Elsevier

Experimental Cell Research

Volume 337, Issue 1, 10 September 2015, Pages 61-67
Experimental Cell Research

Research Article
Histone deacetylase inhibitor valproic acid promotes the induction of pluripotency in mouse fibroblasts by suppressing reprogramming-induced senescence stress

https://doi.org/10.1016/j.yexcr.2015.06.003Get rights and content

Highlights

  • Histone deacetylase inhibitor valproic acid enhances iPSC induction.

  • Valproic acid suppresses reprogramming-induced senescence stress.

  • Valproic acid downregulates the p16/p21 pathway in reprogramming.

  • This study demonstrates a new mechanistic role of valproic acid in enhancing reprogramming.

Abstract

Histone deacetylase inhibitor valproic acid (VPA) has been used to increase the reprogramming efficiency of induced pluripotent stem cell (iPSC) from somatic cells, yet the specific molecular mechanisms underlying this effect is unknown. Here, we demonstrate that reprogramming with lentiviruses carrying the iPSC-inducing factors (Oct4-Sox2-Klf4-cMyc, OSKM) caused senescence in mouse fibroblasts, establishing a stress barrier for cell reprogramming. Administration of VPA protected cells from reprogramming-induced senescent stress. Using an in vitro pre-mature senescence model, we found that VPA treatment increased cell proliferation and inhibited apoptosis through the suppression of the p16/p21 pathway. In addition, VPA also inhibited the G2/M phase blockage derived from the senescence stress. These findings highlight the role of VPA in breaking the cell senescence barrier required for the induction of pluripotency.

Introduction

Terminally-differentiated somatic cells can be reprogrammed into pluripotent stem cells (iPSC) by inducing the expression of a combination of factors associated with pluripotency (Oct4, Sox2, Klf4, and c-Myc) [1]. The iPSCs provide a versatile and ethical alternative to create patient-specific stem cells for regenerative medicine and human disease research with a wide range of biotechnological and therapeutic applications [2]. However, generation of iPSCs from human somatic cells using defined factors is an extremely inefficient process [3], [4]. Methods to improve reprogramming efficiency and to ensure genomic integrity and safety of iPSCs must be warranted before this technology can be translated into clinical application [5], [6].

Numerous attempts have been made to improve the efficiency of iPSC induction, including the use of DNA methylation inhibitors [7], histone modification inhibitors [8], inhibition of tumor suppresser genes [9], [10], [11], antioxidants, vitamin C [12], and signaling pathway inhibitors [8], [13], [14], [15]. Valproic acid (VPA), a well-established histone deacetylase inhibitor [16], [17], [18], has been demonstrated to have the ability of pluripotency-promoting activity [8]. In the presence of cytokine cocktails, VPA enhances long-term engraftment of hematopoietic stem cells [19] and stimulates self-renewal [20], [21]. In the clinic, VPA has been used for the treatment of epilepsy, bipolar mania and migraine prophylaxis [22], [23]. However, little is known about the molecular mechanisms by which VPA mediates the promotion of pluripotency.

Senescence and cellular reprogramming are deeply intertwined processes [5]. Fibroblasts cultured from old mice, which have high levels of the Ink4b/Arf/Ink4a, are less efficiently reprogrammed than are cells from young mice [11]. Lentiviral delivery of iPSC-inducing factors (OSKM) often creates cell stress, cell cycle pause, and apoptosis. Suppression of cell cycle regulator genes, like p16, p21, and p53 [9], [24], [25], promotes cell reprogramming. In the process of cell reprogramming [26], we noticed that supplementation with VPA significantly increased cell proliferation in OSKM lentiviruses-transfected cells, suggesting a possible anti-senescence role of VPA in cellular reprogramming. In this study, we examined whether the VPA treatment improved iPSC induction through a mechanism involved in the suppression of reprogramming-induced senescence stress.

Section snippets

Cell lines and cell culture

MBW2 cells were fibroblast-like cells derived from culturing of M. spretus-Balb/c F1 mouse bone marrow mesenchymal stem cells [27], [28]. Cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acid (NEAA) and 1% antibiotics (penicillin-streptomycin) at 37 °C in an atmosphere containing 5% CO2.

Mouse muscle-derived fibroblasts (MDFs) and brain-derived fibroblasts (BDFs) were cultured from a CF-1 mouse fetus

VPA enhances cell reprogramming

The MBW2 cells were derived from continuous culturing of bone marrow mesenchymal stem cell (MSC) of the M. spretus-Balb/c F1 mouse. To demonstrate if VPA is able to promote cell reprogramming in MBW2 cells, we constructed a polycistronic viral vector, containing the mouse Oct4-Sox2-Klf4-c-Myc (OSKM) [6], [26]. Each of the polycistronic factors was separated by a linker sequence encoding a translation skipping peptide T2A, which allows translation to skip and produces two proteins from a single

Discussion

Valproic acid (VPA) has been used successfully to promote cell reprogramming of neonatal foreskin fibroblasts into iPSCs [8]. VPA is a potent histone deacetylase (HDAC) inhibitor that is widely used as an antiepileptic, anti-mania, and anti-migraine drug. VPA is also currently being evaluated for cancer treatment. In general, histone acetylation is associated with an active gene promoter. It is presumed that its role as an HDAC inhibitor would lead to increased histone acetylation in the

Conflict of interest

No conflicts of interest are declared by the authors.

Acknowledgment

This work was supported by California Institute of Regenerative Medicine (CIRM) Grant (RT2-01942), Jilin International Collaboration Grant (#20120720), the National Natural Science Foundation of China Grant (#81272294, #31430021) to J.F.H.; National Natural Science Foundation of China (#81372835) and Jilin Great Science Grant (#11ZDGG003) to W.L.; the National Natural Science Foundation of China grant (#81302380) and Development Foundation for Youths of Jilin Provincial Science & Technology

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