rapidly cycling transit cell population of oligo- and unipotential lineage precursors. In the small intestine, epithelial cells belonging to the enterocytic, goblet and enteroendocrine cell lineages differentiate as they migrate from a crypt up an adjacent villus, and are removed once they reach the villus tip. In the colon, epithelial cells migrate from a crypt to a flat surface cuff that surrounds its opening.
As in the stomach, there are no confirmed molecular markers of the mouse or human multipotent small intestinal or colonic crypt stem cell. However, studies in mice during the past several years have identified several gene products that help regulate stem cell survival, and/or specification/differentiation of their descendant cell lineages. These genes include Tcf4 (Korinek et al., Nat. Genet. 19:379 (1998)), Nkx2-3 (Pabst et al., Development 126:2215 (1999)), Cdx-1 (Sabramanian et al., Cell 83:641 (1995)), Ihh (Ramalho-Santos et al., Development 127:2763 (2000)), Fkh6 (Kaestner et al., Genes Dev. 11:1583 (1997)), and HFH11 (Ye et al., Mol. Cell Biol. 17: 1626 (1997)).
During the past 5 years transcriptional regulatory elements have been identified that can be used to deliver various gene products to the multipotent small intestinal and colonic stem cell, or its immediate descendants. This has allowed Cre recombinase to be used to knock out genes in crypt progenitor cell populations located a discrete positions along the duodenal-colonic axis. Systems for inducing Cre expression in stem cells at any time point after completion of intestinal morphogenesis have also been reported (Wong et al., Proc. Natl. Acad. Sci. USA 97:12601 (2000)).
The nature of the stem cell hierarchy in small intestinal and colonic crypts, and the fact that the stem cell's progeny are retained in well-defined anatomic units, make the gut an ideal system for conducting in vivo analyses of stem cell biology. For example, it is now possible to create Cre-based genetic mosaic systems in mice where gene function is disrupted in all of the active stem cells and their daughters in one group of crypts, but not in juxtaposed neighboring crypts. These types of engineered focal disruptions of normal biological processes will allow a variety of human GI diseases to be modeled in mice: e.g., from cancer to mucosal barrier injury. Defining molecular interactions that occur at the interface between 'normal' and 'abnormal' cell populations represents an important but under-explored aspect of the pathogenesis of diseases with focal origins.
Clonogenic assays for gut stem cells - These assays are essential for identifying stem cells and studying the factors that regulate their biological properties. For example, the development of a soft agar clonogenic assay for bone marrow progenitor cells led to the discovery of G-CSF, GM-CSF and M-CSF. A clonogenic assay has been described recently for colonic epithelial progenitors, and the source of a potential clonogenic factor has been identified (Whitehead et al, Gastroenterology 117:858 (1999)). However, much work remains to be done.
Gut malignancies are a major cause of morbidity and mortality. It seems likely that these neoplasms arise in long-lived stem cells or their immediate descendants. In addition, radio- and chemotherapy for GI and non-GI related malignancies often produce severe mucositis by disrupting normal mucosal barrier renewal and repair. Thus, one of the goals of gut stem cell research is to decipher how progenitor cells maintain residency (functional anchorage) in their niche, how they avoid replicative scenescence, and how they respond to genotoxic insults. The results should provide important new insights about initiation, progression and treatment of tumorigenesis, and about the process of aging.
The loss or disruption of intestinal absorptive function that occurs with inflammatory bowel diseases and after intestinal resection (e.g., due to vascular disease, trauma, congenital and neonatal disorders) represents a major cause of morbidity and mortality. New strategies to increase the absorptive function of the intestine are needed. This requires research initiatives to identify factors controlling the size of the gut's stem cell population, the proliferative activity of stem cells, and the differentiation of their descendant lineages. To do so, methods must be developed for purifying gut stem cells and characterizing their expressed mRNA and proteins ex vivo and in vivo.
The issue of stem cell 'plasticity' as it pertains to the gut is largely unexplored. For example, can bone marrow stem cell populations home to the gut and establish residency in niches where they can express gut stem cell-like features? A reciprocal question, given the enormity of the gut's stem cell population, is whether these stem cells can re-engineered, and/or placed in niches, so that they can give rise to non-gut cell lineages. Stem cell plasticity should also be considered when considering the mechanisms underlying development of metaplastic states that predispose to cancer, such as Barrett's esophagus (conversion of a squamous to intestinal epithelial phenotype).
The complex interplay between 'environmental' factors (e.g., nutrients, the microflora, mensenchymal-epithelial cross talk) and 'intrinsic' epithelial factors that, together, regulate epithelial renewal remains to be defined.
