Lung progenitors from lambs can differentiate into specialized alveolar or bronchiolar epithelial cells

Abstract

Background: Airways progenitors may be involved in embryogenesis and lung repair. The characterization of these important populations may enable development of new therapeutics to treat acute or chronic lung disease. In this study, we aimed to establish the presence of bronchioloalveolar progenitors in ovine lungs and to characterize their potential to differentiate into specialized cells.

Results: Lung cells were studied using immunohistochemistry on frozen sections of the lung. Immunocytochemistry and flow cytometry were conducted on ex-vivo derived pulmonary cells. The bronchioloalveolar progenitors were identified by their co-expression of CCSP, SP-C and CD34. A minor population of CD34(pos)/SP-C(pos)/CCSP(pos) cells (0.33% ± 0.31) was present ex vivo in cell suspensions from dissociated lungs. Using CD34 magnetic positive-cell sorting, undifferentiated SP-C(pos)/CCSP(pos) cells were purified (>80%) and maintained in culture. Using synthetic media and various extracellular matrices, SP-C(pos)/CCSP(pos) cells differentiated into either club cells (formerly named Clara cells) or alveolar epithelial type-II cells. Furthermore, these ex vivo and in vitro derived bronchioloalveolar progenitors expressed NANOG, OCT4 and BMI1, specifically described in progenitors or stem cells, and during lung development.

Conclusions: We report for the first time in a large animal the existence of bronchioloalveolar progenitors with dual differentiation potential and the expression of specialized genes. These newly described cell population in sheep could be implicated in regeneration of the lung following lesions or in development of diseases such as cancers.

Figure 1

In vivo localization of SP-C^pos/CCSP^pos cells. Frozen lung sections from ten 0 to 3 month old lambs were analyzed by immunohistochemistry for the expression of SP-C (green), CCSP (red) and the co-expression of SP-C and CCSP. The nuclei were stained with DAPI (blue). (A) Lung cross sections showed the expression of CCSP in the bronchiole (b) and SP-C in the alveoli (a) (100x magnification). (B) Immunostaining of rare SP-C^pos/CCSP^pos cells in situ (400x magnification). Inset: enlargement of a SP-C/CCSP double-positive cell. (C) SP-C and CCSP expression from a representative lamb (# 1507) in ex vivo dissociated cells by flow cytometry showing the presence of a SP-C^pos/CCSP^pos population. (D) Frequency of SP-C^pos, CCSP^pos and SP-C^pos/CCSP^pos cells from ten 0 to 3 month old lambs. The data are expressed as the mean (± SD) percentage of the cells expressing the cellular markers.

Figure 2

In vitro expansion of SP-C^pos/CCSP^pos progenitors. Pulmonary epithelial cells from dissociated lung tissues were cultured in vitro on fibronectin-coated plates supplemented with "complete Q286 medium". (A) Lung epithelial cells derived from lamb #1487 and maintained for 2 passages were analyzed by immunostaining for the expression of SP-C (green), CCSP (red) and the co-expression of SP-C and CCSP (400x magnification). Inset: enlargement of double-positive cells. (B) Flow cytometry analysis of a representative animal shows the percentage of SP-C^pos cells and CCSP^pos cells above the background fluorescence. The double staining shows an enriched culture of AECIIs with the presence of SP-C^pos/CCSP^pos bronchioloalveolar progenitors after dissociation, and at the first and second passage in vitro. For each condition, the left graph (smaller) corresponds to controls and the right graph (larger) presents SP-C and CCSP specific immunostaining.

Figure 3

Enrichment in CD34^pos cells by magnetic cell sorting. Lung primary cells from lamb lungs were subjected to magnetic cell sorting with anti-ovine CD34, then analyzed by flow cytometry. A: Representative dot plot from one animal showing expression of CD34^pos cells in the dissociated tissue (Before sorting) and after the positive selection (After sorting) B: Enrichment of CD34^pos cells from independent magnetic cell sorting from 6 lambs shown as the mean (± SD). **: p < 0.01.

Figure 4

Expression of SP-C and CCSP in CD34^pos cells. (A) Dot blot analysis from a representative animal (lamb #1744) showing the expression of SP-C and CCSP in the majority (>90%) of the CD34^pos cells. (B) Combined results from 10 lambs showing the enrichment of SP-C^pos/CCSP^pos cells in the CD34-selected cell fraction. The data are shown as the mean (± SD). **: p < 0.01. Before: before CD34 sorting; After: after CD34 sorting. (C) Expression of SP-C, CCSP, CD34, BMI1, OCT4, NANOG and GAPDH mRNAs in the unselected and in the CD34-selected cells measured by RT-PCR.

Figure 5

In vitro maintenance and differentiation of bronchioloalveolar progenitors. CD34^pos/SP-C^pos/CCSP^pos cells from explanted lungs were maintained either in a. “maintenance conditions” with "complete Q286 medium" on fibronectin and collagen-coated inserts, b. differentiated in “AECII conditions” with "complete Q286 medium" on fibronectin and collagen-coated plates or c. in “Club conditions” with "basic Q286 medium" on fibronectin-coated plates. (A) Representative immunostaining from cells isolated from one lamb for SP-C (green) and CCSP (red). Co-expression was performed on in vitro maintained bronchioloalveolar progenitors (SP-C^pos/CCSP^pos) or differentiated AECIIs (SP-C^pos/CCSP^neg) or club cells (SP-C^neg/CCSP^pos). (B) The population of CD34^pos/SP-C^pos/CCSP^pos cells from a representative lamb grown in maintenance or differentiation media was quantified by flow cytometry for the expression of SP-C or CCSP. The results are presented as dot plots of the expression of both markers. (C) CD34^pos/SP-C^pos/CCSP^pos cells from 6 independent lambs cultured in maintenance or differentiation media were followed over three passages (p0 to p3) by flow cytometry for the expression of SP-C and CCSP. The results are presented as the mean ± SD of the cells expressing each phenotype.

Figure 6

BMI1 and OCT4 mRNA expression in maintained and differentiated CD34^pos/SP-C^pos/CCSP^pos cells. (A) Depending on the culture medium conditions (maintenance, AECII or club medium), the relative proportions of SP-C^pos/CCSP^pos, SP-C^pos/CCSP^neg or SP-C^neg/CCSP^pos were determined at the first passage by flow cytometry and reported in the table as the following: 0% for no cells, + for <15%, ++ ≥15% and ≤50%, and +++ for ≥50%. (B) The BMI1, OCT4 and NANOG mRNAs were detected by RT-PCR from the total cell RNAs extracted from the three culture conditions at passage 1 and compared to the ovine GAPDH housekeeping gene. Prog: progenitors.