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Stem Cells 2004;22:972-980 www.StemCells.com
© 2004 AlphaMed Press

Immortalized Fibroblast-Like Cells Derived from Human Embryonic Stem Cells Support Undifferentiated Cell Growth

Chunhui Xua, Jianjie Jianga, Virginie Sottileb, Jim McWhirb, Jane Lebkowskia, Melissa K. Carpentera

a Geron Corporation, Menlo Park, California, USA;
b Department of Gene Expression and Development, Roslin Institute, Roslin Midlothian, United Kingdom

Key Words. Human embryonic stem cells • Human telomerase reverse transcriptase Telomerase • Immortalization • Differentiation

Correspondence: Chunhui Xu, Ph.D., Geron Corporation, 230 Constitution Drive, Menlo Park, California 94025, USA. Telephone: 650-473-7795; Fax: 650-473-7750; e-mail: cxu{at}geron.com

Human embryonic stem cells (hESCs) have the potential to generate multiple cell types and hold promise for future therapeutic applications. Although undifferentiated hESCs can proliferate indefinitely, hESC derivatives significantly downregulate telomerase and have limited replication potential. In this study we examine whether the replicative lifespan of hESC derivatives can be extended by ectopic expression of human telomerase reverse transcriptase (hTERT), the catalytic component of the telomerase complex. To this end, we have derived HEF1 cells, a fibroblast-like cell type, differentiated from hESCs. Infection of HEF1 cells with a retrovirus expressing hTERT extends their replicative capacity, resulting in immortal human HEF1-hTERT cells. HEF1-hTERT cells can be used to produce conditioned medium (CM) capable of supporting hESC growth under feeder-free conditions. Cultures maintained in HEF1-CM show characteristics similar to mouse embryonic fibroblast CM control cultures, including morphology, surface marker and transcription factor expression, telomerase activity, differentiation, and karyotypic stability. In addition, HEF1-hTERT cells have the capacity to differentiate into cells of the osteogenic lineage. These results suggest that immortalized cell lines can be generated from hESCs and that cells derived from hESCs can be used to support their own growth, creating a genotypically homogeneous system for the culture of hESCs.




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