Isolation of an adult mouse lung mesenchymal progenitor cell population

Abstract

Contained within the adult lung are differentiated mesenchymal cell types (cartilage, smooth muscle, and myofibrobasts) that provide structural support for airways and vessels. Alterations in the number and phenotype of these cells figure prominently in the pathogenesis of a variety of lung diseases. While these cells are thought to arise locally, progenitors have yet to be purified. In previous work, we developed a method for isolating progenitors from lung tissue: this technique takes advantage of the unique ability of cell populations enriched for somatic stem and progenitor activity to efflux the vital dye Hoechst 33342, a feature that permits isolation by flow cytometry-based procedures. Using this method, we determined that a rare population of mesenchymal progenitors resides within the CD45- CD31- Hoechst low fraction of the adult murine lung. Similar to other mesenchymal progenitors, these cells express Sca-1, CD106, and CD44; can be serially passaged; and can differentiate to smooth muscle, cartilage, bone, and fat. Overall, these findings demonstrate that a phenotypically distinct mesenchymal progenitor resides within the adult murine lung, and provide a scheme for their isolation and study.

Figure 1.

Lung SP cells are a heterogeneous population of cells. Left: Density dot plot demonstrating the presence of SP cells in the adult lung. Right: Gated subset demonstrating that lung SP cells consist of at least three fractions: CD45+, CD45− CD31+, and CD45− CD31−. Circled area contains putative mesenchymal progenitor cell population.

Figure 2.

Lung CD45− CD31− SP cells proliferate in mesenchymal cell maintenance media. (A, B) Low- and high-power view of CD45− CD31− SP cells after culture in mesenchymal cell maintenance media. Cells display the morphologic features of mesenchymal cells (long, thin, and stellate appearance). (C, D) CD45+ and CD45− CD31+ SP cell fractions did not grow or differentiate in mesenchymal cell maintenance conditions.

Figure 3.

CD45− CD31− SP cells express genes characteristic of immature mesenchymal cells. RT-PCR was performed on cultured CD45− CD31− SP cells for the mesenchymal genes α-Smooth muscle actin (α-Sma), α-Smooth muscle tropomyosin (α-Tm), the epithelial genes keratin 18 (K18) and keratin 19 (K19), and the endothelial-associated genes CD31 and Tie2. Only expression of the early mesenchymal marker α-smooth muscle actin could be detected in these cells. Markers of epithelial, endothelial, and mature smooth muscle (α-Smooth muscle tropomyosin) were not detected. Whole lung RNA was used as a positive control (Lung).

Figure 4.

CD45− CD31− SP cells can be serially passaed in an undifferentiated state. CD45− CD31− SP cells obtained from pooled lung samples continued to proliferate in culture beyond fifty passages. (A) Right: Cells from early and later passages (5th and 22nd doubling) have similar morphologic features. Left: Doubling time for cells after the first passage was estimated to be ∼ 1.75 d (top). Linear growth kinetics was observed over a range of population doublings (bottom). (B) Propidium iodide staining demonstrates that cells at early (5) and late passages (50) have a similar DNA profile. This profile is in contrast to that seen in immortalized cells, which have a greater fraction of cells in S, and G2 phases and with 4N+ DNA.

Figure 4.

CD45− CD31− SP cells can be serially passaed in an undifferentiated state. CD45− CD31− SP cells obtained from pooled lung samples continued to proliferate in culture beyond fifty passages. (A) Right: Cells from early and later passages (5th and 22nd doubling) have similar morphologic features. Left: Doubling time for cells after the first passage was estimated to be ∼ 1.75 d (top). Linear growth kinetics was observed over a range of population doublings (bottom). (B) Propidium iodide staining demonstrates that cells at early (5) and late passages (50) have a similar DNA profile. This profile is in contrast to that seen in immortalized cells, which have a greater fraction of cells in S, and G2 phases and with 4N+ DNA.

Figure 5.

Phenotype of cultured CD45− CD31−SP cells. (A–E) Flow cytometry analysis of cultured cells after 50 passages demonstrates absence of expression of CD31 (B) and CD45 (C), and uniform expression of stem cell antigen-1 (E, Sca-1). (A, D) Isotype staining. (F) Freshly isolated lung digest (left panel) and cultured CD45− CD31− SP cells were stained with Hoechst dye and analyzed by flow cytometry. Cultured SP cells retain the ability to efflux Hoechst dye (middle panel), a process that could be blocked by pre-treatment with verapamil 50 mM (right panel).

Figure 6.

Cultured CD45− CD31− SP cells uniformly express the markers CD106 (upper right) and CD44 (lower right). Isotype staining for CD106 (upper left) and isotype staining for CD44 (lower left).

Figure 7.

CD45− CD31− SP cells can differentiate to smooth muscle, bone, fat, and cartilage. After exposure to smooth muscle media for 7 d the contractile protein α-smooth muscle tropomyosin was abundantly expressed (low power, A; high power, B). Expression was not detected in cells grown in mesenchymal maintenance conditions (C). In osteogenic conditions, cells readily acquired the features of osteoblasts (rounded shape, D), and demonstrated significant mineralization capacity (orange/red color after Alizarin Red S staining, E). Similar findings were not seen when cells were maintained in mesenchymal cell maintenance media (F). After 21 d in adiopogenic conditions, fat droplets were clearly visible by phase contrast microscopy (H). These features were not observed when cells were grown in mesenchymal cell media for similar periods (G). Consistent with an adipocyte phenotype, droplets stained positive with Oil Red O dye (low and high power view, J and K). RT-PCR demonstrates that cells grown in maintenance media did not express perilipin; however, fresh adipose tissue and cells grown in adipogenic media expressed this adipocyte-specific marker (I). In chondrogenic conditions, CD45− CD31− SP cells formed large cellular structures that contained an abundance of proteoglycans (purple color after Toludine blue stain (L, low power; M, high power). Consistent with a cartilage structure, these cells also stained positive for Type II collagen (N). Negative Isotype staining for Type II collagen (O). Cells grown in mesenchymal cell maintenance media formed smaller, less dense structures (P, low power; Q, high power) that stained weakly to Toludine blue, and negative for Type II collagen (R).

Figure 8.

CD45− CD31− non-SP cells lack multi-potent differentiation capacity. CD45− CD31− non-SP has a limited capacity to form smooth muscle in culture. After 7 d in smooth muscle conditions, only a minority of cells expressed α-smooth muscle tropomyosin. (A) Low-power view. (B) High-power view. Culture in other differentiation conditions demonstrated that cells lacked osteogenic, adipogenic, and chondrogenic potential. In all three conditions, cells failed to demonstrate the morphologic characteristic of these cell types. (C) After 21 d in osteogenic media, cells stain negative for Alizarin Red S. (D) Lipid deposits are not seen after culture of CD45− CD31− non-SP cells in adipogenic media. (E) Proteoglycan staining is absent from CD45− CD31− non-SP grown in chondrogenic media.