Importance of culture conditions during the morula-to-blastocyst period on capacity of inner cell-mass cells of bovine blastocysts for establishment of self-renewing pluripotent cells.
The hypothesis was tested that the pluripotency of the inner cell mass (ICM) of the bovine embryo is enhanced by the glycogen synthase kinase-3β inhibitor CHIR99021 and the MAPK1 and MAPK3 inhibitor PD032591. Treatment with the two inhibitors from Days 6 to 8 after insemination increased blastocyst steady state concentrations of mRNA for NANOG (P < 0.05) and SOX2 (P = 0.055) and tended to decrease (P = 0.09) expression of GATA6. To evaluate pluripotency, the inner cell mass was isolated by immunosurgery at Day 8, seeded on a feeder layer of bovine embryonic fibroblasts, and cultured in the presence of the inhibitors. Ten of 52 (19%) ICM from control embryos had primary outgrowth formation vs. 23 of 50 (46%) of the ICM from embryos cultured with inhibitors (P < 0.01). For ICM outgrowths from embryos cultured without inhibitors, colonies either did not persist through Passage 2 or became differentiated. In contrast, for the inhibitor group, four colonies survived beyond Passage 2, and one line persisted for 19 passages. This cell line possessed alkaline phosphatase activity, expressed several genes characteristically expressed in pluripotent cells, and differentiated into embryoid bodies when cultured in the absence of the signal transduction inhibitors and the feeder layer. Propagation of the cells was difficult due to slow growth and inefficiency in survival through each passage. In conclusion, exposure to inhibitors during the morula-blastocyst transition facilitated formation of self-renewing pluripotent cell lines from bovine blastocysts.
Department of Animal Sciences, University of Florida, Gainesville, Florida, USA.
M
M
Ozawa
M
M
Sakatani
K E
KE
Hankowski
N
N
Terada
K B
KB
Dobbs
P J
PJ
Hansen
D000818:Animals; D001755:Blastocyst / Q000737:chemistry / Q000502:physiology*; D053624:Blastocyst Inner Cell Mass / Q000166:cytology*; D002417:Cattle / Q000196:embryology*; D002469:Cell Separation / Q000662:veterinary; D005576:Colforsin / Q000494:pharmacology; D046149:Embryo Culture Techniques / Q000662:veterinary*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors; D000071679:Glycogen Synthase Kinase 3 beta; D018398:Homeodomain Proteins / Q000235:genetics; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors; D009028:Morula / Q000502:physiology*; D039904:Pluripotent Stem Cells / Q000166:cytology*; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D055748:SOXB1 Transcription Factors / Q000235:genetics
C473711:Chir 99021; D004791:Enzyme Inhibitors; D018398:Homeodomain Proteins; D011725:Pyridines; D011743:Pyrimidines; D012333:RNA, Messenger; D055748:SOXB1 Transcription Factors; D005576:Colforsin; D000071679:Glycogen Synthase Kinase 3 beta; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D038362:Glycogen Synthase Kinase 3