Multimodal label-free imaging to study skeletal stem cells and skeletal regeneration

Abstract

The use of skeletal stem cells (SSCs) in cell-based therapies is currently one of the most promising areas for the treatment of skeletal disease and skeletal tissue repair. The ability to control the modification of SSCs could provide significant therapeutic potential in regenerative medicine, with the prospect to permanently repopulate a host with stem cells. Current limitations in the characterisation of SSC differentiation have led to the development and application of alternative strategies that aim to identify molecules at the subcellular level using their inherent properties, without the use of a dye or label, i.e. label-free.

Label-free imaging methods are emerging as powerful alternatives to conventional techniques in biomedicine. The work presented in this thesis aimed to investigate SSC differentiation and to engineer strategies to generate skeletal tissue and temporally evaluate SSC development using multimodal and label-free imaging approaches.

The differentiation process of adult human bone marrow SSCs into adipocytes was evaluated using coherent anti-Stokes Raman scattering (CARS) imaging. CARS provided an enhanced resolution of lipid droplets compared to conventional staining, detectable as early as 24 hours after adipogenic induction.

The combination of CARS microscopy with second harmonic generation (SHG) and two-photon excited auto-fluorescence (TPEAF) offered a novel insight into the chondrogenic differentiation of human fetal-femur derived skeletal cells. Retrieval of 3D information using a non-invasive and non-destructive platform enabled elucidation of the temporal changes in cartilage development.

Subsequently, multimodal label-free imaging was used to investigate the differentiation of live human fetal-femur derived skeletal cells differentiated into chondrogenic cultures. The results demonstrated that label-free CARS live-cell imaging at the molecular level did not significantly affect cell differentiation and development into cartilage. Moreover, it was demonstrated that CARS microscopy is a suitable platform to image additional key molecules of interest, namely proteins and glycosaminoglycans, with bioengineered cartilage tissue imaged at different wavenumbers.

The ability to dynamically follow the formation of new regenerated tissues in real-time using non-invasive techniques offers exciting opportunities for the design and development of innovative tissue engineering solutions for hard and soft tissues. This thesis indicates that multimodal label-free imaging with non-linear techniques such as CARS and SHG are suitable approaches for the assessment of bioengineered skeletal tissues with potential clinical translation.

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Identifiers

ORCID for Sumeet Mahajan: orcid.org/0000-0001-8923-6666

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