Data for the effect of histone deacetylase inhibitors on voltage- and ligand-gated ion channel gene expression in neurogenic induced-human adipose tissue-derived mesenchymal stem cells

This data article contains descriptive and experimental data on ion channel gene expressions following the histone deacetylase (HDAC) inhibitor treatment of neural induced human adipose tissue-derived mesenchymal stem cells (NI-hADSCs). Following treatment of the HDAC inhibitors, such as MS-275, NaB, TSA, or VPA, the phenotypes of NI-hADSCs exhibit neuron-like features and the neurofilament-L (NFL)-positive cells were increased. The expression of the ion channel marker genes, such as SCN5A, KCNA4, and CACNA1G, was highly increased following treatment with the HDAC inhibitors; however, the expression of others was either decreased or unchanged. For further details and experimental findings please refer to the research article by Jang and Jeong. Histone deacetylase inhibition-mediated neuronal differentiation via the Wnt signaling pathway in human adipose tissue-derived mesenchymal stem cells (Jang and Jeong, 2018) [1].


Subject area
Biology More specific subject area

Stem cell differentiation
Type of data  The effect of HDAC inhibitors in neurogenic differentiation, which is shown as a neurofilament-L positive, was analyzed following the expression of ion channelrelated gene.

Data source location
Stem Cell and Regenerative Medicine Laboratory, Department of Physiology, Chonnam National University Medical School, Gwangju, Republic of Korea Data accessibility The data are available with this article.

Value of the data
This method can be used to control stem cell fate and develop the neurogenic differentiation of the cells.
Phenotypes of NI-hADSCs exhibited distinct neuron-like morphologies with branched processes following HDAC inhibitor treatment.
NFL expressed cells were increased with HDAC inhibitor treatment. The ion channel marker genes were upregulated following treatment with the HDAC inhibitors.

Data
In order to analyze neurogenic differentiation following HDCA inhibitors in hADSCs, we used four HDAC inhibitors; MS-275, NaB, TSA, or VPA. Morphological changes and NFL expressions were observed using phase contrast microscopy and immunofluorescence staining (Fig. 1). Furthermore, we measured the expressions of ion channel marker genes, which are responsible for outward and inward currents (Fig. 2). The primers used in this article are given in Table 1. We cultured hADSCs and differentiated them into NI-hADSCs following our previously published methods [2,3]. Cells were obtained according to the guidelines established by the Ethics Committee at the Chonnam National University Medical School (IRB:I-2009-03-016). The cells were grown as adherent cultures in Dulbecco's modified Eagle's medium (DMEM; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone), 1% penicillin-streptomycin (Gibco BRL, Grand Island, NY, USA), and 0.2% amphotericin B (Gibco) in a 37°C humidified incubator with 5% CO 2 . To induce neural differentiation, the cells were maintained in DMEM containing 1% FBS and supplemented with 100 ng/mL bFGF (Invitrogen Co., Carlsbad, CA, USA), for seven days, and then incubated in 10 µM forskolin (Sigma Chemical Co.) for the next seven days. The HDAC inhibitors; MS-275 (500 nM), NaB (10 µM), TSA (40 µM), or VPA (10 µM); were added individually during the 14-day neural induction.

Phase contrast microscopy
To observe the morphology of cells, the cells were cultured following HDAC inhibitor treatment. Pictures were taken using an inverted microscope (Axio Vert. A1, Carl Zeiss, Germany). Table 1 Sequence of PCR primers.

Immunocytochemistry
The immunocytochemical procedure was modified from our previously published study [2]. Cells were grown on gelatin-coated Aclar plastic coverslips for 2 weeks, fixed for 10 min with chilledmethanol (Burdick & Jackson, Muskegon, MI, USA), and blocked for 20 min with 0.5% Triton X-100 (Sigma Chemical Co.) and 10% normal goat serum (Vector Laboratories Inc., Burlingame, CA, USA) in PBS. Anti-neurofilament (NFL; Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:100) were incubate overnight for 4 � and then added the Alexa 488-conjugated goat anti-mouse antibody (Molecular Probes, Invitrogen Co., CA, USA; 1:200), which was used as a secondary antibody, at room temperature for 1 h. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes; 1 µg/mL) to allow cell counting. Cells were observed using a Zeiss microscope and pictures were taken. Experiments were performed in triplicate, and the number of positive cells was randomly counted. To perform the quantitative analysis, the number of positive cells was counted in each acquired image by ImageJ 1.61 (NIH), and the ratio of the number of positive cells to the number of nuclei was analyzed for the antigen.

Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses
Total RNA was prepared in TriZol (Molecular Research Center, Inc., Cincinnati, OH, USA), according to the manufacturer's instructions, and 1 µg of cDNA was synthesized with the M-MLV Reverse Transcriptase (Gibco BRL) for 90 min at 42°C. The cDNA was amplified by 35 cycles of 94°C for 1 min, appropriate annealing temperatures for each primer for 1 min, and 72°C for 1 min, using the Ex-Taq polymerase (Takara Bio Inc., Shiga, Japan) on PCR machine (Takara Bio Inc.). The forward and reverse PCR oligonucleotide primers that were selected to amplify the cDNA are listed in Table 1 (Bioneer Co., Daejeon, Korea). RT-PCR products were verified by electrophoresis on 2% agarose gels (Sigma Chemical Co.) The amount of cDNA was normalized based on the level of the ubiquitously expressed glyceraldehyde 3-phosphate (GAPDH) to analyze the relative expression of mRNAs [1][2][3][4][5].

Statistics
The levels of mRNA expression were quantified by measuring the optical density of each band by computer-assisted densitometry (NIH Image analysis program, version 1.61). A one-way analysis of variance (ANOVA) test (Bonferroni post-hoc comparison) was performed to analyze the differences between groups, with po 0.05 being considered to indicate statistically significant data. All values are expressed as the mean 7standard error of the mean (SEM).