Purification and functional assessment of smooth muscle cells derived from mouse embryonic stem cells

Objective To obtain a pure population of smooth muscle cells (SMC) derived from mouse embryonic stem cells (ESC) and further assess their functions. Methods A vector, expressing both puromycin resistance gene (puror) and enhanced green fluorescent protein (EGFP) gene driven by smooth muscle 22α (SM22α) promoter, named pSM22α-puror-IRES2-EGFP was constructed and used to transfect ESC. Transgenic ESC (Tg-ESC) clones were selected by G418 and identified by PCR amplification of purorin vivo. After induction of SMC differentiation by all-trans retinoic acid, differentiated Tg-ESC were treated with 10 μg/mL puromycin for three days to obtain purified SMC (P-SMC). Percentage of EGFP+ cells in P-SMC was assessed by flow cytometer. Expressions of smooth muscle specific markers were detected by immunostaining and Western blotting. Proliferation, migration and contractility of P-SMC were analyzed by growth curve, trans-well migration assay, and carbachol treatment, respectively. Finally, both P-SMC and unpurified SMC (unP-SMC) were injected into syngeneic mouse to see teratoma development. Results Tg-ESC clone was successfully established and confirmed by PCR detection of puror+ percentage as high as 98.2% in contrast to 29.47% of unP-SMC. Compared with primary mouse vascular smooth muscle cells (VSMC), P-SMC displayed positive, but lowered expression of SMC-specific markers including SM α-actin and myosin heavy chain (SM-MHC) detected either, by immunostaining, or immunoblotting, accelerated proliferation, improved migration (99.33 ± 2.04 vs. 44.00 ± 2.08 migrated cells/field, P vs. 16.50 ± 3.76 % in cell area reduction, P In vivo injection of unP-SMC developed apparent teratoma while P-SMC did not. Conclusions We obtained a pure population of ESC derived SMC with less mature (differentiated) phenotypes, which will be of great use in research of vascular diseases and in bio-engineered vascular grafts for regenerative medicine.