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. 2017 Dec 21;7(1):17971.
doi: 10.1038/s41598-017-17952-4.

Conditionally reprogrammed primary airway epithelial cells maintain morphology, lineage and disease specific functional characteristics

Kelly M Martinovich  1 , Thomas Iosifidis  2   3 , Alysia G Buckley  4 , Kevin Looi  1 , Kak-Ming Ling  1 , Erika N Sutanto  1 , Elizabeth Kicic-Starcevich  1 , Luke W Garratt  1 , Nicole C Shaw  1 , Samuel Montgomery  1 , Francis J Lannigan  2 , Darryl A Knight  5   6   7 , Anthony Kicic  8   9   10   11   12 , Stephen M Stick  1   2   3   13
Affiliations

Conditionally reprogrammed primary airway epithelial cells maintain morphology, lineage and disease specific functional characteristics

Kelly M Martinovich et al. Sci Rep. .

Abstract

Current limitations to primary cell expansion led us to test whether airway epithelial cells derived from healthy children and those with asthma and cystic fibrosis (CF), co-cultured with an irradiated fibroblast feeder cell in F-medium containing 10 µM ROCK inhibitor could maintain their lineage during expansion and whether this is influenced by underlying disease status. Here, we show that conditionally reprogrammed airway epithelial cells (CRAECs) can be established from both healthy and diseased phenotypes. CRAECs can be expanded, cryopreserved and maintain phenotypes over at least 5 passages. Population doublings of CRAEC cultures were significantly greater than standard cultures, but maintained their lineage characteristics. CRAECs from all phenotypes were also capable of fully differentiating at air-liquid interface (ALI) and maintained disease specific characteristics including; defective CFTR channel function cultures and the inability to repair wounds. Our findings indicate that CRAECs derived from children maintain lineage, phenotypic and importantly disease-specific functional characteristics over a specified passage range.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Epithelial cobblestone morphology is maintained over passage. (a) Healthy CRAEC monolayer at confluence, passage one. (b) Healthy CRAEC monolayer at confluence, passage five. (c) Asthmatic CRAEC monolayer at confluence, passage one. (d) Asthmatic CRAEC monolayer at confluence, passage five. (e) Cystic fibrosis CRAEC monolayer at confluence, passage one. (f) Cystic fibrosis CRAEC monolayer at confluence, passage five. Representative image of n = 4 patients per phenotype/passage. Scale bar: 100 µm.
Figure 2
Figure 2
Conditionally reprogramming improves cell population doubling potential. (a) Population doublings of healthy AECs (n = 4 patients; CRAEC slope 0.52 ± 0.02; pAEC slope 0.08 ± 0.01; p = 0.0001). (b) Population doublings of asthmatic AECs (n = 4 patients; CRAEC slope 0.40 ± 0.03; pAEC slope 0.06 ± 0.02; p = 0.0050). (c) Population doublings of CF AEC (n = 4 patients; CRAEC slope 0.50 ± 0.01; pAEC slope 0.06 ± 0.02; p = 0.0001). CRAEC ■, pAEC □, ***p < 0.0010, **p < 0.0100.
Figure 3
Figure 3
Cell yield is improved by conditional reprogramming. (a) Cell yield of healthy AECs (CRAEC 108.2 ± 50.63 thousand cells per cm2, pAEC 28.6 ± 10.54 thousand cells per cm2, p = 0.0001). (b) Cell yield of asthmatic AECs (CRAEC 97.8 ± 44.68 thousand cells per cm2 pAEC 33.43 ± 13.36 thousand cells per cm2, p = 0.0001) (c) Cell yield of CF AECs (CRAEC 108.1 ± 45.4 thousand cells per cm2, pAEC 36.86 ± 15.97 thousand cells per cm2, p = 0.0001) CRAEC ■, pAEC □, ***p < 0.0010.
Figure 4
Figure 4
Gene expression of cytokeratin 19, cytokeratin 5 and vimentin is maintained over passage. (a) Gene expression profile of healthy CRAECs from passage one and five. (b) Gene expression profile of asthmatic CRAECs from passage one and five. (c) Gene expression profile of CF CRAECs from passage one and five. Passage 1 (black bar), passage 5 (open bar). No significant differences between passages or phenotypes. (n = 4 patients per phenotype/passage, relative expression to housekeeping gene, PPIA).
Figure 5
Figure 5
Expression of the epithelial lineage marker cytokeratin is maintained over passage. Pan-cytokeratin antibody (DAKO) (a,b,e,f,i,j), Vimentin antibody (Abcam), (c,d,g,h,k,l), DAPI nuclear stain represented in blue on all images. Representative images n = 4 patients per phenotype/passage, Scale bar; 30 µm.
Figure 6
Figure 6
Terminal differentiation at the air-liquid interface (ALI). (a) Differentiation expression profile of healthy CRAECs. (b) Differentiation expression profile of asthmatic CRAECs. (c) Differentiation expression profile of CF CRAECs. Cilia shown in green, stained using α-tubulin, tight junction protein ZO-1 shown in red (oil immersion, Scale bar; 50 µm). (d) Cross section of healthy CRAECs. (e) Cross section of asthmatic CRAECs. (f) Cross section of Cystic fibrosis CRAECs. Sections stained with H&E, apical side is the uppermost. (Scale bar; 60 µm; representative images, n = 4 patients per phenotype/per passage).
Figure 7
Figure 7
Disease specific functional characteristics are maintained in non-cryopreserved and cryopreserved CRAECs. (a) Ussing chamber studies utilising differentiated non-cryopreserved ALI cultures from a healthy phenotype have functional CFTR (solid line) whereas CF cultures do not (dotted line). Amiloride treatment (A) blocks sodium ion adsorption, forskolin treatment (F) stimulates CFTR driven chloride ion secretion. Representative tracings of short circuit current (Isc), n = 4 CF patients, n = 4 healthy patients. (b) Change in Isc in Ussing chamber studies, after the addition of forskolin in healthy and CF non-cryopreserved ALI cultures. Floating bars shown of the min and max with line at the mean, n = 4 CF patients, n = 4 healthy patients **p = 0.0060. (c) Asthmatic pAECs and CRAECs have a dysregulated wound repair capacity. Mechanical scratch wounds were performed on pAEC (red) and CRAEC (black) submerged monolayer cultures from non-cryopreserved healthy (solid line & solid squares) and asthmatic children (dashed line & open squares). Wound closure was calculated by manual tracing of the new wound area at each time interval, then expressed as a percentage of total wound recovery. Both pAECs and CRAECs from asthmatic children (dashed line & open squares) failed to repair. (n = 4 healthy patients, asthmatic patients, each performed in technical duplicates at passage two). (d) Ussing chamber studies utilising differentiated cryopreserved ALI cultures from a healthy phenotype have functional CFTR (solid line) whereas CF cultures do not (dotted line). Amiloride treatment (A) blocks sodium ion adsorption, forskolin treatment (F) stimulates CFTR driven chloride ion secretion. Representative tracings of Isc, n = 4 CF patients and n = 4 healthy patients. (e) Change in Isc in Ussing chamber studies, after the addition of forskolin in healthy and CF cryopreserved ALI cultures. Floating bars shown of the min and max with line at the mean, n = 4 CF patients, n = 4 healthy patients ***p = 0.0040. (f) Mechanical scratch wounds were performed on CRAEC submerged monolayer cultures from cryopreserved healthy (solid line & solid squares) and asthmatic children (dashed line & open squares) and wound repair monitored. Cryopreserved asthmatic CRAECs failed to repair. (n = 4 healthy patients, asthmatic patients, each performed in technical duplicates at passage two).
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References

