Derivation and differentiation of haploid human embryonic stem cells

Abstract

Diploidy is a fundamental genetic feature in mammals, in which haploid cells normally arise only as post-meiotic germ cells that serve to ensure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species, but haploid human ES cells have yet to be reported. Here we generated and analysed a collection of human parthenogenetic ES cell lines originating from haploid oocytes, leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics, such as self-renewal capacity and a pluripotency-specific molecular signature. Moreover, we demonstrated the utility of these cells as a platform for loss-of-function genetic screening. Although haploid human ES cells resembled their diploid counterparts, they also displayed distinct properties including differential regulation of X chromosome inactivation and of genes involved in oxidative phosphorylation, alongside reduction in absolute gene expression levels and cell size. Surprisingly, we found that a haploid human genome is compatible not only with the undifferentiated pluripotent state, but also with differentiated somatic fates representing all three embryonic germ layers both in vitro and in vivo, despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics and development.

The X to Autosome Expression Ratio in Haploid and Diploid Human Embryonic Stem Cells

“…They confirmed on the basis of low Xist expression level and low H3K27me3 staining that the single X chromosome in haploid cells is transcriptionally active in both phases. By contrast, only one of the two X chromosomes is active in both phases in diploid cells (Sagi et al 2016). Because Ohno's hypothesis is about genes that existed before the origin of the mammalian X, we followed previous studies (Xiong et al 2010;Lin et al 2012) to focus on human genes having one-to-one orthologs in chicken, and acquired 11,861 autosomal genes and 360 X-linked genes for analysis.…”

“…In other words, the dosage compensation hypothesis predicts a diploid-specific 2-fold upregulation of these genes. Here we address the above questions using RNA-seq data generated from haploid human parthenogenetic embryonic stem (pES) cell lines originating from haploid oocytes (Sagi et al 2016).…”

“…For permissions, please e-mail: journals.permissions@oup.com and EB2 À that were, respectively, from pES10 and pES12, and neural progenitor cells (NPCs) differentiated from pES10 (Sagi et al 2016). Because haploid cells spontaneously and irreversibly diploidize over passages, Sagi et al (2016) used fluorescence-activated cell sorting (FACS) to separate haploids from diploids, followed by validation via visualization of ploidy by DNA fluorescence in situ hybridization (FISH) and quantification of centromere protein foci. To analyze haploid cells and diploidized cells in the same cell cycle phase, the authors used FACS to separately isolate G1-phase cells (before DNA replication) and G2/M-phase cells (after DNA replication).…”

A p53-dependent response limits the viability of mammalian haploid cells

“…Although haploid cell cultures have been established from several invertebrates, including cockroaches and flies (4), similar attempts in vertebrates failed until recently. The isolation of a pseudohaploid human cell line from a patient with chronic myelogenous leukemia (KBM7) (5), along with the development of haploid embryonic stem (haES) cell cultures from zebrafish, mouse, rat, pig, monkey, and human cells (6)(7)(8)(9)(10), have greatly facilitated forward genetic screenings in mammalian cells. Examples of the potential of this approach include identification of the receptors used by the Ebola and Lassa viruses for their entry into the host cell (11,12), and of all genes that are essential for mammalian cell viability (13,14).…”

How human embryonic stem cells sparked a revolution

“…Egli and Nissim Benvenisty at the Hebrew University of Jerusalem overturned long-held concepts of human biology when they derived EScell lines with just half the normal number of chromosomes 1 . Researchers are now starting to use gene-editing tools on these 'haploid' ES cells to understand how genes function in development.…”