term placenta: 1 tissue: amnionic membrane cell type: human amniotic mesenchymal stromal cells (hAMSC) culture conditions: endothelial cell medium EGM-2
Treatment protocol
Aliquots of hAMSC isolated from three different placenta were cultured either in DMEM + 15% FCS (non-induced hAMSC1, 2, 3) or EGM-2 (endothelial-induced hAMSC 1, 2, 3) on gelatin-coated culture flasks until cells reached 90% confluence (about 5-8 d). Then cells were reseeded and cultured in the respective media for 14 d before RNA isolation.
Growth protocol
Human term placentas of normal pregnancies (range 38 to 42 weeks) were obtained after spontaneous delivery or caesarean section with informed consent. Approval of the Ethical Committee of the Medical University of Graz was granted (No. 21-079 ex 09/10). Isolation of amnion-derived mesenchymal stromal cells (hAMSC) was performed according to the protocol of Soncini et al. (J Tissue Eng Regen Med. 2007;1:296-305.). The amnion was manually separated from the chorion and washed with sterile 0.9% saline (Fresenius Kabi, Bad Homburg, Germany) supplemented with 150 IU/ml penicillin, 150 µg/ml streptomycin (both from PAA Laboratories, Pasching, Austria) and 0.4 µg/ml amphotericin B (Gibco, Invitrogen, Paisley, UK). The amnion was cut into small pieces and incubated in 2.5 U/ml dispase (BD Biosciences, Bedford, USA) at 37°C for 9 min. Subsequently, the amnion was transferred to DMEM low glucose (Gibco, Invitrogen) supplemented with 15% FBS Gold (Gibco, Invitrogen), 100 IU/ml penicillin and 100 µg/ml streptomycin for 10 min. Next, the amnion was incubated with 1.0 mg/ml Collagenase A and 0.01 mg/ml DNase (both from Roche, Penzberg, Germany) for 2 h. After centrifugation for 3 min at 150 x g, the supernatant was poured over a 100 µm cell strainer (BD Biosciences) and centrifuged for 10 min at 300 x g. The cell pellet was washed in PBS (Gibco, Invitrogen) and resuspended in either DMEM supplemented with 15% FBS, 100 IU/ml penicillin and 100 µg/ml streptomycin (non-induced hAMSC) or EGM-2 (Lonza, Walkersville, USA) for endothelial induction (induced hAMSC).
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using RNeasy Mini Kit (Qiagen, Hilden, Germany). The integrity of each RNA sample was determined using an Agilent 2100 Bioanalyzer (Agilent, Foster City, CA, USA) and only RNA samples with integrity values of 9.5-10 were used for hybridization.
Label
biotin
Label protocol
GeneChip® Whole Transcript (WT) Sense Target Labeling Assay
Hybridization protocol
GeneChip® Expression Wash, Stain and Scan; For Cartridge Arrays (FS450_0007)
Gene expression data from endothelial-induced hAMSC of placenta no. 1 induced hAMSC_1
Data processing
Geneexpression Console v.1.1, Affymetrix (Expression Console EC_1_1_2) using Affymetrix default analysis settings for generation of CEL-files.
Data Pre-Processing and Filtering (Partek Software, v.6.4): RMA (background correction, quantile normalization accross all chips in the experiment, log2 transformation, median polish summarization); paired t-test