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Long Term Culture of the A549 Cell Line Promotes Differentiation Towards an Alveolar Type II (ATII) Phenotype; Comparison of A549 cells with Primary ATII Cells

ABSTRACT: Pulmonary research requires models that represent the physiology of alveolar epithelium but concerns with reproducibility, consistency and the technical and ethical challenges of using primary or stem cells has resulted in widespread use of continuous cancer or other immortalized cell lines. The A549 ‘alveolar’ cell line has been available for over four decades but there is an inconsistent view as to its suitability as an appropriate model for primary alveolar type II (ATII) cells. Since most work with A549 cells involves short term culture of proliferating cells, we postulated that culture conditions that reduced proliferation of the cancer cells would promote a more differentiated ATII cell phenotype. We examined A549 cell growth in different media over long term culture and then used microarray analysis to investigate temporal regulation of pathways involved in cell cycle and ATII differentiation; we also made comparisons with gene expression in freshly isolated human ATII cells. Analyses indicated that long term culture in Ham’s F12 resulted in substantial modulation of cell cycle genes to result in a quiescent population of cells with significant up-regulation of autophagic, differentiation and lipidogenic pathways. There were also increased numbers of up- and down-regulated genes shared with primary cells suggesting adoption of ATII characteristics and multilamellar body (MLB) development. Subsequent Oil Red-O staining and Transmission Electron Microscopy confirmed MLB expression in the differentiated A549 cells. This work defines a set of conditions for promoting ATII differentiation characteristics in A549 cells that may be advantageous for studies with this cell line.

ORGANISM(S): Homo sapiens

PROVIDER: GSE88880 | GEO | 2016/10/19

SECONDARY ACCESSION(S): PRJNA349019

REPOSITORIES: GEO

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Project description:Alveolar epithelial type II (ATII)-like cells can be generated from murine embryonic stem cells (ESCs), although to date, no robust protocols applying specific differentiation factors are established. We hypothesized that the keratinocyte growth factor (KGF), an important mediator of lung organogenesis and primary ATII cell maturation and proliferation, together with dexamethasone, 8-bromoadenosine-cAMP, and isobutylmethylxanthine (DCI), which induce maturation of primary fetal ATII cells, also support the alveolar differentiation of murine ESCs. Here we demonstrate that the above stimuli synergistically potentiate the alveolar differentiation of ESCs as indicated by increased expression of the surfactant proteins (SP-) C and SP-B. This effect is most profound if KGF is supplied not only in the late stage, but at least also during the intermediate stage of differentiation. Our results indicate that KGF most likely does not enhance the generation of (mes)endodermal or NK2 homeobox 1 (Nkx2.1) expressing progenitor cells but rather, supported by DCI, accelerates further differentiation/maturation of respiratory progeny in the intermediate phase and maturation/proliferation of emerging ATII cells in the late stage of differentiation. Ultrastructural analyses confirmed the presence of ATII-like cells with intracellular composite and lamellar bodies. Finally, induced pluripotent stem cells (iPSCs) were generated from transgenic mice with ATII cell-specific lacZ reporter expression. Again, KGF and DCI synergistically increased SP-C and SP-B expression in iPSC cultures, and lacZ expressing ATII-like cells developed. In conclusion, ATII cell-specific reporter expression enabled the first reliable proof for the generation of murine iPSC-derived ATII cells. In addition, we have shown KGF and DCI to synergistically support the generation of ATII-like cells from ESCs and iPSCs. Combined application of these factors will facilitate more efficient generation of stem cell-derived ATII cells for future basic research and potential therapeutic application. 10 samples in total. mESCs at d8 of differentiation (Control) mESCs at d8 of differentiation with KGF treatment mESCs at d17 of differentiation (Control) mESCs at d17 of differentiation with KGF treatment mESCs at d17 of differentiation with DCI treatment mESCs at d17 of differentiation with KGF and DCI treatment mESCs at d24 of differentiation (Control) mESCs at d24 of differentiation with KGF treatment mESCs at d24 of differentiation with DCI treatment mESCs at d24 of differentiation with KGF and DCI treatment

Project description:Alveoli are thin-walled sacs that serve as the gas exchange units of the lung. They are affected in devastating lung diseases including COPD, Idiopathic Pulmonary Fibrosis, and the major form (adenocarcinoma) of lung cancer, the leading cause of cancer deaths. The alveolar epithelium is composed of two morphologically distinct cell types: alveolar type (AT) 1 cells, exquisitely thin cells across which oxygen diffuses to reach the blood, and AT2 cells, specialized surfactant-secreting cells. Classical studies suggested that AT1 cells arise from AT2 cells during development and following injury, but more recent studies suggest other sources. Here we use histological and marker analysis, lineage tracing, and clonal analysis in mice to identify alveolar progenitor and stem cells and map their locations and potential in vivo. The results show that AT1 and AT2 cells arise independently during development from a bipotential progenitor. After birth, new AT1 cells derive from rare, long-lived, self-renewing AT2 cells, each producing a slowly expanding clonal focus of regenerated alveoli contiguous with the founder AT2 cell. This stem cell function of AT2 cells is broadly activated by diffuse AT1 cell injury, and AT2 self-renewal can be induced in vitro by EGF ligands and permanently activated in vivo by AT2 cell-specific targeting of the oncogenic KrasG12D allele, efficiently transforming AT2 cells into monoclonal adenomatous tumors that rapidly enlarge and prove fatal. Thus, there is a developmental switch in alveolar progenitor cells after birth, when mature AT2 cells function as facultative stem cells that contribute to local alveolar renewal, repair, and cancer. We propose that short-range signals from dying AT1 cells regulate AT2 stem cell activity: a signal transduced by EGFR-KRAS controls AT2 self-renewal and is hijacked during oncogenic transformation, and a separate signal controls reprogramming to AT1 cell fate. To compare expression between ATII and E18 BP populations, RNA was isolated from either population purified by FACS. Two populations are analyzed with 3 biological replicates per population.

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Long Term Culture of the A549 Cell Line Promotes Differentiation Towards an Alveolar Type II (ATII) Phenotype; Comparison of A549 cells with Primary ATII Cells

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