Derivation of a new human embryonic stem cell line, Endeavour-2, and its characterization.
Here, we describe the derivation of a novel human embryonic stem cell (hESC) line, Endeavour-2 (E-2), propagated on human fetal fibroblasts (HFF) in a serum-replacement media. The inner cell mass (ICM) was manually dissected from the blastocyst without using immunodissection and, therefore, antibodies from animal sources. A total of 20 embryos were thawed and cultured, eight embryos were hatched, and five ICMs were obtained. They were transferred onto HFF used as feeder layer, and one colony representing the initial cell proliferation of a new hESC line, E-2, was obtained. The newly emerged hESC colony was passaged first by physical dissection and subsequently by enzymatic dissociation. E-2 has been in culture for over 6 months and has been shown to possess typical features of a pluripotent hESC line including expression of stem cell surface markers (SSEA4, TRA-160, and integrin alpha-6), intracellular alkaline phosphatase, and pluripotency gene markers, OCT4 and NANOG. This hESC line shows lineage-specific differentiation into various representative cell types expressing markers characteristic of the three somatic germ layers under both in vitro and in vivo conditions. E-2 line shows a normal karyotype (46 XX) and has been successfully cryopreserved and thawed several times using slow-freezing procedures. E-2 adds to the repertoire of existing hESC lines for research and development purposes in the field of regenerative medicine.
Sidhu
Kuldip S
KS
Diabetes Transplant Unit, Prince of Wales Hospital & University of New South Wales, Sydney, NSW, Australia. k.sidhu@unsw.edu.au
Ryan
John P
JP
Lees
Justin G
JG
Tuch
Bernard E
BE
D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line / Q000166:cytology*; D003114:Colony-Forming Units Assay; D053595:Embryonic Stem Cells / Q000166:cytology*; D006801:Humans; D039904:Pluripotent Stem Cells / Q000166:cytology