Purified CCE-25–c-Kit^negSca-1^pos and Sca-1^neg cell growth on Tpo-LTMC stroma. Tpo-LTMCs were established according to procedures outlined in “Materials and methods.” Tpo-LTMCs (4-week-old; Ly 5.1 marked) were irradiated (13.0 Gy), nonadherent cells were removed, and the remaining stromal layers were seeded with purified, Ly 5.2^pos, whole bone marrow (positive control), CCE-25–c-Kit^negSca-1^pos, or Sca-1^neg cells. (A) Flow cytometry for c-Kit and “donor” (Ly 5.2) cells after 25 days of culture. Panels are representative of 2 flasks per group in 2 separate experiments. (B) Lineage composition of CCE-25–c-Kit^neg (donor, Ly 5.2) cells grown in the Tpo-LTMCs. After 25 days of culture, the contents of the Tpo-LTMCs ± purified CCE-25–c-Kit^neg (Ly 5.2) cells were analyzed by flow cytometry for Ly 5.2– and lineage-specific markers. Left panel: Tpo-LTMCs alone; middle panel: 4 × 10³ CCE-25–c-Kit^negSca-1^pos cells; and right panel: 4 × 10⁵ CCE-25–c-Kit^negSca-1^neg cells. Data shown are from 1 flask, which represent 2 flasks per group in 2 separate experiments. (C) Cells from the Tpo-LTMC stromal layers were harvested 25 days after CCE-25–c-Kit^negSca-1^pos (Ly 5.2) seeding and then transplanted into lethally irradiated (9.5 Gy) Ly 5.1 recipients with 2 × 10⁵ wBMC competitive host marrow. Peripheral blood was assayed for the presence of donor lymphoid (CD3ϵ and B220) and myeloid (Gr-1 and Mac-1) cells by flow cytometry as described in “Materials and methods.” The top panels show the distribution of donor-derived cells from animals that received transplants of 2 × 10⁵ whole bone marrow cells (positive control). The percentages in the upper right corners of the dot plots represent the number of donor cells in the Lin^pos cell population. For these analyses, background staining, indicated by staining with isotype-matched controls, was subtracted to yield the percentage of cells shown on the dot plots.