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

CORC > 金属研究所 > 中国科学院金属研究所

	Purification and functional assessment of smooth muscle cells derived from mouse embryonic stem cells
	Tian XiaoXiang; Kang Jian; Yan ChengHui; Xu Kai; Tao Jie; Yang GuiTang; Han YaLing
刊名	JOURNAL OF GERIATRIC CARDIOLOGY
	2013
卷号	10 期号:3 页码:272-280
关键词	TRANSGENIC MICE IN-VITRO DIFFERENTIATION EXPRESSION RECRUITMENT SM22-ALPHA PROMOTER ACTIN BETA Smooth muscle cell Embryonic stem cell Differentiation SM22 alpha Phenotype
ISSN号	1671-5411
其他题名	Purification and functional assessment of smooth muscle cells derived from mouse embryonic stem cells
英文摘要	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 22 alpha (SM22 alpha) promoter, named pSM22 alpha-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 puror 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 mu g/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 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 a-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.
资助项目	[National Natural Science Foundation of China] ; [National Prophase Program on Key Basic Research Project of China] ; [Key Technology Research and Development Program of Liaoning Province]
语种	英语
CSCD记录号	CSCD:4948399
内容类型	期刊论文
源URL	[http://ir.imr.ac.cn/handle/321006/155563]
专题	金属研究所_中国科学院金属研究所
作者单位	中国科学院金属研究所
推荐引用方式 GB/T 7714	Tian XiaoXiang,Kang Jian,Yan ChengHui,et al. Purification and functional assessment of smooth muscle cells derived from mouse embryonic stem cells[J]. JOURNAL OF GERIATRIC CARDIOLOGY,2013,10(3):272-280.
APA	Tian XiaoXiang.,Kang Jian.,Yan ChengHui.,Xu Kai.,Tao Jie.,...&Han YaLing.(2013).Purification and functional assessment of smooth muscle cells derived from mouse embryonic stem cells.JOURNAL OF GERIATRIC CARDIOLOGY,10(3),272-280.
MLA	Tian XiaoXiang,et al."Purification and functional assessment of smooth muscle cells derived from mouse embryonic stem cells".JOURNAL OF GERIATRIC CARDIOLOGY 10.3(2013):272-280.