Derivation, characterization, differentiation, and registration of seven human embryonic stem cell lines (VAL-3, -4, -5, -6M, -7, -8, and -9) on human feeder.

Derivation of human embryonic stem cell lines has been a remarkable scientific achievement during the last decade. Human embryonic stem cells are regarded as an unlimited cell source for replacement therapy in regenerative medicine. Clearly, the scientific community requires proper derivation, characterization, and registration with the purpose of making them available for research and future medical applications worldwide. In this paper, we report our derivation work as the Valencian Node of the Spanish Stem Cell Bank in the generation, characterization, and registration of VAL-3, -4, -5, -6M, -7, -8, and 9 (www.isciii/htdocs/terapia/terapia_bancocelular.jsp). The derivation process was performed on microbiologically tested and irradiated human foreskin fibroblasts and designed to minimize contact with xeno-components in knockout Dulbecco's modified Eagle's medium supplemented with knockout serum replacement and basic fibroblast growth factor. Fingerprinting of the cell lines was performed to allow their identification and traceability. All lines were expressed at the mRNA and specific protein markers for undifferentiation and were found to be negative for classical differentiation markers such as neurofilament heavy chain (ectoderm), renin (mesoderm), and amylase (endoderm). All lines displayed high levels of telomerase activity and were shown to successfully overcome cryopreservation and thawing. Finally, we demonstrated the potential to differentiate in vitro (embryoid body formation) and in vivo (teratoma formation) into cell types from all three germ layers. Teratoma derived from all human embryonic stem cell lines present similar morphological features except VAL-8 that display more aggressive tumor behavior with a larger proportion of solid tissues, as opposed to cyst formation in the other cell lines.