Mammalian haploid cells have applications for genetic screening and substituting gametic genomes. Here, we characterize a culture system for obtaining haploid primordial germ cell-like cells (PGCLCs) from haploid mouse embryonic stem cells (ESCs). We find that haploid cells show predisposition for PGCLCs, whereas a large fraction of somatic cells becomes diploid. Characterization of the differentiating haploid ESCs (haESCs) reveals that Xist is activated from and colocalizes with the single X chromosome. This observation suggests that X chromosome inactivation (XCI) is initiated in haploid cells consistent with a model where autosomal blocking factors set a threshold for X-linked activators. We further find that Xist expression is lost at later timepoints in differentiation, which likely reflects the loss of X-linked activators. In vitro differentiation of haploid PGCLCs can be a useful approach for future studies of potential X-linked activators of Xist.

In mammals, one of the two X chromosomes of diploid female cells is inactivated for gene dosage compensation between the sexes. Xist initiates X chromosome inactivation (XCI) in female cells. Although Xist is not expressed in male cells with a single X chromosome, it remains unclear if a single X chromosome in a haploid cell would lead to Xist activation. Haploid embryonic stem cells (haESCs) have been derived from several mammalian species, and maintain pluripotency in culture. They also show a predisposition for diploidization especially during differentiation. Establishing somatic haploid cells has been reported by inhibiting potential factors for diploidization. Considering high diploidization in somatic lineages, we wondered if specification of germline progenitor cells could be less restrictive to a haploid genome. Here, we report that mouse primordial germ cell-like cells (PGCLCs) preferentially maintain a haploid genome. Using this differentiation system, we find that Xist is initially induced in differentiating haESCs but subsides later after germ cell specification. The observation that XCI is initiated in haploid cells demonstrates Xist activation without the requirement of a second X chromosome inactivation center (Xic).