Derivation of Haploid Trophoblast Stem Cells via Conversion In Vitro

Abstract

SummaryOwing to their single genome, haploid cells are powerful to uncover unknown genes by performing genetic screening in mammals. However, no haploid cell line from an extraembryonic lineage has been achieved yet, which limits the application of haploid cells in placental genetic screening. Here, we show that overexpression of Cdx2 can convert haploid embryonic stem cells to trophoblast stem cells (TSCs). p53 deletion reduces diploidization during the conversion and guarantees the generation of haploid-induced TSCs (haiTSCs). haiTSCs not only share the same molecular characterization with trophoderm-derived TSCs but also possess multipotency to placental lineages in various procedures. In addition, haiTSCs can maintain haploidy in the long term, assisted by periodic sorting and with reliance on FGF4 and heparin. Finally, we perform piggyBac-mediated high-throughput mutation in haiTSCs and use them in trophoblast lineage genetic screening. Deep sequencing analysis and validation experiments prove that Htra1 is a blocker for spongiotrophoblast specification.

The milestone of genetic screening: Mammalian haploid cells

“…Except for achievements of haploid cell types in embryonic lineages, mouse haploid trophoblast stem cells (haTSCs) were generated from haploid blastocysts, which could maintain haploidy and differentiation potentials relying on ROCK inhibitor (Y27632) and F4H (FGF4 and heparin) [33] . Meanwhile, mouse haESCs could also be converted to haploid induced trophoblast stem cells (haiTSCs) by inducible overexpression of Cdx2 and knockout of p53 [34] .…”

Current advances in haploid stem cells

“…Shuai lab also reported that overexpression of Cdx2 together with deletion of p53 can convert haESCs to haploid-induced TSCs (haiTSCs). By applying haiTSCs for high-throughput genetic screening, they found that Htra1 is a blocker for spongiotrophoblast specification (Peng et al, 2019). The derivation of haTSCs and haiTSCs represents another interesting avenue that might help to explore fundamental biological roadmaps in the extraembryonic trophoblast lineage at a genomic scale.…”

“…3B and 3C). Usually, mutations in haploid cells are generated by gene trapping and nuclease-mediated gene knockout, including piggyBac and clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease (CRISPR/Cas) systems (Elling et al, 2011;Leeb and Wutz, 2011;Leeb et al, 2014;Monfort et al, 2015;He et al, 2017;Liu et al, 2017;Wang et al, 2018;Cui et al, 2019;Peng et al, 2019). Meanwhile, it was also reported to generate haESC libraries by chemical mutagens (Forment et al, 2016).…”

“…Since 2009, with the constant optimization of derivation and culture techniques, haploid embryonic stem cell (haESC) lines from medaka fish, mouse, rat, monkey and even humans have been successfully established (Yi et al, 2009;Elling et al, 2011;Leeb and Wutz, 2011;Li et al, 2012;Yang et al, 2012Yang et al, , 2013Li et al, 2014;Sagi et al, 2016). Most recently, the repertoire of haploid stem cells further expands to include haploid trophoblast stem cells (Cui et al, 2019;Peng et al, 2019), the extraembryonic counterpart of embryonic stem cells. In this review, we present an overview of existing haploid stem cells, as well as their strengths and limitations.…”

Inhibition of Apoptosis Reduces Diploidization of Haploid Mouse Embryonic Stem Cells during Differentiation

“…Recent studies showed that knockout of p53 ( Olbrich et al., 2017 ) or overexpression of Dnmt3b ( He et al., 2018 ) could stabilize the haploid genome and slow down self-diploidization. In particular, p53 -null haESCs could sustain haploid status well both under daily culture and in cells converting to an extraembryonic cell fate ( Peng et al., 2019 ). p53 is an essential gene related to the cell cycle, DNA damage, and other crucial biological processes ( Aladjem et al., 1998 , Cross et al., 1995 , Fujiwara et al., 2005 ).…”