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Rapid Ex-Vivo Ciliogenesis and Dose-Dependent Effect of Notch Inhibition on Ciliogenesis of Respiratory Epithelia

Zahid, Maliha and Feinstein, Timothy N. and Oro, Anthony and Schwartz, Molly and Lee, Alex D. and Lo, Cecilia W. (2020) Rapid Ex-Vivo Ciliogenesis and Dose-Dependent Effect of Notch Inhibition on Ciliogenesis of Respiratory Epithelia. Biomolecules, 10 (8). p. 1182. ISSN 2218-273X

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Background: Cilia are actin based cellular protrusions conserved from algae to complex multicellular organisms like Homo sapiens. Respiratory motile cilia line epithelial cells of the tracheobronchial tree, beat in a synchronous, metachronal wave, moving inhaled pollutants and pathogens cephalad. Their role in both congenital disorders like primary ciliary dyskinesia (PCD) to acquired disorders like chronic obstructive pulmonary disease (COPD) continues to evolve. In this current body of work we outline a protocol optimized to reciliate human nasal epithelial cells and mouse tracheal cells in vitro. Using this protocol, we knocked down known cilia genes, as well as use a small molecule inhibitor of Notch, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl Ester (DAPT), to assess the effect of these on ciliogenesis in order to show the validity of our protocol. Methods: Tracheas were harvested from wild-type, adult C57B6 mice, pronase digested and sloughed off epithelial cells grown to confluence in stationary culture on rat-tail collagen coated wells. Upon reaching confluence, collagen was digested and cells placed suspension culture protocol to reciliate the cells. Using this suspension culture protocol, we employed siRNA gene knockdown to assay gene functions required for airway ciliogenesis. Knock down of Dynein axonemal heavy chain 5 (Dnah5), a ciliary structural protein, was confirmed using immunostaining. Mouse tracheal cells were treated in suspension with varying doses of DAPT, an inhibitor of Notch, with the purpose of evaluating its effect and dose response on ciliogenesis. The optimum dose was then used on reciliating human nasal epithelial cells. Results: siRNA knockdown of Foxj1 prevented ciliation, consistent with its role as a master regulator of motile cilia. Knockdown of Dnai1 and Dnah5 resulted in immotile cilia, and Cand1 knockdown, a centrosome protein known to regulate centrosome amplification, inhibited airway ciliogenesis. Dnah5 knockdown was confirmed with significantly decreased immunostaining of cilia for this protein. Inhibiting Notch signaling by inhibiting gamma secretase with DAPT enhanced the percentage of ciliation, and resulted in longer cilia that beat with higher frequency in both mouse and human airway epithelia. Conclusions: Modifying existing reciliation protocols to suit both human nasal epithelial and mouse tracheal tissue, we have shown that knockdown of known cilia-related genes have the expected effects. Additionally, we have demonstrated the optimal dosage for significantly improving reciliation of airway epithelia using DAPT. Given that cilia length and function are significantly compromised in COPD, these findings open up interesting avenues for further exploration.


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Item Type: Article
Status: Published
CreatorsEmailPitt UsernameORCID
Zahid, Malihamaz7@pitt.edumaz7
Feinstein, Timothy N.tnf8@pitt.edutnf8
Oro, Anthony
Schwartz, Mollymos24@pitt.edumos24
Lee, Alex D.
Lo, Cecilia W.cel36@pitt.educel36
Date: 14 August 2020
Date Type: Publication
Journal or Publication Title: Biomolecules
Volume: 10
Number: 8
Publisher: MDPI AG
Page Range: p. 1182
DOI or Unique Handle: 10.3390/biom10081182
Schools and Programs: School of Medicine > Developmental Biology
Refereed: Yes
Uncontrolled Keywords: cilia, ciliogenesis, siRNA knockdown, Notch signaling, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl Ester (DAPT)
ISSN: 2218-273X
Official URL:
Funders: National Institutes of Health, Pitt Innovation Challenge
Article Type: Research Article
Date Deposited: 26 May 2021 20:21
Last Modified: 26 May 2021 20:21


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