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 und erstanding 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 expr essed VSMC-specific properties, the differentiated cells were characte rized by 1) morphological analysis, 2) gene expression, 3) immunostain ing for VSMC-specific proteins, 4) expression of characteristic VSMC i on channels, and 5) formation of [Ca2+](i) transients in response to V SMC-specific agonists. Treatment of embryonic stem cell-derived embryo id bodies with retinoic acid and dibutyryl-cyclic adenosine monophosph ate (db-cAMP) induced differentiation of spontaneously contracting cel l clusters in 67% of embryoid bodies compared with 10% of untreated co ntrols. The highest differentiation rate was observed when retinoic ac id and db-cAMP were applied to the embryoid bodies between days 7 and 11 in combination with frequent changes of culture medium. Other proto cols with retinoic acid and db-cAMP, as well as single or combined tre atment with VEGF, ECGF, bFGF, aFGF, fibronectin, matrigel, or hypoxia did not influence the differentiation rate. Single-cell RTPCR and sequ encing of the PCR products identified myosin heavy chain (MHC) splice variants distinguishing: between gut and VSMC isoforms. RT-PCR with VS MC-specific MHC primers and immunostaining confirmed the presence of V SMC transcripts and MHC protein. Furthermore, VSMC expressing MHC had typical ion channels and responded to specific agonists with an increa sed [Ca2+](i). Here we present a retinoic acid + dbcAMP-inducible embr yonic stem cell model of in vitro vasculogenesis. ES cell-derived cell s expressing VSMC-specific MHC and functional VSMC properties may be a suitable system to study mechanisms of VSMC differentiation.