Affymetrix GeneChip Human Genome HG-U133A [HG-U133A]

Isolation and Transcription Profiling of Purified Uncultured Human Stromal Stem Cells: Alteration of Gene Expression after In Vitro Cell Culture

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In order to assess whether culturing adipose-derived adult stem cells (ADASCs) affect their gene expression (see Sample Growth Condition Protocol), we wanted to identify possible genes that were differentially expressed between cultured polyclonal CD31- ADASCs and freshly isolated (uncultured) polyclonal CD31- ADASCs. To that end, RNA was isolated from cultured and uncultured ADASCs from three different donors and analyzed using the Affymetrix Microarray HG-U133A. Then, using the Affymetrix program MAS 5.0 we performed three comparisons and could identify differentially expressed transcripts common between the three donors, using the Affymetrix program DMT 3.0.

growth_condition

Transcription profiling of human freshly isolated adipose-derived adult stem cells (ADASCs) vs cultured cells

In order to assess whether culturing adipose-derived adult stem cells (ADASCs) affect their gene expression (see Sample Growth Condition Protocol), we wanted to identify possible genes that were differentially expressed between cultured polyclonal CD31- ADASCs and freshly isolated (uncultured) polyclonal CD31- ADASCs. To that end, RNA was isolated from cultured and uncultured ADASCs from three different donors and analyzed using the Affymetrix Microarray HG-U133A. Then, using the Affymetrix program MAS 5.0 we performed three comparisons and could identify differentially expressed transcripts common between the three donors, using the Affymetrix program DMT 3.0.


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v4

Title: Fluidics Station Protocol. Description:

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-5112

We started with 100 ng total RNA, using  Affymetrix ”Small Sample Labeling Protocol vII”.  The ”Small Sample Labeling Protocol vII” contains two IVT amplificationsteps (In vitro Transcription). The biotin labeling takes place in the second step using double-stranded cDNA (obtained from 400 ng cRNA) as a template in the presence of biotinylated UTP and CTP. The ENZO BioArray HighYield RNA Transcript Labeling Kit (Affymetrix, Santa Clara, CA, and ENZO Diagnostics, Farmingdale, NY) was used, according to the manufacturer's instructions.  

P-MEXP-5115

When cultured, CD45-CD31- adipose-derived adult stem cells (ADASCs) were maintained in DMEM/F12 (Gibco) containing 20% FBS (Gibco), 100 IU/ml penicillin and 100 IU/ml streptomycin and 2.5 micrograms/ml amphotericin B at 37ºC in an atmosphere of 5% CO2. Cultures were established by plating 105 cells in 25 cm2 cell culture flasks. After 7 days, attached cells were passaged by standard methods of trypsinization and subsequently cultured in medium without amphotericin B. At passage 4, cells were trypsinized, pelleted, snap frozen in liquid nitrogen and RNA could subsequently be extracted (see extraction protocol).

P-MEXP-5116

All materials were supplied by Sigma unless otherwise stated. Adipose tissue was obtained by liposuction from the abdominal, hip and thigh regions of healthy female donors with an average age of 30 (range: 18 to 39). The stromal vascular fraction (SVF) was separated from adipose tissue using a modified procedure as previously described (Zuk, P.A., Zhu, M., Ashjian, P., De Ugarte, D., Huang, J.I., Mizuno, H., Alfonso, Z.C., Fraser, J.K., Benhaim, P. & Hedrick, M.H. Human adipose tissue is a source of multipotent stem cells. Molec. Biol. Cell. 13, 4279-4295 (2002). Briefly, washed lipoaspirate (300-400 ml) was digested with 0.1% collagenase in HBSS containing antibiotics (100 IU/ml penicillin and 100 IU/ml streptomycin) and 2.5 g/ml amphotericin B (Gibco) for 2 h on a shaker at 37ºC. Floating adipocytes were aspirated from pelleted SVF after centrifugation at 400g for 10 min.  Erythrocytes were eliminated by resuspending SVF pellets in lysis buffer (2.06 g/L Tris base, 7.49 g/L NH4Cl, pH 7.2) for 10 min. Resuspended cells in HBSS were then passed through 100 µm, then 40 µm cell sieves (Falcon). Cell suspensions were then applied to Histopaque-1077 gradients. After centrifugation, adipocyte-derived adult stem cells (ADASCs) at the gradient interface were isolated, washed in HBSS and passed through a 30 µm cell mesh. Cell counts and viability assessment were performed. CD45- cells were negatively selected from the freshly isolated ADASCs using anti-CD45 FITC MicroBeads followed by selection with Super MACS (Milteny Biotec, Bergish Gladbach, Germany), according to the manufactures protocol. CD45-CD31+ and CD45-CD31- ADASCs were separated by using anti-CD31 FITC monoclonal antibody followed by anti-FITC MicroBeads and Super MACS (Milteny Biotec) according to the manufactures protocol. Separation was confirmed using flow cytometry. Immediately after separation, aliquots of each cell population, i.e. CD45-CD31+ and CD45-CD31- ADASCs, were snap frozen in liquid nitrogen and RNA could subsequently be extracted (see extraction protocol).

P-MEXP-5118

Total RNA was isolated by use of TRIzol reagent (Invitrogen, Life Technologies) according to the manufactures protocol.  We started with 100 ng total RNA, using  Affymetrix ”Small Sample Labeling Protocol vII”. The ” Small Sample Labeling Protocol vII” contains two cycles of cDNA synthesis and in vitro transcription (IVT).   First cycle: cDNA was synthesised by SuperScript II (Invitrogen, Carlsbad , California) and a primer containing a poly(dT) and a T7 RNA polymerase promotor sequence (Affymetrix, Santa Clara, California). cRNA was obtained using Megascript  High Yield Transcription Kit (Ambion, Austin, Texas). Second cycle: cDNA was synthesised by SuperScript II and Random Primers (Invitrogen, Carlsbad , California) from 400 ng cRNA. Biotinlabeled cRNA was synthesised from cDNA in the presence of biotinylated UTP and CTP, using ENZO BioArray HighYield RNA Transcript Labeling Kit (Affymetrix, Santa Clara, CA, and ENZO Diagnostics, Farmingdale, NY).  

Transcription profiling of human freshly isolated adipose-derived adult stem cells (ADASCs) vs cultured cells

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