Global Genomic Analyses of Human Hematopoiesis
Ihor Lemischka, Ph.D.
Princeton University, Princeton, NJ
Abstract:
The mechanisms that regulate cell fate decisions during the establishment of the hematopoietic stem and progenitor cell hierarchy are poorly understood. As in all developmental systems, hematopoietic regulation is mediated by cell autonomous (stem and progenitor cell derived) and instructive (microenvironmental) mechanisms. Despite many years of effort in the murine and human systems, the mechanistic features of self-renewal versus commitment decisions, lineage partitioning choices, and other aspects of stem and progenitor cell biology, such as proliferative and migratory phenomena, have remained obscure. Most of the available information has been obtained by studies focused on individual gene-products such as transcriptional regulators and cell surface receptor-ligand pairs. While informative, these studies have not provided systems that permit the ex vivo expansion or directed differentiation of primitive hematopoietic cell populations. We have suggested that the regulation of hematopoietic stem cells should be viewed in a collective, systems biology manner. Thus, the control of cell fate choices is a property of numerous interacting regulatory pathways and networks. In order to set the stage for such a systems biology approach, we have attempted to describe the genetic program or molecular "parts list' characteristic of early stages in the murine hematopoietic cell hierarchy, and of its supportive microenvironmental niches. These global genomic efforts are nearing completion, and have yielded numerous functional insights into stem cell regulation. A key feature of our efforts is the development of web-based interactive databases to facilitate information dissemination within the scientific community. In the present application, we will extend our efforts to the human hematopoietic system, and merge the molecular information obtained in the two species. An emphasis will be placed on identifying regulatory features shared by mouse and man, and on initiating functional genomic studies, with these shared features as a guide. We will continue to employ sophisticated computational strategies in our data analysis, and further develop the databases for the scientific community.
Gene Expression and Discovery in Liver, Gut and Hematopoietic Stem Cell
Gretchen J. Darlington, Ph.D.
Baylor College of Medicine, Houston, TX
Abstract:
This project will coordinate several investigators with expertise in hematopoietic stem cell isolation (Goodell), liver cell biology (Darlington, Weiss), biostatistics (Shaw), cDNA microarray manufacture and analysis (Darlington, White), gene profiling by a novel sequence based strategy (Gunaratne), and database management and data distribution (Steffen, Shaw). With the exception of Dr. Weiss, these investigators are faculty at Baylor College of Medicine. The aims of the application are to characterize populations of putative stem cells from the liver and cells obtained from embryonic liver that are able to be maintained in culture and that show the capacity to differentiate in vitro. Phenotypic characterization of these cells will be done using markers of other well described stem cell populations and with markers of mature liver cells. Putative stem cell populations will be assayed by transplantation to the liver using the fumarylacetoacetate hydrolyze (FAH) deficient mouse. Microarray and sequence-based gene profiling will be performed on primitive and differentiated populations. Expression profiles from murine hematopoietic stem cells will be similarly obtained from stem cells in different states of activation. The data from these characterizations will be organized and distributed through a web site using software and interfaces that will permit easy access by the research community. Protocols, custom arrays and cell lines will be distributed on request.
The Osteoprogenitor Lineage
David W. Rowe, M.D.
