title: Functional characterization and transcriptome analysis of embryonic stem cell-derived contractile smooth muscle cells.
abstract: Complete transcriptome profiling of contractile smooth muscle cells (SMCs) differentiated from embryonic stem cells is crucial for the characterization of smooth muscle gene expression signatures and will contribute to defining biological and physiological processes in these cells. We have generated a transgenic embryonic stem cell line expressing both the puromycin acetyl transferase and enhanced green fluorescent protein cassettes under the control of the Acta2 promoter. Applying a specific monolayer culture protocol using retinoic acid, a puromycin-resistant and enhanced green fluorescent protein-positive Acta2(+) SMC population of 95% purity was isolated. Acta2(+) SMCs were characterized by semiquantitative and quantitative RT-PCR profiling of SMC markers and by microarray expression profiling, as well as by immunostaining for SMC-specific cytoskeletal proteins. Patch-clamp electrophysiological characterization of these cells identified SMC-specific channels such as the ATP-sensitive potassium channel and the Ca(2+)-activated potassium channel. Culturing of Acta2(+) SMCs in serum-containing medium resulted in a significant number of hypertrophic and binucleated cells failing to complete cell division. Functional characterization of the cells has been proved by stimulation of the cells with vasoactive agents, such as angiotensin II and endothelin. We concluded that our embryonic stem cell-derived SMC population possesses the contractile and hypertrophic phenotype of SMCs incapable of proliferation. This is the first study describing the complete transcriptome of ES-derived SMCs allowing identification of specific biological and physiological processes in the contractile phenotype SMCs and will contribute to the understanding of these processes in early SMCs derived from embryonic stem cells.
authors.affiliation: University of Cologne, Center of Physiology and Pathophysiology, Institute of Neurophysiology, Robert Koch Str 39, Cologne, Germany.
authors.forename: Shiva Prasad
authors.initials: SP
authors.lastname: Potta
authors.forename: Huamin
authors.initials: H
authors.lastname: Liang
authors.forename: Kurt
authors.initials: K
authors.lastname: Pfannkuche
authors.forename: Johannes
authors.initials: J
authors.lastname: Winkler
authors.forename: Shuhua
authors.initials: S
authors.lastname: Chen
authors.forename: Michael Xavier
authors.initials: MX
authors.lastname: Doss
authors.forename: Kirsten
authors.initials: K
authors.lastname: Obernier
authors.forename: Naidu
authors.initials: N
authors.lastname: Kamisetti
authors.forename: Herbert
authors.initials: H
authors.lastname: Schulz
authors.forename: Norbert
authors.initials: N
authors.lastname: Hübner
authors.forename: Jürgen
authors.initials: J
authors.lastname: Hescheler
authors.forename: Agapios
authors.initials: A
authors.lastname: Sachinidis
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism*; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D003893:Desmin / Q000235:genetics / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology*; D016232:Endothelins / Q000494:pharmacology; D020869:Gene Expression Profiling; D006984:Hypertrophy / Q000473:pathology; D054086:KATP Channels / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D009119:Muscle Contraction / Q000235:genetics; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism; D018408:Patch-Clamp Techniques; D024681:Potassium Channels, Calcium-Activated / Q000235:genetics / Q000378:metabolism; D000096985:Calponins
chemical_list: D000199:Actins; D000804:Angiotensin II; D002135:Calcium-Binding Proteins; D003893:Desmin; D016232:Endothelins; D054086:KATP Channels; D008840:Microfilament Proteins; D009124:Muscle Proteins; D018995:Myosin Heavy Chains; D024681:Potassium Channels, Calcium-Activated; D000096985:Calponins; C051615:transgelin