In addition, surrogate markers should be identified to monitor various elements of this renewal process. Such markers should facilitate more accurate classification of disease states affecting the gut, better design of therapeutic trials since patient populations can be more accurately phenotyped, and new ways for rapid definition of the effectiveness of various therapeutic interventions in these stratified patient populations.Identification of Major Research Issues: Opportunities and Obstacles
- Development of reliable and convenient clonogenic assays for gut epithelial lineage progenitors so that these cells can be purified and characterized, and the factors that influence their biological properties assayed ex vivo.
- Initiation of multicenter G-GAP (gut genome anatomy projects) designed to profile expression of mRNA and proteins in recovered mouse and human gut progenitor cell populations, and in their descendant lineages. G-GAP will encompass normal tissues, harvested at various stages of development and in aging populations, as well as abnormal tissues retrieved from model organisms and humans during the course of evolution of targeted GI diseases. An explicit mission of G-GAP will be generation of searchable, annotated, Internet-accessible databases of expressed genes. G-GAP should therefore partner with other GAP projects sponsored by NIDDK stakeholders to develop new bioinformatic and computational tools for compiling, annotating, searching and comparing databases of genes expressed in normal and abnormal cell populations from model organisms and humans.
- An additional G-GAP agenda will be to support a trans-NIDDK effort to (a) create new and broadly applicable methods for amplifying mRNAs isolated from single or small numbers of recovered epithelial cells so that gene expression profiling can be performed; (b) develop sensitive methods for rapid follow-up confirmation of gene expression in tissues from model organisms and humans; and (c) create economical ways to archive and distribute to the research community mouse and human cDNA clones, microarrays, proteins and antibodies relevant to genes expressed in progenitor cell populations.
- Sponsor research that assesses the contributions of mesenchymal cell populations to creating and maintaining stem cell niches in the gut. This includes, but is not limited to, G-GAP projects, and identification of transcriptional regulatory elements that allow delivery of gene products to these cell populations (e.g. pericryptal fibroblasts).
- Based on work emanating from items 1-4, develop innovative and broadly applicable methods for imaging specified gut cell lineages in vivo.
- To facilitate creation of animal models that simulate human diseases, develop systems where recombination of target genes can be induced in gut progenitor cells and selected descendants lineages at any time during or after completion of gut morphogenesis, under selected physiologic or engineered pathologic conditions.
- Identify cell lines and ex vivo culture conditions that provide useful representations of cell lineages in vivo.
- Sponsor building of interdisciplinary communities of individuals who study gut and other NIDDK-related stem cell populations in a variety of model systems.
Community building can be achieved through (a) NIDDK-sponsored meetings, (b) crafting a diversified portfolio of research funding initiatives including RFAs, expanded financial support for program projects, RO1 supplements, and use of SBA money to sponsor technology development and enhancement of resources available to NIDDK scientists; (c) supporting development of core facilities for functional genomics/proteomics (local and national); (d) Institute sponsorship of bioinformatics resources for its scientists (see item 2 above), and (e) legislative activism in the area of technology transfer policy.
Liver Stem Cells
Overview of current knowledgeDefinitions of liver stem cells
Animals (including humans) can survive surgical removal of up to 75% of the total liver mass. The original number of cells is restored within 1 week and the original tissue mass within 2-3 weeks. This process can occur repeatedly, indicating a very high organ regenerative capacity, which is in contrast to most other parenchymal organs such as kidney or pancreas. The role of liver stem cells in regeneration has been controversial but many of the disagreements can be reconciled by considering the different experimental conditions that have been used to study the process. Liver stem cells can be defined in several different ways that are listed in Table 1. Current evidence strongly suggests that different cell types and mechanisms are responsible for organ reconstitution depending on the type of liver injury. In addition, tissue replacement by endogenous cells (regeneration) must be distinguished from reconstitution by transplanted donor cells (repopulation).
- Cells responsible for normal tissue turnover
- Cells that give rise to regeneration after partial hepatectomy
- Cells responsible for progenitor-dependent regeneration
- Transplantable liver repopulating cells
- Cells that result in hepatocyte and bile duct epithelial phenotypes in vitro
Definitions 1 and 2: In adult mammals normal liver cell turnover and regeneration after tissue removal are affected by the differentiated cell types (hepatocytes and bile duct epithelial cells) and are not stem cell dependent.
Definition 3: Certain kinds of liver injury elicit progenitor dependent liver regeneration. This kind of regeneration occurs when the proliferation of hepatocytes is blocked by chemicals. A novel cell type called "oval cell" emerges which has a phenotype similar to embryonic hepatoblasts and expresses both hepatocellular and biliary markers. Oval cells are not true stem cells and can be considered to be the equivalent of committed progenitors in hematopoiesis. The cell which gives rise to oval cell has not been purified and defined.