    1. Kicic, A., Sutanto, E. N., Stevens, P. T., Knight, D. A. & Stick, S. M. Intrinsic biochemical and functional differences in bronchial epithelial cells of children with asthma. Am J Respir Crit Care Med 174, 1110–1118, 10.1164/rccm.200603-392OC (2006). - PubMed
    1. Sutanto EN, et al. Innate inflammatory responses of pediatric cystic fibrosis airway epithelial cells: effects of nonviral and viral stimulation. Am J Respir Cell Mol Biol. 2011;44:761–767. doi: 10.1165/rcmb.2010-0368OC. - DOI - PubMed
    1. Garratt LW, et al. Alpha-1 Antitrypsin Mitigates the Inhibition of Airway Epithelial Cell Repair by Neutrophil Elastase. Am J Respir Cell Mol Biol. 2016;54:341–349. doi: 10.1165/rcmb.2015-0074OC. - DOI - PubMed
    1. Moheimani F, et al. Disruption of beta-catenin/CBP signaling inhibits human airway epithelial-mesenchymal transition and repair. The international journal of biochemistry & cell biology. 2015;68:59–69. doi: 10.1016/j.biocel.2015.08.014. - DOI - PubMed
    1. Hackett TL, et al. Intrinsic phenotypic differences of asthmatic epithelium and its inflammatory responses to respiratory syncytial virus and air pollution. Am J Respir Cell Mol Biol. 2011;45:1090–1100. doi: 10.1165/rcmb.2011-0031OC. - DOI - PubMed