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Distinct gene expression signatures in human embryonic stem cells differentiated towards definitive endoderm at single-cell level.

Journal article
Authors Karin Norrman
Anna Strömbeck
Henrik Semb
Anders Ståhlberg
Published in Methods
Volume 59
Issue 1
Pages 59-70
ISSN 1046-2023
Publication year 2013
Published at Institute of Biomedicine, Department of Pathology
Sahlgrenska Cancer Center
Pages 59-70
Language en
Links dx.doi.org/10.1016/j.ymeth.2012.03....
Keywords Human embryonic stem cells, Definitive endoderm, Gene expression profiling, RT-qPCR, Single-cell analysis, Single-cell biology
Subject categories Basic Medicine

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.

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