Definition 4: Recently, several animal models have been used to achieve liver repopulation with >90% efficiency. Multiple cell types have been shown to be capable of liver repopulation, but the efficiency and kinetics of repopulation vary widely. The cell types are: a) differentiated hepatocytes; b) fetal hepatoblasts; c) oval cells; d) pancreatic liver precursors and e) hematopoietic stem cells. Of these, hepatocytes have the most favorable properties for liver repopulation. Stem cells are inferior in engraftment and repopulation kinetics in the models currently used.
Definition 5: Several laboratories have developed multiple "liver stem cell lines". These cells isolated from liver are poorly differentiated, but can express properties of bile ducts or hepatocytes in response to certain stimuli.
Orthotopic liver transplantation is the treatment of choice for many liver diseases. Unfortunately, the supply of donor organs is limiting and therefore many patients cannot benefit from this therapy. In contrast, hepatocyte transplantation could potentially overcome the shortage in donor livers by use of cells from a single donor for multiple recipients. In classic hepatocyte transplantation, however, donor cells can replace only 1% of the liver mass or less. Recently though, it has been shown in animal models that >90% of host hepatocytes can be replaced by a small number of transplanted donor cells in a process we term "therapeutic liver repopulation". This phenomenon is analogous to repopulation of the hematopoietic system after bone marrow transplantation. Very recently it has been discovered that transplanted cells from extrahepatic sources such as bone marrow can also be used for liver repopulation. Because bone marrow donors are widely available this finding raises the hope of therapeutic application of these cells in the future.
Identification of Major Research Issues: Opportunities and Obstacles
Therapeutic liver repopulation works in several specialized animal models. For the method to become useful clinically, we will need to find conditions that allow repopulation in a variety of settings. Generally speaking, we need to find the equivalent of preparative conditioning as it is used for bone marrow transplantation (BMT). Liver repopulation fundamentally differs from BMT in that complete ablation of the host hepatocytes cannot work because the patient would not survive the time needed for repopulation (6-12 weeks estimated). In the animal models used, liver repopulation works because the damage to host cells is cell autonomous and occurs gradually, thus permitting continued function during the repopulation period.
Although differentiated hepatocytes have the best repopulation characteristics, the use of stem cells, particularly hematopoietic stem cells, is of great interest because of the wide availability of donors. To make liver repopulation by HSCs more efficient, the factors that permit the conversion of HSCs to hepatocytes need to be discovered.
The relevant questions that need more research are:
- Which chemicals/genetic manipulations are suitable to prepare host liver for repopulation by transplanted donor cells?
- Hepatocyte cell cycle control; block of hepatocyte proliferation
- Maintenance of differentiated hepatocyte functions
- Which chemicals/genetic manipulations are suitable to enhance engraftment and proliferation of transplanted liver repopulating cells?
- Hepatocyte cell cycle control; enhancement of hepatocyte proliferation
- Which genes/proteins are driving the repopulation process in vivo (donor cells/host cells)?
- Genes expressed in repopulating cells, host hepatocytes, host Ito cells during repopulation
- Can hepatocytes and/or liver stem cells be expanded ex vivo prior to transplantation?
- In vitro propagation and differentiation of hepatocytes; organ cultures; artificial organs
- What genes/proteins in the donor HSCs and recipient liver are responsible for the differentiation of HSCs into hepatocytes?
- In vitro studies of factors that can turn differentiated HSCs into hepatocytes; embryonic liver development.
- A method of tracking the differentiation process potentially using in situ hybridization or gene arrays to demonstrate the pattern of gene expression during the progression through lineage from hematopoietic stem cell into hepatocytes or biliary cells
In particular in addition to research areas indicated above, it has been difficult to trace the progression through differentiation process from stem cell to differentiated cells because in vitro methods do not faithfully replicate the differentiation of hepatocytes. In vivo the numbers of actual stem cells are so small so as to have made it impossible to track the differentiation process within the liver.
Ready access to high quality human liver cells (single cell suspension, frozen and shippable). Fetal, adult, normal and diseased should be available.
Frozen and stored human hematopoietic stem cells for in vitro differentiation studies (marrow, cord-blood, peripheral blood mobilized)
Liver gene expression arrays (mouse, rat, human) from adult and fetal liver.
Identification of Major Research Issues: Opportunities and Obstacles
Defining the molecular identity of mesenchymal stem cells in humans and in mice. This includes cloning of the Stro1 gene, identification of transcription factors, cell surface receptors and other molecules that alone or together define a mesenchymal stem cell. How does this molecular identity for mesenchymal stem cells differ from the ones of other stem cell population and from osteoblast progenitors, chondrocyte progenitors, myoblast progenitors and fibroblast progenitors?
