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

TLDR

Human embryonic stem cells can generate diverse cell types, yet their differentiated derivatives downregulate telomerase and have limited replication potential. The study tests whether ectopic expression of hTERT can extend the replicative lifespan of hESC derivatives. Researchers differentiated hESCs into fibroblast‑like HEF1 cells, then transduced them with a retrovirus expressing hTERT to produce immortal HEF1‑hTERT cells that secrete conditioned medium supporting feeder‑free hESC growth. HEF1‑hTERT cells exhibit extended replicative capacity, produce conditioned medium that maintains hESC morphology, marker expression, telomerase activity, differentiation, and karyotype, and can differentiate into osteogenic cells, demonstrating that immortalized hESC‑derived lines can support their own growth in a homogeneous system.

Abstract

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.