In this study, we described the presence of SP-Cpos/CCSPpos progenitors in the lung parenchyma of 0-3 month-old lambs. These rare cells (~0.3% of the pulmonary epithelial cells) have been isolated after tissue dissociation and they co-expressed CD34, SP-C and CCSP. The SP-Cpos/CCSPpos cells were maintained in vitro for up to 2 or 3 passages (30-45 days). Pluripotent progenitors are defined by their ability to maintain and differentiate upon exposure to specific stimuli. To further characterize the ovine SP-Cpos/CCSPpos cells, we first performed positive selection for CD34 expression, a surface protein widely described on various stem cells and present on ovine hematopoietic stem/progenitor cells . A small population of cells (few thousand) was isolated and characterized as SP-Cpos/CCSPpos cells. They expressed mRNAs for the NANOG, OCT4 and BMI1 genes which are molecular markers of stem cells/progenitors. They retained the expression of SP-C, CCSP and CD34 mRNAs over time. Importantly, the CD34pos/SP-Cpos/CCSPpos cells were able to maintain for up to 3 passages in vitro. The CD34pos/SP-Cpos/CCSPpos cell population retained the potential to differentiate at least into AECIIs or club cells in specific culture conditions. The CD34pos/SP-Cpos/CCSPpos cells, maintained in vitro, expressed OCT4 and BMI1 mRNAs, conferring them characteristics inherent to multipotent cells or progenitors.
Bronchioloalveolar progenitors have been reported in mice in an anatomically well-defined location at the bronchioloalveolar junction where conducting and respiratory airways meet [18, 24, 25]. These cells were initially described by their expression of Sca-1 and CD34, and their co-expression of SP-C and CCSP. We succeeded in demonstrating the in vivo presence of rare ovine bronchioloalveolar progenitors in the lungs of newborn or young animals (less than 3 months old), while other groups have difficulties to identify bronchioloalveolar progenitors in sheep upon searching for these cells using CCSP and SP-C immunostaining on lung sections [26, 27]. Interestingly, two studies have reported rare to extremely rare SP-Cpos/CCSPpos cells in virally-induced lung tumors in sheep [26, 28]. Taken together, these results are consistent with the presence of SP-Cpos/CCSPpos cell population, extremely rare in vivo on frozen lung sections and representing a new progenitor population. The discrepancy between our study and previous studies [26, 27] may be explained by the low but clearly above background levels of SP-C and CCSP expression in double-positive cells compared to the expression of these markers in AECIIs and club cells, respectively. Moreover, a recent study on ovine lung sections, have shown that CCSPpos cells were not (preterm lung) or poorly (9 days old lung) detectable in prenatal and early postnatal lung compared to mature lung (91 days) in bronchi, terminal bronchioles and respiratory bronchiole . These elements point the influence of the age of the lung studied, in the search for postnatal bronchioloalveolar CCSPpos/SP-Cpos progenitors.
The visualization of double-positive cells in the complex lung is therefore challenging and the CD34 enrichment strategy enabled us to evidence and to study this progenitor population. In addition, bronchioloalveolar progenitors may be more easily identified in lungs from newborn lambs. Of note, we have previously reported the presence of SP-Cpos/CCSPpos cells in the sheep lung while describing the ex vivo isolation of tumoral AECIIs from a virus-induced lepidic lung adenocarcinoma . While culturing these cells in three-dimensional conditions, better adapted to these polarized and specialized cells, we repeatedly observed SP-Cpos/CCSPpos double-positive cells (data not shown). These results prompted us to go further into their characterization.
SP-Cpos/CCSPpos double-positive bronchioloalveolar progenitors are rare and represent less than 0.4% of the total epithelial cells from dissociated lungs. In comparison, a recent study has established that undifferentiated human stem cells identified by c-kit immunosorting are present at a frequency of 1 per 6,000 cells in the bronchioles and 1 per 30,000 cells in the alveoli in adults . In fetal human lung tissue at 12-36 weeks of gestation, the frequency of stem cells varies from 1 per 11,000 to 1 per 600 cells, with an average of 1 per 4,100 cells . Our CD34-positive selection procedure was crucial to isolate bronchioloalveolar progenitors from the lung even though the absolute number of CD34pos/SP-Cpos/CCSPpos cells was ultimately low (only a few thousands cells) and confirmed the existence of a rare population of SP-Cpos/CCSPpos bronchioloalveolar progenitors. Importantly, all of the selection steps were performed ex vivo, without any cell culture steps and in the absence of fetal calf serum known to induce cell differentiation.
The CD34pos/SP-Cpos/CCSPpos population resembles the now well-characterized murine bronchioloalveolar stem cells in terms of both morphology and cell markers [18, 24, 25]. We clearly demonstrated that ovine bronchioloalveolar progenitors behave as multipotent precursors in culture and have maintenance and differentiation potential upon modulation of the culture conditions.
We then demonstrated that the ex vivo-derived ovine bronchioloalveolar progenitors retained the expression of the BMI1, OCT4 and NANOG genes which are some of the most important markers of undifferentiated pluripotent cells and are major players in embryonic and adult stem cells. BMI1 is a member of the PcG family of transcription repressors that play crucial roles in development, stem cell biology and cancer . BMI1 is also highly expressed in tumors and regulates the cell fate of cancer cells and normal and tumoral stem cells [32, 33]. BMI1 participates in the maintenance of endogenous stem cells, partly by repressing genes involved in cell death and senescence . In mice, BMI1 is necessary for bronchioloalveolar stem cells expansion . Similarly to murine bronchioloalveolar stem cells, ovine bronchioloalveolar progenitors expressed BMI1 under maintenance/self-renewing conditions, and this expression was not modified by exposure to differentiation conditions.
While OCT4, a member of the POU (Pit-Oct-Unc) transcription factor family, is essential for the maintenance of self-renewal capacities, NANOG (a downstream target of OCT4) contributes to the cell fate determination of pluripotent cells during embryogenesis [36, 37]. OCT4 and NANOG are among the few key factors that enable the reprogramming of adult somatic cells into pluripotent stem cells [38–42]. Interestingly, induced pluripotent cells (iPSCs) generated from sheep fibroblasts have recently been demonstrated to exhibit an embryonic stem cell-like morphology and to express OCT4 and NANOG among other intracellular and surface markers associated with undifferentiated cells, as previously demonstrated in humans and mice . The level of OCT4 is critical for the state of a cell and its inactivation results in the loss of pluripotency and induces differentiation . We could observe a loss or reduction of OCT4 gene expression in AECII and club cells compared to progenitors. OCT4 and NANOG are expressed in lung stem cells, and their co-expression may enhance malignancy by inducing cancer stem cell-like properties [45, 46]. Surprisingly, NANOG gene expression was higher in AECII and club cells than in progenitors in our in vitro culture conditions. NANOG expression is known to be regulated by a number of pluripotent transcription factors (FoxD3, SOX2, OCT4) . It is likely that this complex regulation is also dependent of the in vitro context (growth factors, monolayer or 2D culture conditions) and others active signaling cascades . With respect to the recently described human c-kit lung stem cell , it would be interesting to study the expression of c-kit in lamb lung sections and in the SP-Cpos/CCSPpos bronchioloalveolar progenitors population to better understand the hierarchical organization of these progenitors in this complex tissue.