Figure 3. Epithelial multilineage differentiation of Ccsp⁺ BMCs.

(A) Isotype mix control staining of airway epithelial cells showed no cross-reactivity of the antibodies. (B) Control staining of isolated airway epithelial cells for each antibody. Bleed-through of signal is visualized in depicted representative images. Scale bars: 20 μm. (C) After 4 weeks in ALI culture, Ccsp⁺ cells gave rise to a multitude of epithelial lineage cells including proSpc⁺, proSpc⁺Aqp5⁺, proSpc⁺K14⁺, K14⁺, acetylated α-tubulin⁺, and acetylated α-tubulin⁺proSpc⁺ cells, whereas Ccsp^– cells only gave rise to proSpc⁺ cells. White arrows point to double-positive cells. Hoechst counterstain was used to visualize nuclei (blue). Numbers in parentheses indicate the fluorescent emission wavelengths for the stains. Scale bars: 10 μm. Original magnification, ×40. (D) Real-time PCR confirmed expression of various epithelial genes in ALI-cultured Ccsp⁺ cells. Ccsp⁺ cells expressed various epithelial genes including Ccsp, K5, K14, K18, Aqp5, and proSpc, whereas Ccsp^– cells only expressed proSpc. Gapdh was used as housekeeping gene for normalization of expression levels. Ccsp-sorted cells were cultured in a mixture of bone marrow– and epithelial cell–specific media and in ALI conditions to induce epithelial differentiation. Each bar represents normalized relative levels compared with tracheal epithelial cells. n = 4 sets of cells from 4 different animals.