Collection and culture of primordial germ cells from cynomolgus monkeys (Macaca fascicularis)


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Abstract

Aim:To clarify the location of primordial germ cells (PGC) in an embryo of target-age and to examine the culture environment of the PCG.Methods:The days of ovulation and fertilization were estimated by measuring the serum concentration of estrogen. Pregnancy was confirmed by measurement of the serum concentration of the beta subunit of macaque chorionic gonadotropin and by ultrasonography We also examined the location of PGC in the embryo at the time of retrieval.Results:Results showed that PGC in an embryo were in the hindguts at day 30 postfertilization, arrived at the genital ridges via mesenteries at approximately day 33 postfertilization, and colonized the gonads by day 36 postfertilization.Conclusions:In conclusion, embryos collected on day 33 postfertilization are more suitable for obtaining PGC from cynomolgus monkeys. The PGC collected from cynomolgus monkey fetuses were cultured under conditions for the derivation and culture of human embryonic germ cells; enzymatically dispersed single cells were cultured on a SIM thioguanine-resistant ouabain-resistant cells (STO) feeder layer with recombinant human leukemia inhibitory factor, recombinant human basic fibroblast growth factor and forskolin. The cells from genital ridges and mesenteries at day 33 postfertilization had alkaline phosphatase (ALP) activity in vitro for a maximum of 13 days. In contrast, ALP activity had been held for 2 months under the same culture condition when the cells were derived from the gonads at day 66 postfertilization. Derivation of an embryonic germ cell from a cynomolgus monkey was not achieved from these cultures.

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