Simplified Derivation of Stable Early Passage Murine Embryonic and Parthenogenetic Stem Cells

Abstract

Aneuploidy in cultured embryonic stem (ES) cells severely limits research and therapeutic potential. To begin to understand when chromosomal imbalances arise, primary ES cell lines were derived in feeder-free, fully defined culture media from 44% of two-cell CF1 embryos (5 lines), 37% of CF1 multi-drug resistance (Mdr1/Abcb1) mutant embryos (19 lines), and 2 parthenogenetic stem (mPS) lines from Mdr1 mutant eggs for array comparative genomic hybridization analysis. By passage 2, trivial somatic chromosome euploidy was detected in all wild-type lines and in 9 (64%) of the 14 mutant cell lines. Five Mdr1 mutant ES cell lines and both mPS cell lines exhibited mosaic gain of chromosome 8 in at least 30% of cells, a common finding in mouse ES cells. X chromosomes exhibited more aneuploidy than somatic chromosomes. Five of the Mdr1 mutant lines were male, while the nine female ES cell lines and both mPS lines exhibited almost complete loss of one X chromosome. Two ES lines analyzed at passage 3 exhibited partial gains and losses in chromosomes 15 and 19, suggesting that aneuploidy in murine ES cells begins by passage 3 in standard culture conditions. The somatic cell euploidy in early passage wild-type CF1 ES lines, in contrast to the early accumulation of chromosome 8 triploidy in the Mdr1 mutant lines, suggests that component(s) of the culture system not well tolerated by ES cells with the Mdr1 mutation may be responsible for triploid chromosome 8 in multipassaged mouse ES cells. The ease of deriving euploid mouse stem cell lines in fully defined culture conditions from individual females encourages the use of primary ES cell lines for a variety of purposes. Adjusting culture components to correct early chromosome 8 accumulation in primary Mdr1 mutant lines may reveal pathways critical to maintaining euploidy in cultured ES cells.