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

In Vitro Expansion of Human Mesenchymal Stem Cells: Choice of Serum Is a Determinant of Cell Proliferation, Differentiation, Gene Expression, and Transcriptome Stability

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The purpose of this study was to determine whether the serum condition affected the gene expression in mesenchymal stem cells (MSCs). To that end, we compared gene expression in MSCs maintained in regular growth medium supplemented with fetal calf serum (FCS) with gene expression of MSCs cultured in regular growth medium supplemented with autologous serum (AS) for 3 different donors (i.e. donor2, donor3 and donor4). MSCs were cultured in FCS- or AS-supplemented medium and were analyzed at passage 4.

growth_condition

Transcription profiling of Mesenchymal stem cells (MSC) maintained in FCS vs autologous serum analysed at passage 4

The purpose of this study was to determine whether the serum condition affected the gene expression in mesenchymal stem cells (MSCs). To that end, we compared gene expression in MSCs maintained in regular growth medium supplemented with fetal calf serum (FCS) with gene expression of MSCs cultured in regular growth medium supplemented with autologous serum (AS) for 3 different donors (i.e. donor2, donor3 and donor4). MSCs were cultured in FCS- or AS-supplemented medium and were analyzed at passage 4.


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-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).  

P-MEXP-6244

After isolation, mesenchymal stem cells were washed and allowed to adhere overnight in DMED/F12 supplemented with 20% FCS (Gibco Lot no. 3959132), 100unit/ml penicillin, 100µg/ml streptomycin and 2.5µg amphotericin B in a 175-cm2 culture flask (Falcon) and incubate at 37º C with 5% humidified CO2. The following day, non adherent cells were discarded, and adherent cells washed with PBS (Gibco-BRL). Fresh complete medium was added and replaced every 3 or 4 days for about 10-14 days (passage 0). The cells were harvested by trypsin-EDTA and subsequently replated at about 5000 cells/cm2 in 75 cm2 culture flasks (Falcon) until 50% confluence. In subsequent passages amphotericin B was removed and 10% FCS was used instead of 20%. 

P-MEXP-6245

After isolation, mesenchymal stem cells were washed and allowed to adhere overnight in DMED/F12 supplemented with 20% autologous serum (AS), 100unit/ml penicillin, 100µg/ml streptomycin and 2.5µg amphotericin B in a 175-cm2 culture flask (Falcon) and incubate at 37º C with 5% humidified CO2. The following day, non adherent cells were discarded, and adherent cells washed with PBS (Gibco-BRL). Fresh complete medium was added and replaced every 3 or 4 days for about 10-14 days (passage 0). The cells were harvested by trypsin-EDTA and subsequently replated at about 5000 cells/cm2 in 75 cm2 culture flasks (Falcon) until 50% confluence. In subsequent passages amphotericin B was removed and 10% AS was used instead of 20%. 

P-MEXP-6246

MSCs were obtained from 100 ml bone marrow aspirates from iliac crest of normal voluntary donors. Each 100 ml aspirate was diluted 1:3 with DMEM/F12 (Gibco-BRL). Mononuclear cells were isolated by density-gradient centrifugation, i.e. diluted aspirates layered over 12 ml Lymphoprep (Axis-Shield, Oslo, Norway) in eight 50 ml tubes. After centrifugation at 750 g for 20min, the mononuclear cell layer was removed from the interface and suspended in DMEM/F12. CD14+ cells were removed using magnetic beads coupled to mouse anti-human CD14 MAb (Milteny Biotech) and superMACS magnet and LS columns, according to manufactures recommendations. Then cells were put into culture (see sample growth condition protocol). At passage 4, MSC were pelleted and snap-frozen in liquid nitrogen and subsequently RNA was extracted (see extraction protocol).

P-MEXP-6248

Total RNA was extracted from the cells with Trizol (Invitrogen) following the manufacturers protocol. 

P-MEXP-6250

In vitro transcription by using double-stranded cDNA as a template 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)according to the manufacturer's instructions

In vitro expansion of human mesenchymal stem cells: choice of serum is a determinant of cell proliferation, differentiation, gene expression, and transcriptome stability.

Transcription profiling of Mesenchymal stem cells (MSC) maintained in FCS vs autologous serum analysed at passage 4

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