ESTABLISHMENT OF FEEDER-FREE CULTURE SYSTEM OF HUMAN PARTHENOGENETIC EMBRYONIC STEM CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIANG Rui ¹ , WANG Zhiqiang ² , CHEN Tianxing ¹ , ZHU Jing ¹ , ZHU Shu ³ , LI Ying ¹ , YANG Long ¹ , ZHU Baosheng ³

1Department of Pathology, 3Genetic Diagnosis Center, the First People’s Hospital of Yunnan Province, Kunming Yunnan, 650032, P.R.China;;
2Department of Oncology, the First Affiliated Hospital of Kunming Medical University. Corresponding author: WANG Zhiqiang, E-mail: wzqcy2000@126.com;

Corresponding author：

Keywords：

Human parthenogenetic embryonic stem cells; Feeder-free; Conditional medium; Human foreskin fibroblasts

DOI：

10.7507/1002-1892.20130124

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Objective To establish a safe, effective, and economic feeder-free culture system which is suitable for the culture of human parthenogenetic embryonic stem cells (hPESCs) in vitro. Methods hPESCs were cultured with mTeSRTMl medium (control group) and human foreskin fibroblasts-conditional medium (hFFs-CM) (experimental group). The growth status of hPESCs in both feeder-free culture systems were observed with inverted microscope. Alkaline phosphatase (ALP) analysis and karyotype analysis were used to study the biological characteristics of hPESCs. The expression of hPESCs pluripotent marker Oct-4 was analyzed by RT-PCR. Differentiation experiment in vivo and in vitro was applied to observe the differentiation potential of hPESCs into three germ layers. Results hPESCs had regular morphology with difficulty in differentiation in both culture systems. No obvious difference was observed in morphology and expansion speed of hPESCs between 2 groups. After subcultured for 15 passages in vitro, hPESCs in 2 groups could maintain normal female diploid karyotype 46, XX and pluripotency. The expression of Oct-4 mRNA was positive in 2 groups. hPESCs in 2 groups could form embryonic body in differentiation experiment in vitro and could develop into teratomas containing three germ layers in nude mice. Conclusion Feeder-free culture system of hFFs-CM can sustain the growth of hPESCs and keep hPESCs undifferentiated state for long. A feeder-free culture system of hPESCs is successfully established, which can support the growth of hPESCs, reduce the contamination from animals, decrease the cost of culture, and satisfy the clinical large-scale application.

Citation： LIANG Rui,WANG Zhiqiang,CHEN Tianxing,ZHU Jing,ZHU Shu,LI Ying,YANG Long,ZHU Baosheng. ESTABLISHMENT OF FEEDER-FREE CULTURE SYSTEM OF HUMAN PARTHENOGENETIC EMBRYONIC STEM CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(5): 559-564. doi: 10.7507/1002-1892.20130124 Copy

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4.	Cobo F, Navarro JM, Herrera MI, et al. Electron microscopy reveals the presence of viruses in mouse embryonic fibroblasts but neither in human embryonic fibroblasts nor in human mesenchymal cells used for hESC maintenance: toward an implementation of microbiological quality assurance program in stem cell banks. Cloning Stem Cells, 2008, 10(1): 65-74.
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8.	Richards M, Fong CY, Chan WK, et al. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol, 2002, 20(9): 933-936.
9.	Lee JB, Lee JE, Park JH, et al. Establishment and maintenance of human embryonic stem cell lines on human feeder cells derived from uterine endometrium under serum-free condition. Biol Reprod, 2005, 72(1): 42-49.
10.	王志强, 梁锐, 陈明清, 等. 胰蛋白酶消化法和组织块法原代培养人包皮成纤维细胞的比较. 生命科学研究, 2012, 16(3): 242-247.
11.	王志强, 梁锐, 陈明清, 等. 人孤雌胚胎干细胞在人包皮成纤维细胞饲养层上的生长状态. 生命科学研究, 2012, 16(4): 314-318.
12.	胡智兴, 罗敏, 周轶平, 等. 四种胞外基质支持人胚胎干细胞的生长: 效果有差异吗? 中国组织工程研究与临床康复, 2010, 14(6): 1027-1030.
13.	罗敏, 胡智兴, 梁道明. 无血清无饲养层人胚胎干细胞向胶质前体细胞的分化. 解剖学报, 2011, 42(2): 279-282.
14.	Ludwig TE, Levenstein ME, Jones JM, et al. Derivation of human embryonic stem cells in defined conditions. Nat Biotechnol, 2006, 24(2): 185-187.
15.	何丽娟, 习佳飞, 曾泉, 等. 一种新的人胚胎干细胞无饲养层培养方法的建立. 生物技术通讯, 2010, 21(5): 660-665.
16.	杨阿聪, 金颖. 人胚胎干细胞优化培养的进展. 生命科学, 2006, 18(4): 402-406.
17.	Chin AC, Padmanabhan J, Oh SK, et al. Defined and serum-free media support undifferentiated human embryonic stem cell growth. Stem Cells Dev, 2010, 19(6): 753-761.
18.	李向东, 王纪文, 魏国峰. 人胚胎干细胞在无血清mTeSR1培养基中维持培养和向内皮细胞的诱导分化. 中国组织工程研究与临床康复, 2011, 15(10): 1827-1831.
19.	许慧芳, 陈红, 焦淑洁, 等. 人胚胎干细胞在3种培养体系中的生长. 生命科学研究, 2009, 13(1): 1-5.
20.	Bigdeli N, Andersson M, Strehl R, et al. Adaptation of human embryonic stem cells to feeder-free and matrix-free culture conditions directly on plastic surfaces. J Biotechnol, 2008, 133(1): 146-153.
21.	Montes R, Ligero G, Sanchez L, et al. Feeder-free maintenance of hESCs in mesenchymal stem cell-conditioned media: distinct requirements for TGF-beta and IGF-II. Cell Res, 2009, 19(6): 698-709.

