The following protocol was performed using human Embryonic Stem Cell line (hESC) H9. This cell line was routinely cultured on mitotically inactivated mouse embryonic fibroblasts (MEFs) in hESC culture media supplemented with bFGF and then cultured in stem cell media on 6 cm Petri plates coated with basement membrane matrix such as matrigel, to get rid of MEFs. The H9 cells from >80% confluent plates were used for further passage. H9 cells cultured on basement membrane matrix plates were used for EBs formation. All procedures mentioned in the following protocol have been performed using standard methods for aseptic and good cell culture practices.
Part 1. UKK test System
1. Human Embryonic Stem Cell Culturing
2. Embryoid Bodies (EBs) Formation
Perform all procedure mentioned below as per aseptic precautions and in the biosafety cabinet.
3. Cytotoxicity Assay for IC10 Determination
4. Biomarker Study Based on Microarrays
5. RNA Isolation and Integrity Testing
6. Microarray Studies
Part 2. UKN 1 Test System
1. Maintenance of hESC
2. Differentiation of hESC towards Neuroectodermal Progenitor Cells (NEP)
3. Chromatin Immunoprecipitation (ChIP) of hESC and NEP
Methyl mercury exposure in UKK test system
The cytotoxicity assay was performed with H9 EBs to obtain an IC10 value (reduction of viability by 10%) for the cytotoxicity of methyl mercury (Figure 1). We also performed a microarray based (affymetrix platform) biomarker study. The H9 EBs have been exposed to methyl mercury (0.25 and 1 µM) for 14 days. On day 14, samples have been collected using TRIzol and RNA was isolated. Transcriptional profiling was performed using Human Genome U133 plus 2.0 array chips. The data have been analyzed with Partek Genomic SuiteTM 6.6. First data overview was obtained by Principle Component Analysis (Figure 2A), generation of Venn diagrams (Figure 2B) and construction of heat maps (Figure 2C). The principle component analysis represents the overall distribution of gene expression and it clearly visualized segregation of MeHg 1 µM from the vehicle control and MeHg 0.25 µM groups (PC # 25.2) (Figure 2A). A list of differentially-expressed genes (DEG) was obtained after statistical treatment (one-way ANOVA) and filtering of the data using a fold change cut-off of ± 2 and a multiplicity-corrected (Benjamini-Hochberg method) p-value < 0.05 (Table 1). The 1 µM MeHg treatment resulted in 276 DEGs and 0.25 µM in 31 DEGs (Figure 2B). The heat map showed that MeHg 1 µM treatment mainly reduced gene expression (Figure 2C). Information on overrepresented gene ontology terms was obtained by using the DAVID bioinformatics tool. Table 2 represents the significantly overrepresented GO gene categories that contained more than 5 genes. The down-regulated transcription factors related to the nervous system development were identified. SEPP1, DDIT4, AK4, FRZB (brain development), PITX (neural nucleus development) and ERBB3, UGT8, APOB, APOA1 (nervous system development) were down-regulated in a dose dependent manner for methyl mercury treatment (Table 3).
UKN 1 test system
This differentiation protocol uses dual SMAD inhibiton6 to generate a pure population of NEP within six days of differentiation. The resultant cells are characterized by an up-regulation of the neural precursor genes PAX6 and OTX2. The stem cell markers OCT4 and Nanog are down regulated during the differentiation towards NEP (Figure 3A). Due to the highly synchronous and homogenous differentiation, it is also possible to get information on the histone modifications during this early stage of development. We adapted the protocol for chromatin immuno-precipitation (ChIP) using the cells either at the beginning of differentiation or after 6 days of differentiation. A switch of methylation sites on the promoter regions of PAX6 and OTX2 was evident from these studies (Figure 3B). The investigated methylation sites histone 3 lysine 4 trimethylation (H3K4me3) and histone 3 lysine 27 trimethylation (H3K27me3) were highly dynamic during the differentiation. Also on protein level a down regulation of Oct4 could be observed (Figure 4). The up-regulation of Pax6 and the neural stem cell marker Nestin was observed by immunofluorescence microscopy on protein level (Figure 4). The cell population showed a homogeneous and pure differentiation after six days of differentiation. Therefore the cultures can be easily used for analysis of RNA and protein. The system provides also the possibility to test substances and the effect they have on early neural development 16,29.
