Basic Study
Copyright ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Jul 26, 2023; 15(7): 734-750
Published online Jul 26, 2023. doi: 10.4252/wjsc.v15.i7.734
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
Hai-Song Wang, Xin-Rui Ma, Wen-Bin Niu, Hao Shi, Yi-Dong Liu, Ning-Zhao Ma, Nan Zhang, Ying-Pu Sun, Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Zhengzhou 450052, Henan Province, China
Zi-Wei Jiang, Basic Medical School, Zhengzhou University, Zhengzhou 450052, Henan Province, China
Author contributions: Sun YP and Wang HS conceived, designed, and supervised the study; Wang HS and Ma XR performed the experiments; Shi H, Zhang N, and Ma NZ analyzed the data; Wang HS, Ma XR, and Jiang ZW wrote the manuscript; Sun YP, Niu WB, and Liu YD revised the manuscript; all authors have read and approve the final manuscript.
Supported by the National Natural Science Foundation of China, No. 81901476.
Institutional review board statement: This study did not involve any human or animal subjects.
Conflict-of-interest statement: The authors declare having no conflicts of interest.
Data sharing statement: The data supporting the results of this research are available from the corresponding author upon reasonable request.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Hai-Song Wang, Doctor, PhD, Assistant Professor, Center for Reproductive Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou University, No. 40 Daxue Road, Zhengzhou 450052, Henan Province, China. hswang813@zzu.edu.cn
Received: March 21, 2023
Peer-review started: March 21, 2023
First decision: May 22, 2023
Revised: June 1, 2023
Accepted: June 21, 2023
Article in press: June 21, 2023
Published online: July 26, 2023
Processing time: 125 Days and 15.7 Hours
ARTICLE HIGHLIGHTS
Research background

Recently, haploid embryonic stem cells (haESCs) have been established in many species and are widely used in forward and reverse genetic screening. Differentiated haploid cell line types in mammals are lacking due to spontaneous diploidization during differentiation that compromises lineage-specific screens.

Research motivation

No human haploid somatic cell line has been generated, although there is an urgent need for lineage-specific screens.

Research objectives

This study aimed to derive haploid neural stem cells (haNSCs) from human extended haESCs by optimizing differentiation methods and verifying human haNSCs to easily carry out lineage-specific screens.

Research methods

Human haploid NSCs were differentiated from human extended haESCs with the help of Y27632 to reduce the diploidization. Neuronal differentiation of haNSCs was performed to examine their neural differentiation potential. Global gene expression analysis was performed to compare haNSCs with diploid NSCs and haESCs. Fluorescence activated cell sorting was conducted 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.

Research results

haNSCs can be generated by differentiation in vitro, and they maintain long-term haploidy with a complete genome and multipotency towards neurons and glia. Human haESCs cultured in extended pluripotency medium formed more compact colonies and exhibited more efficient neural differentiation and more stable maintenance of haploidy, which effectively facilitated the derivation of haNSCs. After PiggyBac transfection, there were multiple insertion sites in the haNSC genome and the insertion sites were evenly spread across all chromosomes.

Research conclusions

In this work, we demonstrated that human haNSCs can be generated by differentiation from extended haESCs. This is the first human haploid somatic cell line with a complete genome, acceptable proliferative ability and neural differentiation potential, which provides a cell resource for studies of recessive inheritance and drug targeted screening.

Research perspectives

This cell line will be valuable for genetic screening relevant to the function of the human nervous system and drug targeting research. Future research will be related to identifying key neurogenesis genes with the help of this cell line. Another research focus is to identify the underlying mechanisms of diploidization.