title: Bulk cell density and Wnt/TGFbeta signalling regulate mesendodermal patterning of human pluripotent stem cells.
abstract: In vitro differentiation of human pluripotent stem cells (hPSCs) recapitulates early aspects of human embryogenesis, but the underlying processes are poorly understood and controlled. Here we show that modulating the bulk cell density (BCD: cell number per culture volume) deterministically alters anteroposterior patterning of primitive streak (PS)-like priming. The BCD in conjunction with the chemical WNT pathway activator CHIR99021 results in distinct paracrine microenvironments codifying hPSCs towards definitive endoderm, precardiac or presomitic mesoderm within the first 24 h of differentiation, respectively. Global gene expression and secretome analysis reveals that TGFß superfamily members, antagonist of Nodal signalling LEFTY1 and CER1, are paracrine determinants restricting PS progression. These data result in a tangible model disclosing how hPSC-released factors deflect CHIR99021-induced lineage commitment over time. By demonstrating a decisive, functional role of the BCD, we show its utility as a method to control lineage-specific differentiation. Furthermore, these findings have profound consequences for inter-experimental comparability, reproducibility, bioprocess optimization and scale-up.
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG), Hannover Medical School, 30625 Hannover, Germany.|REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Henning
authors.initials: H
authors.lastname: Kempf
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG), Hannover Medical School, 30625 Hannover, Germany.|REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany.|Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), German Center for Lung Research (DZL), 30625 Hannover, Germany.
authors.forename: Ruth
authors.initials: R
authors.lastname: Olmer
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG), Hannover Medical School, 30625 Hannover, Germany.|REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Haase
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG), Hannover Medical School, 30625 Hannover, Germany.|REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Annika
authors.initials: A
authors.lastname: Franke
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG), Hannover Medical School, 30625 Hannover, Germany.|REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Emiliano
authors.initials: E
authors.lastname: Bolesani
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG), Hannover Medical School, 30625 Hannover, Germany.|REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Kristin
authors.initials: K
authors.lastname: Schwanke
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG), Hannover Medical School, 30625 Hannover, Germany.|REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Diana
authors.initials: D
authors.lastname: Robles-Diaz
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG), Hannover Medical School, 30625 Hannover, Germany.|REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Michelle
authors.initials: M
authors.lastname: Coffee
authors.affiliation: REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany.|Institute of Human Genetics, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Gudrun
authors.initials: G
authors.lastname: Göhring
authors.affiliation: REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany.|Institute of Organic Chemistry, Leibniz University Hannover, 30167 Hannover, Germany.|Center of Biomolecular Drug Research (BMWZ), Leibniz University Hannover, 30167 Hannover, Germany.
authors.forename: Gerald
authors.initials: G
authors.lastname: Dräger
authors.affiliation: Natural and Medical Sciences Institute at the University of Tuebingen (NMI), 72770 Reutlingen, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Pötz
authors.affiliation: Natural and Medical Sciences Institute at the University of Tuebingen (NMI), 72770 Reutlingen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Joos
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, 48824-1319 Michigan, USA.
authors.forename: Erik
authors.initials: E
authors.lastname: Martinez-Hackert
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG), Hannover Medical School, 30625 Hannover, Germany.|REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Haverich
authors.affiliation: REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany.|Institute for Cellular Chemistry, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Falk F R
authors.initials: FFR
authors.lastname: Buettner
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG), Hannover Medical School, 30625 Hannover, Germany.|REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany.|Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), German Center for Lung Research (DZL), 30625 Hannover, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Martin
authors.affiliation: Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG), Hannover Medical School, 30625 Hannover, Germany.|REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Robert
authors.identifier: http://orcid.org/0000-0002-4656-0770
authors.initials: R
authors.lastname: Zweigerdt
mesh_terms: D002452:Cell Count; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D039904:Pluripotent Stem Cells / Q000502:physiology*; D040081:Protein Array Analysis; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D034741:RNA, Small Interfering; D015398:Signal Transduction / Q000502:physiology; D059467:Transcriptome; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*; D051153:Wnt Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C473711:Chir 99021; D011725:Pyridines; D011743:Pyrimidines; D034741:RNA, Small Interfering; D016212:Transforming Growth Factor beta; D051153:Wnt Proteins