Single Cell Phospho-Flow Analysis of Cytokine Stimulation in Human Hematopoietic Progenitors Reveals That G-CSF Acts Directly On Human Hematopoietic Stem Cells.

Abstract

Abstract 3617Poster Board III-553Hematopoietic stem and progenitor cells (HSPC) give rise to all blood cell lineages for the lifetime of an individual. However, their low frequency has precluded the use of traditional biochemical methods to study their signaling biology. We developed a phospho-specific flow cytometry approach that allows for the simultaneous interrogation of multiple elements of signaling networks in primary human HSPC at the single cell level without prospective purification by cell sorting. We found that HSPC contain biochemically distinct signaling subpopulations, and that, although considered quiescent, HSC from cord blood and adult bone marrow respond directly to several cytokine stimuli through multiple signaling nodes including JAK/Stat, Erk, and Akt. Surprisingly, we found that a subset of HSC respond rapidly and directly to in vitro G-CSF stimulation through Stat3 signaling. The addition of G-CSF to purified cultures of HSC significantly increased the percentage of cells that had entered the cell cycle. Moreover, when G-CSF was added to cultures of purified human HSC spatially segregated in hydrogel microwell arrays, we observed a significant increase in the frequency of HSC proliferation. These results demonstrate that contrary to the accepted paradigm, G-CSF acts directly on human HSC. Cytometric single cell characterization of the molecular behavior of HSPC in a wide variety of hematopoietic malignancies can now be applied to study the signaling behavior of other primary and rare stem cell populations. Disclosures:Weissman:U.S. Patent Application 11/528,890 entitled “Methods for Diagnosing and Evaluating Treatment of Blood Disorders.”: Patents & Royalties; Stem Cells Inc.: Cofounder and Director; Cellerant Inc.: Cofounder; Amgen: Equity Ownership. Nolan:BD Biosciences: Consultancy.