Int J Stem Cells.  2023 Feb;16(1):27-35. 10.15283/ijsc22110.

Inhibition of Class I Histone Deacetylase Enhances Self-Reprogramming of Spermatogonial Stem Cells into Pluripotent Stem Cells

• Lee Y
• Lee SW
• Jeong D
• Lee HJ
• Choi NY
• Bang JS
• Ham S
• Ko K

• KMID: 2539618
• DOI: http://doi.org/10.15283/ijsc22110

Abstract

Background and Objectives
Spermatogonial stem cells (SSCs) are the most primitive cells in spermatogenesis and are the only adult stem cells capable of passing on the genome of a given species to the next generation. SSCs are the only adult stem cells known to exhibit high Oct4 expression and can be induced to self-reprogram into pluripotent cells depending on culture conditions. Epigenetic modulation is well known to be involved in the induction of pluripotency of somatic cells. However, epigenetic modulation in self-reprogramming of SSCs into pluripotent cells has not been studied.
Methods and Results
In this study, we examined the involvement of epigenetic modulation by assessing whether selfreprogramming of SSCs is enhanced by treatment with epigenetic modulators. We found that second-generation selective class I HDAC inhibitors increased SSC reprogramming efficiency, whereas non-selective HDAC inhibitors had no effect.
Conclusions
We showed that pluripotent stem cells derived from adult SSCs by treatment with small molecules with epigenetic modulator functions exhibit pluripotency in vitro and in vivo. Our results suggest that the mechanism of SSC reprogramming by epigenetic modulator can be used for important applications in epigenetic reprogramming research.

Keyword

SSCs; Spermatogonial stem cells; HDAC inhibitor; Epigenetic modulator; Epigenetic reprogramming; Self-re- programming

Figure

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  Fig. 1 Pluripotency induction into gPSCs using small molecule CI-994. (A) Schematic of pluripotency induction into GFP-positive gPSCs using CI-994 treatment. (B) Time frame to convert SSCs to gPSCs. (C) Phase contrast and positive for alkialine phosphatase (AP), SSEA-1, and Nanog of CI-gPSCs. (D) Number of Oct4-GFP-positive gPSC colonies depending on CI-994 treatment. Scale bars: 200 μm (B) and 100 μm (C).

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  Fig. 2 Cellular and molecular characterization of CI-gPSCs. (A) RT-PCR analysis of pluripotency marker genes in ESCs, SSCs, gPSCs, FF-gPSCs (clone 1∼2), CI-gPSCs (clone 1∼3), and MEFs. β-actin was used as a loading control. (B) Comparison of global gene expression between CI-gPSCs and SSCs (left), and between CI-gPSCs and ESCs (right). (C) Hierarchical clustering and a heatmap analysis of MEFs, SSCs, FF-gPSCs (clone 1 and 6), CI-gPSCs (clone 1, 2, and 3), gPSCs, and ESCs. (D) Bisulfite genomic sequencing of promoter regions of Oct4 and Nanog in SSCs, gPSCs, FF-gPSCs, CI-gPSCs, and ESCs. Open and filled circles indicate unmethylated and methylated CpGs, respectively.

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  Fig. 3 Pluripotency of CI-gPSCs. (A∼C) In vitro differentiation into three germ layers. Stained cells positive in AFP, a-SMA, and Tuj1. (D∼F) H&E staining of teratoma-derived tissues. (G) Staining for β-galactosidase of chimeric mouse. (H∼I) Fetal gonads from embryo. White arrows indicate Oct4-GFP-positive SSCs in the gonad. Scale bars: 25 μm (A∼G), 100 μm (D∼F).

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  Fig. 4 Conversion of SSCs into gPSCs using Class I HDAC inhibitors mocetinostat and entinostat. (A) Experimental scheme for comparison of self-reprogramming efficiency of SSCs by HDAC inhibitor types. (B) Phase contrast photomicrograph and fluorescence images of Oct4-GFP in 0.5 μM mocetinostat and 1 μM entinostat treated SSCs. (C) Number of GFP-positive gPSC colonies depending on class I HDAC inhibitor type. Scale bars: 100 μm.

Reference

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