Conceived and designed the experiments: AU DK. Performed the experiments: DK HM RI KM. Analyzed the data: AU AN DK YT RI MT MW. Contributed reagents/materials/analysis tools: IK AN IS HS. Wrote the paper: AU DK.
The authors have declared that no competing interests exist.
The critical event in heart formation is commitment of mesodermal cells to a cardiomyogenic fate, and cardiac fate determination is regulated by a series of cytokines. Bone morphogenetic proteins (BMPs) and fibroblast growth factors have been shown to be involved in this process, however additional factors needs to be identified for the fate determination, especially at the early stage of cardiomyogenic development.
Global gene expression analysis using a series of human cells with a cardiomyogenic potential suggested
Grem1 enhances the determined path to cardiomyogenesis in a stage-specific manner, and inhibition of the BMP signaling pathway is involved in initial determination of Grem1-promoted cardiomyogenesis. Our results shed new light on renewal of the cardiovascular system using Grem1 in human.
The critical event in heart formation is commitment of mesodermal cells to a cardiomyogenic fate and their migration into anterolateral regions of the embryo during late gastrulation. In this process, morphogenic movements and cardiac fate determination are regulated by cytokines such as bone morphogenetic proteins (BMPs)
In this study, we performed GeneChip analysis to identify multiple extracellular determinants, such as cytokines, cell membrane-bound molecules and matrix responsible for cardiomyogenic differentiation, and evaluated the statistical significance of differential gene expression by NIA array analysis (
To identify cytokines and transcription factors responsible for cardiomyogenic differentiation, 69 human cells were analyzed, depending on gene expression levels, by GeneSpringGX software, and clustered into 30 groups (
(A) Hierarchical clustering analyzed by GeneSpring. Based on gene expression pattern, 69 human cells were clustered into 30 sub-groups. The raw data from the GeneChip analysis are available at the GEO database with accession number GSE8481, GSM41342- GSM41344, and GSM201137- GSM201145. (B) Hierarchical clustering analysis was performed by NIA array (
Group | Title | Description | GSM | |
1 | Normal epithelial cell,primary | NHEK-Neo1 | Normal epidermal keratinocyte, neonate, primary | GSM210361 |
NHBE-1 | Normal bronchial epithelial cell, primary | GSM210362 | ||
2 | Pulmonary epithelial cell line | A549 | Pulmonary epithelial cell line | GSM210363 |
BEAS-2B control (6hr) | Bronchial epithelial cell line | GSM210364 | ||
3 | Lymphocyte | RPMI8226control (6hr) | B cell line | GSM210365 |
Raji-1 | B cell line | GSM210366 | ||
NK92 | NK cell line | GSM210367 | ||
4 | Myelomonocytic leukemia | U937c | U937 control | GSM210368 |
U937h | U937+HRF | GSM210369 | ||
U937ha | U937+HRF+antibody | GSM210370 | ||
U937a | U937+antibody | GSM210371 | ||
5 | Embryonal carcinoma, cancer | NCR-G3 | Embryonal carcinoma, NCR-G3, non-adherent | GSM201141 |
NCR-G2NAd | Embryonal carcinoma, NCR-G2, non-adherent | GSM210373 | ||
NCR-G4Ad | Embryonal carcinoma, NCR-G4, adherent | GSM201142 | ||
NCR-G3Ad | Embryonal carcinoma, NCR-G3, adherent | GSM210375 | ||
6 | ES cell | H1_P43 | Undifferentiated hES | GSM41342 |
H1-P46 | Undifferentiated hES | GSM41343 | ||
H1-P41 | Undifferentiated hES | GSM41344 | ||
7 | Embryonal carcinoma, cancer | NCR-G2Ad | Embryonal carcinoma, NCR-G2, adherent | GSM201140 |
NCR-G1 | Embryonal carcinoma, NCR-G3, non-adherent | GSM201139 | ||
8 | Ewing, cancer | NCR-EW2 | Ewing, cancer | GSM210378 |
NCR-EW3 | Ewing, ETV4, cancer | GSM210379 | ||
9 | Ewing, cancer | GST6 | Ewing, POU5F1, cancer | GSM201137 |
GST6-extra | Ewing, POU5F1, cancer | GSM210381 | ||
10 | Ewing, cancer | GST6-5az | Ewing, POU5F1, 5azaC, cancer | GSM201138 |
GST6-5az-extra | Ewing, POU5F1, 5azaC, cancer | GSM210383 | ||
11 | Bone marrow cell, primary | H4-1 | Bone marrow cell, primary | GSM201143 |
UBT5 | Bmi-1, hTERT, bone marrow cell | GSM210385 | ||
UBET7 | Bmi-1, E6, hTERT, bone marrow cell | GSM210386 | ||
12 | Ligament-derived cells | #10 | Ligament, primary | GSM210387 |
Marrow stromal cells | H10-2Vec | Vector, bone marrow cell | GSM210388 | |
H10-2TERT | hTERT, bone marrow cell | GSM210389 | ||
H10-2Bmi1 | Bmi-1, bone marrow cell | GSM210390 | ||
13 | Placenta, primary | PL90 | Placenta, primary | GSM210391 |
14 | De-differentiated chondrocyte | TdHC1 | E6, E7, hTERT, de-differentiated chondrocyte | GSM210392 |
15 | Neural differentiated marrow stromal cell | UET13 Neural differentiation | E7, hTERT, neural differentiation, bone marrow cell | GSM210393 |
