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

Core Tip: Human embryonic stem cells are widely used in preclinical and genetic screening studies. Haploid cells are ideal tools to perform genetic screening. To date, no human haploid somatic cell lines have been successfully created. We converted human haploid embryonic stem cells to an extended pluripotency state by optimizing the culture medium. The derived haploid neural stem cells can proliferate as a haploid genome and maintain multipotency to generate functional neurons and glia. The haploid neural stem cells can also be easily used for gene editing to generate numerous homozygous mutations for lineage-specific screens.