title: Distinct gene expression signatures in human embryonic stem cells differentiated towards definitive endoderm at single-cell level.
abstract: Characterization of directed differentiation of pluripotent stem cells towards therapeutically relevant cell types, including pancreatic beta-cells and hepatocytes, depends on molecular markers and assays that resolve the signature of individual cells. Pancreas and liver both have a common origin of anterior definitive endoderm (DE). Here, we differentiated human embryonic stem cells towards DE using three different activin A based treatments. Differentiation efficiencies were evaluated by gene expression profiling over time at cell population level. A panel of key markers was used to study DE formation. Final DE differentiation was also analyzed with immunocytochemistry and single-cell gene expression profiling. We found that cells treated with activin A in combination with sodium butyrate and B27 serum-free supplement medium generated the most mature DE cells. Cell population studies were useful to monitor the temporal expression of genes involved in primitive streak formation and endoderm formation, while single-cell analysis allowed us to study cell culture heterogeneity and fingerprint individual cells. In addition, single-cell analysis revealed distinct gene expression patterns for the three activin A based protocols applied. Our data provide novel insights in DE gene expression at the cellular level of in vitro differentiated human embryonic stem cells, and illustrate the power of using single-cell gene expression profiling to study differentiation heterogeneity and to characterize cell types and subpopulations.
authors.affiliation: Stem Cell and Pancreas Developmental Biology, Stem Cell Center, Department of Laboratory Medicine, Lund University, BMC B10, Klinikgatan 26, SE-22184 Lund, Sweden. Karin.Norrman@cellartis.com
authors.forename: Karin
authors.initials: K
authors.lastname: Norrman
authors.forename: Anna
authors.initials: A
authors.lastname: Strömbeck
authors.forename: Henrik
authors.initials: H
authors.lastname: Semb
authors.forename: Anders
authors.initials: A
authors.lastname: Ståhlberg
mesh_terms: D028341:Activins / Q000494:pharmacology / Q000502:physiology; D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D048430:Cell Shape; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000378:metabolism* / Q000502:physiology; D004707:Endoderm / Q000166:cytology; D020869:Gene Expression Profiling; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055760:SOXF Transcription Factors / Q000235:genetics / Q000378:metabolism; D059010:Single-Cell Analysis; D059467:Transcriptome
chemical_list: D000943:Antigens, Differentiation; C109188:SOX17 protein, human; D055760:SOXF Transcription Factors; C422405:activin A; D028341:Activins