Haploid Mouse ESC Lines Generated For Developmental Studies and Genetic Screening

Researchers are reporting on the generation of stable haploid embryonic stem cells (ESCs) from haploid mouse embryos. They say that resulting ESCs can be expanded over numerous passages and maintain an intact haploid genome without amplification or losses.

The scientists add that the haploid mouse ESCs also maintain a wide differentiation potential, contribute substantially to the development of chimeric embryos and live-born chimeric mice, and can be used for forward genetic screening studies.

Led by Martin Leeb, Ph.D., and Anton Wutz, Ph.D., at the U.K.’s Wellcome Trust Centre for Stem Cell Research, the team describes their achievement in Nature. Their paper is titled “Derivation of haploid embryonic stem cells from mouse embryos.”

Drs. Leeb  and Wutz generated haploid mouse embryos from activated unfertilized mouse oocytes taken from superovulated B6CBAF1 hybrid female mice. Thirty resulting blastocysts were recovered, and after removal of the zona and trophectoderm, inner cell masses (ICMs) were cultured in 2i medium in the presence of leukemia inhibitory factor (LIF). Twenty seven ESC lines obtained were expanded individually and their DNA content evaluated.

In six of these ESC lines at least 10% of the cells had a haploid DNA content, although, the proportion of haploid cells reached a conservatively estimated 60%, the authors note. Haploid ESC lines were expanded for over 35 passages.

Interestingly, the researchers found that generating haploid ESCs could be achieved without removal of the trophectoderm from blastocysts and by culturing using DMEM medium supplemented with knockout serum replacement and LIF.  Haploid ESCs were also generated in repeat experiments using a different inbred mouse strain and genetically modified mouse lines.

Further analysis showed that haploid ESCs exhibited a typical mouse ESC colony morphology and expressed pluripotency markers. Genome-wide expression analysis showed a high correlation between haploid ESCs and control diploid male ESCs, suggesting that the cells “largely maintain a mouse ES cell transcription profile,” the team states.

When GFP-tagged haploid ESCs were injected into C57BL/6 blastocysts, the majority of GFP-expressing cells in the developing chimeric embryo had a diploid content, indicating that the haploid ESCs contributed significantly to development after diploidization.

Live-born male and female chimeric mice were in addition generated, and these also showed substantial contribution from haploid ESCs and developed normally with evidence of coat color chimerism. “Taken together these findings demonstrate that haploid ES cells maintain a wide differentiation potential,” the researchers write. 

The haploid ESCs could in addition have utility in genetics studies, they continue. In a pilot screen for mismatch repair genes the haploid ESCs were employed in a forward genetics screening approach, based on a previously described piggyBac transposon gene trap technique, to identify mutations in autosomal genes.

“It is interesting to speculate whether differentiated haploid lineages can be generated, perhaps through suppression of X-inactivation, and whether it is possible to derive haploid human ES cells,” they conclude.