Investigating the origins and consequences of cell-to-cell variability in stem cell populations

Abstract

It is becoming increasingly recognised that stem cell populations from both the embryo and the adult are highly heterogeneous in their molecular expression patterns. However, the underlying causes and consequences are not well understood. This thesis examines cell-to-cell variability in both adult and embryonic stem cell populations, using both experimental and theoretical models to better understand stem cell biology at the single cell level.

The first part of this thesis investigates how combinatorial control of signalling pathways, and transcription regulatory networks centred around Nanog can lend robustness to the pluripotent state, despite the observed variability in individual gene expression of core regulatory factors. In order to decouple functional variability from artefacts associated with reporter constructs, a novel theoretical framework is developed to model transcriptional co-regulation, and investigate how variability of regulatory inputs can coordinate stochastic gene expression, and in particular how extrinsic factors can regulate allelic expression in heterozygous knock-in reporter cell lines. This novel theoretical model captures the co-expression characteristics of pluripotency genes and explains the abnormal expression behaviour in widely used Nanog reporter cell lines, as well as illustrating general pitfalls when designing reporter cell lines for single-cell based assays.

In the second part of this thesis, cell-to-cell variability in adult stem cell populations is examined. While functionally homogeneous embryonic stem cells are readily available, obtaining and purifying adult stem cells from primary tissue samples is a substantial challenge. This problem is particularly apparent for skeletal stem cells, which are exceptionally rare and cannot be reliably identified in situ through surface makers expression (although populations of cells enriched for skeletal stem cells may be obtained). Since these cells play a central role mediating hematopoietic stem cell activity, and are essential for bone regeneration and therefore skeletal tissue engineering strategies, understanding their molecular identity is a pressing current problem. To address this issue, current skeletal stem cell purification and recently developed high-throughput single cell profiling technologies that are able to quantify the expression of multiple transcripts in a large number of individual cells are combined. This method allows previously inaccessible detail on skeletal cell populations in situ to be obtained. To further investigate the role of cell-to-cell variability, the final part of this thesis explores how cells derived from bone marrow can be reverted to the pluripotent state, a process that is highly reliant on the individual cell fate and thereby strongly affected by cell-to-cell variability.

In summary, this thesis gives examples of how variability affects the specification of cellular identities and contributes to the understanding of how variability is regulated across cell populations.

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Identifiers

ORCID for Patrick Stumpf: orcid.org/0000-0003-0862-0290

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

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