For modulating cellular fate, small compound cocktails were applied to the cultured samples for 3 days. At first, the combination of 0.25 mM valproic acid (an inhibitor of histone deacetylase; VPA; Wako), 1.5 uM CHIR99021 (an inhibitor of glycogen synthase kinase 3 (GSK3); CHIR; Cayman Chemical Company), 0.5 uM 616452 (an inhibitor of transforming growth factor-beta [TGFb] RI kinase; Merck Millipore), 5 uM Forskolin (an agonist of cyclic adenosine monophosphate; FSK; Wako) and 2.5 uM Tranylcypromine (an inhibitor of lysine-specific demethylase 1; Tranyl; Abcam) was used. These small molecular compounds were previously identified and used for converting mouse somatic cells to pluripotent state. The cocktail was mixed in DMEM supplemented with 20% FBS, 1% NEAA, 1% PS, 50 ng/ml bFGF and a culture supernatant from CHO cells producing leukemia inhibitory factor (LIF). The CHO cells producing LIF were kindly gifted from Prof. Toru Nakano (Osaka University, Japan). The chemical medium was applied to the culture samples for 2 days. Then, another small compound cocktail, which was composed of 0.25 mM VPA, 5 uM FSK, 2.5 uM Tranyl, 2.5 uM dorsomorphin (an inhibitor of bone morphogenic protein (BMP) signaling; DM; Sigma-Aldrich) and 2.5 uM SB431542 (an inhibitor of TGFb/Activin/Nodal signaling; SB; Sigma-Aldrich), was applied for one day. This small molecular cocktail was also mixed in DMEM-based medium same as above. After exposure to small molecular cocktails for 3 days, cells were collected and transferred to poly-L-ornithine (PLO; Sigma-Aldrich) and Laminin (Sigma-Aldrich) coated dishes for neuronal differentiation. Briefly, cells were rinsed with PBS and then dissociated with 0.025% Trypsin-EDTA for 5 min at 37 oC. After stopping trypsin reaction with FBS-containing DMEM, cells were collected by centrifugation at 1000 rpm for 3 min. Cells were suspended into Differentiation Medium consisted of DMEM/F-12 (Life Technologies), 25 ug/ml Insulin (Wako), 50 ug/ml Human Transferrin (Sigma-Aldrich), 30 nM Sodium Selenite (Sigma-Aldrich), 20 nM Progesterone (Sigma-Aldrich), 100 nM Putrescine (Sigma-Aldrich) and 1% PS. For neuronal differentiation, the differentiation medium was further supplemented with 1 uM Retinoic Acid (RA; Sigma-Aldrich), 10 M FSK, 10 ng/ml Brain-derived neurotrophic factor (BDNF; Wako), 10 ng/ml Glial cell-derived neurotrophic factor (GDNF; Wako) and 50 ug/ml Ascorbic Acid (Wako), which was defined as the neuronal differentiation medium (NDM). Culture dishes were previously coated with 20 ug/ml PLO for 1 hour at 37 oC and subsequent 5 ug/ml Laminin for 2 hours at 37 oC. Finally, cells were plated onto coated dishes at a density of 5 x 10^4 cells/cm2. Half of NDM was changed twice a week for maintaining the cultures.
Growth protocol
MEFs were isolated from 12.5 days old embryos of C57BL/6NCrSlc mice. Briefly, the pregnant female mouse was sacrificed by cervical dislocation. The uterus was dissected out and placed into a φ100mm dish containing phosphate buffered saline (PBS; Wako). Each embryo was separated from the uterus. Then, head and visceral tissues were removed from each embryo with forceps and tweezers. The remaining portion of the embryo was transferred into a single well of 12-well plate containing PBS and minced into pieces. The pieces were treated with 1 ml/well 0.05% Trypsin-EDTA (Wako) for 5 min at 37 oC. After trypsinization, 1 ml/well Dulbecco's modified eagle medium (DMEM; Wako) supplemented with 10% fetal bovine serum (FBS; Hyclone), 1% Non-essential amino acid solution (NEAA; Wako) and 1% Penicillin-Streptomycin solution (PS; Wako), was added and the pieces were suspended by pipetting. Cells were collected by centrifugation (1000 rpm, 3 min) and resuspended with DMEM supplemented with 20% FBS, 1% NEAA and 1% PS (defined as P0 MEFs). These P0 MEFs were harvested on T175 flasks and cultured at 37 oC with 5% CO2. The P0 MEFs were grown to confluent, then passed and cryopreserved. The P1 MEFs were used for further neuronal induction.
Extracted molecule
total RNA
Extraction protocol
Total RNAs extracted from the day10 chemical-treated MEFs and the control MEFs were used for microarray analysis.
Label
Cy3
Label protocol
cDNA synthesis from 140 ng total RNAs and hybridization were performed with SurePrint G3 Mouse GE microarray 8x60K (Agilent).
Hybridization protocol
cDNA synthesis from 140 ng total RNAs and hybridization were performed with SurePrint G3 Mouse GE microarray 8x60K (Agilent).
Scan protocol
Agilent DNA microarray scanner (Agilent) was used to obtain fluorescence intensity of the microarray.
Description
Gene expression of MEFs before exposure to small molecular cocktails (ctrl) replicate 2
Data processing
Signal intensities were measured by Feature Extraction Software (Agilent), and were intra- and inter-array normalized using the limma package in the R statistical computing environment. Signal intensities for non-unique probe names were averaged.