Unversity of Connecticut SCH of Med/DNT, Farmington, CT
Abstract:
The osteoprogenitor lineage provides a renewing source of osteoblast for embryogenesis, somatic growth, bone fracture repair and states of estrogen deficiency. Many diseases of bone can be viewed as a failure of the lineage to provide sufficient numbers of functional to meet the mechanical load place on the skeleton. This resource development application is designed to implement a comprehensive strategy for recognizing stages of differentiation within the lineage and to appreciate the RNA expression profile in relatively homogeneous population of cells at defined levels of differentiation. There are nine elements of the grant submitted by a complementary group of cell and molecular, mouse geneticists, computational engineers and computer scientists. The project is based on the use of promoter-GFP transgenes that active at defined stages within the osteoblast lineage. This allows the lineage to be characterized in vitro (project 1) or in vivo (project 2). Both approaches use a computer controlled microscopic work station that allows repetitive images to be recorded in tissue culture or histological section. Digital analysis of the images will allow the lineage to be defined in quantitative terms. A bone focused microarray library will be assembled (project 3) so that informative expression studies can be performed of GFP positive population of cells isolated by FAC sorting (project 1). This library will be annotated with particular reference to molecular pathway and its relationship to bone biology (project 4). Data from a microarray study will be tested for statistical significance prior to being place into standard and newly developed clustering algorithms (project 5). A second feature of the strategy is that all the murine models will be assessed in a uniformly heterozygous environment found in F1 mice of C57/B16 and C3H mice. Project 6 provides the discipline to build the congenic inbred lines from which the testor F1 mice will be produced. The applicability of the reagents and protocols for general use by bone biologists will be evaluated by 3 investigators at the Health Center using murine models that perturb the osteoprogenitor pathway. The data that is generated from control and mutant mouse models will be organized in a cartoon based query database (project 8). The strategies, reagents and protocols that prove to be valuable to defining lineage performance and molecular pathways will be accessible from a web interface.
Urologic Epithelial Stem Cells
Alvin Y. Liu, Ph.D.
University of Washington, Seattle, WA
Abstract:
A population of epithelial progenitor cells with self-renewal capability resides in the adult human prostate and urinary bladder. Induced by diffusible signals, and cell-cell interaction with the proper stromal mesenchyme cells; the progenitor cells undergo differentiation to produce functionally mature cells. Thus, progenitor cells from either the prostate or the bladder will develop into prostatic structures when triggered by prostate-inducing stromal cells. For cell type analysis we will use the expression of cluster designation (CD) cell surface molecules to identify cell types in lineages. CD molecules are differentially expressed among the component cell types of organs, and are therefore ideally suited to such analysis. We propose to study the process of prostatic epithelial cell differentiation and to demonstrate possible genetic disorder that leads to abnormal cell proliferation characteristic of the common prostatic diseases of benign hyperplasia and cancer. Our objectives are: (1) to use fluorescent CD antibodies and confocal laser scanning microscopy to show that the progenitor cells found in diverse organs are phenotypically alike whereas the differentiated cells are phenotypically dissimilar with respect to their CD expression, and that distinct intermediate cell types can be identified; (2) to use flow cytometry to characterize and sort by CD expression (in particular, CD49b; CD49f, and CD71) progenitor cells, which are postulated to comprise a similar proportion in these and other organs; (3) to show by in vitro 3-D cell cultures that prostate morphogenesis (formation of ducts and glands) can be modeled with progenitor cells and stromal cell derived factors to give rise to the differentiated cell type of secretory luminal cells (one of the principal stromal factors is hepatocyte growth factor (HGF), which activates the morphogenetic program through its binding to tyrosine kinase receptor c-met on responsive epithelial cells); (4) to show that functional differentiation (as indicated by the synthesis of the abundant secretory protein, prostate-specific antigen (PSA), by luminal cells) requires epithelial/stromal cell-cell interaction; (5) to show that the differentially expressed CD10 and CD13 ectopeptidases play a role in luminal cell differentiation, since cancer cells, which are luminal-like, lack CD10 and CD13 expression; (6) to determine the transcriptome of progenitor cells, to identify differentially expressed genes between progenitor and differentiated cells, to screen for genes expressed by stromal cells that induce prostate epithelial differentiation; and (7) to build a public database of the experimental results obtained.
Towards Renal Regeneration
Melissa Little, Ph.D.
