Using the approach shown in Figure 2, we have identified genes which illustrate different principles of stem cell gene expression. We first identify expression profiles in adult and fetal murine hematopoietic stem cells (HSCs). Together, these define a common HSC profile. This profile should contain gene products needed for HSC function throughout development. We have also generated expression profiles for human HSC and murine neural stem cells (NSCs) and embryonic stem cells (ESCs). The common HSC profile above was compared to each of these SC profiles. The murine-human comparison should indicate regulatory functions that are conserved through evolution. The comparison to other SC fractions should indicate SC-shared biological processes.

To assign probes to an expression cluster, we defined a collection of hypothetical expression patterns for BM and FL hematopoietic hierarchies as shown in Figure 1. These patterns correlate with the functional properties of key hematopoietic stages. Combinatorial patterns were built from these tissue-specific patterns. The Pearson correlation coefficient of the normalized expression profile to each of the hypothetical patterns. Each probe was assigned to the expression cluster that had the highest correlation coefficient with the normalized readings of that probe. Additional criteria were used to increase the stringency of selection. For example, HSC-specific probes must shown at least two-fold increase in expression relative to the corresponding MBC fraction.

Gene products were grouped into categories according to their function as reported in the literature or as predicted on the basis of the presence of diagnostic protein motifs. Regulatory molecules, such as transcription factors, proteins involved in intracellular signaling, cell-surface receptors, and ligands account for 45% of the HSC-related gene-products.

Sorted cell populations were plated into cytokine-enriched semi-solid media, 100 cells per ml, in triplicate. Colonies were scored 10-12 days after plating as High-Proliferative-Potential (HPP; > 1 mm in diameter)-Granulocyte Macrophage (GM), GM, MIX (3 or more lineages; G, M, megakaryocyte, erythrocyte), and HPP-MIX. Media containing 50 ng/ml recombinant mouse (rm) Stem Cell Factor, 01 ng/ml rm IL-3, 10 ng/ml rhuman (rh) IL-6, and 3 units/ml rh Erythropoietin, (Methocult™ GF M3434) was obtained from Stem Cell Technologies Inc., Vancouver, British Columbia.