Index sorting resolves heterogeneous murine hematopoietic stem cell populations

Abstract

Recent advances in the cellular and molecular biology of single stem cells have uncovered significant heterogeneity in the functional properties of stem cell populations. This has prompted the development of approaches to study single cells in isolation, often performed using multiparameter flow cytometry. However, many stem cell populations are too rare to test all possible cell surface marker combinations, and virtually nothing is known about functional differences associated with varying intensities of such markers. Here we describe the use of index sorting for further resolution of the flow cytometric isolation of single murine hematopoietic stem cells (HSCs). Specifically, we associate single-cell functional assay outcomes with distinct cell surface marker expression intensities. High levels of both CD150 and EPCR associate with delayed kinetics of cell division and low levels of differentiation. Moreover, cells that do not form single HSC-derived clones appear in the 7AADdim fraction, suggesting that even low levels of 7AAD staining are indicative of less healthy cell populations. These data indicate that when used in combination with single-cell functional assays, index sorting is a powerful tool for refining cell isolation strategies. This approach can be broadly applied to other single-cell systems, both to improve isolation and to acquire additional cell surface marker information.

Simultaneous tracking of division and differentiation from individual hematopoietic stem and progenitor cells reveals within-family homogeneity despite population heterogeneity

“…SLAM-HSCs preferentially differentiated without dividing into SLAM + MEP, as reported previously [10]. On comparing the surface marker expression of differentiated and not-differentiated nondivided cells ( Figure S1B), Sca-1 high SLAM-HSCs were more likely not to differentiate, in agreement with a previous report [30]. The addition of IL-3 and IL-6 only significantly impacted the differentiation pattern of the progeny of MPPs.…”

Single-cell transcriptome analysis of fish immune cells provides insight into the evolution of vertebrate immune cell types

“…Propidium iodide (PI) staining was used to exclude dead cells. Individual cells were sorted, using a Becton Dickinson influx sorter with 488-and 561-nm lasers (Schulte et al 2015) and collected in single wells of 96-well plates containing 2.3 µL of 0.2% Triton X-100 supplemented with 1 U/µL SUPERase·In RNAse inhibitor (Ambion). The size, granularity, and level of fluorescence for each cell were simultaneously recorded.…”

Heterogeneity of hypothalamic Pro-opiomelanocortin-expressing neurons revealed by single-cell RNA sequencing

“…Fluorescence-activating cell sorting was performed using an Influx Cell Sorter (BD Biosciences, San Jose, CA, USA) utilising a protocol that was previously used for single-cell isolation (Macaulay et al, 2016;Schulte et al, 2015). The cell gating was set according to cell size (FSC), cell granularity (SSC), FSC pulse-width for singlets, fluorescence at 488nm/532nm for GFP and 647/670nm for nuclear stain with DraQ5 (Biostatus, Shepshed, Leicester, UK).…”