1. 欧阳琦, 林戈, 周晓樱, 等. 人孤雌胚胎干细胞与正常胚胎干细胞分化能力的比较. 解剖学报, 2010, 41(6): 785-789.
2. Lagarkova MA, Eremeev AV, Svetlakov AV, et al. Human embryonic stem cell lines isolation, cultivation, and characterization. In Vitro Cell Dev Biol Anim, 2010, 46(3-4): 284-293.
3. Venu P, Chakraborty S, Inamdar MS. Analysis of long-term culture properties and pluripotent character of two sibling human embryonic stem cell lines derived from discarded embryos. In Vitro Cell Dev Biol Anim, 2010, 46(3-4): 200-205.
4. Cobo F, Navarro JM, Herrera MI, et al. Electron microscopy reveals the presence of viruses in mouse embryonic fibroblasts but neither in human embryonic fibroblasts nor in human mesenchymal cells used for hESC maintenance: toward an implementation of microbiological quality assurance program in stem cell banks. Cloning Stem Cells, 2008, 10(1): 65-74.
5. Zhou HS, Yong J, Sun XM, et al. A human endothelial cell feeder system that efficiently supports the undifferentiated growth of mouse embryonic stem cells. Differentiation, 2008, 76(9): 923-930.
6. Lu Z, Zhu W, Yu Y, et al. Derivation and long-term culture of human parthenogenetic embryonic stem cells using human foreskin feeders. J Assist Reprod Genet, 2010, 27(6): 285-291.
7. Genbacev O, Krtolica A, Zdravkovic T, et al. Serum-free derivation of human embryonic stem cell lines on human placental fibroblast feeders. Fertil Steril, 2005, 83(5): 1517-1529.
8. Richards M, Fong CY, Chan WK, et al. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol, 2002, 20(9): 933-936.
9. Lee JB, Lee JE, Park JH, et al. Establishment and maintenance of human embryonic stem cell lines on human feeder cells derived from uterine endometrium under serum-free condition. Biol Reprod, 2005, 72(1): 42-49.
10. 王志强, 梁锐, 陈明清, 等. 胰蛋白酶消化法和组织块法原代培养人包皮成纤维细胞的比较. 生命科学研究, 2012, 16(3): 242-247.
11. 王志强, 梁锐, 陈明清, 等. 人孤雌胚胎干细胞在人包皮成纤维细胞饲养层上的生长状态. 生命科学研究, 2012, 16(4): 314-318.
12. 胡智兴, 罗敏, 周轶平, 等. 四种胞外基质支持人胚胎干细胞的生长: 效果有差异吗? 中国组织工程研究与临床康复, 2010, 14(6): 1027-1030.
13. 罗敏, 胡智兴, 梁道明. 无血清无饲养层人胚胎干细胞向胶质前体细胞的分化. 解剖学报, 2011, 42(2): 279-282.
14. Ludwig TE, Levenstein ME, Jones JM, et al. Derivation of human embryonic stem cells in defined conditions. Nat Biotechnol, 2006, 24(2): 185-187.
15. 何丽娟, 习佳飞, 曾泉, 等. 一种新的人胚胎干细胞无饲养层培养方法的建立. 生物技术通讯, 2010, 21(5): 660-665.
16. 杨阿聪, 金颖. 人胚胎干细胞优化培养的进展. 生命科学, 2006, 18(4): 402-406.
17. Chin AC, Padmanabhan J, Oh SK, et al. Defined and serum-free media support undifferentiated human embryonic stem cell growth. Stem Cells Dev, 2010, 19(6): 753-761.
18. 李向东, 王纪文, 魏国峰. 人胚胎干细胞在无血清mTeSR1培养基中维持培养和向内皮细胞的诱导分化. 中国组织工程研究与临床康复, 2011, 15(10): 1827-1831.
19. 许慧芳, 陈红, 焦淑洁, 等. 人胚胎干细胞在3种培养体系中的生长. 生命科学研究, 2009, 13(1): 1-5.
20. Bigdeli N, Andersson M, Strehl R, et al. Adaptation of human embryonic stem cells to feeder-free and matrix-free culture conditions directly on plastic surfaces. J Biotechnol, 2008, 133(1): 146-153.
21. Montes R, Ligero G, Sanchez L, et al. Feeder-free maintenance of hESCs in mesenchymal stem cell-conditioned media: distinct requirements for TGF-beta and IGF-II. Cell Res, 2009, 19(6): 698-709.

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