title: 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 7AAD(dim) 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.
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Reiner
authors.initials: R
authors.lastname: Schulte
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom; Department of Haematology, University of Cambridge, Cambridge, United Kingdom; Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Nicola K
authors.initials: NK
authors.lastname: Wilson
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom; Department of Haematology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Janine C M
authors.initials: JC
authors.lastname: Prick
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Chiara
authors.initials: C
authors.lastname: Cossetti
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Michal K
authors.initials: MK
authors.lastname: Maj
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom; Department of Haematology, University of Cambridge, Cambridge, United Kingdom; Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Berthold
authors.initials: B
authors.lastname: Gottgens
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom; Department of Haematology, University of Cambridge, Cambridge, United Kingdom; Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom. Electronic address: dgk23@cam.ac.uk.
authors.forename: David G
authors.initials: DG
authors.lastname: Kent
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology*; D002455:Cell Division / Q000502:physiology*; D000074283:Endothelial Protein C Receptor; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism; D051379:Mice; D011956:Receptors, Cell Surface / Q000378:metabolism*; D000071177:Signaling Lymphocytic Activation Molecule Family Member 1
chemical_list: D015703:Antigens, CD; D000074283:Endothelial Protein C Receptor; C080248:Procr protein, mouse; D011956:Receptors, Cell Surface; D000071177:Signaling Lymphocytic Activation Molecule Family Member 1