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 (micro environmental) mechanisms. We have suggested that the regulation of hematopoietic stem cells should be viewed in a collective, systems biology manner. Taking this 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.

The major challenges facing the hematopoietic society field are

1. to identify the cell autonomous and microenvironmental molecular "parts lists" that define the stem and progenitor cell "states", and their developmental abilities;
2. to assemble these individual components into interacting regulatory pathways and networks.

For over five years we have addressed these challenges largely in the murine system. In the present application, we will extend our efforts to the human hematopoietic system, and merge the molecular information obtained in the two species, with these shared features as a guide. We will continue to employ sophisticated computational strategies in our data analysis, and further develop the database for the scientific community.

Using well-characterized purified populations of human stem and progenitor cells, gene expression profiles that correlate with distinct biological activities will be identified. In order to sharpen our focus on the functionally most important gene products, we will use four distinct sources of biologically similar hematopoietic stem and projector cells:

• Bone Marrow (BM)
• Umbilical Cord Blood (UCB)
• Fetal Liver (FL)
• Mobilized Peripheral Blood (MPB).

We will also integrate the molecular phenotype data sets from the human and routine systems. Our studies will combine diverse functional, molecular and computational approaches. A major and immediate goal will be to provide an on-line infrastructure for the rapid dissemination of experimental data and analytical results as they are acquired during the courses of the project.

High-density microarray and chip technologies will be employed to analyze how the molecular phenotypes (defined in Specific Aim 1) are modulated when stem and progenitor cells undergo functionally defined changes in their biological properties. One consequence of these efforts will be the construction of the human "stem cell chip" that will be made available to other investigators. For our experiments we will employ distinct in vitro situations that induce differentiation, stem cell maintenance, and possibly self-renewal. A particular emphasis will be placed on the elucidating the molecular "cross-talk" or "dialogue" that occurs between stem cells and their supportive microenvironments. The utility of gain and loss-of-function strategies to modulate the expression levels of selected gene products in stem cells will be explored: For all of the studies in this Aim, major emphases will be placed on computational analyses, on the integration of microarray data with global molecular phenotypes, and on the rapid on-line provision of experimental results.