Identifying the skeletal stem cell for regeneration - harnessing single-cell RNA-sequencing

Abstract

As global life expectancy increases, the rising incidence of age-related skeletal degeneration emerges as a major public health concern. The ability of bone to regenerate declines with age and there remains an unmet need to develop robust regenerative strategies, to reduce bone loss and improve quality of life. Cell-based strategies aim to harness the regenerative potential of skeletal stem cells (SSCs); a population of specialised stem cells residing in human post-natal bone marrow that possess the capacity for self-renewal and tri-lineage stromal cell differentiation. However, the application of SSCs in cell-based therapies is currently significantly limited by the lack of a defined unique, or set of, marker(s) that can be used to define the SSC population. Recent advancements in high-throughput single-cell RNA-sequencing (scRNA-Seq) have enabled the simultaneous discovery and assessment of genetic markers across hundreds and thousands of cells, offering invaluable insights into cellular heterogeneity and subpopulation biomarkers. This thesis investigates the hypothesis that scRNA-seq technology can be applied to develop SSC marker profiles, which, coupled with innovative cell isolation strategies, enables enrichment of the SSC from human bone marrow. Consequently, this thesis aimed to characterise SSCs to identify new candidate biomarkers for the development of robust SSC enrichment strategies. Moreover, the current study employed a novel mRNA-based cell sorting technology whereby spherical nucleic acids (SNAs) fluorescently label live cells for isolation based on expression of target mRNA.
Analysis of unfractionated and enriched human bone marrow scRNA-seq datasets enabled the identification of candidate markers, including SPARC-Like 1 (SPARCL1) and TRANSFERRIN (TF), to serve as SNA targets for SSC enrichment strategies. When targeting molecular markers identified by scRNA-seq, the sorted cell populations displayed enhanced colony formation (CFU-F), indicative of SSCenrichment. SPARCL1 and TF enriched CFU-F 3.75- and 4.5-fold respectively, and dual-target sorting of SPARCL1+TF+ cells enriched CFU-F 13.38-fold. Furthermore, the enriched SSC populations displayed a capacity for multi-lineage differentiation in vitro; one of the general requirements for SSC classification. In a preliminary in vivo study, TF+ populations were encapsulated in alginate/chitosan polysaccharide capsules, and heterotopic bone formation was examined using the subcutaneous implant model in athymic immuno-deficient mice. Micro-computed tomography revealed mineral deposition and dense bone formation in scaffolds after 8 weeks in vivo.
Overall, the reported findings describe novel markers that enable the isolation of an enriched pool of SSCs from human adult bone marrow. The current study examined the transcriptomic profiles, and the in vitro and in vivo characteristics of enriched SSC populations, which were assessed in over 100 patient samples. The enriched SSC populations demonstrate an enhanced CFU-F capacity and multipotentiality in vitro, and heterotopic mineralisation in vivo. The unique populations identified offer an invaluable resource for further characterisation of SSCs to progress SSC isolation strategies and ultimately, allow for the development of SSC-based therapies for the treatment of skeletal damage and disease.

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

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