Gene expression profiling of trophoblast cells - GSE9773
Invasion of cytotrophoblasts into uterine tissues is essential for placental development. To identify molecules regulating trophoblast invasion, mRNA signatures of purified villous (CTB, poor invasiveness) and extravillous (EVT, high invasiveness) trophoblasts isolated from first trimester human placentae and villous explant cultures, respectively, were compared using GeneChip analyses yielding 991 invasion/migration related transcripts. Several genes involved in physiological and pathologic cell invasion, including ADAM-12,-19,-28 as well as Spondin-2, were upregulated in EVT. Pathway prediction analyses identified several functional modules associated with either the invasive or the non-invasive trophoblast phenotype. One of the genes which were downregulated in the invasive mRNA pool, heme oxygenase-1 (HO-1), was selected for functional analyses. Real-time PCR analyses, Western blottting, and immunofluorescene of first trimester placentae and differentiating villous explant cultures demonstrated downregulation of HO-1 in invasive EVT as compared to CTB. Modulation of HO-1 expression in loss-of as well as gain-of function cell models (BeWo and HTR8/SVneo, respectively) demonstrated an inverse relationship of HO-1 expression with trophoblast migration in transwell and wound healing assays. Importantly, HO-1 expression led to an increase in protein levels and activity of the nuclear hormone receptor PPARgamma. Pharmacological inhibition of PPARgamma abrogated the inhibitory effects of HO-1 on trophoblast migration. Collectively, our results demonstrate that gene expression profiling of EVT and CTB can be used to unravel novel regulators of cell invasion. Accordingly, we identify heme oxygenase-1 as a negative regulator of trophoblast motility acting via upregulation of PPARgamma.
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To identify genes potentially regulating cell invasion trophoblast cells of early human gestation with distinct invasive properties were profiled.Distinct gene expression signatures of highly invasive EVT (n = 6) and poorly invasive CTB (n = 5) of different first trimester placentae using Affymetrix U133A GeneChips interrogating >20,000 genes were determined.
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| Sample ID | !Sample Title | Group | Dissected mesenchymal villous tissue of early pregnancy was arranged radially on 80 μl drops of growth-factor-reduced Matrigel™ (BD, Bedford, MA) in 24 well plates (SFM; DMEM | Trophoblasts were cultured for 24 h in DMEM (GIBCO) | With 10% FCS, 100 mg/ml streptomycin and 100 IU/ml penicillin |
|---|---|---|---|---|---|
| GSM246492 | EVT-1 | extravillous trophoblasts | Ham's F12 (Gibco) 1:1, supplemented with 100 µg/ml streptomycin/penicillin (Gibco) and incubated for 4 hours at 37°C, 5% CO2 to allow attachment. Wells were then carefully flooded with 500 µl SFM, incubated at 37°C, 5% CO2 and subsequent growth characteristics were monitored by light microscopy. Total RNA or tissue sections were prepared after 72 hours of incubation. | ||
| GSM246493 | EVT-2 | extravillous trophoblasts | Ham's F12 (Gibco) 1:1, supplemented with 100 µg/ml streptomycin/penicillin (Gibco) and incubated for 4 hours at 37°C, 5% CO2 to allow attachment. Wells were then carefully flooded with 500 µl SFM, incubated at 37°C, 5% CO2 and subsequent growth characteristics were monitored by light microscopy. Total RNA or tissue sections were prepared after 72 hours of incubation. | ||