Figure 1. Cytotoxicity Assay (H9 differentiation) for MeHg. The assay has been performed as per the protocol to define the IC10 value for methyl mercury.
Figure 2. Representative analysis of the differential expressed genes induced by 0.25 and 1 µM MeHg after application of the UKK test system. The hESCs were treated with 0.25 and 1 µM MeHg according to the UKK test system. Analysis of the differential expressed transcripts in 14-day differentiated EBs has been performed using the Partek Genomic SuiteTM 6.6 software. (A) Principal component analysis (3-Dimenional) of the microarray data. (B) Venn diagram obtained from microarray analysis of gene expression. The diagram shows the number of genes modulated by the MeHg treatment (fold change > ± 2, p value < 0.05). (C) Hierarchical clustering of the gene expression data (fold change > ± 2, p value < 0.05). The highly expressed genes in vehicle control group are repressed by 1 µM MeHg treatment. The 1 µM MeHg treatment resulted in 233 transcripts with lower expression and 43 probes with higher expression as compare to vehicle control group. Please click here to view a larger version of this figure.
Figure 3. Gene expression and histone methylation pattern during differentiation from hESC towards NEP. For all experiments, hESC were differentiated to neuroectodermal precursor cells (NEP). (A) Samples were taken at day 6 of differentiation, and transcript levels of marker genes of neural differentiation were determined by RT-qPCR. Data (gene expression relative to hESC) are means ± SEM of 5 experiments. (B) Samples for chromatin immunoprecipitation (ChIP) were prepared at day 6 of differentiation. ChIP was performed with antibodies specific for H3K4me3 or H3K27me3 or control IgG. The enrichment factors of promoter sequences are given as % input for H3K4me3 (grey) and H3K27me3 (black). Data are means ± SEM of 3 independent cell preparations. Please click here to view a larger version of this figure.
Figure 4. Protein expression during differentiation from hESC towards NEP. Cells were fixed and stained for the stem cell marker Oct4 (green) at day 0 of differentiation (DoD0) and for NEP markers Pax6 (red) and Nestin (green) at day 6 of differentiation (DoD6). Scale bar indicates 50 µm. Please click here to view a larger version of this figure.
Table 1. List of differentially expressed genes (> ± 2 fold, p value < 0.05) of MeHg treatment versus vehicle control in 14 day old EBs. Please click here to view this table.
Table 2. List of significantly enriched and selected GO categories (p value < 0.05, > 5 genes) with dysregulated transcripts for MeHg versus vehicle control in 14 day old EBs.
GO Term | Count | P value | Genes |
Regulation of Apoptosis | 18 | 0.0068 | ARHGEF3, TBX3, ERBB3, MITF, BNIP3, CDH1, IGF2, IFI16, HGF, GCH1, AMIGO2, SERPINB9, KRT18, MSX1, ETS1, VEGFA, PERP, IGFBP3 |
Regulation of Cell Proliferation | 17 | 0.0123 | RBP4, LYN, TBX3, ERBB3, MITF, IGF2, KDR, RERG, MSX1, ADM, ETS1, VEGFA, BNC1, ADAMTS1, FABP1, IGFBP3, FIGF |
Vasculature Development | 12 | 0.0001 | PLAT, APOB, HAND1, TBX3, EPAS1, FOXF1, LEPR, VEGFA, COL3A1, LOX, FIGF, KDR |
Skeletal System Development | 12 | 0.0008 | RBP4, MSX1, LGALS3, TBX3, HOXB6, COL3A1, STC1, IGF2, POSTN, FRZB, IGFBP3, AHSG |
Heart Development | 11 | 0.0001 | RBP4, ACTC1, MSX1, HAND1, TBX3, ADM, PKP2, ERBB3, GATA6, COL3A1, ADAMTS1 |
Glucose Metabolic Process | 9 | 0.0003 | PDK1, RBP4, LDHA, PGM5, PYGL, HK2, PFKP, IGF2, PGK1 |
Lung Development | 7 | 0.0008 | RBP4, EPAS1, GATA6, FOXF1, VEGFA, LOX, KDR |
Epithelium Development | 7 | 0.0386 | F11R, FREM2, GATA6, FOXF1, VEGFA, DSP, KDR |
Mesoderm Development | 5 | 0.0088 | HAND1, TBX3, FOXF1, VEGFA, SNAI2 |
Table 3. List of significantly down-regulated transcripts related to the developmental nervous system with MeHg treatment in 14 day old EBs.
