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Examination of MicroRNAs in skeletal stem cell differentiation

Examination of MicroRNAs in skeletal stem cell differentiation
Examination of MicroRNAs in skeletal stem cell differentiation
MicroRNAs (miRNA/miRs) play a crucial role in a variety of biological processes including stem cell differentiation and function. Foetal femur-derived skeletal stem cells (SSCs) display enhanced proliferation and multipotential capacity, indicating excellent potential as candidates for tissue engineering applications. This thesis has identified and characterised subpopulations of skeletal stem cells found within the foetal femur. Cells isolated from the epiphyseal region of the foetal femur expressed higher levels of genes associated with chondrogenesis while cells from the diaphyseal region expressed higher levels of genes associated with osteogenic differentiation. In addition to the difference in osteogenic and chondrogenic gene expression, epiphyseal and diaphyseal cell populations displayed distinct miRs expression profiles. To examine the role of miRNA during skeletogenesis, a robust cell culture model containing differentiating SSCs and an effective transfection protocol was developed. Spermine-pullulan complex, a potential delivery system for miRNA-based gene therapy, was shown to be able to transfect SSCs with miRNA mimics and inhibitors but with lower efficacy compared to liposome base transfection reagent. Through miRNA gene expression profiling and mRNA targets analysis, miR-146a was found to be expressed by diaphyseal cell populations at a significantly enhanced level compared to epiphyseal populations and was predicted to target various components of the TGF-? pathway. Examination of miR-146a function in foetal femur cells confirmed regulation of protein translation of SMAD2 and SMAD3 following transient overexpression in epiphyseal cells. The down-regulation of SMAD2 and SMAD3 following overexpression of miR-146a resulted in an up-regulation of the osteogenesis-related gene RUNX2 and down-regulation of the chondrogenesis-related gene SOX9.In conclusion, this thesis has identified subpopulations of skeletal stem cells with enhanced osteogenic and chondrogenic potential and has explored new miRNA targets involved in skeletogenesis in the attempt to develop novel treatments for patients requiring reparation of the skeletal system.
Cheung, Kelvin
4466c036-4252-4eb8-919a-a299742e0786
Cheung, Kelvin
4466c036-4252-4eb8-919a-a299742e0786
Oreffo, Richard
ff9fff72-6855-4d0f-bfb2-311d0e8f3778
Sanchez-Elsner, Tilman
b8799f8d-e2b4-4b37-b77c-f2f0e8e2070d

(2015) Examination of MicroRNAs in skeletal stem cell differentiation. University of Southampton, Faculty of Medicine, Doctoral Thesis, 155pp.

Record type: Thesis (Doctoral)

Abstract

MicroRNAs (miRNA/miRs) play a crucial role in a variety of biological processes including stem cell differentiation and function. Foetal femur-derived skeletal stem cells (SSCs) display enhanced proliferation and multipotential capacity, indicating excellent potential as candidates for tissue engineering applications. This thesis has identified and characterised subpopulations of skeletal stem cells found within the foetal femur. Cells isolated from the epiphyseal region of the foetal femur expressed higher levels of genes associated with chondrogenesis while cells from the diaphyseal region expressed higher levels of genes associated with osteogenic differentiation. In addition to the difference in osteogenic and chondrogenic gene expression, epiphyseal and diaphyseal cell populations displayed distinct miRs expression profiles. To examine the role of miRNA during skeletogenesis, a robust cell culture model containing differentiating SSCs and an effective transfection protocol was developed. Spermine-pullulan complex, a potential delivery system for miRNA-based gene therapy, was shown to be able to transfect SSCs with miRNA mimics and inhibitors but with lower efficacy compared to liposome base transfection reagent. Through miRNA gene expression profiling and mRNA targets analysis, miR-146a was found to be expressed by diaphyseal cell populations at a significantly enhanced level compared to epiphyseal populations and was predicted to target various components of the TGF-? pathway. Examination of miR-146a function in foetal femur cells confirmed regulation of protein translation of SMAD2 and SMAD3 following transient overexpression in epiphyseal cells. The down-regulation of SMAD2 and SMAD3 following overexpression of miR-146a resulted in an up-regulation of the osteogenesis-related gene RUNX2 and down-regulation of the chondrogenesis-related gene SOX9.In conclusion, this thesis has identified subpopulations of skeletal stem cells with enhanced osteogenic and chondrogenic potential and has explored new miRNA targets involved in skeletogenesis in the attempt to develop novel treatments for patients requiring reparation of the skeletal system.

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Published date: September 2015
Organisations: University of Southampton, Faculty of Medicine

Identifiers

Local EPrints ID: 397111
URI: http://eprints.soton.ac.uk/id/eprint/397111
PURE UUID: db404e9d-5160-4a14-b263-77d2f27f51ec
ORCID for Richard Oreffo: ORCID iD orcid.org/0000-0001-5995-6726
ORCID for Tilman Sanchez-Elsner: ORCID iD orcid.org/0000-0003-1915-2410

Catalogue record

Date deposited: 15 Jul 2016 13:45
Last modified: 23 Jun 2020 00:30

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Contributors

Author: Kelvin Cheung
Thesis advisor: Richard Oreffo ORCID iD
Thesis advisor: Tilman Sanchez-Elsner ORCID iD

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