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Isolation and long-term culture of mink and bovine embryonic stem-like cells
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Theriogenology ISOLATION AND LONG-TERM CULTURE OF MINK AND BOVINE EMBRYONIC STEM-LIKECELLS I.A. Polejaevat, K.L. Whitel, L.C. Ellis2 and W.A. Reed1 1 Department of Animal, Dairy and Veterinary Sciences and ZDepartment of Biology, Biotechnology Center, Utah State University, Logan, UT 84322 USA
The objective of this study was to establish mink and bovine embryonic stem -(ES) like cell lines and test them for the presence of alkaline phosphatase (AP), a biochemical marker characteristic of the earliest developmental stages and also mouse embryonic stem cells. Expanded blastocysts and 4 to &cell embryos were flushed from the uteri and oviducts of naturally mated mink (wild type). Bovine expanded blastocysts were produced by IVF procedures. Pronase was used to remove the zonae pellucidae from embryos and they were individually seeded into 4-well culture dishes onto feeder layers of mitomycin C-treated mouse primary embryonic fibroblasts. Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % FBS, 0.1 mM g-mercaptoethanol, 0.1 mM non-essential amino acids and antibiotics was used for embryo culture. The mink reproductive cycle includes a period of embryonic diapause (delayed implantation 30 to 40 d). Therefore, blastocysts remained intact in suspension for 10 d following removal from the reproductive tract and prolactin (PRL) was used to “reactivate” blastocysts. Within 24 h after the addition of 5pg/ml prolactin, all blastocysts attached to the feeder layers . Three mink ES- (MES) like cell lines were obtained from 4 blastocysts. No MESlike cell lines were established from 4 to 8 cell embryos. Seven bovine ES- (BES) like cell lines were established from 10 blastocysts. MES and BES cells have a high nuclei/cytoplasm ratio. Simple embryonic bodies were produced from all these cell lines. MES-like cells were positive for AP staining. MES-like cells differentiated into fibroblast-like and epithelial-like cells when cultured without feeder layers. Differentiated cells were AP negative. BES-like cells were tested and found to be negative for alkaline phosphatase activity. BES-like cells spontaneously differentiated into neuron-like, epithelial-like and fibroblast-like cells. The MES and BES-like lines were maintained in culture for approximately 5 mo without morphological differentiation. These data suggest that PRL stimulates blastocyst “reactivation” of mink in vitro and that AP activity is a specific and convenient marker for mink ES-like cells. However, AP activity may not be a useful marker for BES cells. Evidence for pluripotency is the ability of the MES and BES-like cells to form embryonic bodies and their differentiation into several different cell types,
Theriogenology ISOLATION AND LONG-TERM CULTURE OF MINK AND BOVINE EMBRYONIC STEM-LIKECELLS I.A. Polejaevat, K.L. Whitel, L.C. Ellis2 and W.A. Reed1 1 Department of Animal, Dairy and Veterinary Sciences and ZDepartment of Biology, Biotechnology Center, Utah State University, Logan, UT 84322 USA
The objective of this study was to establish mink and bovine embryonic stem -(ES) like cell lines and test them for the presence of alkaline phosphatase (AP), a biochemical marker characteristic of the earliest developmental stages and also mouse embryonic stem cells. Expanded blastocysts and 4 to &cell embryos were flushed from the uteri and oviducts of naturally mated mink (wild type). Bovine expanded blastocysts were produced by IVF procedures. Pronase was used to remove the zonae pellucidae from embryos and they were individually seeded into 4-well culture dishes onto feeder layers of mitomycin C-treated mouse primary embryonic fibroblasts. Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % FBS, 0.1 mM g-mercaptoethanol, 0.1 mM non-essential amino acids and antibiotics was used for embryo culture. The mink reproductive cycle includes a period of embryonic diapause (delayed implantation 30 to 40 d). Therefore, blastocysts remained intact in suspension for 10 d following removal from the reproductive tract and prolactin (PRL) was used to “reactivate” blastocysts. Within 24 h after the addition of 5pg/ml prolactin, all blastocysts attached to the feeder layers . Three mink ES- (MES) like cell lines were obtained from 4 blastocysts. No MESlike cell lines were established from 4 to 8 cell embryos. Seven bovine ES- (BES) like cell lines were established from 10 blastocysts. MES and BES cells have a high nuclei/cytoplasm ratio. Simple embryonic bodies were produced from all these cell lines. MES-like cells were positive for AP staining. MES-like cells differentiated into fibroblast-like and epithelial-like cells when cultured without feeder layers. Differentiated cells were AP negative. BES-like cells were tested and found to be negative for alkaline phosphatase activity. BES-like cells spontaneously differentiated into neuron-like, epithelial-like and fibroblast-like cells. The MES and BES-like lines were maintained in culture for approximately 5 mo without morphological differentiation. These data suggest that PRL stimulates blastocyst “reactivation” of mink in vitro and that AP activity is a specific and convenient marker for mink ES-like cells. However, AP activity may not be a useful marker for BES cells. Evidence for pluripotency is the ability of the MES and BES-like cells to form embryonic bodies and their differentiation into several different cell types,