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

IMR OpenIR

	Purification and functional assessment of smooth muscle cells derived from mouse embryonic stem cells
其他题名	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
	2013
发表期刊	JOURNAL OF GERIATRIC CARDIOLOGY
ISSN	1671-5411
卷号	10 期号:3 页码:272-280
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.
其他摘要	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 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.
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
收录类别	CSCD
语种	英语
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]
CSCD记录号	CSCD:4948399
引用统计
文献类型	期刊论文
条目标识符	http://ir.imr.ac.cn/handle/321006/155564
专题	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.