Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.733349
Title: Smooth muscle differentiation from human umbilical cord derived mesenchymal stem cells : miRNA-involved mechanism and potential application for vascular tissue engineering
Author: Gu, Wenduo
ISNI:       0000 0004 6497 7726
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2017
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Abstract:
Tissue engineered vascular grafts with long term patency are in great need in the clinics. An accessible source of human smooth muscle cell (SMC) is important for constructing functional vascular grafts. Human mesenchymal stem cells from the umbilical cord (UCMSCs) exhibit multi-lineage differentiation abilities, including the potential to differentiate towards vascular lineages such as SMCs. MicroRNAs (miRNAs) are short non-coding regulatory RNAs. They widely participate in regulation of stem cell differentiation and may play an important role in SMC differentiation. Understanding how to generate SMCs from UCMSCs as well as its underlying mechanism might greatly contribute to our knowledge of manufacturing functional vascular grafts. We hypothesise that vascular grafts could be generated with SMCs differentiated from human UCMSCs, and further explore the role of miRNAs in the differentiation process. We utilised transforming growth factor β 1 (TGFβ1) to stimulate the UCMSCs differentiation towards SMCs. A panel of SMC markers including αSMA, SM22, Calponin and SMMHC were highly upregulated both at the gene expression and the protein level at day 5 of TGFβ1 treatment. Micro-RNA (miR) array analysis showed that miR-503 was increased at early time points (6 h and 24 h) after TGFβ1 treatment, which was confirmed by TaqMan microRNA assay. We further demonstrated that miR-503 mimics promoted SMC differentiation both at the gene expression and the protein level and miR-503 inhibitors downregulated SMC markers at the protein level. Smad7, which is a negative regulator of TGFβ1-related signalling pathways, was identified to be a direct target of miR-503 by luciferase reporter experiments. The expression level of miR-503 was Smad4-dependent as shown by the Smad4 knockdown experiments. Also, Smad4 was demonstrated to be enriched at the promoter region of miR-503 as shown by Chromatin immunoprecipitation experiments. In addition to miR-503, miR-222-5p was also downregulated in the differentiation process. The gain-of-function study with the treatment of miR-222-5p mimics significantly inhibited the induction of SMC markers Calponin and αSMA both at the gene expression and protein level during differentiation. αSMA was confirmed to be a direct target of miR-222-5p. Moreover, ROCK2, which could mediate SMC differentiation through RhoA/ROCK pathway, was downregulated by miR-222-5p mimics both at the gene expression and protein level. The 3’-UTR segment of ROCK2 was identified to be a direct target of miR-222-5p. Finally, SMCs differentiated from UCMSCs exhibited the ability to migrate into decellularised mouse aorta grafts. Seeding of the cells onto the decellularised scaffold gave rise to vascular graft with smooth muscle layer that is comparable to its analog of the native vessel. In conclusion, we demonstrated the potential of using hUCMSCs-derived SMCs to generate vascular grafts which are in critical need in the clinics.
Supervisor: Xu, Qingbo ; Hu, Yanhua Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.733349  DOI: Not available
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