Use of synthetic mRNA transfection encoding telomerase to improve the in-vitro lifespan of dental pulp stem cells

Kinteh, Fatoumata (2025) Use of synthetic mRNA transfection encoding telomerase to improve the in-vitro lifespan of dental pulp stem cells. MSc(R) thesis, University of Glasgow.

Abstract

Dental pulp stem cells (DPSCs), like most stem cells, have the potential to be used in regenerative medicine. However, one of the limitations to the usage of DPSCs is their inability to maintain a long term, in-vitro lifespan due to replicative senescence. A key contributing factor to this senescence is telomere erosion.

Our research aimed to generate and use a synthetic messenger RNA (mRNA) encoding the human telomerase, a key protein responsible for telomere maintenance. mRNA was chosen instead of reprogramming cells using DNA constructs as the latter carries the risk of unwanted genomic integrations. Successes with the highly effective COVID-19 vaccines and other RNA drugs, such as small interfering RNA (siRNA) drugs, has driven substantial improvements in RNA delivery technologies and greatly increased the potential of mRNA approaches.

After correcting a mutation found in the telomerase mRNA sequence, we synthesised telomerase mRNA from the corrected plasmid using CleanCapAGTM to add cap1 structure to the 5’ end, substituting the normal uridine triphosphate (UTP) with the N1-methylpseudouridine triphosphate and polyadenylating the final product. We used eGFP-encoding mRNA made the same way as a transfection control. We showed that both DPSCs and a control cancer cell line, TR146, could be transfected with enhanced green fluorescent protein (eGFP) mRNA at high efficiency with a simple procedure and an inexpensive commercially available RNA transfection reagent.

Telomere lengths (TL) in DPSCs (both wild type and transfected) were quantified as a relative measurement using the quantitative polymerase chain reaction (qPCR) method originally designed by Richard Cawthon and later modified by O’Callaghan and Fenech. We found that, indeed, telomere erosion occurs in DPSCs as the cells age by increasing passage numbers. However, DPSCs with shorter telomere lengths transfected with the telomerase mRNA showed no sign of telomere extension.

In contrast, TR146 cancer cells which had shorter telomeres than DPSCs had their telomeres significantly extended after transfection with the same synthetic telomerase mRNA indicating the mRNA was functional. It is possible that the synthetic telomerase mRNA is not translated in DPSCs, and Western blot evaluation is needed to test this. Initial experiments suggested other genes required for telomere extension were transcribed in DPSCs. However further works is also needed to test if these are expressed strongly enough.

• Item Type: Thesis (MSc(R))
• Qualification Level: Masters
• Keywords: DPSCs, synthetic mRNA, telomeres, telomerase, q-PCR.
• Subjects: R Medicine > R Medicine (General); R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer); R Medicine > RM Therapeutics. Pharmacology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Medicine, Dentistry & Nursing
• Supervisor's Name: Hamilton, Dr. Andrew
• Date of Award: 2025
• Depositing User: Theses Team
• Unique ID: glathesis:2025-84971
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 01 Apr 2025 15:17
• Last Modified: 01 Apr 2025 15:55
• Thesis DOI: 10.5525/gla.thesis.84971
• URI: https://theses.gla.ac.uk/id/eprint/84971