title: Purification and functional assessment of smooth muscle cells derived from mouse embryonic stem cells.
abstract: 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 (puro(r) ) and enhanced green fluorescent protein (EGFP) gene driven by smooth muscle 22α (SM22α) promoter, named pSM22α-puro(r)-IRES2-EGFP was constructed and used to transfect ESC. Transgenic ESC (Tg-ESC) clones were selected by G418 and identified by PCR amplification of puro(r) gene. The characteristics of Tg-ESC were detected by alkaline phosphatase (ALP) staining, SSEA-1 immunofluorescence and teratoma formation test in 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 puro(r) gene in its genomic DNA. The Tg-ESC was positive for ALP staining, SSEA-1 staining and formed teratoma containing tissues derived from three germ layers. After retinoic acid induction, large amount of EGFP positive cells outgrew from differentiated Tg-ESC. Three days of puromycin treatment produced a population of P-SMC with an EGFP(+) 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 < 0.05), and decreased contractility in response to carbachol (7.75 ± 1.19 % vs. 16.50 ± 3.76 % in cell area reduction, P < 0.05). 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.
authors.affiliation: Department of Cardiology, Chinese PLA Cardiovascular Research Institute, General Hospital of Shenyang Military Region, 83 Wenhua Road, Shenhe District, Shenyang, Liaoning 110840, China.
authors.forename: Xiao-Xiang
authors.initials: XX
authors.lastname: Tian
authors.forename: Jian
authors.initials: J
authors.lastname: Kang
authors.forename: Cheng-Hui
authors.initials: CH
authors.lastname: Yan
authors.forename: Kai
authors.initials: K
authors.lastname: Xu
authors.forename: Jie
authors.initials: J
authors.lastname: Tao
authors.forename: Gui-Tang
authors.initials: GT
authors.lastname: Yang
authors.forename: Ya-Ling
authors.initials: YL
authors.lastname: Han
keywords: Differentiation; Embryonic stem cell; Phenotype; SM22α; Smooth muscle cell