Biology subjects

Awatade, N. T.

Publications and source records attributed to Awatade, N. T..

4 recordsLinked to original sources

bioRxiv2023

Comparison of commercially available differentiation media on morphology, function, and virus-host interaction in conditionally reprogrammed human bronchial epithelial cells

IntroductionPrimary air liquid interface (ALI) cultures of bronchial epithelial cells are used extensively to model airway responses. A recent advance is the development of conditional reprogramming that enhances proliferative capability. Several different media and protocols are utilized, yet even subtle differences may influence cellular responses. We compared the morphology and functional responses, including innate immune responses to rhinovirus infection in conditionally reprogrammed primary bronchial epithelial cells (pBECs) differentiated using two commonly used culture media. MethodspBECs from healthy participants (n = 5) were CR using {gamma}-irradiated 3T3 fibroblasts and Rho Kinase inhibitor. CRpBECs were differentiated at ALI in either PneumaCult (PN-ALI) or Bronchial Epithelial Growth Medium (BEGM)-based differentiation media (BEBM:DMEM, 50:50, Lonza) - (AB-ALI) for 28 days. Transepithelial electrical resistance (TEER), immunofluorescence, histology, cilia activity, ion channel function, and expression of cell markers were analyzed. Viral load was assessed by RT-qPCR and anti-viral factors quantified by Legendplex following Rhinovirus-A1b (RVA1b) infection. ResultsCRpBECs differentiated in PneumaCult were smaller and had a lower TEER and cilia beat frequency (CBF) compared to BEGM media. PneumaCult media cultures exhibited significantly increased FOXJ1 expression, more ciliated cells with a larger active area, increased intracellular mucins, and increased calcium-activated chloride channel current. However, there were no significant changes in viral RNA or host antiviral responses. ConclusionThere are distinct structural and functional differences in CRpBECs cultured in the two commonly used ALI differentiation media. Such factors need to be taken into consideration when designing and comparing CRpBECs ALI experiments.

cell biology↗

bioRxiv2021

Molecular dynamics and theratyping in airway and gut organoids reveal R352Q-CFTR conductance defect

A significant challenge to making targeted CFTR modulator therapies accessible to all individuals with cystic fibrosis (CF) are many mutations in the CFTR gene that can cause CF, most of which remain uncharacterized. Here, we characterized the structural and functional defects of the rare CFTR mutation R352Q - with potential role contributing to intrapore chloride ion permeation - in patient-derived cell models of the airway and gut. CFTR function in differentiated nasal epithelial cultures and matched intestinal organoids was assessed using ion transport assay and forskolin-induced swelling (FIS) assay respectively. Two CFTR potentiators (VX-770, GLPG1837) and a corrector (VX-809) were tested. Data from R352Q-CFTR were compared to that of participants with mutations with known impact on CFTR function. R352Q-CFTR has residual CFTR function which was restored to functional CFTR activity by CFTR potentiators but not the corrector. Molecular dynamics (MD) simulations of R352Q-CFTR were carried out which indicated the presence of a chloride conductance defect, with little evidence supporting a gating defect. The combination approach of in vitro patient-derived cell models and in silico MD simulations to characterize rare CFTR mutations can improve the specificity and sensitivity of modulator response predictions and aid in their translational use for CF precision medicine.

molecular biology↗

bioRxiv2020

Significant functional differences despite morphological and molecular similarity in fully differentiated matched Conditionally Reprogrammed (CRC) and Feeder free dual SMAD inhibited expanded human nasal epithelial cells

BackgroundPatient-derived airway cells differentiated at Air Liquid Interface (ALI) are valuable models for Cystic fibrosis (CF) precision therapy. Advances in culture techniques have improved expansion capacity of airway basal cells, while retaining functional airway epithelium physiology. However, considerable variation in response to CFTR modulators is observed even when using similar ALI culture techniques. We aimed to address if variation in response reflects true biological differences between patients or technical differences as a consequence of different culture expansion methods. MethodsNasal epithelial brushings from 14 individuals (CF=9; non-CF=5) were collected, then equally divided and expanded under conditional reprogramming culture (CRC) and feeder-serum-free "dual-SMAD inhibition" (SMADi) methods. Expanded cells from each culture were differentiated with proprietary PneumaCult-ALI media. Morphology (Immunofluorescence), global proteomics (LC-MS/MS) and function (barrier integrity, cilia motility, and ion transport) were compared in CRCALI and SMADiALI under basal and CFTR corrector treated (VX-809) conditions. ResultsNo significant difference in the structural morphology or global proteomics profile were observed. Barrier integrity and cilia motility were significantly different, despite no difference in cell junction morphology or cilia abundance. Epithelial Sodium Channels and Calcium-activated Chloride Channel activity did not differ but CFTR mediated chloride currents were significantly reduced in SMADiALI compare to their CRCALI counterparts. ConclusionAlteration of cellular physiological function in vitro occurs were more prominent than structural and differentiation potential in airway ALI. Since culture conditions significantly influence CFTR activity, this could lead to false conclusions if data from different labs are compared against each other without specific reference ranges.

physiology↗

bioRxiv2020

Novel antioxidant therapy with the immediate precursor to glutathione, γ-glutamylcysteine (GGC), ameliorates LPS-induced cellular stress in an in vitro cystic fibrosis model

IntroductionGlutathione deficiency and chronic bacterial inflammation exacerbates the oxidative stress damage to airways in cystic fibrosis. Improvements to current antioxidant therapeutic strategies are needed. Dietary supplement, {gamma}-glutamylcysteine (GGC), the immediate precursor to glutathione, rapidly boosts cellular glutathione levels following a single dose in healthy individuals. Efficacy of GGC against Pseudomonas aeruginosa derived lipopolysaccharide (LPS), a prominent factor in mediating both bacterial virulence and host responses, in CF remains unassessed. MethodsPrimary F508del/F508del mucociliary differentiated bronchial and nasal epithelial cells were created to model LPS-induced oxidative stress and inflammation of CF. The proteomic signature of GGC treated cells was resolved by qLC-MS/MS. Parameters including cell redox state (glutathione, ROS), anti-inflammatory mediators (IL-8, IDO-1) and cellular health (membrane integrity, stress granule formation and cell viability) were assayed. ResultsProteomic analysis identified perturbation of several pathways related to cellular respiration and stress responses upon LPS challenge. Most of these were resolved when cells were treated with GGC. While GGC did not resolve LPS-induced IL-8 and IDO-1 activity, it effectively attenuated LPS-induced ROS and stress granule formation, while significantly increasing intracellular glutathione levels and improving epithelial cell barrier integrity. Moreover, we compared the effect of GGC with thiols NAC and glutathione on cell viability. GGC was the only thiol that increased cell viability; protecting cells against LPS induced cell death. Both therapeutic and prophylactic treatments were successful. ConclusionTogether, these findings indicate that GGC has therapeutic potential for treatment and prevention of oxidative stress related damage to airways in Cystic Fibrosis.

cell biology↗