Epigenetic Heterogeneity across Single Cells within Functionally Defined Hematopoietic Stem Cell Populations
Tony Hui1,2, Kieran O’Neil2,3, Joanna Wegrzyn Woltosz2,4, Emma Laks4, Michelle Moksa1, Sam Aparicio3,4, Aly Karsan2,3, and Martin Hirst1,2*
1Department of Microbiology and Immunology, Michael Smith Laboratories, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada; 2Canada’s Michael Smith Genome Science Center, BC Cancer Agency, Vancouver, BC, V5Z 4S6, Canada; 3Department of Pathology and Laboratory Medicine, University of British Columbia 4Department of Molecular Oncology, British Columbia Cancer Agency
Hematopoietic stem cells (HSC) are heterogeneous in their ability to durably reconstitute the blood system. Epigenome surveys along the hematopoietic hierarchy and within the HSC compartment have revealed distinct epigenomic states associated with functionally defined hematopoietic cell types. However, the degree to which epigenomic states vary within functionally defined hematopoietic populations is still largely unknown. To address this question we performed single-cell genome-wide DNA methylation profiling of linage negative Sca+cKit+ (LSK) and EPCR+CD45+CD48-CD150+ (ESLAM) hematopoietic stem cell populations purified from adult mouse bone marrow. For this we developed a bisulfite based whole genome protocol suitable for use on single index sorted mammalian cells. Analysis of the resulting data from 148 single cells revealed that the methylomes of single HSCs show characteristics consistent with those defined from bulk populations. Comparing methylation states across single HSCs revealed a significant reduction in CpG state adjacency compared to bulk estimates and that on average 13% of CpGs exist in the opposite methylation state between any two cells within both surface markers defined HSC containing populations. Utilizing a novel analytical approach designed to address missing data inherent in single cell assays we identified an epigenetically distinct subpopulation of cells present in both the LSK and ESLAM populations. Enrichment analysis of genes associated with this distinct epigenetic state revealed pathways consistent with hematopoietic stem cell function, and integration with single-cell RNA-seq data defined putative surface proteins that mark this population, including the previously identified hematopoietic marker, CD82. We further discuss a recent improvement to our protocol which allows, on average, the recovery of 2 million CpGs per cell.
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