16 | Neural differentiated marrow stromal cell | UET13 Neural differentiation1 | E7, hTERT, neural differentiation, bone marrow cell | GSM210394 |
UET13 Neural differentiation4 | E7, hTERT, neural differentiation, bone marrow cell | GSM210395 | ||
UET13 Neural differentiation5 | E7, hTERT, neural differentiation, bone marrow cell | GSM210396 | ||
17 | Cord blood-derived cells | UET13 | E7, hTERT, bone marrow cell | GSM210397 |
UCB408 | Cord blood, primary | GSM210398 | ||
UCB408E6E7-31 | E6, E7, umbilical cord blood | GSM210399 | ||
Adipocyte cell, primary | HAdPC1(5/21) | HAdpc1E6E7TERT28 | GSM210400 | |
18 | Marrow mesenchymal cell, primary | UEET12 | E6, E7, hTERT, bone marrow cell | GSM210401 |
UEE16 | E6, E7, bone marrow cell | GSM210402 | ||
EPC hTERT+1 | E6, E7, hTERT, endometrial cell | GSM201144 | ||
19 | Cord blood, primary | UCB302 | Cord blood, primary | GSM210382 |
UCB302-D7 | Cord blood, primary | GSM210405 | ||
UCB302TERT | hTERT, cord blood | GSM210406 | ||
UET9 | E7, hTERT, bone marrow cell | GSM210407 | ||
20 | Cord blood, primary | UCB408E7-32 | E7, hTERT, cord blood | GSM210408 |
21 | Fetal fibroblast, primary | HFDPC cont. | Normal follicular dermal papillar cell, primary | GSM210409 |
PL112 | Placenta, primary | GSM210410 | ||
HF7-3 | Fetal fibroblast, primary | GSM210411 | ||
22 | Bone marrow cell, primary | 3F0664 | Bone marrow cell (commercial item), primary | GSM201145 |
BM-MSC | Bone marrow-derived mesenchymal stem cells | GSM38627 | ||
23 | ES cell-derived mesenchymal cell | H1 clone 2 | ES cell-derived mesenchymal precursor | GSM38628 |
H9 clone 1 | ES cell-derived mesenchymal precursor | GSM38629 | ||
24 | Endometrial cell | EPC100 | E6, E7, hTERT, endometrial cell | GSM210413 |
25 | Bone marrow cell, primary | Yub10F | Bone marrow cell, primary | GSM210414 |
26 | Endometrial cell | EPC hTERT+2 | E6, E7, hTERT, endometrial cell | GSM210415 |
EPC Control | E6, E7, hTERT, endometrial cell | GSM210416 | ||
27 | Endometrial cell | EPC214 | E6, E7, hTERT, endometrial cell | GSM210417 |
28 | Menstruation blood-derived mesenchymal cell, primary | #E4 | Menstruation blood, primary | GSM210418 |
#E4HRF | Menstruation blood, HRF treatment, primary | GSM210419 | ||
#E5HRF | Menstruation blood, HRF treatment, primary | GSM210420 | ||
29 | Menstruation blood-derived mesenchymal cell, primary | #E6 | Menstruation blood, primary | GSM210421 |
#E6HRF | Menstruation blood, HRF treatment, primary | GSM210422 | ||
30 | Menstruation blood-derived mesenchymal cell, primary | #E5 | Menstruation blood, primary | GSM210423 |
To investigate cardiomyogenic activity of Grem1, P19CL6 embryonal carcinoma cells (CL6 cells) were used for assessment of
(A) Phase contrast micrograph of CL6 cells with exposure to DMSO alone (a), Grem1 (125 ng/ml) and DMSO (b) for 14 days. The medium, including Grem1 and DMSO, was changed every day. CL6 cells exhibited apparent spontaneous beating between days 9–11. Beating CL6 cell colonies are outlined by white lines. (B) Percentage of beating area in differentiated CL6 cells. CL6 cell treated with Grem1 (125 ng/ml) and DMSO exhibited the strongest contraction. (C) RT-PCR analysis of the genes encoding cardiac-specific transcriptional factors (
To investigate gene expression as well as morphological analysis, i.e. beating, during cardiomyogenic differentiation, RT-PCR analysis was performed to detect expression of cardiomyocyte-specific/associate transcription factors, and structural genes (
To examine CL6 cells for expression of cardiomyocytic protein, immunocytochemical analysis was performed. CL6 treated with Grem1 (125 ng/ml) and DMSO exhibited clear striation with immunostain using anti-cTnT or anti-α-actinin (
To determine if Grem1 (125 ng/ml) functions during the early or the late stage of differentiation, CL6 cells were treated with Grem1 for different time periods (
(A) Protocol for treatment of Grem1 and DMSO. CL6 cells were passaged at 1.8×105 cells in 6-well plate on Day 0. CL6 cells were exposed to Grem1 (125 ng/ml) and/or DMSO on the indicated day. Day when the cells were exposed to the inducers is shown by “+” (in gray cells for clarity). The medium including Grem1 and DMSO was changed every day. On day 14, the cells were immunocytochemically stained with MF20 antibody. (B) Myogenic differentiation of CL6 cells was estimated by sarcomeric myosin (MF20)-positive area. CL6 cells were treated with Grem1 (125 ng/ml) and DMSO for the indicated days. (C) Myogenic differentiation of CL6 cells was estimated by beating area. CL6 cells treated with DMSO and Grem1 (125 ng/ml) were incubated at indicated days.
