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Sample GSM2080550 Query DataSets for GSM2080550
Status Public on Mar 22, 2017
Title MEF_day0_rep3
Sample type RNA
 
Source name Mouse embryonic fibroblasts (MEFs) before treatment (ctrl) replicate 3
Organism Mus musculus
Characteristics tissue: embryonic fibroblast
strain: C57BL/6NCrSlc
Treatment protocol 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 3
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.
 
Submission date Mar 04, 2016
Last update date Mar 22, 2017
Contact name Yasuyuki S Kida
E-mail(s) y-kida@aist.go.jp
Organization name National Institute of Advanced Industrial Science and Technology (AIST)
Street address Central 5-41, Higashi 1-1-1
City Tsukuba
State/province Ibaraki
ZIP/Postal code 305-8565
Country Japan
 
Platform ID GPL10787
Series (1)
GSE78890 Brief exposure to small molecules allows induction of mouse embryonic fibroblasts into neural crest-like precursors

Data table header descriptions
ID_REF
VALUE Normalized signal intensity

Data table
ID_REF VALUE
A_55_P2051983 11.94958559
A_52_P169082 35.56495395
A_30_P01028193 18.52235465
A_52_P237997 17.43840921
A_51_P414243 2035.460344
A_55_P2136348 6.202978788
A_51_P108228 48.81641284
A_30_P01033363 36.25695797
A_55_P2049737 13.69772304
A_30_P01024440 146.4398118
A_30_P01025554 4300.145536
A_30_P01031558 13.86404924
A_30_P01030675 8.53631006
A_51_P328014 1140.401377
A_30_P01019108 93.17557365
A_55_P2056220 1329.387268
A_55_P1985764 31437.70389
A_52_P108321 108.8201891
A_55_P2018002 87.77341184
A_52_P123354 399.5588228

Total number of rows: 55681

Table truncated, full table size 1408 Kbytes.




Supplementary file Size Download File type/resource
GSM2080550_MEF_day0_rep3.txt.gz 3.2 Mb (ftp)(http) TXT
Processed data included within Sample table

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