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Enrichment and characterisation of the skeletal stem cell population

Enrichment and characterisation of the skeletal stem cell population
Enrichment and characterisation of the skeletal stem cell population
Throughout the world, the average population age is increasing and, in many areas, nearly a quarter of the population will be over the age of 65 within the next 15 years, 40% of the total NHS budget is spent on people over the age of 65. The UK spends to £13 billion on musculoskeletal treatments, a cost which will increase as the population over 65 also increases. Skeletal stem cells are a rare cell population within the bone marrow, which facilitate bone repair and homeostasis. With no identified markers capable of isolating skeletal stem cells, characterisation, understanding, and subsequent use is limited. Understanding these cells within the skeletal system may help us understand disease states, the causes and, importantly, offer therapeutic solutions.

This thesis approached the study of skeletal stem cells from two alternative angles. The first, a marker discovery direction using aptamers (synthetic binding ligands, normally nucleic acid based) to identify and exploit potential skeletal stem cell-specific markers. The second approach using current enrichment techniques and the application of Drop-Seq to characterise the enriched cell gene expression profiles at a single cell level. Aptamer selection was first applied against a bonederived cell line, SAOS-2, to ensure a robust methodology was established before the use of precious primary human skeletal tissue. The SAOS-2 selection proved successful, providing ten aptamers which preferentially bound to the SAOS-2 cells over a counter selection target of the Raji cell line. Unfortunately, when carrying out the same aptamer selection method using bone marrow and foetal femur samples unwanted amplification took place within all the attempted selections, resulting in failure of selection. This was narrowed down to amplification products from the cell that was not detected in QC steps.

The use of enrichment markers provided a population (STRO-1BRIGHT+ CD146+) highly enriched for Colony Forming Units - Fibroblastic (CFU-F) capacity. This population was too small for use with Drop-Seq. STRO-1+ CD146+ cells were therefore analysed instead. The analysis highlighted four main cell types within the population, monocytes, lymphocytes, erythrocyte progenitors and CXC chemokine ligand (CXCL) 12-abundant reticular (CAR) cells.

The failure of the aptamer selection against the primary cells presented a setback to the characterisation of the skeletal stem cells. However, with a redesign of the primers and aptamer pool, this methodology should prove fruitful. The identification of the CAR cells is highly intriguing as these cells are studied within the haematopoietic stem cell field, as the CAR cells act as regulatory cells of the haematopoietic stem cells. The CAR cells have been shown to be multipotent, differentiating into adipocytes, chondrocytes and osteocytes, and are a rare population. These characteristics are shared with the characteristics attributed to the skeletal stem cell, and therefore the identification of the CAR cells from a population enriched for the skeletal stem cells suggests a relationship between these cells, whether the SSCs are the CAR cells or are closely related is unclear at this point, with further protein localisation studies this may be elucidated.
University of Southampton
Noble, Timothy John
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Noble, Timothy John
aa282744-0b13-4550-9117-a9dcb0b57124
Oreffo, Richard
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Sanchez-Elsner, Tilman
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Noble, Timothy John (2018) Enrichment and characterisation of the skeletal stem cell population. University of Southampton, Doctoral Thesis, 303pp.

Record type: Thesis (Doctoral)

Abstract

Throughout the world, the average population age is increasing and, in many areas, nearly a quarter of the population will be over the age of 65 within the next 15 years, 40% of the total NHS budget is spent on people over the age of 65. The UK spends to £13 billion on musculoskeletal treatments, a cost which will increase as the population over 65 also increases. Skeletal stem cells are a rare cell population within the bone marrow, which facilitate bone repair and homeostasis. With no identified markers capable of isolating skeletal stem cells, characterisation, understanding, and subsequent use is limited. Understanding these cells within the skeletal system may help us understand disease states, the causes and, importantly, offer therapeutic solutions.

This thesis approached the study of skeletal stem cells from two alternative angles. The first, a marker discovery direction using aptamers (synthetic binding ligands, normally nucleic acid based) to identify and exploit potential skeletal stem cell-specific markers. The second approach using current enrichment techniques and the application of Drop-Seq to characterise the enriched cell gene expression profiles at a single cell level. Aptamer selection was first applied against a bonederived cell line, SAOS-2, to ensure a robust methodology was established before the use of precious primary human skeletal tissue. The SAOS-2 selection proved successful, providing ten aptamers which preferentially bound to the SAOS-2 cells over a counter selection target of the Raji cell line. Unfortunately, when carrying out the same aptamer selection method using bone marrow and foetal femur samples unwanted amplification took place within all the attempted selections, resulting in failure of selection. This was narrowed down to amplification products from the cell that was not detected in QC steps.

The use of enrichment markers provided a population (STRO-1BRIGHT+ CD146+) highly enriched for Colony Forming Units - Fibroblastic (CFU-F) capacity. This population was too small for use with Drop-Seq. STRO-1+ CD146+ cells were therefore analysed instead. The analysis highlighted four main cell types within the population, monocytes, lymphocytes, erythrocyte progenitors and CXC chemokine ligand (CXCL) 12-abundant reticular (CAR) cells.

The failure of the aptamer selection against the primary cells presented a setback to the characterisation of the skeletal stem cells. However, with a redesign of the primers and aptamer pool, this methodology should prove fruitful. The identification of the CAR cells is highly intriguing as these cells are studied within the haematopoietic stem cell field, as the CAR cells act as regulatory cells of the haematopoietic stem cells. The CAR cells have been shown to be multipotent, differentiating into adipocytes, chondrocytes and osteocytes, and are a rare population. These characteristics are shared with the characteristics attributed to the skeletal stem cell, and therefore the identification of the CAR cells from a population enriched for the skeletal stem cells suggests a relationship between these cells, whether the SSCs are the CAR cells or are closely related is unclear at this point, with further protein localisation studies this may be elucidated.

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T J Noble Thesis - Enrichment and Characterisation of SSCs - Version of Record
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Published date: September 2018

Identifiers

Local EPrints ID: 435543
URI: http://eprints.soton.ac.uk/id/eprint/435543
PURE UUID: 795d91f3-4c44-4268-b1d2-b031d152c44c
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: 08 Nov 2019 17:30
Last modified: 23 Jun 2020 00:30

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Contributors

Author: Timothy John Noble
Thesis advisor: Richard Oreffo ORCID iD
Thesis advisor: Tilman Sanchez-Elsner ORCID iD

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