University of Queensland, Queensland, Australia
Abstract:
Chronic renal failure is both devastating to the individual and expensive to treat. In 1998, there were 87,000 new cases of end-stage renal disease (ESRD) in the US, taking the total patient number to 400,000. This resulted in 63,000 deaths and cost $US16.74 billion. Current treatment options for ESRD are dialysis and renal transplantation. Dialysis is expensive (($USD15-25,000/year/patient), results in a poor quality of life and a high yearly mortality rate ((16%). Kidney transplantation, although requiring immunosuppression, is preferable to dialysis, but due to a decrease in cadaveric donors worldwide only a quarter of patients awaiting transplantation will receive this treatment. Compounding the problem is a steady increase in the rate of ESRD worldwide primarily due to an increase in Type II diabetes. Several alternative treatment options are being investigated to treat chronic renal disease, including pig xenotransplantation and bioartificial kidney devices. In this application we will investigate the ability to treat chronic renal disease using stem cells. Two long-term approaches to renal regeneration will be investigated: de novo renal generation and endogenous renal repair. Both of these will require the induction of embryonic or adult stem cells to adopt a renal progenitor fate and then the isolation of these cells via specific markers. De novo generation of a replacement organ would then involve aggregation of renal progenitors and implantation of these aggregates into the omentum for vascularisation, together with reconnection to the excretory tract via a replacement ureter. Endogenous renal repair would involve the reintroduction and integration of induced and isolated renal progenitor cells into the damaged kidney. To reach these clinical objectives, we propose the following basic research objectives: (1) Use expression profiling to further dissect the processes of commitment to a renal fate during normal development;(2) Examine the potential for embryonic and adult stem cells to be differentiated into the lineages necessary for renal regeneration; (3) Identify novel renal progenitor cell markers and growth factors to assist in the identification, isolation and / or reactivation of renal stem cells; and (4) Utilize pathological and functional assays to determine the in vivo outcomes of de novo organ generation and renal repair. Human ES cell work will be performed using ES01, 02, 03, 04, 05 & 06 listed on the NIH ES cell line registry.
Gastric and Intestinal Epithelial Progenitor Cell GAP
Jeffrey I. Gordon, M.D.
Washington University, St. Louis, MO
Abstract:
The human gut epithelium undergoes continuous rapid renewal throughout life. A major unfulfilled goal of GI research is to define the properties of the stem cells that fuel this renewal. The results will have important therapeutic and diagnostic implications for diseases as varied as GI neoplasia, the severe mucositis that accompanies radio- or chemotherapy, and the loss or disruption of intestinal absorptive function due to inflammatory bowel diseases, or surgical resection. We are proposing a gut epithelial cell progenitor GAP that uses two gnotobiotic transgenic mouse models developed in the laboratory of the Principal Investigator as part of its long-standing examination of gut stem cell biology. The representation of multipotent gastric stem cells and their immediate committed daughters is dramatically increased in one of these models through ablation of acid-producing parietal cells. In another model, the representation of intestinal epithelial progenitors is augmented markedly through removal of Paneth cells from the base of crypts of Lieberkuhn. Each model provides a unique opportunity to directly recover progenitors from anatomically distinct structures by laser capture microdissection (LCM) in sufficient numbers and purity to profile expression of their component mRNAs. Our goal is to provide the most comprehensive profile of progenitor cell gene expression possible: i.e., one that includes low abundance transcripts. To accomplish this goal, we have formed a consortium that includes members of Washington University's Genome Sequencing Center, and Michael Lovett (Professor of Genetics), an expert in the development of methods for creating normalized cDNA libraries from small numbers of cells. This GAP has 2 aims: (I) Generate and sequence normalized and subtracted cDNA libraries from LCM mouse gastric and intestinal epithelial progenitors. We will provide the public with annotated, Internet-accessible databases of expressed genes, and create tools that will allow our databases to be used as probes for examining other expression profiles. Sequenced clones will be distributed to the research community through the I.M.A.G.E. Consortium. (2) Relate what we find in mice to the human gut. Lessons learned from the adult mouse during the course of this GAP will serve as a template for a directed and focused exploration of the features of adult human gut epithelial progenitors. This will be accomplished using a 3-tier expression profiling scheme. (1) A subset of genes will be selected from our three normalized and two subtracted libraries and their expression patterns analyzed in normal adult FVBIN mouse gut using sensitive SYBR-Green based real time quantitative RT-PCR (qRT-PCR) assays of LCM epithelial cells harvested from progenitor and non-progenitor compartments within the gastric unit, the small intestinal crypt-villus unit, and the colonic crypt. (2) A subset of genes from (1) will be selected for LCM/qRT-PCR expression profiling of human gastric units, small intestinal crypt-villus units, and colonic crypts. (3) In situ hybridization studies of the adult mouse and human gut will be used to obtain a more refined view of the cellular basis of expression of genes selected from (1) and (2).
