title: Single-cell phospho-specific flow cytometric analysis demonstrates biochemical and functional heterogeneity in human hematopoietic stem and progenitor compartments.
abstract: The low frequency of hematopoietic stem and progenitor cells (HSPCs) in human BM has precluded analysis of the direct biochemical effects elicited by cytokines in these populations, and their functional consequences. Here, single-cell phospho-specific flow cytometry was used to define the signaling networks active in 5 previously defined human HSPC subsets. This analysis revealed that the currently defined HSC compartment is composed of biochemically distinct subsets with the ability to respond rapidly and directly in vitro to a broader array of cytokines than previously appreciated, including G-CSF. The G-CSF response was physiologically relevant-driving cell-cycle entry and increased proliferation in a subset of single cells within the HSC compartment. The heterogeneity in the single-cell signaling and proliferation responses prompted subfractionation of the adult BM HSC compartment by expression of CD114 (G-CSF receptor). Xenotransplantation assays revealed that HSC activity is significantly enriched in the CD114(neg/lo) compartment, and almost completely absent in the CD114(pos) subfraction. The single-cell analyses used here can be adapted for further refinement of HSPC surface immunophenotypes, and for examining the direct regulatory effects of other factors on the homeostasis of stem and progenitor populations in normal or diseased states.
authors.affiliation: Program in Immunology, Stanford University School of Medicine, Stanford, CA, USA; Baxter Laboratories for Stem Cell Biology, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Gibbs
authors.forename: Penney M
authors.initials: PM
authors.lastname: Gilbert
authors.forename: Karen
authors.initials: K
authors.lastname: Sachs
authors.forename: Feifei
authors.initials: F
authors.lastname: Zhao
authors.forename: Helen M
authors.initials: HM
authors.lastname: Blau
authors.forename: Irving L
authors.initials: IL
authors.lastname: Weissman
authors.forename: Garry P
authors.initials: GP
authors.lastname: Nolan
authors.forename: Ravindra
authors.initials: R
authors.lastname: Majeti
mesh_terms: D000328:Adult; D000818:Animals; D002453:Cell Cycle; D016207:Cytokines / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D016179:Granulocyte Colony-Stimulating Factor / Q000276:immunology; D006410:Hematopoiesis; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology*; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000276:immunology; D059010:Single-Cell Analysis / Q000379:methods*; D014183:Transplantation, Heterologous
chemical_list: D016207:Cytokines; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D016179:Granulocyte Colony-Stimulating Factor