Establishment of standard protocols for the culture of mesenchymal stem cells of either human or mouse origin. This includes but is not limited to the elucidation of the role of the various family of growth factors i.e. Wnts, hedgehog, FGFs and TGFs/Bmps and of cell matrix interactions in maintaining multipotency of mesenchymal stem cells. Defining the signal transduction pathways used for the differentiation of mesenchymal stem cells in osteoblasts versus adipocytes or myoblasts.
Study of the plasticity and location of mesenchymal stem cells. Is differentiation in one lineage irreversible? If reversible how "dedifferentiation" can be achieved? Are mesenchymal stem cells restricted to skeleton elements or can they be identified in other organs or in blood?
Study and improvement of the homing of the mesenchymal stem cells. Establishment of the most efficient and clinically acceptable way to infuse stem cells. Establishment of culture conditions for patients mesenchymal stem cells including genetic manipulations such as homologous recombination, before reintroducing them in a patient. The use of animal models to define the most appropriate and the safest clinical indications of the mesenchymal stem cells.
Prostate, Bladder, Kidney Stem Cells
Overview of current knowledge
Urologically associated cells, such as renal and prostate stem cell populations, have been recently identified, although their characterization has been limited. Stem cells have been classically defined as a subpopulation of cells that are present in self-renewing tissues and are responsible for the long-term maintenance and acute repair of the organ. The main criteria that have been used to identify such cells include slow-cycling, high in vivo and in vitro proliferative potential, primitive cytological and ultrastructural features, expression of certain "stem cell markers", well-protected location and specialized "stem cell niche", and associations with predominant sites of tumor initiation. Recent findings have shown that stem cells are also present in some tissues that are not traditionally regarded as self-renewing, such as liver, brain and skeletal muscle. Moreover, unexpected flexibility in some stem cell populations have been demonstrated, including the ability of hematopoietic and mesenchymal stem cells to be directed to the formation of other cell types, such as renal and smooth muscle stem cells. Stem cells can also be derived from embryos and from nuclear transfer using somatic cells. Stem cells may be unipotent, multipotent or pluripotent and may be capable of generating different cell types of single or multiple organs (or tissues).
An estimated 2.8 million Americans suffer from prostate disease, including prostate cancer, the second most common malignancy in men. Approximately 24 million Americans suffer from bladder disease, including 3.1 million with chronic conditions, and an estimated 10.9 million Americans suffer from kidney disease, including more than 360,000 who depend on dialysis or a kidney transplant to survive. The identification of urologic stem cells is of major importance because (i) they may be the targets of chemical and viral oncogenesis and would therefore be a predominant source of malignancy which may lead to neoplastic conditions such as kidney, bladder, and prostate cancer.(ii) their deficiency may lead to a variety of tissue abnormalities, including acquired abnormalities, such as hypercontractile bladders and nephrotic syndromes, and developmental abnormalities involving the metanephric ridge, ureteric bud, urogenital sinus, mullerian, wolffian and genital structures. (iii) the stem cells may be ideal targets for gene therapy; (iv) they may play a major role in wound repair secondary to injury or acquired inflammatory conditions such as interstitial cystitis and prostatitis; and (v) they are needed for the construction of artificial tissues and organs. Development and applications of tissue bioengineering techniques using stem cells would enable a patient to "donate" an organ to himself, such as a bladder or kidney, starting with non-diseased cells that could be grown into a full or partial organ.
Identification of Major Research Issues: Opportunities and Obstacles During the past 20 years, remarkable progress has been made in elucidating the tissue interactions and molecules regulating prostate, bladder and kidney formation. From this work, it is now clear that epithelial-mesenchymal interactions are essential for the formation of normal tissues and organs. These seminal discoveries may soon be exploited to generate sizable quantities of tissue suitable for study and transplantation, provided that strategies can be developed to isolate, expand and differentiate stem cells in vitro and in vivo.
The stem cell biology of the prostate, bladder and kidney is largely unknown. The features and location of these stem cells are mostly undefined. There is a need for the establishment of reproducible conditions and protocols for the identification, retrieval, and maintenance of stem cells. Several approaches should be used to identify and isolate urologic stem cells. These include the use of cell surface antigens to sort putative stem cells from tissues, the evaluation of in vitro and in vivo growth and differentiation capabilities, and the use of morphologic, molecular and clonogenic assays. Once the lineage relationship between disparate cell types is established, signaling molecules that direct stem cell fate decisions must be identified. Integral to these studies will be the generation of mononclonal antibodies and cDNA probes that discriminate between pluripotent stem cells and developmental intermediary epithelial, vascular and mesenchymal progenitor cell types. In addition, novel approaches to characterization, such as determining the profile of expressed genes using micro-array methods, may be helpful.
