Bovine trophectoderm cells induced from bovine fibroblasts with induced pluripotent stem cell reprogramming factors.
Thirteen independent induced bovine trophectroderm (iBT) cell lines were established by reprogramming bovine fetal liver-derived fibroblasts after viral-vector transduction with either six or eight factors, including POU5F1 (OCT4), KLF4, SOX2, MYC, NANOG, LIN28, SV40 large T antigen, and hTERT. Light- and electron-microscopy analysis showed that the iBT cells had epithelial cell morphology typical of bovine trophectoderm cells. Reverse-transcription-PCR assays indicated that all of the cell lines expressed interferon-tau (IFNT) at passages 1 or 2. At later passages (≥ passage 8), however, immunoblot and antiviral activity assays revealed that more than half of the iBT cell lines had stopped expressing IFNT. Messenger RNAs specific to trophectoderm differentiation and function were found in the iBT cell lines, and 2-dimensional-gel analysis for cellular proteins showed an expression pattern similar to that of trophectoderm cell lines derived from bovine blastocysts. Integration of some of the human reprogramming factors, including POU5F1, KLF4, SOX2, MYC, NANOG, and LIN28, were detected by PCR, but their transcription was mostly absent in the iBT cell lines. Gene expression assessment of endogenous bovine reprogramming factor orthologs revealed endogenous bLIN28 and bMYC transcripts in all; bSOX2 and bNANOG in none; and bKLF4 and bPOU5F1 in less than half of the iBT cell lines. These results demonstrate that bovine trophectoderm can be induced via reprogramming factor expression from bovine liver-derived fibroblasts, although other fibroblast populations-e.g., derived from fetal thigh tissue-may produce similar results, albeit at lower frequencies.
U.S. Department of Agriculture, Agricultural Research Service, Animal Biosciences and Biotechnology Laboratory, Beltsville, Maryland.
Neil C
http://orcid.org/0000-0002-9204-817X
NC
Talbot
U.S. Department of Agriculture, Agricultural Research Service, Animal Biosciences and Biotechnology Laboratory, Beltsville, Maryland.
Wendy O
WO
Sparks
U.S. Department of Agriculture, Agricultural Research Service, Animal Biosciences and Biotechnology Laboratory, Beltsville, Maryland.
Caitlin E
CE
Phillips
Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, Virginia.
Alan D
AD
Ealy
U.S. Department of Agriculture, Agricultural Research Service, Animal Biosciences and Biotechnology Laboratory, Beltsville, Maryland.
Anne M
AM
Powell
U.S. Department of Agriculture, Agricultural Research Service, Animal Biosciences and Biotechnology Laboratory, Beltsville, Maryland.
Thomas J
TJ
Caperna
U.S. Department of Agriculture, Agricultural Research Service, Animal Biosciences and Biotechnology Laboratory, Beltsville, Maryland.
Wesley M
WM
Garrett
U.S. Department of Agriculture, Agricultural Research Service, Animal Biosciences and Biotechnology Laboratory, Beltsville, Maryland.
David M
DM
Donovan
U.S. Department of Agriculture, Agricultural Research Service, Animal Biosciences and Biotechnology Laboratory, Beltsville, Maryland.
Le Ann
LA
Blomberg
D000818:Animals; D002417:Cattle; D065150:Cellular Reprogramming; D000067470:Cellular Reprogramming Techniques; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D000090062:Kruppel-Like Factor 4; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics
C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D014157:Transcription Factors
bovine; cell culture; iPSC; induced; reprogramming; trophectoderm