Durable pluripotency and haploidy in epiblast stem cells derived from haploid embryonic stem cellsin vitro

Abstract

Haploid pluripotent stem cells, such as haploid embryonic stem cells (haESCs), facilitate the genetic study of recessive traits. In vitro, fish haESCs maintain haploidy in both undifferentiated and differentiated states, but whether mammalian haESCs can preserve pluripotency in the haploid state has not been tested. Here, we report that mouse haESCs can differentiate in vitro into haploid epiblast stem cells (haEpiSCs), which maintain an intact haploid genome, unlimited self-renewal potential, and durable pluripotency to differentiate into various tissues in vitro and in vivo. Mechanistically, the maintenance of self-renewal potential depends on the Activin/bFGF pathway. We further show that haEpiSCs can differentiate in vitro into haploid progenitor-like cells. When injected into the cytoplasm of an oocyte, androgenetic haEpiSC (ahaEpiSCs) can support embryonic development until midgestation (E12.5). Together, these results demonstrate durable pluripotency in mouse haESCs and haEpiSCs, as well as the valuable potential of using these haploid pluripotent stem cells in high-throughput genetic screening.

Asymmetric Expression of LincGET Biases Cell Fate in Two-Cell Mouse Embryos

“…Mouse ESCs were cultured on a feeder layer in N2B27 medium supplemented with 2i (1 mM PD0325901 and 3 mM CHIR99021) and 20 ng/mL leukemia inhibitory factor (LIF). The mouse epiblast stem cells (mEpiSCs) was cultured in fibronectin coated dish in N2B27 medium plus 12 ng/mL bFGF (R&D, 233-FB-001MG/CF) and 10 ng/mL Activin A (R&D, 338-AC-01M) as described previously (Shuai et al, 2015). The culture medium was changed every day, and the mEpiSCs was digested to single cells by 0.05% trypsin/EDTA (GIBCO, 25300062) and passaged every 2 to 3 days.…”

Generation and application of mammalian haploid embryonic stem cells

“…The recent finding, by Shuai et al . , that haploidy can be well maintained in mouse epiblast stem cells derived from in vitro differentiation of haESCs brings us closer to answering this question.…”

“…In summary, mammalian haploid cell lines are in a pluripotent state, and differentiation of haESCs in chimeric embryos correlates with the gain of a diploid karyotype [46] whereas haploid cells from the Medaka fish can be in both undifferentiated and differentiated states [5], raising the interesting question of whether haploid cell lines in different differentiation states can be generated. The recent finding, by Shuai et al [50], that haploidy can be well maintained in mouse epiblast stem cells derived from in vitro differentiation of haESCs brings us closer to answering this question.…”

Derivation and application of pluripotent stem cells for regenerative medicine

“…In addition, cynomolgus monkey ESCs with higher pluripotency with which monkey chimeric fetuses were generated have been obtained . Human parthenogenetic ESCs Shuai et al, 2015) and homozygous human androgenetic ESCs (Ding et al, 2015) were derived, providing important tools for future therapeutic use in a clinical setting.…”