Skeletal stem cell isolation and differentiation: Interdisciplinary strategies for skeletal tissue engineering

Abstract

Stem cell based tissue engineering is viewed as a promising approach for orthopaedic reparative medicine and the application of microfluidic techniques for isolation and characterisation of individual skeletal stem cells is considered a potential source of cells for regenerative medicine. The studies described in this thesis aim to develop original techniques for isolation and characterisation of mesenchymal stem cells and to examine their possible uses in skeletal tissue engineering. These studies developed novel microfluidic technology using dielectrophoretic ring traps and sorting gates for isolation and recovery of specific cells according to immunofluorescent intensity. To date, the devices outlined in this work are limited by the small number of cells that can be isolated, but are capable of recovering established and primary cell populations with 100% purity for specific markers such as STRO-1, while also displaying potential for on-chip analysis and culture due to the ability to precisely control the device's microenvironment. This study has also investigated 28 day organotypic culture of 3D fetal femur-derived cell pellets at an air-liquid interface. It was demonstrated that addition of serum, ascorbate, dexamethasone and BMP-2 resulted in mimicry of in vivo femur development, while addition of ascorbate and TGF- phenotype, thus offering potential models for both cartilage and early bone development. Analysis of pellets demonstrated that significant pellet diameter at day 1 (greater than 0.8mm) is crucial for maintaining reproducible results in osteogenic and chondrogenic conditions. Furthermore, addition of BMP-2 to fetal femur-derived cells cultured in chemically defined media induced formation of a novel cobblestone cell morphology. Characterisation of the cobblestone cells demonstrated a primitive adipogenic phenotype as indicated by the lack of endothelial and haematopoietic marker expression including CD146, TIE2, CD34, and CD105 and upregulation of mesenchymal differ lipid. Overall these studies have offered a novel approach to stem cell isolation for characterisation and have furthered the knowledge of fetal femur-derived cell and their potential as an alternative cell source for skeletal tissue engineering

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Identifiers

ORCID for Richard O.C. Oreffo: orcid.org/0000-0001-5995-6726

ORCID for Hywel Morgan: orcid.org/0000-0003-4850-5676

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