Bovine embryonic stem cell-like cell lines cultured over several passages

Abstract

A total of 14 microsurgically produced zona pellucida-free bovine demi-blastocysts were cultured for 3 days in tissue culture medium (TCM) 199 supplemented with 10% heat-inactivated newborn calf serum (NBCS). Developing embryos were continuously cultured in TCM 199 plus 10% NBCS on a feeder-layer of murine embryonic fibroblasts, that had been incubated with mitomycin C (10 μg/ml) for 3 h prior to the onset of embryo cultivation to block mitotic activity of the fibroblasts. After 2 days, 3 expanded blastocysts were attached to the feeder-layer and both trophoblastic cells and inner cell mass (ICM) cells became apparent on the 9th day of culture in 2 out of the 3 expanded blastocysts. Five days later, the ICM cells were disaggregated by a short-term trypsin treatment. The resulting dissociated clumps were seeded on a new murine embryonic fibroblast feeder-layer and covered with modified minimum essential medium (MEM)-Alpha with 10% fetal calf serum (FCS), 0.1 mm mercaptoethanol, 4.5 g/l glucose and 20 mM HEPES-buffer (=passage 0). To prevent differentiation of the cells, approximately 1/3 of the MEM-Alpha was replaced by MEM previously incubated on cell line 5637 containing leucaemia inhibitory factor (LIF) for 3 days. Colonies of embryonic stem cell (ES)-like cells were observed 5 days after the 1st passage. These colonies were repeatedly passaged at approximately 2-week intervals. Two bovine ES-like cell lines were established, which grew considerably slower than murine ES cells, but were lost after the 4th passage, possibly because of toxic effects of a new FCS batch. After cytogenetic analysis, 16 out of 18 metaphase plates contained an euploid number of chromosomes with 2 X-chromosomes and 58 autosomes. Distribution of G-banding on the chromosomes of ES-like cells was in accordance with the diploid set of the bovine genome. ES-like cells were fused to in vitro matured bovine oocytes and, upon successful fusion, cultured in vitro over 5 days. Successful fusion was observed in 79.8% (67/84), 31.3% initiated cleavege and 10.4% reached the 8-16 cell stage at termination of culture.

In-Vitro Development of Aggregates of Bovine Inner Cell Mass Cells or Bovine Mammary Cells and Putative Tetraploid Embryos Produced by Electrofusion.

“…There are several reports about the establishment of bovine ES-like cells from ICM cells [5][6][7][8] and birth of calves after nuclear transfer of these cells [6]. From the results obtained in the present study, if bovine ES-like cells form a clump to some extent after the trypsinization like mammary cells, they would contribute to ICMs of aggregates as well as in mice.…”

“…Furthermore there have been several reports on the establishment of bovine ES cell-like cell lines [5][6][7][8]. Therefore there are some possibilities to produce cloned calves efficiently, if bovine ES cell-like cells can be treated with this aggregation technique using tetraploid embryos.…”

Derivation and Characterization of Bovine Induced Pluripotent Stem Cells by Transposon-Mediated Reprogramming

“…The availability of bovine pluripotent stem cells could facilitate the application of advanced reproductive technologies in this species. Numerous attempts to establish bovine ESC lines have been undertaken; however, they have had limited success (Gong et al, 2010;Jin et al, 2012;Maruotti et al, 2012;Saito et al, 1992;Saito et al, 2003;Solter et al, 2000). Most ESC-like cultures proliferated slowly, lost pluripotency markers, and ceased to divide at early passages (Cao et al, 2009;Malaver-Ortega et al, 2012;Maruotti et al, 2012;Munoz et al, 2008;Saito et al, 1992).…”

Effects of Feeder Cells on the Establishment of ES Cells from Mice and Rats. The Difference between Allogenetic and Xenogenetic Feeders.

“…There have been many attempts to gain ES cell lines in such mammalian species as hamster [10], pig [11][12][13], cow [11,14], mink [15], sheep [12], rabbit [16], rat [17], monkey [18] and human [19]. However, no ES-like cells mentioned distinctly contributed chimeras.…”