To examine cardiomyogenic differentiation, immunocytochemical analysis was performed on CL6 cells treated with the inducers. CL6 cells treated with DMSO and BMP2 for the first 3 days were negative for sarcomeric myosin (MF20) at 14 days, but became positive for sarcomeric myosin, following exposure to DMSO alone during days 1–3 (
(A) RT-PCR analysis of the gene encoding
To investigate BMP signaling on cardiomyogenic differentiation, we used the
Since Wnt/β-catenin signaling is involved in CL6 cardiomyogenesis
Our bioinformatics study using the results from the global gene expression analysis of human cells (GSM412342-41344 and GSM201137-201145 at
CL6 embryonic cells start to differentiate into mesodermal cells through Wnt/β-catenin signaling pathway at the early stage (days 1–3), and mesodermal CL6 cells differentiate into mature cardiomyocytes by BMP pathway at the late stage (days 4–14). Grem1 accelerates DMSO-induced cardiomyogenesis through inhibition of the BMP-signaling pathway.
Many studies have indicated that Grem1 is involved in cell differentiation and development, such as osteogenesis
The stage specificity of the Grem1 effect is possibly correlated with the biphasic and antagonistic effect of Wnt/β-catenin signaling on cardiomyogenesis, depending on the stage of development
In conclusion, we have demonstrated that Grem1 enhances the commitment or determined path to cardiogenic differentiation of CL6 teratocarcinoma cells. Apart from a role in development, Grem1 may serve a clinical use in cardiology, like granulocyte colony-stimulating factor that accelerates production of granulocytes in both peripheral blood and bone marrow. Nomination of
GeneChip analysis was performed (
CL6 cells were grown on 100 mm dishes (Becton Dickinson) in α-MEM (Gibco) supplemented with 10% fetal bovine serum (FBS) (JRH Bioscience, Inc.), penicillin, and streptomycin, and were maintained in a 5% CO2 atmosphere at 37°C. To induce differentiation, CL6 cells were plated at a density of 1.8×105 cells in a 6-well plate (Becton Dickinson) or gelatin-coated 35 mm glass base dishes (IWAKI) with α-MEM containing Grem1 (63 or 125 ng/ml: R&D system) and/or 1% dimethyl sulfoxide (DMSO) for 14 days. Recombinant human bone morphogenetic protein-2 (BMP2) was purchased from R&D systems.
