title: A model of early human embryonic stem cell differentiation reveals inter- and intracellular changes on transition to squamous epithelium.
abstract: The molecular events leading to human embryonic stem cell (hESC) differentiation are the subject of considerable scrutiny. Here, we characterize an in vitro model that permits analysis of the earliest steps in the transition of hESC colonies to squamous epithelium on basic fibroblast growth factor withdrawal. A set of markers (GSC, CK18, Gata4, Eomes, and Sox17) point to a mesendodermal nature of the epithelial cells with subsequent commitment to definitive endoderm (Sox17, Cdx2, nestin, and Islet1). We assayed alterations in the transcriptome in parallel with the distribution of immunohistochemical markers. Our results indicate that the alterations of tight junctions in pluripotent culture precede the beginning of differentiation. We defined this cell population as "specified," as it is committed toward differentiation. The transitional zone between "specified" pluripotent and differentiated cells displays significant up-regulation of keratin-18 (CK18) along with a decrease in the functional activity of gap junctions and the down-regulation of 2 gap junction proteins, connexin 43 (Cx43) and connexin 45 (Cx45), which is coincidental with substantial elevation of intracellular Ca2+ levels. These findings reveal a set of cellular changes that may represent the earliest markers of in vitro hESC transition to an epithelial phenotype, before the induction of gene expression networks that guide hESC differentiation. Moreover, we hypothesize that these events may be common during the primary steps of hESC commitment to functionally varied epithelial tissue derivatives of different embryological origins.
authors.affiliation: Developmental Biology Program, iPS and Human Stem Cell Core Facility, Children's Memorial Research Center, Northwestern University, Feinberg School of Medicine, Chicago, Illinois 60614-3394, USA. v-galat@northwestern.edu
authors.forename: Vasiliy
authors.initials: V
authors.lastname: Galat
authors.forename: Sergey
authors.initials: S
authors.lastname: Malchenko
authors.forename: Yekaterina
authors.initials: Y
authors.lastname: Galat
authors.forename: Alex
authors.initials: A
authors.lastname: Ishkin
authors.forename: Yuri
authors.initials: Y
authors.lastname: Nikolsky
authors.forename: Steven T
authors.initials: ST
authors.lastname: Kosak
authors.forename: Bento Marcelo
authors.initials: BM
authors.lastname: Soares
authors.forename: Philip
authors.initials: P
authors.lastname: Iannaccone
authors.forename: John D
authors.initials: JD
authors.lastname: Crispino
authors.forename: Mary J C
authors.initials: MJ
authors.lastname: Hendrix
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D019070:Cell Lineage; D016000:Cluster Analysis; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism*; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D015726:Giant Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008954:Models, Biological; D010750:Phosphoproteins / Q000378:metabolism; D062826:Zonula Occludens-1 Protein
chemical_list: D015415:Biomarkers; D008565:Membrane Proteins; D010750:Phosphoproteins; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein