Bovine trophectoderm cells induced from bovine fibroblasts with induced pluripotent stem cell reprogramming factors

Abstract

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.

Analyzing bovine OCT4 and NANOG enhancer activity in pluripotent stem cells using fluorescent protein reporters

“…We wondered whether the b OCT4 -GFP reporters we generated might be used as live indicators in reprogramming cells for iPSC generation. To test this we used the MEF reprogramming system with doxycycline (Dox)-inducible OKSM expression[ 40 ] as bovine iPSC generation remains a challenge[ 16 ]. B6/129 MEFs were infected with lentiviral OKSM plus lentivirus of empty pGreenFire vector, b OCT4 -GFP1, b OCT4 -DE-GFP, or b OCT4 -DE2-GFP.…”

“…These bovine iPSCs are not completely reprogrammed as shown by their dependence on the continued expression of the viral transgenes for self-renewal[ 12 – 15 ]. Also, there is a profound trophectoderm lineage generation or differentiation during bovine iPSC induction and ESC derivation[ 16 , 17 ]. These problems negatively impact the practical applications of bovine ESCs/iPSCs.…”

The past, present and future of bovine pluripotent stem cells: a brief overview

“…The reported naive-like bovine iPSCs [25][26][27][28] did not seem to fare better in either activation of pluripotent circuitry (i.e., they were dependent on continuous transgene expression), passage capacity or differentiation potential (i.e., they failed to form teratomas in nude mice) [28] . Furthermore, using the reprogramming conditions for iPSCs generation, two research teams independently obtained bovine trophectoderm cells [29,30] . These observations suggest that the reported, presumptive bovine iPSCs are either incompletely reprogrammed or are of trophectoderm-lineage while expressing some pluripotent features, as was also reported in pigs [31] .…”

Establishment of Bovine-Induced Pluripotent Stem Cells

“…Meanwhile, bovine trophectoderm cells were reported when reprogramed using conditions for biPSCs generation [ 15 , 21 ]. The problems of persistent transgene activity [ 22 ], limited propagation capacity of the biPSCs [ 20 ], and the unexpected generation of trophectoderm-lineage stem cells [ 23 ] indicate that the majority of reported biPSCs were partially reprogrammed. Overall, the lack of completely reprogrammed biPSCs signifies the existence of unidentified reprogramming hurdles in bovine somatic cells that prevent the generation of bona fide biPSCs [ 24 ].…”