Isolation and cultivation of blastocyst-derived stem cell lines from American mink (Mustela vison)

Abstract

Ten embryonic stem (ES) cell lines from mink blastocysts were isolated and characterized. All the lines had a normal diploid karyotype; of the ten lines studied, five had the XX and five had the XY constitution. Testing of the pluripotency of the ES-like cells demonstrated that 1) among four lines of genotype XX, and X was late-replicating in three; both Xs were active in about one-third of cells of line MES8, and analysis of glucose-6-phosphate dehydrogenase revealed no dosage compensation for the X-linked gene; 2) when cultured in suspension, the majority of lines were capable of forming "simple" embryoid bodies (EB), and two only showed the capacity for forming "cystic" multilayer EBs. However, formation of ectoderm or foci of yolk sac hematopoiesis, a feature of mouse ES cells, was not observed in the "cystic" EB; 3) when cultured as a monolayer without feeder, the ES cells differentiated into either vimentin-positive fibroblast-like cells or cytokeratin-positive epithelial-like cells (less frequently); neural cells appeared in two lines; 4) when injected into athymic mice, only one of the four tested lines gave rise to tumors. These were fibrosarcomas composed of fibroblast-like cells, with an admixture of smooth muscular elements and stray islets of epithelial tissue; (5) when the ES cells of line MES1 were injected into 102 blastocyst cavities and subsequently transplanted into foster mothers, we obtained 30 offspring. Analysis of the biochemical markers and coat color did not demonstrate the presence of chimaeras among offspring. Thus the cell lines derived from mink blastocysts are true ES cells. However, their pluripotential capacities are restricted.