Kinetic appearance of actively cycling cells from human BM and MPB CD34⁺ Thy-1⁺ Lin⁻ and CD34⁺ Thy-1⁻ Lin⁻ subsets during in vitro culture. BM CD34⁺Thy-1⁺Lin⁻ (a through e), BM CD34⁺Thy-1⁻Lin⁻ (f through j), MPB CD34⁺Thy-1⁺Lin⁻ (k through o), and MPB CD34⁺Thy-1⁻Lin⁻ (p through t) cells were isolated and were cultured on a murine BM stromal layer SyS-1 in the presence of IL-3, IL-6, SLF, GM, and FLK2L. At each time point, these cells were stained with Hoechst 33342 for analysis of DNA content and with MoAbs specific for CD34 and Thy-1. The FACS plots show the expression of Thy-1 versus Hoechst 33342 staining in viable cells. Cells were analyzed at t = 0 (a, f, k, and p), 12 (b, g, l, and q), 24 (c, h, m, and r), 36 (d, l, n, and s), and 48 (e, j, o, and t) hours. The percentage of cells in each quadrant of the plot is shown. At 48 hours, approximately 94%, 74%, 97%, and 94% of cells in the culture initiated from BM CD34⁺Thy-1⁺Lin⁻ (e), BM CD34⁺Thy-1⁻Lin⁻ (j), MPB CD34⁺Thy-1⁺Lin⁻ (o), and MPB CD34⁺Thy-1⁻Lin⁻ (t), respectively, were CD34^hi.