178 ESTABLISHMENT OF MOUSE PLURIPOTENT STEM CELLS GENERATED FROM PRIMORDIAL GERM CELLS

Abstract

Pluripotent stem cells have been generated from two embryonic sources: ES cells generated from ICM of blastocyst stage embryos, and embryonic germ (EG) cells generated from primordial germ cells (PGCs). Both ES and EG cells are pluripotent and exhibit important characteristics such as high alkaline phosphatase (AP) activity, multicellular colony formation, normal and stable karyotype, continuous passaging ability, and capacity to differentiate into three embryonic germ layers. This study was performed to establish the culture system for mouse EG cells derived from mouse PGCs. PGCs collected from the genital ridge of Day 11.5, 12.5, and 13.5 mouse embryos (C57BL/6 × DBA/2) were cultured and subsequently passaged on mitotically inactivated STO feeder cell layer. Cells were grown in Dulbecco's modified eagle medium (DMEM) supplemented with 15% fetal bovine serum (FBS), 0.1 mM nonessential amino acids, 0.1 mM 2-mercaptoethanol, 2 mM glutamine, 100 IU/mL of penicillin, 100 μg/mL of streptomycin, 1,000 units/mL of leukemia inhibiting factor (LIF), 6 ng/mL of SCF, and 10 ng/mL of bFGF at culture conditions of 5% CO₂ in air, 95% relative humidity, 37°C temperature. Cells were routinely passaged every 3–4 days. Over a period of 7–10 days in primary culture, PGCs proliferated to form small, densely packed, multicellular colonies consisting of AP-positive cells that morphologically resembled undifferentiated ES cells. RT-PCR analysis confirmed mRNA expression of transcription factors Oct-4 and Nanog in these cells. Cultured cells could be maintained on the feeder cell layer for at least 10 passages and still retain normal karyotype. These results suggest that cell lines derived from mouse primordial germ cells are presumably EG cell lines and could be useful for transgenic animal production and ES cell study.