FIG. 2.

Serial cloning analysis for measuring self-renewal of human endometrial large and small primary (1°) epithelial and stromal CFU. A) Schematic showing serial cloning strategy. The initial cloning plate (seeded at 10–20 cells/cm²), second panel with two cloning rings selecting the largest (>4000 cells), and a small to medium (<2000 cells) CFU. These clones were individually replated at 5–10 cells/cm² and cultured for 14 days. Serial clonal passaging (2°–5°) is depicted as cloning plates containing representative clones, with selection of typical large clones for the subsequent round of cloning indicated within cloning rings, until CFU activity was exhausted (4°/5°). Typical endometrial epithelial (B) and stromal (C) colonies formed at each round of serial cloning. Endometrial epithelial colonies were cytokeratin⁺ (CK) and stromal colonies THY1 (CD90⁺) (second panels). Inserts are isotype controls. Rate of clonal extinction is shown for both large and small CFU for each round of serial cloning for epithelial (D) and stromal (E) CFU. Percentage of CFU in large and small epithelial (F) and stromal (G) CFU at each round of serial cloning. Results are means ± SEM (n = 3 patient samples [from nos. 7–9 for epithelial and nos. 1–4 for stromal); averages of three to five small and large CFU/cell type/patient sample). *Significant difference between large and small CFU (P < 0.05). Bars = 1 mm for clones (C); bars = 50 μm for immunostained images, including insets (B and C).