A variety of adult organs have been identified as containing stem cells that might be sufficiently pluripotent that they could generate cells in organs other than the ones from which they were derived. For instance, bone marrow stem cells could be induced to differentiate into neuronal, hepatic or renal cells. These findings need to be documented further and to be applied rigorously to the identification of prostate, bladder and kidney stem cells. The genes controlling stem cell division (asymmetric/symmetric) and the commitment of progeny to a particular fate need to be defined. In addition, the interaction of these cells with the microenvironment that helps define its stem origin needs to be determined.
The developmental potential of prostate, bladder and renal stem cells, and the properties of immediate committed daughter cells need to be defined. Above, the generation and maintenance of stem cells was addressed. Parallel studies are needed to determine the developmental potential of these cell types and their progeny throughout embryogenesis and morphogenesis in post-natal life. For example, which stem cells are present in adult tissues? When and how do stem cells generate epithelial, mesenchymal, and vascular progenitors? What is the normal function of these stem cells in urologic organs? What is the role of these stem cells in maintaining homeostasis in self-renewing cell populations? Current fate mapping techniques (i.e., retroviral mediated gene transfer approaches) may be utilized to address these questions. Functional genomics, including gene expression profiling and surface markers need to be determined.
As described above, stem cells may be involved in pathogenesis throughout the genitourinary tract. The role of stem cells in urologic tissues needs to be defined. The stem cells could be therapeutic targets for gene therapy, and their activity could be modulated for therapeutic benefit, in combination with radiotherapy and chemotherapy, for a variety of urologic conditions, such as neoplasia and inflammatory disease.
Advances in cell biology and stem cell research will enable scientists to grow tissues and organs in the laboratory, starting from only a few cells. For example, cells could theoretically be "instructed" to develop into a bladder or kidney, under the appropriate conditions. This would eliminate many of the current obstacles to organ replacement, such as donor-recipient mismatches and limited organ availability. The conditions required for urologic tissue and organ formation need to be defined. It is now established that tissue formation is dependent on the presence of organ-specific cells and biocompatible matrices. However, the full extent of tissue-inducing parameters remains to be identified. The precise role of stem cells in the formation of tissues and organs remains to be determined. Finally, a possible requirement for vascular and neuronal elements in regulating tissue and organ formation must be addressed.
Several training awards should be directed to the field of urologic stem cell research. These awards may be given to: (1) newly independent scientists, who can demonstrate the need for a period of intensive research focus as a means of enhancing their research directives in the area of stem cell research; (2) research scientists who have identified a mentor with extensive stem cell research experience; (3) health scientists who have the opportunity to receive research training in the area of urologic stem cells; and (4) outstanding scientists who have demonstrated a sustained level of productivity and whose expertise, research accomplishments, and contributions to the field have been and will continue to be critical to the mission outlined in this white paper.
It is expected that bioinformatics will have a major role in the area of urologic stem cell research and clinical application. Self-renewing stem cells usually originate from a rare multipotent cell population. Genome-wide gene expression analyses should be performed in order to define regulatory pathways in urologic cells, as well as their global genetic programs. Subtracted complementary DNA libraries from highly purified stem cell populations should be analyzed with bioinformatic and array hybridization strategies. It would be expected that a large percentage of the several thousand gene products that would be characterized would correspond to previously undescribed molecules, some involved in regulatory functions. As stem cell research proceeds, the ability to rapidly access previously observed stem cell behavior will prove valuable. This behavior should be tracked at the levels of molecular expression and whole cell responses - and it will be a function of the cell's environmental context. In this way, data can be aggregated to predict stem cell responses and thereby control their roles in tissue regeneration. These sets of complete data should be made available in biologically processed-oriented databases, representing the molecular phenotype of specific stem cell populations. Training and equipment in the area of bioinformatics will be needed to accomplish the above. In addition, access to deposited searchable databases generated from urologic associated microarray and proteomics studies needs to be made available to the scientific community. Efforts need to be coordinated with the current NIH committees working on functional genomic and bioinformatics initiatives.
With highest priority, a urologic stem cell bank should be established, which would include a depository and central distribution center for the sharing of cells, reagents and model systems. An academic center with access to human and animal urologic tissue sources would be preferable as a selected site. The stem cell and reagent bank would operate under the auspices of the NIDDK.
Technologies such as gene therapy, genetic engineering, and bioengineering are also essential for the eventual translation of stem cells into the urologic spectrum of diseases. An appropriate focus is expected in these areas of research, as they relate to urologic stem cells. Interactions with the biotechnology and large pharmaceutical industry should be encouraged.
