Generation of a human haploid neural stem cell line for genome-wide genetic screening

Hai-Song Wang; Xin-Rui Ma; Wen-Bin Niu; Hao Shi; Yi-Dong Liu; Ning-Zhao Ma; Nan Zhang; Zi-Wei Jiang; Ying-Pu Sun

doi:10.4252/wjsc.v15.i7.734

Generation of a human haploid neural stem cell line for genome-wide genetic screening

World J Stem Cells. 2023 Jul 26;15(7):734-750. doi: 10.4252/wjsc.v15.i7.734.

Authors

Hai-Song Wang¹, Xin-Rui Ma², Wen-Bin Niu², Hao Shi², Yi-Dong Liu², Ning-Zhao Ma², Nan Zhang², Zi-Wei Jiang³, Ying-Pu Sun²

Affiliations

¹ Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Zhengzhou 450052, Henan Province, China. hswang813@zzu.edu.cn.
² Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Zhengzhou 450052, Henan Province, China.
³ Basic Medical School, Zhengzhou University, Zhengzhou 450052, Henan Province, China.

Abstract

Background: Haploid embryonic stem cells (haESCs) have been established in many species. Differentiated haploid cell line types in mammals are lacking due to spontaneous diploidization during differentiation that compromises lineage-specific screens.

Aim: To derive human haploid neural stem cells (haNSCs) to carry out lineage-specific screens.

Methods: Human haNSCs were differentiated from human extended haESCs with the help of Y27632 (ROCK signaling pathway inhibitor) and a series of cytokines to reduce diploidization. Neuronal differentiation of haNSCs was performed to examine their neural differentiation potency. Global gene expression analysis was con-ducted to compare haNSCs with diploid NSCs and haESCs. Fluorescence activated cell sorting was performed to assess the diploidization rate of extended haESCs and haNSCs. Genetic manipulation and screening were utilized to evaluate the significance of human haNSCs as genetic screening tools.

Results: Human haESCs in extended pluripotent culture medium showed more compact and smaller colonies, a higher efficiency in neural differentiation, a higher cell survival ratio and higher stability in haploidy maintenance. These characteristics effectively facilitated the derivation of human haNSCs. These human haNSCs can be generated by differentiation and maintain haploidy and multipotency to neurons and glia in the long term in vitro. After PiggyBac transfection, there were multiple insertion sites in the human haNSCs' genome, and the insertion sites were evenly spread across all chromosomes. In addition, after the cells were treated with manganese, we were able to generate a list of manganese-induced toxicity genes, demonstrating their utility as genetic screening tools.

Conclusion: This is the first report of a generated human haploid somatic cell line with a complete genome, proliferative ability and neural differentiation potential that provides cell resources for recessive inheritance and drug targeted screening.

Keywords: Cell therapy; Extended pluripotency; Genetic screening; Haploid; Neural stem cells.