Derivation and characterization of three new Spanish human embryonic stem cell lines (VAL -3 -4 -5) on human feeder and in serum-free conditions.
A total of 184 human embryos, frozen for >5 years, were donated; informed consent was obtained according to Spanish law 45/2003. Survival rate was 40% and three out of 24 blastocysts (12.5%) developed into putative hESC lines, named VAL-3, VAL-4, and VAL-5. The derivation process was performed on microbiologically tested and irradiated human foreskin fibroblasts and designed to minimize contact with xeno-components in knockout DMEM supplemented with knockout serum replacement, and basic fibroblast growth factor. Fingerprinting and HLA typing of the cell lines allowed their identification and traceability. Karyotype was normal for VAL-3 (46XY), VAL-4 (46XX) and VAL-5 (46XX). All three hESC lines expressed specific markers for non-differentiation (Nanog, stage-specific embryonic antigen-4 [SSEA-4], tumour-related antigen [TRA]-1-60, and TRA-1-81) and were negative for SSEA-1. RT-PCR further demonstrated the expression of Oct-4, Sox2, Rex-1, Nanog, Cripto, Thy-1, and Lefty-A. Furthermore, they were found to be negative for classical differentiation markers such as neurofilament heavy chain (ectoderm), renin (mesoderm), and amylase (endoderm). All three cell lines displayed high levels of telomerase activity, and were shown to successfully overcome cryopreservation and thawing. Finally, these three new hESC lines have demonstrated the potential to differentiate in vitro and in vivo (teratoma formation) into cell types originating from all three germ layers.
Valbuena
Diana
D
Banco Nacional de Líneas Celulares, Nodo de Valencia, Centro de Investigación Príncipe Felipe (CIPF), Valencia, Spain.
Galán
Amparo
A
Sánchez
Eva
E
Poo
M Eugenia
ME
Gómez
Eva
E
Sánchez-Luengo
Silvia
S
Melguizo
Darío
D
García
Aìda
A
Ruiz
Verónica
V
Moreno
Rubén
R
Pellicer
Antonio
A
Simón
Carlos
C
D000818:Animals; D001483:Base Sequence; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line; D018920:Coculture Techniques; D015925:Cryopreservation; D016895:Culture Media, Serum-Free; D017931:DNA Primers / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005260:Female; D005347:Fibroblasts; D015870:Gene Expression; D006801:Humans; D007621:Karyotyping; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D013030:Spain; D033581:Stem Cell Transplantation; D014183:Transplantation, Heterologous
D015415:Biomarkers; D016895:Culture Media, Serum-Free; D017931:DNA Primers