Functional characterization and transcriptome analysis of embryonic stem cell-derived contractile smooth muscle cells.
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.
Potta
Shiva Prasad
SP
University of Cologne, Center of Physiology and Pathophysiology, Institute of Neurophysiology, Robert Koch Str 39, Cologne, Germany.
Liang
Huamin
H
Pfannkuche
Kurt
K
Winkler
Johannes
J
Chen
Shuhua
S
Doss
Michael Xavier
MX
Obernier
Kirsten
K
Kamisetti
Naidu
N
Schulz
Herbert
H
Hübner
Norbert
N
Hescheler
Jürgen
J
Sachinidis
Agapios
A
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
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