Recommended First Steps:
In the 21st century, there will be many opportunities to use stem cells for the treatment of a wide range of urologic diseases. As the American population ages and life expectancy increases, the number of applications will also increase.
- NIDDK is committed to developing new approaches to prevent and treat urologic disease with an expanding knowledge of stem cell biology.
- Successful applications of urologic stem cell biology should be pursued through NIH-solicited and investigator-initiated research programs, training and scientist awards. The identification of urologic stem cells is of major importance. Furthermore, understanding the basic processes that control cell progeny will facilitate new approaches to the development of new approaches to wound repair and the creation of tissues and organs for transplantation. In addition, clinical applications for stem cells will improve preventive and therapeutic approaches, including gene transfer, for a wide range of urologic diseases. Examples include: cancer, functional disorders, end-stage organ disease (e.g., kidney, bladder) and prostate disease. Thus, findings generated from this research will be highly relevant to many NIH components.
- NIDDK should hold a workshop to discuss scientific issues relevant to genitourinary stem cell research. This workshop should also include a discussion regarding the National guidelines that must be followed and required of all experimenters and institutions that undertake stem cell research.
- A mechanism should be established within NIH-NIDDK to coordinate all endeavors in urologic stem cell research and to ensure proper development, oversight and evaluation of established guidelines.
- A urologic stem cell bank should be established, preferably in an academic institution, which would include a depository and central distribution center for the sharing of cells, reagents and model systems.
- While the science of urologic stem cells continues to be developed, it must not be overlooked that particular experimental therapies will raise ethical concerns among some members of the public. Although the scientific rationale for stem cell research may be quite clear to researchers, these areas may be quite controversial to the public as a whole. Forums that will discuss the ethical, legal, and social issues in biomedical research will guide policy decisions in these areas. These forums include: (1) The NIH Office of Biotechnology Activities; (2) The NIH Office for Protection from Research Risks; and (3) The President's National Bioethics Advisory Committee. Ongoing communication among scientists, physicians, educators, ethicists, theologians, elected officials and the public is essential to guide the future of stem cell research and to ensure that America continues to invest judiciously and responsibly in biomedical research.
Development of the Pancreas
Overview of Current Knowledge Processes fundamental to the development of all organ systems include germ layer specification, cell fate determination, pattern formation, proliferation, and differentiation. In general, the activation of a specific developmental pathway is initiated by an extracellular signal. Signaling molecules that are utilized during development are very diverse and include members of the transforming growth factor superfamily (TGFs, including bone morphogenetic proteins), the epidermal growth factor family (EGFs), the fibroblast growth factor family (FGFs), the wingless family (Wnts) sonic hedgehog family (shh), Notch-like signals, and cytokines such as interleukin-3 (IL-3). Additionally, signaling cascades can be triggered by cell-cell or cell-matrix contact (i.e. integrins). These signaling cascades culminate in the temporal and spatial expression of an array of negative and positive transcriptional regulators. Ultimately, the "readout" of these signaling events will produce a defined cellular phenotype. Applying basic knowledge of these developmental processes will lead to an increased understanding of pancreatic cancer and pancreatitis, and will allow the rational design of protocols to regenerate key endocrine tissues devastated by diseases like diabetes mellitus, such as the endocrine pancreas.
In early pancreatic development, signals produced by the notochord such as the TGFß signal, activin, or the FGF ligand, FGF2, repress the expression of shh, and promote pancreatic development. Development of endocrine organs involves the "budding morphogenesis" of an epithelial layer in order to create a specific organ. The dorsal pancreatic bud expresses a number of transcriptional regulators, including Isl1, pdx1/IDX1, nkx2.2, pax 4, pax6, Hlxb9, and HNF 3ß. Many of these transcriptional regulators are required for proper development of the endocrine pancreas. In mice, at least one transcriptional regulator, P48, is necessary for formation of the exocrine pancreas. TGFß, hedgehog, and notch signaling pathways control the appearance and number of pancreatic precursor cells, regulating both endocrine and exocrine cell fate. For the endocrine pancreas, this ultimately means differentiation into cell types responsible for production of insulin, glucagon, somatostatin, and pancreatic polypeptide. Secreted factors such as the insulinotropic hormone, glucagon-like peptide-1 or hepatocyte growth factor, can regulate pancreatic islet cell mass and may be critical in determining new endocrine cell formation. Extracellular matrix proteins such as Ep-CAM can affect endocrine cell differentiation and cell adhesion molecules such as the integrins can mediate adhesion and migration of pancreatic precursor cells, thus influencing islet morphogenesis.