| GSM246494 | EVT-3 | extravillous trophoblasts | Ham's F12 (Gibco) 1:1, supplemented with 100 µg/ml streptomycin/penicillin (Gibco) and incubated for 4 hours at 37°C, 5% CO2 to allow attachment. Wells were then carefully flooded with 500 µl SFM, incubated at 37°C, 5% CO2 and subsequent growth characteristics were monitored by light microscopy. Total RNA or tissue sections were prepared after 72 hours of incubation. | ||
| GSM246495 | CTB-1 | cytotrophoblasts | TRUE | TRUE | |
| GSM246497 | CTB-2 | cytotrophoblasts | TRUE | TRUE |
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| Sample ID | !Sample Title | Group | Dissected mesenchymal villous tissue of early pregnancy was arranged radially on 80 μl drops of growth-factor-reduced Matrigel™ (BD, Bedford, MA) in 24 well plates (SFM; DMEM | Trophoblasts were cultured for 24 h in DMEM (GIBCO) | With 10% FCS, 100 mg/ml streptomycin and 100 IU/ml penicillin |
|---|---|---|---|---|---|
| GSM246492 | EVT-1 |
extravillous trophoblasts |
Ham's F12 (Gibco) 1:1, supplemented with 100 µg/ml streptomycin/penicillin (Gibco) and incubated for 4 hours at 37°C, 5% CO2 to allow attachment. Wells were then carefully flooded with 500 µl SFM, incubated at 37°C, 5% CO2 and subsequent growth characteristics were monitored by light microscopy. Total RNA or tissue sections were prepared after 72 hours of incubation. |
||
| GSM246493 | EVT-2 |
extravillous trophoblasts |
Ham's F12 (Gibco) 1:1, supplemented with 100 µg/ml streptomycin/penicillin (Gibco) and incubated for 4 hours at 37°C, 5% CO2 to allow attachment. Wells were then carefully flooded with 500 µl SFM, incubated at 37°C, 5% CO2 and subsequent growth characteristics were monitored by light microscopy. Total RNA or tissue sections were prepared after 72 hours of incubation. |
||
| GSM246494 | EVT-3 |
extravillous trophoblasts |
Ham's F12 (Gibco) 1:1, supplemented with 100 µg/ml streptomycin/penicillin (Gibco) and incubated for 4 hours at 37°C, 5% CO2 to allow attachment. Wells were then carefully flooded with 500 µl SFM, incubated at 37°C, 5% CO2 and subsequent growth characteristics were monitored by light microscopy. Total RNA or tissue sections were prepared after 72 hours of incubation. |
||
| GSM246495 | CTB-1 |
cytotrophoblasts |
TRUE |
TRUE |
|
| GSM246497 | CTB-2 |
cytotrophoblasts |
TRUE |
TRUE |
|
| GSM246499 | CTB-3 |
cytotrophoblasts |
TRUE |
TRUE |
|
| GSM307639 | EVT-4 |
extravillous trophoblasts |
Ham's F12 (Gibco) 1:1, supplemented with 100 µg/ml streptomycin/penicillin (Gibco) and incubated for 4 hours at 37°C, 5% CO2 to allow attachment. Wells were then carefully flooded with 500 µl SFM, incubated at 37°C, 5% CO2 and subsequent growth characteristics were monitored by light microscopy. Total RNA or tissue sections were prepared after 72 hours of incubation. |
||
| GSM307640 | EVT-5 |
extravillous trophoblasts |
Ham's F12 (Gibco) 1:1, supplemented with 100 µg/ml streptomycin/penicillin (Gibco) and incubated for 4 hours at 37°C, 5% CO2 to allow attachment. Wells were then carefully flooded with 500 µl SFM, incubated at 37°C, 5% CO2 and subsequent growth characteristics were monitored by light microscopy. Total RNA or tissue sections were prepared after 72 hours of incubation. |
||
| GSM307641 | EVT-6 |
extravillous trophoblasts |
Ham's F12 (Gibco) 1:1, supplemented with 100 µg/ml streptomycin/penicillin (Gibco) and incubated for 4 hours at 37°C, 5% CO2 to allow attachment. Wells were then carefully flooded with 500 µl SFM, incubated at 37°C, 5% CO2 and subsequent growth characteristics were monitored by light microscopy. Total RNA or tissue sections were prepared after 72 hours of incubation |
||
| GSM307642 | CTB-4 |
cytotrophoblasts |
TRUE |
TRUE |
|
| GSM307643 | CTB-5 |
cytotrophoblasts |
TRUE |
TRUE |
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