title: Identification of factors promoting ex vivo maintenance of mouse hematopoietic stem cells by long-term single-cell quantification.
abstract: The maintenance of hematopoietic stem cells (HSCs) during ex vivo culture is an important prerequisite for their therapeutic manipulation. However, despite intense research, culture conditions for robust maintenance of HSCs are still missing. Cultured HSCs are quickly lost, preventing their improved analysis and manipulation. Identification of novel factors supporting HSC ex vivo maintenance is therefore necessary. Coculture with the AFT024 stroma cell line is capable of maintaining HSCs ex vivo long-term, but the responsible molecular players remain unknown. Here, we use continuous long-term single-cell observation to identify the HSC behavioral signature under supportive or nonsupportive stroma cocultures. We report early HSC survival as a major characteristic of HSC-maintaining conditions. Behavioral screening after manipulation of candidate molecules revealed that the extracellular matrix protein dermatopontin (Dpt) is involved in HSC maintenance. DPT knockdown in supportive stroma impaired HSC survival, whereas ectopic expression of the Dpt gene or protein in nonsupportive conditions restored HSC survival. Supplementing defined stroma- and serum-free culture conditions with recombinant DPT protein improved HSC clonogenicity. These findings illustrate a previously uncharacterized role of Dpt in maintaining HSCs ex vivo.
authors.affiliation: Research Unit Stem Cell Dynamics, Helmholtz Center Munich-German Research Center for Environmental Health (GmbH), Neuherberg, Germany; Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zurich, Basel, Switzerland;
authors.forename: Konstantinos D
authors.identifier: 0000-0002-9952-3463
authors.initials: KD
authors.lastname: Kokkaliaris
authors.affiliation: Research Unit Stem Cell Dynamics, Helmholtz Center Munich-German Research Center for Environmental Health (GmbH), Neuherberg, Germany;
authors.forename: Erin
authors.initials: E
authors.lastname: Drew
authors.affiliation: Research Unit Stem Cell Dynamics, Helmholtz Center Munich-German Research Center for Environmental Health (GmbH), Neuherberg, Germany; Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zurich, Basel, Switzerland;
authors.forename: Max
authors.initials: M
authors.lastname: Endele
authors.affiliation: Research Unit Stem Cell Dynamics, Helmholtz Center Munich-German Research Center for Environmental Health (GmbH), Neuherberg, Germany; Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zurich, Basel, Switzerland;
authors.forename: Dirk
authors.initials: D
authors.lastname: Loeffler
authors.affiliation: Research Unit Stem Cell Dynamics, Helmholtz Center Munich-German Research Center for Environmental Health (GmbH), Neuherberg, Germany; Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zurich, Basel, Switzerland;
authors.forename: Philipp S
authors.initials: PS
authors.lastname: Hoppe
authors.affiliation: Research Unit Stem Cell Dynamics, Helmholtz Center Munich-German Research Center for Environmental Health (GmbH), Neuherberg, Germany; Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zurich, Basel, Switzerland;
authors.forename: Oliver
authors.initials: O
authors.lastname: Hilsenbeck
authors.affiliation: Research Unit Stem Cell Dynamics, Helmholtz Center Munich-German Research Center for Environmental Health (GmbH), Neuherberg, Germany;
authors.forename: Bernhard
authors.initials: B
authors.lastname: Schauberger
authors.affiliation: Institute of Clinical Molecular Biology and Tumor Genetics, Helmholtz Center Munich-German Research Center for Environmental Health (GmbH), Munich, Germany;
authors.forename: Christoph
authors.initials: C
authors.lastname: Hinzen
authors.affiliation: Research Unit Stem Cell Dynamics, Helmholtz Center Munich-German Research Center for Environmental Health (GmbH), Neuherberg, Germany; Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zurich, Basel, Switzerland;
authors.forename: Stavroula
authors.initials: S
authors.lastname: Skylaki
authors.affiliation: Institute for Developmental Genetics, Helmholtz Center Munich-German Research Center for Environmental Health (GmbH), Neuherberg, Germany; and.
authors.forename: Marina
authors.initials: M
authors.lastname: Theodorou
authors.affiliation: Institute of Clinical Molecular Biology and Tumor Genetics, Helmholtz Center Munich-German Research Center for Environmental Health (GmbH), Munich, Germany;
authors.forename: Matthias
authors.initials: M
authors.lastname: Kieslinger
authors.affiliation: Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY.
authors.forename: Ihor
authors.initials: I
authors.lastname: Lemischka
authors.affiliation: Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY.
authors.forename: Kateri
authors.initials: K
authors.lastname: Moore
authors.affiliation: Research Unit Stem Cell Dynamics, Helmholtz Center Munich-German Research Center for Environmental Health (GmbH), Neuherberg, Germany; Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zurich, Basel, Switzerland;
authors.forename: Timm
authors.initials: T
authors.lastname: Schroeder
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D011508:Chondroitin Sulfate Proteoglycans / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D017154:Stromal Cells / Q000166:cytology / Q000378:metabolism; D013997:Time Factors
chemical_list: D011508:Chondroitin Sulfate Proteoglycans; C504453:Dpt protein, mouse; D016326:Extracellular Matrix Proteins