Genetic screening and multipotency in rhesus monkey haploid neural progenitor cells

Abstract

Haploid embryonic stem cells (haESCs) have been extensively applied in forward and reverse genetic screening. However, a mammalian haploid somatic cell line is difficult to achieve because of spontaneous diploidization in differentiation. As a non-human primate species, monkeys are widely used in basic and pre-clinical research in which haploid cells are restricted to ESCs. Here, we report that rhesus monkey haESCs in an optimized culture medium show naïve-state pluripotency and stable haploidy. This model facilitated the derivation of haploid neural progenitor cells (haNPCs), which maintained haploidy and differentiation potential into neurons and glia for a long period High-throughput trapping mutations can be efficiently introduced into haNPCs via transposons. This system proves useful when identifying gene targets of neural toxicants via a proof-of-concept experiment. Using CRISPR/Cas9 editing, we confirmed that , from the candidate gene list, is a resistance gene of A803467 (a tetrodotoxin-like toxicant). This model is the first non-human primate haploid somatic cell line with proliferative ability, multipotency and an intact genome, thus providing a cellular resource for recessive genetic and potential drug screening.

Structural basis of seamless excision and specific targeting by piggyBac transposase

“…pB has proved to be extremely versatile and the lack of a DNA footprint left behind after its transposition is a unique and valuable property 13 – 16 . It is used in non-viral vectors for transgenesis 17 , 18 , gene therapy 7 , 19 , insertional mutagenesis 20 , and genetic screens 21 – 23 . It has also found application in novel therapeutic strategies including CAR T-cell engineering 24 – 26 , CRISPR/Cas-mediated gene therapy 27 – 29 , and human induced pluripotent stem cells (iPSC) engineering 30 – 32 .…”

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

“…Previous studies reported that primate haESCs maintain haploidy more stably than rodent haESCs ( Sagi et al., 2016 , Yang et al., 2013 ), and whether the mechanisms involved correlate with p53 or p73 warrants further investigation. Another study showed that although rhesus monkey haESCs maintained haploidy well in the ESC state, they underwent severe diploidization during differentiation for reasons related to apoptosis ( Wang et al., 2018 ). Therefore, apoptosis may be the major cause of the diploidization of mammalian haploid cells.…”

The milestone of genetic screening: Mammalian haploid cells

“…Nevertheless, haploid epiblast stem cells-like cells (haEpiLCs) were generated by differentiation of haESCs in vitro , assisting with optimized culture medium and FACS [9] , [28] . Mouse haploid neural stem cells [29] and monkey haploid neural progenitor cells [30] were also derived via modified differentiation protocols. Besides, haploid neurons were generated by differentiation of haESCs in mouse [31] and human [25] .…”

“…Resistant gene of neurotoxin Mn 2+ ( Park2 ) was figured out with mouse haNSCLCs mutant libraries [32] . Target genes of a tetrodotoxin-like toxicant A803467 ( B4GALT6 ) were uncovered using monkey mutant haploid neural progenitors [30] . In addition, Peng et al screened out the blocker gene ( Htra1 ) for spongiotrophoblast specification with mouse haiTSCs [34] .…”