Chinese Journal of Tissue Engineering Research ›› 2010, Vol. 14 ›› Issue (2): 223-226.doi: 10.3969/j.issn.1673-8225.2010.02.008

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Construction of immortalized human precartilaginous stem cell lines

Yin De-long, Chen An-min, Guo Feng-jin, Wang Jun-fang, Cheng Hao   

  1. Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan   430030, Hubei Province, China
  • Online:2010-01-08 Published:2010-01-08
  • Contact: Cheng Hao, Doctor, Attending physician, Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430030, Hubei Province, China chenghao@tjh.tjmu.edu.cn
  • About author:Yin De-long☆, Studying for doctorate, Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430030, Hubei Province, China yindelong607@sina.com
  • Supported by:

     the National Natural Science Foundation of China, No. 30650007*

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Abstract:

BACKGROUND: The precartilaginous stem cells are limited regarding in vitro proliferative capacity, but the immortalized cell lines can provide a large number of stable immortalized cells, and simian virus 40 large T antigen gene (SV40Tag) is one of gene fragments which are commonly used and effective in vitro immortalized cells.
OBJECTIVE: To construct human immortalized precartilaginous stem cells (IPSCs) using human precartilaginous stem cells induced by SV40LTAg gene.
METHODS: The human immortalized precartilaginous stem cells were isolated from aborted fetus and purified with enzyme digestion and immunomagnetic beads screening method. By using liposome-mediated gene transfection technology, plasmid pCMVSV40T/PUR containing SV40Tag was transfected in primary embryonic precartilaginous stem cells, while non-transfected cells served as negative controls. Positive clones were cultured to observe the cell morphology and the passage recovery, to calculate cell survival rate and population doubling time, to draw cell growth curve. Immunofluorescence cytochemistry was used to detect the expression of IPSCs fibroblast growth factor receptor 3, the expressions of SV40Tag and fibroblast growth factor receptor 3 in the human precartilaginous stem cells were determined by RT-PCR.
RESULTS AND CONCLUSION: Morphology of human IPSCs seemed coincidence with primary human precartilaginous stem cells. The survival rate of human IPSCs was not influenced by subculture, freezing and recovery, but the survival rate was descended in the human precartilaginous stem cells at the 6th and 10th passages (P < 0.01). Compared with cells at the 6th and 10th passages, the proliferation of human IPSCs was greater, with short population doubling time and high growth rate (P < 0.01). The immunofluorescence showed that fibroblast growth factor receptor 3 was positive in human IPSCs at the second passage, and the RT-PCR results of fibroblast growth factor receptor 3 revealed a specific amplification band at 400 bp, while that of SV40Tag revealed at 560 bp. No band was seen in the primary cells. It is indicated that SV40Tag human IPSCs can be constructed successfully using immunomagnetic bead screening technology and liposome transfection technique.

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