Derivation of a xeno-free human embryonic stem cell line.

Elimination of all animal material during both the derivation and long-term culture of human embryonic stem cells (hESCs) is necessary prior to future application of hESCs in clinical cell therapy. The potential consequences of transplanting xeno-contaminated hESCs into patients, such as an increased risk of graft rejection [Stem Cells 2006; 24:221-229] and the potential transfer of nonhuman pathogens, make existing hESC lines unsuitable for clinical applications. To avoid xeno-contamination during derivation and culture of hESCs, we first developed a xeno-free medium supplemented with human serum, which supports long-term (>50 passages) culture of hESCs in an undifferentiated state. To enable derivation of new xeno-free hESCs, we also established xeno-free human foreskin fibroblast feeders and replaced immunosurgery, which involves the use of guinea pig complement, with a modified animal-product-free derivation procedure. Here, we report the establishment and characterization (>20 passages) of a xeno-free pluripotent diploid normal hESC line, SA611.

Large-scale propagation of four undifferentiated human embryonic stem cell lines in a feeder-free culture system.

We describe an improved and more robust protocol for transfer and subsequent propagation of human embryonic stem cells under feeder-free conditions. The results show that mechanical dissociation for transfer of the human embryonic stem cells to Matrigel resulted in highest survival rates. For passage of the cultures on the other hand, enzymatic dissociation was found to be most efficient. In addition, this method reduces the time, work, and skills needed for propagation of the human embryonic stem cells. With the present protocol, the human embryonic stem cells have been cultured under feeder-free conditions for up to 35 passages while maintaining a normal karyotype, stable proliferation rate, and high telomerase activity. Furthermore, the feeder-free human embryonic stem cell cultures express the transcription factor Oct-4, alkaline phosphatase, and cell surface markers SSEA-3, SSEA-4, Tra 1-60, Tra 1-81, and formed teratomas in severe combined immunodeficient mice. This method provides distinct advantages compared with previous protocols and make propagation of human embryonic stem cells less laborious and more efficient.