Hibernating HSCs present a striking contrast to cycling progenitors in their lipid raft organization and cytokine signaling. (A) Cell cycle status of hematopoietic stem and progenitor cells. Freshly isolated BM KSL cells were analyzed for their expression of CD34 and Ki-67 or CD34 expression and Pyronin Y incorporation on a FACS Vantage. (B) Lipid raft organization and cytokine signaling in HSCs. Freshly isolated CD34⁻KSL HSCs and CD34⁺KSL hematopoietic progenitor cells were stained with DAPI (blue) and with an anti-c-Kit, an anti-phospho-Akt, or an anti-FOXO3a antibody (red). Lipid raft distribution was assessed by detecting GM1 ganglioside, a component of lipid rafts, with FITC-conjugated CTB, which marks endogenous GM1 ganglioside (green). Proportions of cells that showed lipid raft clustering or cluster formation are indicated as gray bars (Cluster+) (right panel).

Inhibition of lipid raft clustering attenuates cytokine signals and maintains p57 expression in HSCs. (A) Inhibition of cytokine-induced lipid raft clustering maintains repression of the Akt–FOXO pathway. Freshly isolated CD34⁻KSL HSCs were incubated for 30 min in the presence or absence of SCF and TPO (S+T and Cyto−, respectively), or were preincubated with MβCD for 30 min and further incubated in the presence of SCF and TPO for 30 min (MβCD+S+T). The cells were stained with DAPI (blue), CTB (green), and either an anti-phospho-Akt (red) or an anti-FOXO3a antibody (red). The proportions of CD34⁻KSL HSCs that showed lipid raft cluster formation are indicated as gray bars (Cluster+) (right panel). (B) Subcellular localization of cyclin D1, cyclin D2 and cyclin D3 in HSCs. Freshly isolated CD34⁻KSL HSCs (CD34−) and CD34⁺KSL hematopoietic progenitor cells (CD34+) were incubated for 30 min in the presence or absence of SCF and TPO (S+T and Cyto−, respectively) or were preincubated with MβCD for 30 min and further incubated in the presence of SCF and TPO for another 30 min (MβCD+S+T). The cells were stained with DAPI (blue) and with an anti-cyclin D1 antibody (red). (C) Subcellular localization of p57 in HSCs. Freshly isolated CD34⁻KSL HSCs (CD34−) were preincubated with and without MβCD for 30 min and further incubated in the presence of SCF and TPO for 12 h (MβCD+S+T and S+T, respectively). The incubated cells, together with freshly isolated CD34⁻KSL HSCs (CD34−, Cyto−) and CD34⁺KSL hematopoietic progenitor cells (CD34+, Cyto−), were stained with DAPI (blue) and with an anti-p57 antibody (green)(upper panel). mRNA expression for mouse Cip/Kip genes is presented (lower panel). Cells analyzed are BM CD34⁻KSL HSCs (CD34−), CD34⁺KSL progenitors (CD34+), and Lineage marker⁻ cells (Lin⁻).

Inhibition of lipid raft clustering attenuates cytokine signals and induces HSC hibernation ex vivo. Freshly isolated CD34⁻KSL HSCs were sorted clonally into 96-well micro-titer plates and were incubated in the presence of SCF, TPO, and 1 mM MβCD (STM). At the indicated times, surviving single HSCs that had not divided during 5- to 7-day culture were selected. Single HSCs or pools of 20 single HSCs (B6-Ly5.1) were mixed with ‘competitor cells from B6-Ly5.1 XLy5.2 F1 mice and injected into lethally irradiated B6-Ly5.2 recipient mice. As a control, freshly isolated single HSCs or 20-HSC pools were similarly transplanted into recipient mice. Percentage chimerism of donor cells in recipient mice 12 weeks after transplantation is plotted as dots, with mean values indicated as bars. Recipient mice with donor cell chimerism >1.0% for myeloid and for B- and T-lymphoid lineages were considered multilineage-reconstituted (positive mice).

Cytokine stimulation induces lipid raft clustering and activation of the Akt-FOXO signaling pathway in HSCs. Freshly isolated CD34⁻KSL HSCs were incubated for 30 min in the presence or absence of cytokines (SCF and/or TPO) and were stained with DAPI (blue), CTB (green), and an anti-c-Kit (red) (A), an anti-phospho-Akt (red), or an anti-FOXO3a antibody (red) (B). Proportions of CD34⁻KSL HSCs that showed lipid raft cluster formation in response to the indicated cytokines are depicted as gray bars (Cluster+) (A: right panel).

Lipid raft reorganization is essential for hibernating HSCs to re-enter the cell cycle. (A) Freshly isolated CD34⁻KSL HSCs (CD34−) and CD34⁺KSL hematopoietic progenitor cells (CD34+) were sorted clonally into 96-well micro-titer plates and incubated in the presence of SCF, TPO, and 1 mM MβCD (STM). After the indicated culture periods, cell viability and cell numbers were assessed under an inverted microscope. (B) Freshly isolated CD34⁻KSL HSCs (CD34−) were sorted clonally into 96-well micro-titer plates and incubated in the presence of SCF, TPO, and 1 mM MβCD (STM). At the indicated times, surviving single HSCs that had not divided were selected. Culture medium was replaced with one supplemented with SCF, TPO, IL-3, and EPO, permitting colony formation. After 14 subsequent days of culture, the colonies were recovered for morphological examination. As a control, freshly isolated CD34⁻KSL HSCs were sorted clonally into 96-well micro-titer plates supplemented with SCF, TPO, IL-3, and EPO and cultured for 14 days. Proportions of colony types are depicted: n, neutrophils; m, macrophages; E, erythroblasts; M, megakaryocytes.