Total RNAs were extracted from differentiated and undifferentiated CL6 cells and mouse embryonic stem (ES) cells with RNeasy minikit and DNase I treatment (QIAGEN). Mouse ES cell (129 strains) RNA, mouse heart total RNA (Clontech) and mouse skeletal muscle/total RNA (UNITECH. Co., Ltd.) were used as a positive control for each primer. Total RNA (2.0 µg each) for RT-PCR was converted to cDNA with Superscript™ III RNase H– reverse transcriptase (Invitrogen), according to the manufacturer's manual. PCR conditions were optimized and linear amplification range was determined for each primer by varying annealing temperature and cycle number. PCR products were identified by positive control size. RT-PCR was performed using the primers of the genes of cardiac specific transcription factors:
Quantitative real-time RT-PCR was performed on an ABI Prism 7700 Sequence Detection System (Applied Biosystems), using 100 ng of cDNA in 25 µl reaction volume with 10 nmol/l of each primer, and 12.5 µl SYBR Green Realtime PCR Master Mix (TOYOBO). PCR primers for the genes of
A laser confocal microscope (LSM510, Zeiss) was used for immunocytochemical analysis. Differentiated and undifferentiated CL6 cells were fixed with 4% paraformaldehyde (Wako) for 5 min at 4°C and treated with 0.1% triton X-100 (Sigma) in PBS for 20 min at room temperature, then incubated for 20 min at room temperature in a protein-blocking solution consisting of PBS supplemented with 5% normal goat serum (DakoCytomation). These CL6 cells were then incubated overnight with primary antibody monoclonal anti-sarcomeric myosin antibody (MF20, mouse IgG2b isotype, 1 mg/ml, University of Iowa Hybridoma Bank) and Troponin T, and Cardiac Isoform Ab-1 clone 13-11 (cTnT, mouse IgG1 isotype, 1∶300, Lab Vision Corp), or the monoclonal anti-α-actinin (SARCOMERIC) CLONE EA-53 (α-actinin, mouse IgG1 isotype, 1∶300, Sigma) in PBS at 4°C. The cells were extensively washed in PBS and incubated at room temperature with Alexa Fluor 568-conjugated goat anti-Mouse IgG2b (anti-MF20) (Molecular Probe; diluted 1∶300), Alexa Fluor 488-conjugated goat anti-mouse IgG1 (anti-cTnT) (Molecular Probe; diluted 1∶300), Alexa Fluor 546-conjugated goat anti-mouse IgG(H+L) (anti-α-actinin) (Molecular Probe; diluted 1∶300), and nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) (Wako; diluted 1∶300) for 45 min. To prevent fading, cells were then mounted in DakoCytomation Fluorescent Mounting Medium (DakoCytomation).
Cells (8.0×105) seeded and cultured in 60 mm dishes (Becton Dickinson) were transfected 18 h after plating using Lipofectamine 2000 (Invitrogen) and PLUS reagent (Invitrogen) in Opti-MEM (Gibco). Transfection contained 1.0 µg of TOPflash plasmid (Upstate Biotechnology) for measurement of Wnt/β-catenin activity, or 5.0 µg of the
The regions of interest (beating area, immunostaining area) were defined in Photoshop (Adobe systems) using the ‘magic wand’ tool. The total numbers of pixels identified were then counted using the histogram function. At least five different fields were measured for each dish.
Results, shown as the mean±SE, were compared by ANOVA followed by Scheffé's test, with
A semi-quantitative RT-PCR of cardiomyocyte-specific genes. To investigate expression level of cardiomyocyte-specific genes (Csx/Nkx2.5, Gata4, MyLC-2a, and MyLC-2v), a semi-quantitative RT-PCR was performed from CL6 cells treated with 1% DMSO and the indicated concentration of Grem1 for 14 days. Each RT-PCR product was electrophoresed in 2% agarose gel, and was measured using ImageJ software (
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Grem1 enhanced cardiomyogenic differentiation of mouse ES cells. Mouse ES cells (NCH1.5, C57BL/6J×129ter/Sv) were cultured on a mouse embryonic fibroblast feeder layer inactivated with 30 Gy γ-irradiation in gelatin-coated 60 mm dishes (Becton, Dickinson). Cells were grown in KnockOut DMEM (Gibco) supplemented with 15% fetal bovine serum (Cell Culture Technologies), 2 mM GlutaMAX (Gibco), 0.1 mM non-essential amino acid (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), penicillin, streptomycin, and 2,000 U/ml mouse leukemia inhibitory factor (LIF) (Chemicon). For cardiomyogenic differentiation, ES cells were exposed to 125 ng/ml Grem1 (R&D systems) for the three days. The cells were then trypsinized and cultured to form embryonic bodies (EBs) from a single cell using a three-dimensional culture system (without LIF) on low cell binding dishes (96-well plate round bottom). This represented day 0 of EB formation. On the next day, the medium was replaced with the same medium without LIF. EBs were re-seeded on gelatin-coated 48-well plates with one EB per well, on day 8 after the start of EB formation. The cardiomyogenic induction was estimated by the beating EB number per total EB number, measured on day 12 under a phase-contrast microscope. Grem1 increased the percentage of beating EBs to 69.2%, as compared with 26.7% in EBs without Grem1 treatment. The numbers in parentheses indicate the EB numbers counted.
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Primer sequences.
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CL6 cells treated with DMSO alone. P19CL6 cells are reproducibly and stably induced into beating cardiomyocytes with DMSO.
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CL6 cells treated with Grem1 (125 ng/ml) and DMSO. Grem1 dramatically promotes DMSO-induced cardiomyogenic differentiation of P19CL6 cells at a concentration of 125 ng/ml.
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We would like to express our sincere thanks to T. Imamura and K. Miyazono for the