Characterization of side population cells from human airway epithelium

Abstract

The airway epithelium is the first line of contact with the inhaled external environment and is continuously exposed to and injured by pollutants, allergens, and viruses. However, little is known about epithelial repair and in particular the identity and role of tissue resident stem/progenitor cells that may contribute to epithelial regeneration. The aims of the present study were to identify, isolate, and characterize side population (SP) cells in human tracheobronchial epithelium. Epithelial cells were obtained from seven nontransplantable healthy lungs and four asthmatic lungs by pronase digestion. SP cells were identified by verapamil-sensitive efflux of the DNA-binding dye Hoechst 33342. Using flow cytometry, CD45(-) SP, CD45(+) SP, and non-SP cells were isolated and sorted. CD45(-) SP cells made up 0.12% +/- 0.01% of the total epithelial cell population in normal airway but 4.1% +/- 0.06% of the epithelium in asthmatic airways. All CD45(-) SP cells showed positive staining for epithelial-specific markers cytokeratin-5, E-cadherin, ZO-1, and p63. CD45(-) SP cells exhibited stable telomere length and increased colony-forming and proliferative potential, undergoing population expansion for at least 16 consecutive passages. In contrast with non-SP cells, fewer than 100 CD45(-) SP cells were able to generate a multilayered and differentiated epithelium in air-liquid interface culture. SP cells are present in human tracheobronchial epithelium, exhibit both short- and long-term proliferative potential, and are capable of generation of differentiated epithelium in vitro. The number of SP cells is significantly greater in asthmatic airways, providing evidence of dysregulated resident SP cells in the asthmatic epithelium. Disclosure of potential conflicts of interest is found at the end of this article.

Figure 1

Identification of side population (SP) cells in human airways. Isolated airway epithelial cells from the trachea and bronchus, as shown by FSC and SSC plots (A, E), were 90% viable by propidium iodide staining (B, F). Cells from both the trachea and bronchus were stained with 5 µg/ml Hoechst 33342 alone ([C, G], respectively) or in combination with 50 µg/ml verapamil ([D, H], respectively). Results indicate that a SP cells exist in both the trachea ([C], region R1) and bronchus ([G], region R1) and represent 0.12% ± 0.01% of the epithelial population (n = 7). Detection of SP cells was inhibited by the calcium channel blocker verapamil ([D, H], gate R1). The data shown are representative of all the normal airways analyzed in this study. Following incubation with Hoechst 33342, cells were stained and analyzed by fluorescence-activated cell sorting (FACS) for Bcrp-1 and pan-cytokeratin to confirm epithelial SP phenotype (I, J). Airway SP cells were further segregated using FACS by positive staining for CD45; 93% of airway SP cells were CD45⁻ compared with 7% that were CD45⁺ (K). Abbreviations: Bcrp-1, breast cancer resistance protein; FSC, forward scatter; SSC, side scatter.

Figure 2

Clonogenic capacity and phenotypic characterization of airway SP cells. CD45⁻ SP and non-SP colonies were characterized as either cobblestone (A) or stellate (B) in appearance. CD45⁻ SP colonies were stained with antibodies for basal epithelial markers ΔNp63 (C), cytokeratin-5 (D), CD151 (E), tissue factor (F), E-cadherin (G), ZO-1 (H), and breast cancer resistance protein 1 (I). There was no expression of mesenchymal marker vimentin, as determined by quantitative polymerase chain reaction (J). (K): Colony-forming capacity of 1 × 10² CD45⁻ SP cells over five passages. This staining pattern was consistent over 16 passages. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p, passage; SP, side population; -ve, negative.

Figure 3

Telomere length is maintained in SP cells. Telomere length normalized to control was analyzed in CD45⁻ SP (black bars) and non-SP cells (gray bars) over three consecutive passages. Values are expressed as mean ± SEM, as results were done in triplicate (n = 3). Abbreviations: SP, side population; -ve, negative.

Figure 4

Capacity of CD45⁻ side population (SP) cells to generate differentiated epithelium. CD45⁻ SP cells seeded at 1 × 10² cells in transwell insert (0.45 µm pore, 12 mm diameter) were able to form a stratified epithelium in air-liquid-interface culture. Differentiation of CD45⁻ SP cells was confirmed by immunostaining with antibodies for basal cell markers ΔNp63 (A), cytokeratin-5 (B), cytokeratin-18 (C), Muc5AC (D), tissue factor (E), and CD151 (F), and the staining was compared with patient matched airway sections (G–L).

Figure 5

Identification of lung SPs in asthmatic airways. Representative fluorescence-activated cell sorting FSC and SSC plots of airway epithelial SP cells in normal and asthmatic pediatric patients ([A, D], region R1) demonstrate a distinct SP that represented 4.01% of the epithelial population in asthmatic pediatric airways ([B], region R1) and 0.11% in normal pediatric airways ([E], region R1). Detection of SP cells was inhibited in the presence of 50 µg/ml verapamil ([C, F], region R1). (G): Mean percentages of SP cells for all normal (■) and asthmatic (▲) individuals in the study; * indicates p < .05. (H, I): Representative images demonstrating ΔNp63 and E-cadherin staining in asthmatic CD45⁻ SP colonies. Abbreviations: FSC, forward scatter; SP, side population; SSC, side scatter.