In the last decade, studies in non-vertebrate genetic models of C. elegans and Drosophila, have provided a wealth of scientific advances in the definition of early developmental pathways. In the next decade, the emerging genetic model, zebrafish, can be exploited for dissecting events in endoderm development and morphogenesis of the pancreas. Non-genetic models such as Xenopus and chick have proven useful in identifying and characterizing novel signals, and associated components of signaling pathways, as well as transcriptional regulators in endoderm development and organ morphogenesis. BETA2/NeuroD, a basic helix-loop-helix transcription factor involved in pancreatic endocrine development was isolated using insulin gene regulation as a paradigm. The study of how specific cell- and tissue specific gene expression in pancreatic cell development is regulated may lead to the identification of cell-specific promoters and the generation of useful animal models of pancreatic disease.
Overview of Current Knowledge
Type 1 and type 2 diabetes and chronic pancreatitis result from the anatomical and functional loss of insulin-producing beta cells and the ductal and acinar cells, respectively, while uncontrollable proliferation of the ductal cells leads to pancreatic carcinogenesis. Characterization, isolation and experimentation with pancreatic stem cells could provide powerful practical and conceptual tools to solve problems of the pancreas organogenesis, function and carcinogenesis. The replacement of these cells through regeneration or transplantation could offer lifelong treatment for diabetics and for patients with chronic pancreatitis. However, a major problem in implementing treatment is the lack of sufficient pancreatic/ islet cell tissue for transplantation, and a lack of understanding whether and how beta cells regenerate. Embryonic stem cells and other tissue-specific stem cells could potentially provide a limitless source of islet cells for transplantation therapies. Stem cells are capable of self-renewal and can give rise to multiple different lineages. To characterize stem/multipotential progenitor cells, it is necessary to have quantitative assays (both in vivo and in vitro) in which to assess the ability of these cells to: (1) give rise to multiple lineages, and (2) self-renew, and (3) reconstitute a cellular compartment. Despite major advances in stem cell research and in defining specific developmental pathways in neurobiology and hematopoiesis, research focused on isolating and characterizing potential pancreatic stem/progenitor cells and on understanding the developmental pathways that lead to a pancreatic islet are still in their infancy.
Identification of Major Research Issues: Opportunities and Obstacles More basic research in the following areas is needed to enhance our fundamental understanding of development and stem cells of the pancreas:
- What are the specific signals, signaling pathway components, and transcriptional factors that regulate endoderm specification, dorsal and ventral pancreatic bud formation, pancreatic duct formation and pancreatic cell fate determination?
- What and where are the specific cells that secrete or are responsive to these signals throughout pancreatic development?
- What are the molecules that participate in cell-cell interactions, differential cell adhesion and cell motility, and extracellular matrix in islet cell morphogenesis?
- What are the molecular markers that define all stages of pancreatic development including markers of stem cell/progenitors of the pancreas?
- What is the mechanism of transdifferentiation events in the adult pancreas, including ductal to islet cells, acinar to ductal cells, and islet to ductal cells?
- What is the potential use of embryonic stem cells, hematopoietic, neural and other stem cells for the formation of differentiated cells of the endocrine pancreas?
- What are the growth conditions required to generate differentiated cells of the endocrine pancreas from stem/progenitor cells?
- How is neogenesis, proliferation, or regeneration in the adult pancreas regulated?
A major question is whether we can identify and isolate stem/progenitor cell populations of the pancreas? The following methodology, reagents and assays would be essential to this process:
- Methods for examining endocrine and exocrine pancreatic cell lineage, including those that use the activation of inheritable markers.
- Developing reagents such as antibodies to specific cell surface markers for purification of pancreatic stem/progenitor cells.
- Developing clonogenic assays, both in vivo and in vitro, for characterizing potential pancreatic stem/progenitor cells.
- Recruitment of well-trained young investigators into the field of pancreatic development. Fellowships, grants for junior faculty, and grants for meetings specifically targeted at recruiting young scientists and bringing new technology into the field.
- Collaboration among stem cell biologists, developmental biologists, and diabetes investigators.
- Enabling technology including gene expression arrays from islets and all stages of pancreatic development (mouse and human).
- Continued support for an annotated database for pancreas.
Stem Cell and Developmental Biology Writing Groups
Brigid L. M. Hogan, Ph.D.
Professor, Department of Cell Biology
Room U-2219 Medical Center North
1161 21st Avenue South
Vanderbilt University Medical Center
Nashville, Tennessee 37232-2175
Fax (615) 343-2033
Kenneth Zaret, Ph.D.
Institute for Cancer Research
Dept. Cell and Developmental Biology
7701 Burholme Avenue
Philadelphia, PA 19111
PH: (215) 728-7066
FAX: (215) 379-4305
Douglas Melton, Ph.D.
Professor in the Natural Sciences
Dept. Molecular and Cellular Biology
7 Divinity Avenue
Cambridge, Massachusetts 02138-2019
Fax (617) 495-8557
Leonard Zon, M.D.
