TGF-β induces HSC hibernation ex vivo. (A) Freshly isolated CD34⁻KSL HSCs and CD34⁺KSL hematopoietic progenitor cells were sorted clonally into 96-well microtiter plates and incubated in the presence of SCF, TPO, and TGF-β1. At day 5 of culture, cell viability and cell numbers were assessed under an inverted microscope. (B) Freshly isolated CD34⁻KSL HSCs were sorted clonally into 96-well microtiter plates and incubated in the presence of SCF, TPO, and TGF-β1. At the indicated time points, 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 morphologic examination. As a control, freshly isolated CD34⁻KSL HSCs were sorted clonally into 96-well microtiter plates supplemented with SCF, TPO, IL-3, and EPO and cultured for 14 days. Proportions of colony types are depicted: n indicates neutrophils; m, macrophages; E, erythroblasts; and M, megakaryocytes. (C) Freshly isolated CD34⁻KSL HSCs were sorted clonally into 96-well microtiter plates and were incubated in the presence of SCF, TPO, and TGF-β1. At day 5 of culture, surviving single HSCs that had not divided during 5-day culture were selected. Single HSCs or pools of 20 single HSCs (indicated by STT day 5; B6-Ly5.1) were mixed with B6-Ly5.2 competitor cells and injected into lethally irradiated B6-Ly5.2 recipient mice. As a control, freshly isolated single-HSC or 20-HSCs 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 more than 1.0% for myeloid and for B- and T-lymphoid lineages were considered multilineage reconstituted (positive mice).