Int J Stem Cells.  2023 Feb;16(1):44-51. 10.15283/ijsc22138.

Embryonic Stem Cells Lacking DNA Methyltransferases Differentiate into Neural Stem Cells that Are Defective in Self-Renewal

• Seo BJ
• Hong TK
• Yoon SH
• Song JH
• Uhm SJ
• Song H
• Hong K
• Schöler HR
• Do JT

• KMID: 2539620
• DOI: http://doi.org/10.15283/ijsc22138

Abstract

Background and Objectives
DNA methyltransferases (Dnmts) play an important role in regulating DNA methylation during early developmental processes and cellular differentiation. In this study, we aimed to investigate the role of Dnmts in neural differentiation of embryonic stem cells (ESCs) and in maintenance of the resulting neural stem cells (NSCs).
Methods and Results
We used three types of Dnmt knockout (KO) ESCs, including Dnmt1 KO, Dnmt3a/3b double KO (Dnmt3 DKO), and Dnmt1/3a/3b triple KO (Dnmt TKO), to investigate the role of Dnmts in neural differentiation of ESCs. All three types of Dnmt KO ESCs could form neural rosette and differentiate into NSCs in vitro. Interestingly, however, after passage three, Dnmt KO ESC-derived NSCs could not maintain their self-renewal and differentiated into neurons and glial cells.
Conclusions
Taken together, the data suggested that, although deficiency of Dnmts had no effect on the differentiation of ESCs into NSCs, the latter had defective maintenance, thereby indicating that Dnmts are crucial for self-renewal of NSCs.

Keyword

Neural stem cells; DNA methyltransferases; Neuronal and glial differentiation

Figure

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  Fig. 1 Effect of Dnmt inhibitor 5-AzaC on the proliferation of neural stem cells (NSCs). (A) Representative images of neural stem cells cultured in various concentrations of 5-AzaC; scale bars: 200 μm. (B) Cell proliferation assay to compare the rate of cell proliferation in control (DMSO) and 5-AzaC-treated NSCs; Student’s t-test; ***p-value<0.001.

• 

  Fig. 2 Pluripotency of three types of Dnmt knockout (KO) ESCs and their differentiation potential into neural rosette and neural stem cells (NSCs). (A) Immunocytochemical analysis of control (Olig2-GFP) and three types of Dnmt KO ESCs, including Dnmt1 KO, Dnmt3a/3b double KO (Dnmt3 DKO), and Dnmt1/3a/3b triple KO (Dnmt TKO). All ESC lines expressed the pluripotency markers Oct4 and Nanog. Nuclei were counter-stained with Hoechst (blue); scale bars: 200 μm. (B) Embryoid body formation of control (Olig2-GFP) and three types of Dnmt KO ESCs; scale bars: 200 μm. (C) Neural rosette formation in control and three types of Dnmt KO ESCs. White arrows indicate neural rosettes, which stained positive for the NSC markers Sox2 and Nestin. Nuclei were counter-stained with Hoechst (blue). Control (Olig2-GFP) ESCs expressed Olig2-GFP, which is expressed in NSCs after commitment from pluripotent stem cells; scale bars: 100 μm. (D) Established NSC lines from control and three types of Dnmt KO ESCs; scale bars: 100 μm.

• 

  Fig. 3 Immunocytochemical analysis to determine the maintenance of neural stem cells derived from Dnmt knockout (KO) ESCs. After passage three, NSCs from three types of Dnmt KO ESCs, including Dnmt1 KO, Dnmt3a/3b double KO (Dnmt3 DKO), and Dnmt1/3a/3b triple KO (Dnmt TKO), were no longer immunoreactive to the neural stem cell markers Sox2 and Nestin. Control NSCs derived from Olig2-GFP ESCs maintained NSC marker ex-pression. Nuclei were counter-stained with Hoechst (blue); scale bars: 50 μm.

• 

  Fig. 4 Differentiation of Dnmt knockout (KO) NSCs into neural subtypes. (A) Immunocytochemical analysis of control (Olig2-GFP) and three types of Dnmt KO NSCs, including Dnmt1 KO, Dnmt3a/3b double KO (Dnmt3 DKO), and Dnmt1/3a/3b triple KO (Dnmt TKO). During the culture, Dnmt KO NSCs spontaneously differentiated into neurons (Tuj1＋), astrocytes (GFAP＋), and oligodendrocytes (O1＋). Nuclei were counter-stained with Hoechst (blue); scale bars: 200 μm for Tuj1 and GFAP, and 100 μm for O1. Control NSCs derived from Olig2-GFP ESCs maintained self-renewal and were not stained by antibodies for Tuj1, GFAP, and O1; scale bar: 200 μm. (B) Representative karyotype of control and three types of Dnmt KO cell lines. Karyotyping revealed no chromosome abnormalities in all groups; scale bar: 5 μm.

Reference

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