Associate Professor of Pediatrics
Howard Hughes Medical Institute
320 Longwood Avenue
Boston, Massachusetts 02115
Fax (617) 355-7262
Irving Weissman, M.D.
Professor of Pathology and Developmental Biology
Department of Pathology
Stanford University Medical School
B257 Beckman Center
Stanford, California 94305-5323
Fax (650) 723-4034
Judith A. Shizuru, M.D.
Assistant Professor of Medicine
Stanford University Medical School
B257 Beckman Center
Stanford, California 94305-5623
Fax: (650) 725-8950
Ihor R. Lemischka, Ph.D.
Department of Molecular Biology
210 Lewis Thomas Laboratory
Princeton, New Jersey 08544
Fax (609) 258-2759
Peter Quesenberry, M.D.
Professor of Medicine
Department of Medicine
University of Massachusetts Medical School
NRI Building, Room 211
55 Lake Avenue North
Worchester, Massachusetts 01655
Fax (508) 856-6955
Jeffrey I. Gordon, M.D.
Professor and Head
Department of Molecular Biology and Pharmacology
Washington University School of Medicine
660 South Euclid Avenue
St. Louis, Missouri 63110
Fax (314) 362-7047
Daniel K. Podolsky, M.D.
Chief, Gastrointestinal Unit
Massachusetts General Hospital
Boston, MA 02114
Fax (617) 724-2136
Robert J. Coffey, Jr., M.D.
Department of Medicine
Vanderbilt Univ Sch of Med
2201 West End Avenue
Nashville, TN 37235
Fax (615) 343-1591
Robert P. Whitehead, M.D.
Department of Medicine
Vanderbilt Univ Sch of Med
2201 West End Avenue
Nashville, TN 37235
Rebecca Taub, M.D.
Professor of Genetics
705a Stellar-Chance Laboratories/6100
University of Pennsylvania School of Medicine
Philadelphia, Pennsylvania 19104
Fax (215) 573-5892
Markus Grompe, MD
Oregon Health Sciences University
Molecular & Medical Genetics, L103
3181 SW Sam Jackson Pk Rd.
Portland, OR 97201-3098
PH: (503) 494-6888
FAX: (503) 494-6886
Gerard Karsenty, M.D., Ph.D.
Professor of Molecular and Human Genetics
BCM-Smith Medical Research Bldg
Room BCMS S930
One Baylor Plaza, 630E
Houston, Texas 77030
Steven L. Teitelbaum, M.D.
Department of Pathology Mailstop 8050
Washington University School of Medicine
Barnes-Jewish Hospital North, 216 South Kingshighway
St. Louis, Missouri 63110
Fax: (314) 454-5505
Henry M. Kronenberg, M.D.
Chief, Endocrine Unit
Massachusetts General Hospital
50 Blossom Street, Wellman 501
Boston, Massachusetts 02114
FAX (617) 726-7543
Prostate, Bladder and Kidney
Anthony Atala, M.D.
Assistant in Surgery (Urology)
Department of Urology
300 Longwood Avenue
Boston, Massachusetts 02115
Fax (617) 355-8336
Marc R. Hammerman, M.D.
Chromalloy Prof of Renal Diseases In Medicine
Campus Box 8126
Washington University School of Medicine
One Brookings Drive, St. Louis, MO 63130
Fax (314) 362-8237
Qais Al-Awqati, M.D.
Robert F Loeb Professor of Medicine
Division of Nephrology
Department of Medicine
630 W 168 ST
New York, NY 10032
Doris A. Herzlinger, Ph.D.
Associate Professor of Physiology and Biophysics
Weill Medical College of Cornell University
1300 York Avenue
New York, NY 10021
Don Coffey, M.D.
Department of Urology
School of Medicine
Johns Hopkins University
Baltimore, MD 21827
Fax (410) 955-0833
Donna M Peehl, Ph.D.
Associate Professor (Research) of Urology
Stanford, CA, 94305-5118
Fax (650) 723-0765
Tung-tien (Henry) Sun
Department of Dermatology
New York University School of Medicine
70 Washington Square South
New York, NY 10012
212 263 5685
Fax: 212 263 8561
Sheryl M. Sato, Ph.D.
Division of Diabetes, Endocrinology, and Metabolism
45 Center Drive MSC 6600
Bethesda, MD 20892-6600
FAX: (301) 480-3503
Jose Serrano, M.D., Ph.D.
Division of Digestive Diseases and Nutrition
National Institute of Diabetes and Digestive and Kidney Diseases
6707 Democracy Blvd RM 671 MSC5450
Bethesda MD 20892-5450
FAX: (301) 480-8300