title: Combined Single-Cell Functional and Gene Expression Analysis Resolves Heterogeneity within Stem Cell Populations.
abstract: Heterogeneity 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.
authors.affiliation: Department of Haematology, Wellcome Trust and MRC Cambridge Stem Cell Institute and Cambridge Institute for Medical Research, Cambridge University, Cambridge CB2 0XY, UK.
authors.forename: Nicola K
authors.initials: NK
authors.lastname: Wilson
authors.affiliation: Department of Haematology, Wellcome Trust and MRC Cambridge Stem Cell Institute and Cambridge Institute for Medical Research, Cambridge University, Cambridge CB2 0XY, UK.
authors.forename: David G
authors.initials: DG
authors.lastname: Kent
authors.affiliation: Institute of Computational Biology, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764 Neuherberg, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Buettner
authors.affiliation: Department of Oncology and Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge CB2 0RE, UK.
authors.forename: Mona
authors.initials: M
authors.lastname: Shehata
authors.affiliation: Single Cell Genomics Centre, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.
authors.forename: Iain C
authors.initials: IC
authors.lastname: Macaulay
authors.affiliation: Department of Haematology, Wellcome Trust and MRC Cambridge Stem Cell Institute and Cambridge Institute for Medical Research, Cambridge University, Cambridge CB2 0XY, UK.
authors.forename: Fernando J
authors.initials: FJ
authors.lastname: Calero-Nieto
authors.affiliation: Department of Haematology, Wellcome Trust and MRC Cambridge Stem Cell Institute and Cambridge Institute for Medical Research, Cambridge University, Cambridge CB2 0XY, UK.
authors.forename: Manuel
authors.initials: M
authors.lastname: Sánchez Castillo
authors.affiliation: Department of Haematology, Wellcome Trust and MRC Cambridge Stem Cell Institute and Cambridge Institute for Medical Research, Cambridge University, Cambridge CB2 0XY, UK.
authors.forename: Caroline A
authors.initials: CA
authors.lastname: Oedekoven
authors.affiliation: Department of Haematology, Wellcome Trust and MRC Cambridge Stem Cell Institute and Cambridge Institute for Medical Research, Cambridge University, Cambridge CB2 0XY, UK.
authors.forename: Evangelia
authors.initials: E
authors.lastname: Diamanti
authors.affiliation: Head of Flow Cytometry, Cambridge Institute for Medical Research, Cambridge University, Cambridge CB2 0XY, UK.
authors.forename: Reiner
authors.initials: R
authors.lastname: Schulte
authors.affiliation: Single Cell Genomics Centre, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK; MRC Computational Genomics Analysis and Training Programme, MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, UK.
authors.forename: Chris P
authors.initials: CP
authors.lastname: Ponting
authors.affiliation: Single Cell Genomics Centre, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK; Laboratory of Reproductive Genomics, Department of Human Genetics, KU Leuven, 3000 Leuven, Belgium.
authors.forename: Thierry
authors.initials: T
authors.lastname: Voet
authors.affiliation: Department of Oncology and Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge CB2 0RE, UK.
authors.forename: Carlos
authors.initials: C
authors.lastname: Caldas
authors.affiliation: Department of Oncology and Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge CB2 0RE, UK.
authors.forename: John
authors.initials: J
authors.lastname: Stingl
authors.affiliation: Department of Haematology, Wellcome Trust and MRC Cambridge Stem Cell Institute and Cambridge Institute for Medical Research, Cambridge University, Cambridge CB2 0XY, UK.
authors.forename: Anthony R
authors.initials: AR
authors.lastname: Green
authors.affiliation: Institute of Computational Biology, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764 Neuherberg, Germany; Department of Mathematics, Technische Universität München, Boltzmannstraße 3, 85748 Garching, Germany.
authors.forename: Fabian J
authors.initials: FJ
authors.lastname: Theis
authors.affiliation: Department of Haematology, Wellcome Trust and MRC Cambridge Stem Cell Institute and Cambridge Institute for Medical Research, Cambridge University, Cambridge CB2 0XY, UK. Electronic address: bg200@cam.ac.uk.
authors.forename: Berthold
authors.initials: B
authors.lastname: Göttgens
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D019070:Cell Lineage / Q000235:genetics; D049109:Cell Proliferation; D002999:Clone Cells; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D016678:Genome; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D008810:Mice, Inbred C57BL; D059010:Single-Cell Analysis / Q000379:methods*