Human embryonic stem cells derived without feeder cells

Abstract

Human embryonic stem cells are likely to play an important role in the future of regenerative medicine. However, exposure of existing human embryonic stem-cell lines to live animal cells and serum risks contamination with pathogens that could lead to human health risks. We aimed to derive an embryonic stem-cell line without exposure to cells or serum. Frozen cleavage-stage embryos were thawed and cultured to the blastocyst stage. Inner cell masses were isolated by immunosurgery and plated onto extracellular-matrix-coated plates that can be easily sterilised. Six established human embryonic stem-cell lines were also maintained with this serum and feeder free culture system. A new stem-cell line was derived from human embryos under completely cell and serum free conditions. The cells maintained normal karyotype and markers of pluripotency, including octamer binding protein 4 (Oct-4), stage-specific embryonic antigen (SSEA)-3, SSEA-4, tumour-rejection antigen (TRA)-1-60, TRA-1-81, and alkaline phosphatase. After more than 6 months of undifferentiated proliferation, these cells retained the potential to form derivatives of all three embryonic germ layers both in vitro and in teratomas. These properties were also successfully maintained (for more than 30 passages) with the established stem-cell lines. This system eliminates exposure of human embryonic stem cells and their progeny to animal and human feeder layers, and thus the risk of contamination with pathogenic agents capable of transmitting diseases to patients.