From totipotent embryonic stem cells to spontaneously contracting smooth muscle cells: a retinoic acid and db-cAMP in vitro differentiation model.
Vascular smooth muscle cell (VSMC) differentiation is important in understanding vascular disease; however, no in vitro model is available. Totipotent mouse embryonic stem (ES) cells were used to establish such a model. To test whether the ES cell-derived smooth muscle cells expressed VSMC-specific properties, the differentiated cells were characterized by 1) morphological analysis, 2) gene expression, 3) immunostaining for VSMC-specific proteins, 4) expression of characteristic VSMC ion channels, and 5) formation of [Ca2+]i transients in response to VSMC-specific agonists. Treatment of embryonic stem cell-derived embryoid bodies with retinoic acid and dibutyryl-cyclic adenosine monophosphate (db-cAMP) induced differentiation of spontaneously contracting cell clusters in 67% of embryoid bodies compared with 10% of untreated controls. The highest differentiation rate was observed when retinoic acid and db-cAMP were applied to the embryoid bodies between days 7 and 11 in combination with frequent changes of culture medium. Other protocols with retinoic acid and db-cAMP, as well as single or combined treatment with VEGF, ECGF, bFGF, aFGF, fibronectin, matrigel, or hypoxia did not influence the differentiation rate. Single-cell RT-PCR and sequencing of the PCR products identified myosin heavy chain (MHC) splice variants distinguishing between gut and VSMC isoforms. RT-PCR with VSMC-specific MHC primers and immunostaining confirmed the presence of VSMC transcripts and MHC protein. Furthermore, VSMC expressing MHC had typical ion channels and responded to specific agonists with an increased [Ca2+]i. Here we present a retinoic acid + db-cAMP-inducible embryonic stem cell model of in vitro vasculogenesis. ES cell-derived cells expressing VSMC-specific MHC and functional VSMC properties may be a suitable system to study mechanisms of VSMC differentiation.
Drab
M
M
Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany.
Haller
H
H
Bychkov
R
R
Erdmann
B
B
Lindschau
C
C
Haase
H
H
Morano
I
I
Luft
F C
FC
Wobus
A M
AM
D000818:Animals; D001483:Base Sequence; D003994:Bucladesine / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology*; D051379:Mice; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D018995:Myosin Heavy Chains / Q000032:analysis; D015221:Potassium Channels / Q000502:physiology; D013234:Stem Cells / Q000166:cytology*; D014212:Tretinoin / Q000494:pharmacology*
D015221:Potassium Channels; D014212:Tretinoin; D003994:Bucladesine; D018995:Myosin Heavy Chains; D002118:Calcium