OBJECTIVE: Research into the biology of human trophoblast invasion has been hampered by a lack of in vitro models. The aim of this study was to establish and characterize a human extravillous trophoblast cell line from the first-trimester placenta. METHODS: Human papillomavirus type 16 (HPV16) E6/E7 genes were stably expressed in primary cultures of first-trimester placenta via a retroviral vector (pLXSN-E6/E7). Several clones were characterized for extravillous trophoblastic properties by reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry. The activities of matrix metalloproteinase (MMP)-2 and MMP-9 were examined with gelatin zymography. One clone (TEV-1), which retains all the established criteria for extravillous trophoblasts, was used in microarray analysis with Stanford Human cDNA chip (41, 421 cDNA features) to examine the differential gene expression after treatment of transforming growth factor beta 1 (TGFbeta1). The responsive gene to TGFbeta1 treatment was confirmed by quantitative real-time PCR. RESULTS: The clonal TEV-1 has been passaged for more than 105 population doublings with no sign of senescence, the activation of telomerase at early passages, and a near-diploid karyotype. TEV-1 cells expressed cytokeratin 7, HLA-G (a histocompatibility antigen, class IB), and CD9 (the cluster of differentiation antigen 9), and secreted active MMP-2 and MMP-9. TGFbeta1 treatment altered the gene expression profile of TEV-1 cells with a marked up-regulation of insulin-like growth factor binding protein 3 (IGFBP3), which was confirmed by quantitative real-time PCR. In addition, the TEV-1 was nontumorigenic when injected into nude mice and unable to form colonies in soft agar. CONCLUSION: Phenotypic and biologic characteristics of TEV-1 were shown as the properties of extravillous trophoblasts; thus, the TEV-1 cell line may be used as a cell model in extravillous trophoblast studies.
OBJECTIVE: Research into the biology of human trophoblast invasion has been hampered by a lack of in vitro models. The aim of this study was to establish and characterize a human extravillous trophoblast cell line from the first-trimester placenta. METHODS:Human papillomavirus type 16 (HPV16) E6/E7 genes were stably expressed in primary cultures of first-trimester placenta via a retroviral vector (pLXSN-E6/E7). Several clones were characterized for extravillous trophoblastic properties by reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry. The activities of matrix metalloproteinase (MMP)-2 and MMP-9 were examined with gelatin zymography. One clone (TEV-1), which retains all the established criteria for extravillous trophoblasts, was used in microarray analysis with Stanford Human cDNA chip (41, 421 cDNA features) to examine the differential gene expression after treatment of transforming growth factor beta 1 (TGFbeta1). The responsive gene to TGFbeta1 treatment was confirmed by quantitative real-time PCR. RESULTS: The clonal TEV-1 has been passaged for more than 105 population doublings with no sign of senescence, the activation of telomerase at early passages, and a near-diploid karyotype. TEV-1 cells expressed cytokeratin 7, HLA-G (a histocompatibility antigen, class IB), and CD9 (the cluster of differentiation antigen 9), and secreted active MMP-2 and MMP-9. TGFbeta1 treatment altered the gene expression profile of TEV-1 cells with a marked up-regulation of insulin-like growth factor binding protein 3 (IGFBP3), which was confirmed by quantitative real-time PCR. In addition, the TEV-1 was nontumorigenic when injected into nude mice and unable to form colonies in soft agar. CONCLUSION: Phenotypic and biologic characteristics of TEV-1 were shown as the properties of extravillous trophoblasts; thus, the TEV-1 cell line may be used as a cell model in extravillous trophoblast studies.
Authors: Boris Novakovic; Lavinia Gordon; Nicholas C Wong; Ashley Moffett; Ursula Manuelpillai; Jeffrey M Craig; Andrew Sharkey; Richard Saffery Journal: Mol Hum Reprod Date: 2011-02-02 Impact factor: 4.025
Authors: Kevin K W Lam; Philip C N Chiu; Cheuk-Lun Lee; Ronald T K Pang; Carmen O N Leung; Hannu Koistinen; Markku Seppala; Pak-Chung Ho; William S B Yeung Journal: J Biol Chem Date: 2011-08-31 Impact factor: 5.157