You are hereNovember 5, 2012 | Pluripotent Stem Cells
Mature airway epithelia formation breakthrough for Cystic Fibrosis
“Directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional CFTR protein”
Previous attempts to differentiate human pluripotent cells into lung epithelia have generated mixed results; studies in human embryonic stem cells (hESCs) have produced cells that express distal airway epithelial phenotypes at low efficiency (Van Haute et al, Samadikuchaksaraei et al and Wang et al) while studies in human induced pluripotent stem cells (hiPSCs) generated lung endoderm progenitors but not mature proximal and distal lung epithelial cells (Mou et al). However, in a study published recently in Nature Biotechnology, researchers from the laboratories of James Ellis and Janet Rossant at the Department of Molecular Genetics, University of Toronto, Canada have now developed a method to generate functional proximal conducting airway epithelia expressing the cystic fibrosis transmembrane conductance regulator gene (CFTR) from human pluripotent stem cells (hESCs and hiPSCs). Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in CFTR and the researchers also demonstrate that the treatment of CF patient iPSC–derived epithelial cells with a small-molecule compound to correct for the common CF processing mutation results in enhanced plasma membrane localization of mature CFTR protein (Wong et al).
Initially, hESCs were induced to differentiate toward definitive endoderm using a previously described method (D’Amour et al). Treatment with Activin-A and WNT3A for 4 days led to a large proportion of cells to express several endodermal markers (CXCR4, cKIT, FOXA2 and SOX17) and upon treatment with FGF2 (Fibroblast Growth Factor 2) and SHH (Sonic HedgeHog)for 5 days, to induce promote anterior foregut identity and specify lung cell fate (Ameri et al and Serls et al), the majority of cells expressed the pan-endoderm marker FOXA2, and pan-epithelial marker EpCAM. mRNA analysis of these cells also found an upregulation of genes associated with anterior foregut endoderm (SOX2 and NKX2.1), pharyngeal endoderm (FOXG) and thyroid (TG and PAX9). Subsequently, these cells were exposed to growth factors known to be important in lung development; FGF7 (epithelial cell growth (Shiratori et al)), FGF10 (lung bud outgrowth and organogenesis (Bellusci et al)) and BMP4 (drives distal or proximal bud tip outgrowth dependent on concentration (Weaver et al)). Exposure of these cells to low levels of BMP4, to induce a proximal fate, in combination with FGF7 and FGF10 led to the upregulation of airway cell genes (KRT5 and TRP63), glial cells genes (FOXJ1 and SOX17), NKX2.1, CFTR and FOXA2. Proximal airway differentiation was then induced by the addition of FGF18 (Whitsett et al), resulting in the further upregulation of airway genes (KRT5, TRP63, FOXJ1, SOX17, MUC5AC and CFTR). Subsequent flow cytometric analysis suggested that at least one-third of the cells in the culture were of the ciliated CFTR-expressing airway phenotype. Additionally, over 50% of the cells were P63+, suggesting that the vast majority of the cells were potentially basal cell progenitors which give rise to other proximal airway lineages.
Next, to mature the cells towards functional airway epithelium, the use of primary bronchial epithelial cell growth medium was pared with an air-liquid interface (ALI) growth condition to mimic the post-natal airway epithelial niche and promote differentiation, maturation and polarization of the epithelium. After 5 weeks of growth in these conditions, 50% of cells expressed CFTR as well as KRT, FOXJ1 and LHS28, a marker of the basal bodies of cilia, and reduced levels of P63 suggesting that the basal progenitors had differentiated. Additionally, the cells expressed conducting airway epithelia markers; acetylated tubulin TUBA1A (cilia), MUC1 (Goblet cells), KRT14 (basal epithelia) and FOXA2. The presence of proximal airway epithelium was shown through the presence of mucin 16 and cytokeratin 16 proteins and the upregulation of proximal airway lineage mRNAs (SOX17, FOXJ1, MUC5AC, TRP63, KRT5, ARG2, SOX2, CFTR, KRT16, MUC16, and NGFR). Immunofluorescence staining and confocal analysis found contiguous patches of epithelial cells typified by membrane expression of Zona Occludin-1 (ZO1), a protein associated with tight junctions, and co-staining with pan-KRT and apical plasma membrane localisation of CFTR. Furthermore, these cells were found to be ciliated and to produce mucin, suggesting that the ALI allows for the maturation and polarization of a ciliated large airway epithelium with proper localization of the CFTR protein.
Next, iPSCs were generated from primary human fibroblasts carrying a CF mutation (F508del) (CF-iPSCs), which were shown to resemble hESCs, to be functionally pluripotent while still carrying the initial mutation, and which were subsequently differentiated towards mature airway epithelium in a similar manner to hESCs. However, while the hESC cultures displayed CFTR channel activity, the CF-iPSC cultures lacked this activity, and while some hESC cultures expressed functional expression of CFTR, as observed by cAMP-activated efflux, the CF-iPSC cultures did not. Lack of CFTR protein due to this specific gene mutation (F508del) occurs due to mis-folding in the endoplasmic reticulum and subsequent rapid degradation (Lewis et al). However, recent studies have shown that small molecules called ‘corrector’ compounds can partially rescue this trafficking defect (Van Goor et al), and excitingly, treatment of CF-iPSC cultures with one of these small molecules (C18) led to the expression of cell surface CFTR on patches of cells and a small increase in CFTR channel activity, although no substantial changes in cAMP-regulated efflux were observed.
This study represents a potentially important step forward in cell replacement therapy for the lung. In addition to the first precise delineation of a protocol which generates specific functional cell type, the ability to recapitulate this for a relevant mutation bearing iPSC line are also described, and more excitingly, the ability to reverse this mutation pharmacologically is demonstrated. Hopefully, this system will allow the validation of new and existing drugs for the treatment of Cystic Fibrosis and also to allow a more specific understanding of lung disease.
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Article originally appeared in Nature Biotechnology.
STEM CELLS correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.