A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells

Abstract

Alveolar epithelial type II (ATII) cells are small, cuboidal cells that constitute approximately 60% of the pulmonary alveolar epithelium. These cells are crucial for repair of the injured alveolus by differentiating into alveolar epithelial type I cells. ATII cells derived from human ES (hES) cells are a promising source of cells that could be used therapeutically to treat distal lung diseases. We have developed a reliable transfection and culture procedure, which facilitates, via genetic selection, the differentiation of hES cells into an essentially pure (>99%) population of ATII cells (hES-ATII). Purity, as well as biological features and morphological characteristics of normal ATII cells, was demonstrated for the hES-ATII cells, including lamellar body formation, expression of surfactant proteins A, B, and C, alpha-1-antitrypsin, and the cystic fibrosis transmembrane conductance receptor, as well as the synthesis and secretion of complement proteins C3 and C5. Collectively, these data document the successful generation of a pure population of ATII cells derived from hES cells, providing a practical source of ATII cells to explore in disease models their potential in the regeneration and repair of the injured alveolus and in the therapeutic treatment of genetic diseases affecting the lung.

Fig. 1.

Structure of the SPC promoter-NEO transgene 3′-HPRT vector. A 3.8-kb human genomic DNA fragment containing the SPC promoter and 170 bp of noncoding sequence of exon 1 was cloned into the 3′-hprt targeting vector containing the puromycin-resistance gene. The Neo^r gene was added downstream of the SPC promoter. The EcoRI site located between the Agouti and Neo^r gene was used to linearize the plasmid before transfection.

Fig. 2.

Relative RNA levels of SPC in G418-selected and nonselected differentiating hES cells. SPC-specific RT-PCR was performed by using total RNA isolated from differentiating cultures of hES cell lines H9.2 and SPCP/NEO.74. (Left) Data obtained from differentiating cells subjected to EB formation. (Right) Data obtained from differentiating cells without EB formation. (Lower) The results from G418-selected SPCP/NEO.74 hES cells. Lanes M, 1-kb DNA ladder. Lanes C, the SPC-specific RT-PCR positive control using RNA isolated from the ATII cell line A549. The lower blot in each panel shows the 18S-specific RT-PCR, demonstrating that changes in the amount of SPC-specific 327-bp RT-PCR product was due to corresponding changes in SPC RNA expression. Total days of differentiation at which the RNA samples were obtained are indicated.

Fig. 3.

Flow cytometry examining SPC expression in G418-selected and nonselected cultures of differentiating hES cells. hES cell lines H9.2 and SPCP/NEO.74 were induced to differentiate by culturing on Matrigel-coated plates with DM for 10 days (Left) or 15 days (Right). The differentiated cells were dissociated and immunostained by rabbit anti-human SPC antibody for flow cytometry analysis as described in Methods. Results using the SPC antibody are depicted by solid lines, and nonimmune rabbit serum controls are illustrated by dashed lines.

Fig. 4.

Immunofluorescence of SPA, SPB, and SPC in SPCP/NEO.74-derived ATII cells. The hES cell-derived ATII cells generated by G418 selection from the SPCP/NEO.74 cell line and A549 cells were immunostained by rabbit anti-human SPA, SPB, and SPC antibodies (red) and nuclear-counterstained with DAPI (blue). (Magnification: ×400.)

Fig. 5.

Transmission electron micrographs of A549 and hES-ATII cells. (A) A549 cells with characteristic cytoplasmic electron-dense and loose lamellar bodies. (B and C) Magnified views of regions ∗ and ∗∗, respectively, more clearly showing the structure of the lamellar bodies. (D) hES cell-derived ATII cells showing similar lamellar bodies and other morphological characteristics as the A549 cells. (E and F) Magnified views of regions ∗ and ∗∗ in D, respectively, showing clear lamellar structures. (Scale bars: 5 μm in A and D and 0.5 μm in B, C, E, and F.)

Fig. 6.

Expression of CFTR, α-1AT, and the complement proteins C3 and C5 by hES-ATII cells. Total RNA isolated from hES cell-derived ATII cells was used to examine CFTR and α-1AT expression by RT-PCR as described in Methods. The RNA expression levels of CFTR and α-1AT in hES cell-derived ATII cells were comparable with that in A549 cells but were not detectable in the undifferentiated hES cell lines H9.2 and SPCP/NEO.74 (Left). C3 and C5 proteins produced by hES cell-derived ATII cells were determined by ELISA as described in Methods. (Upper Right) Bar graph depicting C3 protein levels from the hES cell-derived ATII cultures on days 10, 12, and 15. The numerical values on these days were 33 ± 3, 32 ± 3, and 35 ± 3 ng/mg total protein per 24 h, respectively. (Lower Right) Bar graph depicting C5 protein levels from the hES cell-derived ATII cultures. C5 protein in the hES cell-derived ATII cells was measurable in the day-15 culture (1.6 ± 0.1 ng/mg total protein per 24 h).