Term | Gene Symbol | Fold Change* | |
0.25 μM MeHg vs VC | 1 μM MeHg vs VC | ||
Brain Development | SEPP1 | -2.17 | -4.13 |
DDIT4 | -1.20 | -3.11 | |
AK4 | -1.41 | -3.08 | |
FRZB | -1.29 | -2.19 | |
Neuronal nucleus development | PITX2 | -2.08 | -4.90 |
Nervous system development | ERBB3 | -1.86 | -2.89 |
UGT8 | -1.67 | -2.14 | |
APOB | -3.59 | -5.72 | |
APOA1 | 2.63 | -2.90 | |
VEGFA | -1.28 | -3.06 |
* p value < 0.05
Table 4. Composition of culture media.
Sr. No. | Medium / Buffer Name | Composition | |
Contents | Amount | ||
1 | MEF Medium | DMEM High glucose | |
FCS | 10% | ||
Penicillin | 100 units/ml | ||
Streptomycin | 100 μg/ml | ||
L-Glutamine | 2 mM | ||
2 | H9 Culture Medium | DMEM F12 | |
KOSR | 20% | ||
NEAA | 1% | ||
Glutamax | 1x | ||
β-mercaptoethanol | 0.1 mM | ||
Penicillin | 100 units/ml | ||
Streptomycin | 100 μg/ml | ||
bFGF | 4 ng /ml | ||
3 | RD Medium | H9 culture medium without bFGF | |
4 | Wash Medium | DMEM/F12 | |
Knockout Serum Replacment | 20% | ||
1x GlutaMAX | 1x | ||
MEM non-essential amino acids | |||
HEPES | 15 mM | ||
β-mercaptoethanol | 90 μM | ||
5 | KCM Medium | DMEM | |
FBS | 10% | ||
incubated for 24 hr on MEFs | |||
6 | Knockout Serum Replacement (KSR) |
Knockout DMEM/F12 | |
Knockout serum replacement | 15% | ||
1x GlutaMAX | |||
1x MEM non-essential amino acids | |||
β-mercaptoethanol | 15 μm | ||
Noggin | 35 ng/ml | ||
Dorsomorphin | 600 nM | ||
SB431542 | 10 μM | ||
7 | N2-S | DMEM/F-12 | |
Apotransferin | 100 μg/ml | ||
Glucose | 1.55 mg/ml | ||
Putrescine | 10 mM | ||
Selenium | 500 μM | ||
Progesteron | 20 μM | ||
GlutaMAX | 200 μM | ||
Insulin | 25 μg/ml | ||
8 | L1 Buffer | Tris pH 8 | 50 mM |
EDTA | 2 mM | ||
NP-40 | 0.10% | ||
Glycerol | 10% | ||
9 | L2 Buffer | Tris pH 8 | 50 mM |
EDTA | 10 mM | ||
SDS | 1% | ||
10 | Elution Buffer | NaHCO3 | 100 mM |
SDS | 1% | ||
11 | Wash Buffer | Tris | 20 mM |
EDTA | 2 mM | ||
SDS | 0.10% | ||
NP-40 | 0.50% | ||
NaCl | 150 mM | ||
12 | Final Wash Buffer | Tris | 20 mM |
EDTA | 2 mM | ||
SDS | 0.10% | ||
NP-40 | 0.50% | ||
NaCl | 500 mM | ||
13 | Stem Cell Medium | mTESARTM basal medium | 400 ml |
mTESARTM supplement | 100 ml |
DMEM/F-12 | Life Technologies | 11320082 | Dulbecco's Modified Eagle Medium:Nutrient Mixture F-12 |
KOSR | Life Technologies | 10828028 | Knockout Serum Replacement |
GlutaMAX | Life Technologies | 35050061 | GlutaMAX supplement |
NEAA | Life Technologies | 11140050 | MEM Nonessential Amino Acids Solution |
DPBS | Life Technologies | 14190-0144 | Dulbecco's Phosphate-Buffered Saline, without calcium, without magnesium |
mTeSR medium | Stemcell Technologies | 5850 | |
Pluronic F-127 | Sigma | P2443-250G | |
