3003 – IDENTIFYING NEW DRIVERS OF HAEMATOPOIETIC STEM CELL FATE CHOICE USING PROTEOMICS

Abstract

De-regulation of cell fate choices in adult stem cells has been implicated in ageing and tumorigenesis. Recent advances in single cell technologies have equipped researchers with unprecedented resolution of both the functional properties and transcriptional profile of single adult haematopoietic stem cells (HSCs). Whether these transcriptional programmes are reflected at the protein level remains poorly understood. Moreover, poly-A based capture methods can introduce biases or completely miss critical molecular changes. To date, the low frequency and number of functional HSCs, the inability to prospectively isolate distinct HSC subtypes, and the technical limitations of large scale protein profiling have precluded extensive analyses of molecular networks at the protein level. Here we describe an optimized mass spectrometry-based workflow to characterise the proteome from as few as 10,000 HSCs using tandem mass tag (TMT) labelling across 10 cell fractions. Our approach reduced the required cell input 30-fold when compared to conventional protocols and permitted the identification and quantification of over 4,000 proteins. We applied this technology to probe the HSC compartment from wild-type and TET2 deficient cells, identifying protein networks potentially involved in the regulation of HSC fate choice. In HSC fractions with different self-renewal durability and HSC content (Lin-Kit+CD150+CD48- compared to Lin-Kit+CD150+CD48-EPCR+) we observed progressive re-shaping of protein networks that implicate the physical role of neighbouring cells in the bone marrow niche. Together our results indicate that HSCs obtained from the bone marrow retain molecular features of the niche from which they were extracted and offer a new potential avenue to study HSC fate choice in normal and malignant cells.