Direct RT-qPCR Assay for the Detection of SARS-CoV-2 in Saliva Samples

Since mid-2020 there have been complexities and difficulties in the standardisation and administration of nasopharyngeal swabs. Coupled with the variable and/or poor accuracy of lateral flow devices, this has led to increased societal ‘testing fatigue’ and reduced confidence in test results. Consequently, asymptomatic individuals have developed reluctance towards repeat testing, which remains the best way to monitor COVID-19 cases in the wider population. On the other hand, saliva-based PCR, a non-invasive, highly sensitive, and accurate test suitable for everyone, is gaining momentum as a straightforward and reliable means of detecting SARS-CoV-2 in symptomatic and asymptomatic individuals. Here, we provide an itemised list of the equipment and reagents involved in the process of sample submission, inactivation and analysis, as well as a detailed description of how each of these steps is performed

Novel Culture Strategies and Signal Transduction Pathways of Pluripotent Stem Cells

Pluripotent stem cells (PSCs) can self-renew indefinitely in culture while maintaining their capacity to differentiate into any cell type of an organism, thus offering novel sources for drug screening, in vitro disease modelling, and cell replacement therapies. However, due to their sensitive nature, many PSC lines are still cultured using undefined components such as serum or serum-derived components, on either feeder cells or complex protein mixes such as Matrigel or gelatine. In order to fully realize the potential of these cells we need controlled, completely defined and xeno-free culturing conditions that maintain growth and survival of homogenous, non-differentiated colonies. This thesis focuses on the in vitro maintenance of both mouse and human PSCs, analysing the media and substrate requirements of these cells and linking them to the intracellular signalling pathways involved in the maintenance of pluripotency and self-renewal. Benchmarking of commercially available culture methods for PSCs has been performed, evaluating their capacity to maintain pluripotency and growth of undifferentiated PSCs over several passages and reporting new characteristics, like the tendency of mouse PSCs to grow as floating spheres in 2i medium, a novel media formulation that uses two inhibitors to hinder differentiation capacity and subsequently induce pure, undifferentiated cultures. The major finding in this thesis is the identification of Inter-α-Inhibitor (IαI) as a protein able to activate the previously described signal-transduction pathway Yes/YAP/TEAD in mouse PSCs and to induce transcription of the well-known stem cell transcription factors Nanog and Oct3/4. IαI is a serum protein found in high concentration in human serum that had been traditionally described as an extracellular matrix remodelling protein. For the first time, we describe IαI to have signalling capacity on PSCs. Moreover, IαI is demonstrated to induce attachment, growth and long-term survival of undifferentiated mouse and human PSCs when added to serum-free, chemically defined media. IαI is the first molecule described to date to induce attachment of human PSCs on uncoated, standard tissue-culture treated plastic, just by supplementation as a soluble molecule at the seeding step. Following this discovery, we evaluate a novel culture method using the completely defined, serum-free E8 medium supplemented with IαI (E8:IαI) for long-term propagation of four different human PSC lines and discover that IαI can indeed support long-term culture with maintained pluripotency, differentiation capacity, growth rate and genetic stability. Moreover, in contrast to the control culture method using a commercially available surface coating, IαI supplementation can support single cell passaging of human PSCs, and adapt feeder-dependent cultured human PSCs to E8:IαI with high efficiency. A mouse PSC line is also grown for over 20 passages in IαI with retained pluripotency, differentiation capacity and genetic stability. IαI is inexpensive to produce and derived from human plasma, and could therefore be produced in compliance with Good Manufacturing Practices. Ultimately, our group aims to develop and test large-scale, completely defined, xeno-free culturing methods for PSCs, suitable for pharmacological and medical applications.

Phenotypc analysis of free-floating mouse ES cell sphere morphology as assessed by phase-contrast microscopy and immunochemistry.

<p>Phase-contrast micrographs of spheroid morphology (A) and fluorescence micrographs of Oct4 (B) and Ki67 (C) immunohistochemical detection of Oct4 and Ki67 in cryosectioned spheroids of mES cells grown in 2i or ESN2 media.</p

Analysis of ES cells induced to differentiate as assessed by quantitative PCR.

<p>Quantitative PCR analysis for the expression of Oct4 and FGF4 and indicator genes for the three germ layers in mES cells that were induced to differentiate for 6(A) or on gelatin (B) in SCM, mES Prime Kit or 2i media, and in suspension (C) in 2i or ESN2 media. 18S expression is used for normalization, and results are comparative (expression levels at passage 15 set as control) Ct value means ± sd (n = 3).</p

Characterisation of mouse ES cells cultured in 2i media.

<p>Fluorescence micrographs of GFP-expression E14 mES cells, pre-transfection cultured in 2i or SCM, transfected with Lipofectamine 2000 and pmaxGFP (A). Luciferase reporter assay for Oct4 and Nanog promoter activation, as well as Tead-enhanced promoter activation, 24 hrs post-transfection in E14 cells cultured without LIF in serum-free SCM with or without PD0325901 (1 μM) and CH99021 (3 μM), SCM, or 2i media. Results are mean ± sd (n = 3) p(*) <0.05 (B). Phase-contrast micrographs of colony morphology and alkaline phosphatase activity staining (C), and qPCR analysis (D) of E14 cell 96 hrs after 2i rescue and clean-up of partially differentiated mES cells cultures. 18S expression is used for normalization, and results are comparative (expression levels at passage 15 set as control) Ct value means ± sd (n = 3) p(*) <0.05.</p

Analysis of ES cells cultured using different protocols as assessed by quantitative PCR.

<p>Quantitative PCR analysis at passage 15 for genes associated with undifferentiated mES cells, i.e. Oct4 and Fgf4, as well as early indicator genes for the three germ layers (endoderm: Dab2 and Gata6; ectoderm: Fgf5 and Pax6; mesoderm: Brc and Actc1). Graphs represent pooled results for mES cell lines grown on feeders (A) or on gelatin (B) in SCM, mES Prime Kit or 2i media, and in suspension (C) in 2i or ESN2 media. 18S expression is used for normalization, and results are comparative (SCM values for feeders and gelatin, and 2i media for suspension, were set as control) Ct value means ± sd (n = 3) p(*) <0.05.</p

Adherent mouse ES cell colony morphology and alkaline phosphatase activity.

<p>Phase-contrast micrographs of colony morphology and alkaline phosphatase activity staining for mES cells grown on feeders (A) and mES cells grown on gelatin (B) for 15 passages in SCM, mES Prime Kit or 2i media. Noteworthy is that the feeder-cell dependent mES cell line C57 was maintained in 2i medium on gelatin for at least 15 passages without showing morphological signs of spontaneous differentiation or loss in alkaline phosphatase activity (C).</p

Proliferation rates of mouse ES cells cultured using different protocols.

<p>Cell doubling time assessment of mES cells grown on feeders (A) in SCM, mES Prime Kit and 2i media; on gelatin (B) in SCM, mES Prime Kit and 2i media supplemented with 2% FBS; or in suspension (C) in 2i and ESN2 media. Results are means ± sd for 5 consecutive passages (n = 3).</p