Isolation of stem/progenitor cells from normal lung tissue of adult humans

Abstract

Objectives: This study aimed to isolate and characterize stem/progenitor cells, starting from normal airway epithelia, obtained from human adults.

Materials and methods: Cultures of multicellular spheroids were obtained from human lung tissue specimens after mechanical and enzymatic digestion. Tissue-specific markers were detected on their cells by immunohistochemical and immunofluorescent techniques. Ultrastructural morphology of the spheroids (termed as bronchospheres) was evaluated by electron microscopy, gene expression analysis was performed by reverse transcription-polymerase chain reaction, and gene down-regulation was analysed by an RNA interference technique.

Results: Bronchospheres were found to be composed of cells with high expression of stem cell regulatory genes, which was not or was only weakly detectable in original tissues. Morphological analysis showed that bronchospheres were composed of mixed phenotype cells with type II alveolar and Clara cell features, highlighting their airway resident cell origin. In addition to displaying specific pulmonary and epithelial commitment, bronchospheres showed mesenchymal features. Silencing of the Slug gene, known to play a pivotal role in epithelial-mesenchymal transition processes and which was highly expressed in bronchospheres but not in original tissue, led bronchospheres to gain a differentiated bronchial/alveolar phenotype and to lose the stemness gene expression pattern.

Conclusions: Ours is the first study to describe ex vivo expansion of stem/progenitor cells resident in human lung epithelia, and our results suggest that the epithelial-mesenchymal transition process, still active in a subset of airway cells, may regulate transit of stem/progenitor cells towards epithelial differentiation.

Figure 1

Multicellular bronchospheres can be reliably generated from normal adult lung tissues. Inverted phase‐contrast microscopy (Olympus IX51) of primary bronchospheres (I) obtained from lung tissue, and secondary bronchospheres (II) derived from primary bronchospheres cultured in serum‐free medium. Scale bar, 100 µm.

Figure 2

Bronchospheres (BS) express higher levels of stem cell regulatory genes than original tissue. (A) Number of primary BS and original tissue samples expressing stem cell markers (P‐value according to McNemar's test) and mean expression levels of stem cell markers (P‐value according to paired sample t‐test). (B) mRNA levels of stem cell markers analysed by RT‐PCR in original tissue and in BS (representative samples). GAPDH was used as a loading control. Images represent similar results. (C) Immunofluorescence analysis of Oct 3/4 and CD133 in BS. Scale bar, 20 µm.

Figure 3

Ultrastructural morphology and molecular characterization of bronchospheres (BS) reveal features of type II pneumocytes, Clara cells and mesenchymal cells. (A) Electron microscopical analysis: (a, b) features of type II pneumocytes: lipid droplets (L), intracellular lamellar bodies (arrow) and extracellular concentric lamellae (arrow head); (c) nondescript peripheral cells with a flat surface, cytoplasmic projections and microvilli (Mv) not ascribable to any lung cell type previously described; (d) features of Clara cells: dilated cisternae of rough endoplasmic reticulum with large round electron‐dense granules (arrow). Scale bar, 1 µm. (B) Electron microscopical analysis of BS. Left panel shows cells with mesenchymal features: large euchromatic nucleus (N), cytoplasmic vimentin‐like intermediate filaments (v), paired subplasmalemmal linear densities (arrow) with converging actin microfilaments (arrow head); middle and right panels show luminal cells with well‐developed tight junctions (arrow head), microvilli (Mv), lipid droplets (L) and lamellar bodies (arrows). Scale bar, 1 µm. (C) Immunofluorescence and immunohistochemical analyses of vimentin and immunofluorescence analysis of TTF‐1 protein expression in BS. Scale bar, 20 µm (D) Immunofluorescence analysis of CD44, cytokeratin 5, SP‐A, CC10 protein expression in BS. Scale bar, 20 µm. (E) Representative RT‐PCR analysis of levels of CD44, cytokeratin 5, and SP‐A mRNA, proportion of BS and original tissue expressing these markers, and comparison of mean expression levels of CD44 in primary BS and in original tissue, performed by real‐time RT‐PCR.

Figure 4

Bronchospheres (BS) show a mixed mesenchymal/epithelial phenotype. (A) Immunofluorescence analysis of CD105, CD90, CD34, and CD45, and immunohistochemical analysis of factor VIII and CD45 protein. (B) Immunofluorescence and RT‐PCR analyses of cytokeratins 8 and 19.

Figure 5

Bronchospheres (BS) differentiate into bronchial/alveolar cells. (A) Comparison between BS and BS‐derived epithelia (BSDE): phase contrast analysis (a, e), IHC analysis of TTF‐1, SP‐A, and CC10 in BS (b, c, d) and in BSDE (f, g, h). (B) mRNA levels of E‐cadherin, cytokeratin 8, cytokeratin 19, TTF‐1, and SP‐A analysed by RT‐PCR in BS and BSDE. β2‐microglobulin was used as loading control.

Figure 6

Slug governs transition from epithelial to mesenchymal phenotype in bronchospheres (BS). (A) RT‐PCR and real‐time RT‐PCR analyses of Slug mRNA expression in BS and original tissues. (B) RT‐PCR analysis of Slug mRNA expression in BS and BSDE. (C) RT‐PCR analysis of cytokeratin 8, cytokeratin 18, TTF‐1, SP‐A, Oct 3/4,CD133 and Lef‐1 mRNA levels in BS exposed to Slug‐specific or control (SCR) siRNA for 72 h. (D) Inverted phase‐contrast microscopy of 10‐day secondary BS obtained from primary BS exposed to Slug‐specific or control (SCR) siRNA.