Identification of Novel Regulators of Hematopoietic Stem Cell Mobilization.

Abstract

In an effort to identify the molecular changes during hematopoietic stem cell (HSC) mobilization we have analyzed genome wide changes in gene expression in HSCs in response to the chemotherapeutic agent cyclophosphamide (Cy) and the growth factor granulocyte colony stimulating factor (G). Cy/G treatment leads to an initial loss of proliferating precursors, followed by a rapid expansion of stem and progenitor cells and their migration to the peripheral blood. While this approach has been capitalized on for harvesting hematopoietic stem cells in clinical therapy, the molecular regulation of this process remains less well understood. To analyze changes in gene expression that occur in vivo as HSCs proliferate and prepare to migrate out of the bone marrow, we enriched for HSC fractions by isolating cells that express c-kit (K), Sca-1(S) and low levels of Thy1.1(T) but do not express lineage (L) markers (KTLS) from untreated mice and mice treated with Cy, G or Cy/G. The gene expression profile of these cells was examined by microarray analysis, relative to HSCs isolated from untreated mice. Interestingly, among the genes upregulated by Cy/G, a large majority (>60%) were a consequence of synergistic activity between Cy and G, while the rest of the upregulated genes could be attributed to activation by Cy or G alone. To test whether this screen allowed identification of novel candidates that regulate HSC function we analyzed the role of transforming growth factor beta induced gene (Big-h3), a gene highly upregulated after Cy/G treatment. Big-h3 is an extracellular matrix protein that mediates the adhesion and migration of various cell types, however its role in HSCs is unknown. Our experiments indicate that overexpression of Big-h3 in HSCs leads to enhanced adhesion to fibronectin as well as upregulation of specific integrins. These experiments suggest that changes in adhesion are an integral component of HSC mobilization, and that Big-h3 is one regulator of such processes. Further functional analysis of other candidates will likely lead to identification of other novel regulators of HSC development and mobilization.