Identification of Novel Drivers of Haematopoietic Stem Cell Fate Using Low Cell Number Proteomics and Single Cell Profiling
The abrogation and de-regulation of cellular decisions in adult haematopoietic stem cells (HSCs) have been widely recognised as key contributing factors in ageing and disease. In particular, acquired mutations altering HSC self-renewal contribute to the formation of pre-leukaemic disorders, such as myeloproliferative neoplasms (MPNs), and ultimately to their transformation to acute myeloid leukaemia. Therefore, we urgently require a complete characterisation of the underlying molecular pathways of HSC self-renewal in order to provide crucial insights into disease progression and to help inform novel strategies for ex vivo expansion for gene therapy applications.
To date, advances in functional and molecular single cell technologies have provided unprecedented resolution of heterogeneous HSC populations and their transcriptional landscapes. In contrast, the scarcity of functional HSCs and technical limitations of unbiased proteomic screening technology have prevented comprehensive characterisation of protein networks governing HSC fate. To overcome these technical limitations, we developed an optimised mass spectrometry workflow to interrogate as few as 10,000 HSCs and multiplex up to 16 cell fractions. Using this approach, we were able to quantify in excess of 4,000 proteins, while reducing the required cell input 30-fold. To identify key molecular drivers of in vivo HSC self-renewal, we probed HSC populations with increasing self-renewal potency, including TET2-deficient HSCs exhibiting a self-renewal advantage in MPN mouse models. Here, we observed reshaping of extracellular matrix protein networks, indicating a potential physical role of the neighbouring bone marrow niche cells for regulating HSC fate. Next, we integrated proteomic and transcriptomic to characterise the molecular pathways underlying HSC self-renewal across numerous -omics technologies. Here, we identified a wide range of intrinsic regulatory pathways and described molecular mechanisms regulating intracellular calcium levels in HSCs.
In the final results chapter, we explored the ex vivo expansion of HSCs, since this is of paramount importance for the delivery of gene and cellular therapies against a plethora of monogenic haematological diseases. Recently pioneered HSC expansion protocols greatly enhanced the yield of phenotypic HSCs, but such cultures exhibit significant clone-to-clone variability in long-term self-renewal potency and differentiation. By linking the transplantation outcomes with transcriptional profiles of individual single cell-derived clones, we derived a novel gene signature for ex vivo expanded HSCs with long-term self-renewal potency and characterised key molecular pathways governing HSC fate throughout expansion. Furthermore, we identified a reporter strategy for prospectively isolating expanded HSCs.
Together, these findings provided an insight into the molecular machinery underlying HSC self-renewal within the native bone marrow niche and during ex vivo expansion. Comprehensive multi-omic profiling also revealed the intricate relationship of gene expression profiles with the proteomic phenotype within the HSC compartment. Finally, we propose an optimised workflow for performing comprehensive proteomics on any rare cell populations which will be of use to researchers investigating a wide-range of cellular biology questions.