Combined Single-Cell Functional and Gene Expression Analysis Resolves Heterogeneity within Stem Cell Populations

Abstract

SummaryHeterogeneity within the self-renewal durability of adult hematopoietic stem cells (HSCs) challenges our understanding of the molecular framework underlying HSC function. Gene expression studies have been hampered by the presence of multiple HSC subtypes and contaminating non-HSCs in bulk HSC populations. To gain deeper insight into the gene expression program of murine HSCs, we combined single-cell functional assays with flow cytometric index sorting and single-cell gene expression assays. Through bioinformatic integration of these datasets, we designed an unbiased sorting strategy that separates non-HSCs away from HSCs, and single-cell transplantation experiments using the enriched population were combined with RNA-seq data to identify key molecules that associate with long-term durable self-renewal, producing a single-cell molecular dataset that is linked to functional stem cell activity. Finally, we demonstrated the broader applicability of this approach for linking key molecules with defined cellular functions in another stem cell system.

Sca-1 expression level identifies quiescent hematopoietic stem and progenitor cells

“…we confirmed the recent finding (Säwén et al, 2016;Wilson et al, 2015) (Vazquez et al, 2015). Our result that CD201 expression level correlates with quiescence of HSPCs similar to SCA-1 facilitates identification and isolation of quiescent subsets among lin…”

Epigenetic Memory Underlies Cell-Autonomous Heterogeneous Behavior of Hematopoietic Stem Cells

“…Hematopoietic stem cells have been demonstrated to exhibit bias toward myeloid, lymphoid, or megakaryocytic lineage upon transplantation of single cells (Dykstra et al, 2007(Dykstra et al, , 2011Morita et al, 2010), on ex vivo barcoding and transplantation of populations (Aiuti et al, 2013;Gerrits et al, 2010;Jordan and Lemischka, 1990;Lemischka, 1993;Lemischka et al, 1986;Lu et al, 2011;Mazurier et al, 2004;Shi et al, 2002;Snodgrass and Keller, 1987), or by retrotransposon tagging of endogenous cells (Sun et al, 2014b). Further, single-cell transplant data have been coupled with single-cell gene expression analysis on different cells to resolve subpopulations with corresponding gene expression and repopulation potential (Wilson et al, 2015). Overlaying in vivo functional behavior of endogenous HSC clones with their gene expression and epigenetic characteristics represents a key unresolved challenge.…”

Reconstructing blood stem cell regulatory network models from single-cell molecular profiles

“…The sorting strategies used to isolate each population are displayed to the right of the lineage tree. The three cell types with starred sorting strategies were collected and profiled specifically for this study; unstarred populations were profiled in our previous study, and the lineage tree diagram is also adapted from this paper (2). ( can capture a variety of more complex structures.…”

“…Hematopoiesis is an extensively studied and well-characterized system (1), and yet it is only with the recent development of high-throughput single-cell technologies that we are understanding how heterogeneity within hematopoietic stem and progenitor cell (HSPC) populations is related to fate choices in the blood (2,3). Unlike bulk population studies, which measure average states of expression and assume homogeneity within a population, single-cell assays can resolve the molecular basis of cell type heterogeneity.…”