Optimization of culture conditions to support long-term self-renewal of buffalo (Bubalus bubalis) embryonic stem cell-like cells.
A culture system capable of sustaining self-renewal of buffalo embryonic stem (ES) cell-like cells in an undifferentiated state over a long period of time was developed. Inner cell masses were seeded on KO-DMEM+15% KO-serum replacer on buffalo fetal fibroblast feeder layer. Supplementation of culture medium with 5 ng/mL FGF-2 and 1000 IU/mL mLIF gave the highest (p<0.05) rate of primary colony formation. The ES cell-like cells' colony survival rate and increase in colony size were highest (p<0.05) following supplementation with FGF-2 and LIF compared to other groups examined. FGF-2 supplementation affected the quantitative expression of NANOG, SOX-2, ACTIVIN A, BMP 4, and TGFβ1, but not OCT4 and GREMLIN. Supplementation with SU5402, an FGFR inhibitor (≥20 μM) increased (p<0.05) the percentage of colonies that differentiated. FGFR1-3 and ERK1, K-RAS, E-RAS, and SHP-2, key signaling intermediates of FGF signaling, were detected in ES cell-like cells. Under culture conditions described, three ES cell lines were derived that, to date, have been maintained for 135, 95, and 85 passages for over 27, 19, and 17 months, respectively, whereas under other conditions examined, ES cell-like cells did not survive beyond passage 10. The ES cell-like cells were regularly monitored for expression of pluripotency markers and their potency to form embryoid bodies.
Embryo Biotechnology Lab, Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana, India.
Ruchi
R
Sharma
Aman
A
George
Nitin Manchindra
NM
Kamble
Karn Pratap
KP
Singh
Manmohan Singh
MS
Chauhan
Suresh Kumar
SK
Singla
Radhey Sham
RS
Manik
Prabhat
P
Palta
D000818:Animals; D000943:Antigens, Differentiation / Q000096:biosynthesis; D002020:Buffaloes; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D049109:Cell Proliferation; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D061252:Feeder Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
D000943:Antigens, Differentiation; D047428:Protein Kinase Inhibitors; D011758:Pyrroles; C105686:SU 5402