| Abstrakt: |
Vero and Cos7 cell lines derived from the kidney of the African green monkey (Cercopithecus aethiops) are invaluable for testing viral infections and for introducing foreign genes. Here, we established a embryonic stem (ES) cell line in African green monkey that expected to be precious resources for the researches as well as other primate ES cell lines. A piezo-driven micromanipulation system was used for intracytoplasmic sperm injection in male and female African green monkeys maintained in our research center. A total of 68 fertilized eggs with two pronuclei and a second polar body were produced and then cultured. Of 68 zygotes cultured with or without buffalo rat liver cell (BRL) or mouse embryonic fibroblast cell (MEF) monolayer, 9 developed to the blastocyst stage. Culture with or without MEF yielded development to the blastocyst stage, where culture with BRL did not yield any development (3/14, 21.4% and 6/31, 19.4% vs. 0/23, 0%, respectively; P < 0.05). Parts of the inner cell mass isolated by removing trophectoderm and the zona pellucida (ZP) with needles (1 blastocyst cultured with MEF, hereafter designated I; and 3 without MEF, designated II) or the blastocysts dissolved ZP with an enzyme (2 blastocysts with MEF, designated III; and 3 without MEF, designated IV) were transferred to ES culture medium, containing 20% FBS or knockout serum replacement, with mitomycin C-treated STO cell or MEF monolayer. Of 9 blastocysts, 4 formed outgrowth (1 of I blastocysts, 2 of III, and 1 of IV). At the first passage using collagenase and pipetting or dividing into small clusters with needles, 3 colonies (1 each of I, III and IV) appeared and passaged further. The colony I comprised one ES-like cell line of flat-formed colonies consisting of cells with large nuclei similar to those exhibited by other primate ES cell lines. Immunocytochemistry revealed that this cell line had the expression of the undifferentiated markers (Oct-3, SSEA4, TRA-1-60, TRA-1-81 and Nanog, although SSEA3 was weakly expressed) and that spontaneous differentiated cells from embryoid bodies plated onto dishes had the expression of -smooth muscle actin, -tubllin III, nestin, -fetoprotein and cdx2. In addition, teratomas derived from the cells transferred into immunodeficient mice exhibited tissues from three embryonic germ layers (ectoderm, mesoderm and endoderm). Karyotyping analysis revealed that 88% of cells examined had a normal chromosome number of 60 and sex chromosomes of XX. For further characterization, we examined whether this cell line can be passaged by dividing into single cells with trypsin. The average replating efficiency and rate of colonies showing undifferentiated morphology until 37 passages were 20.7% and 15.5%, respectively. These results demonstrate that a novel non-human primate ES cell line in African green monkey was established. |
