Induced pluripotent stem cell generation from bovine somatic cells indicates unmet needs for pluripotency sustenance

Abstract

Mechanisms that direct reprogramming of differentiated somatic cells to induced pluripotent stem cells (iPSCs), albeit incomplete in understanding, are highly conserved across all mammalian species studied. Equally, proof of principle that iPSCs can be derived from domestic cattle has been reported in several publications. In our efforts to derive and study bovine iPSCs, we encountered inadequacy of methods to generate, sustain, and characterize these cells. Our results suggest that iPSC protocols optimized for mouse and human somatic cells do not effectively translate to bovine somatic cells, which show some refractoriness to reprogramming that also affects sustenance. Moreover, methods that enhance reprogramming efficiency in mouse and human cells had no effect on improving bovine cell reprogramming. Although use of retroviral vectors coding for bovine OCT4, SOX2, KLF4, cMYC, and NANOG appeared to produce consistent iPSC‐like cells from both fibroblasts and cells from the Wharton's jelly, these colonies could not be sustained. Use of bovine genes could successfully reprogram both mouse and human cells. These findings indicated either incomplete reprogramming and/or discordant/inadequate culture conditions for bovine pluripotent stem cells. Therefore, additional studies that advance core knowledge of bovine pluripotency are necessary before any anticipated iPSC‐driven bovine technologies can be realized.

Establishment of bovine expanded potential stem cells

“…These cells, in general, however, lack the standard pluripotent stem cell criteria: poor derivation efficiencies; inability of maintaining pluripotency in long-term culture; limited developmental potential in the in vitro and in vivo assays such as chimera generation. The reported bovine iPSC lines often have leaky expression of the reprogramming genetic factors (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38).…”

Strategy to Establish Embryo-Derived Pluripotent Stem Cells in Cattle

“…It has been reported that iPSCs can be established not only in humans and mice but also in several other animals [ 62 , 68 , 69 , 70 , 71 ]. Interestingly, the number of species for which iPSCs has be established is greater than the number of species for which ESCs can be established.…”

Establishment of Bovine-Induced Pluripotent Stem Cells

“…However, like their bESC counterparts, all these cells in general suffer limited self-renewal capacity. Also, most of the biPSCs, including those reported recently [ 19 , 20 ] still relied on the expression of exogenous transgenes for continuous propagation. Meanwhile, bovine trophectoderm cells were reported when reprogramed using conditions for biPSCs generation [ 15 , 21 ].…”

“…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 ].…”