Culture conditions and enzymatic passaging of bovine ESC-like cells.
The goals of the current study were to (1) improve culture conditions and (2) chemical passaging of bovine embryonic stem cell-like (bESC-like) cells. Specifically, the effects of human leukemia inhibitory factor (hLIF), two types of feeders, mouse embryonic fibroblast (MEF) and bovine embryonic fibroblast (BEF), as well as three different enzymatic treatments including Trypsin-EDTA, TrypLE, and Liberase Blendzymes 3 were investigated. The addition of hLIF at 1000 U/mL to the culture medium (41.2 and 36.9%), and the use of either MEF or BEF feeders (40.3 and 38.1%) had no significant effect on the ability of inner cell masses (ICMs) to form primary cell colonies compared to controls. All bESC-like cells were first dissociated mechanically for three passages followed by enzymatic dissociation. The ability to maintain ESC morphology to passage 10 was compared among the three enzymes above. More bESC-like cell lines survived beyond passage 10 when treated with TrypLE compared to Trypson-EDTA (28.8 and 12.6%; p < 0.05), and bESC-like cells differentiated quickly when treated with Liberase Blendzyme 3. The bESC-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as SSEA-1, AP, OCT-4, and Nanog. When removed from feeders, these bESC-like cells formed embryoid bodies (EBs) in a suspension culture. When EBs were cultured on tissue culture plates, they differentiated into various cell types. In summary, we were able to culture bESC-like cells more than 10 passages by enzymatic dissociation, which is important in gene targeting, maintenance, and banking of bESC lines.
Gong
Guochun
G
Department of Animal Science and Center for Regenerative Biology, University of Connecticut , Storrs, CT 06269-4243, USA.
Roach
Marsha L
ML
Jiang
Le
L
Yang
Xiangzhong
X
Tian
Xiuchun Cindy
XC
D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002478:Cells, Cultured / Q000166:cytology; D003470:Culture Media / Q000494:pharmacology; D046149:Embryo Culture Techniques / Q000379:methods*; D053595:Embryonic Stem Cells / Q000166:cytology*; D005260:Female; D005307:Fertilization in Vitro; D006801:Humans; D053605:Leukemia Inhibitory Factor / Q000378:metabolism; D051379:Mice; D015398:Signal Transduction; D013997:Time Factors
D003470:Culture Media; D053605:Leukemia Inhibitory Factor