V bottom plate | VWR | 734-0483 | Plate,Microwell,V BTTM,96 Well,Sterile 1 * 50 ST |
Vbottom plate lid | VWR | 634-0011 | Lid, Microtitre plates, Cond. Ring 1 * 50 ST |
Pen/Strep | Life Technologies | 15140-122 | Penicillin- Streptomycin, Liquid |
Distilled Water | Life Technologies | 15230-089. | Sterile Distilled Water |
Human FGF-2 (bFGF) | Millipore | GF003AF-100UG | Fibroblast Growth Factor basic, human recombinant, animal-free |
Filter 0.22 μm | Millipore | SCGPU02RE | Stericup-GP, 0.22 μm, polyethersulfone, 250 ml, radio- sterilized |
StemPro EZPassageTM Disposablte | Invitrogen | 23181010 | |
BD MatrigelTM, hESC qualified Matrix | Stemcell Technologies | 354277 | 5 ml vial |
DMSO | Sigma | D-2650 | |
RNAlater Stabilization Solution | Life Technologies | AM7020 | It stabilizes and protect the RNA integrity in unfrozen samples. |
70 μm Cell Strainer | Becton Dickinson | 352350 | Cell strainer with 70 μm Nylon mesh |
35 μm Lid cell strainer, 5 ml tube | Becton Dickinson | 352235 | 5 ml polystyrene round bottom test tube, with a cell strainer cap (35 μm) |
50 ml sterile Polypropylene tube | Greiner Bio-One | 227261 | 50 ml Polypropylene tube with conical bottom, Sterile |
T75 flask | Greiner Bio-One | 658175 | CELLSTAR Filter Cap Cell Culture 75 cm2 Flasks |
TRIzol | Life Technologies | 10296010 | |
96 well optical bottom plates | Thermo Scientific | 165305 | |
CellTiter-Blue | Promega | G8081 | |
Accutase | PAA | L11-007 | |
Apotransferin | Sigma-Aldrich | T-2036 | |
Dispase | Worthington Biochemicals | LS002104 | |
Dorsomorphin | Tocris Bioscience | 3093 | |
EDTA | Roth | 8043.2 | |
FBS | PAA | A15-101 | |
FGF-2 | R&D Systems | 233-FB | |
Gelatine | Sigma-Aldrich | G1890-100G | |
Glucose | Sigma-Aldrich | G7021-100G | |
GlutaMAX | Gibco Invitrogen | 35050-038 | |
HEPES | Gibco Invitrogen | 15630-056 | |
Insulin | Sigma-Aldrich | I-6634 | |
Knockout DMEM | Gibco Invitrogen | 10829-018 | |
Matrigel | BD Biosciences | 354234 | |
Noggin | R&D Systems | 719-NG | |
PBS | Biochrom AG | L1825 | |
Progesteron | Sigma-Aldrich | P7556 | |
Putrescine | Sigma-Aldrich | P-5780 | |
ROCK inhibitor Y-27632 | Tocris Biosciences | 1254 | |
SB431542 | Tocris Biosciences | 1614 | |
SDS | Bio-Rad | 161-0416 | |
Selenium | Sigma-Aldrich | S-5261 | |
β-Mercaptoethanol | Gibco Invitrogen | 31350-010 | |
List of Kits | |||
RNeasy Mini Kit (250) | QIAGEN | 74106 | |
GeneChip Hybridization, Wash, and Stain Kit | Affymetrix | 900721, 22, 23 | This kit provides all reagents required for hybridization wash and staining of microarrays. |
Rnase-Free DNase Set | QIAGEN | 79254 | |
List of equipment. | |||
Inverted microscope | Olympus | IX71 | |
Genechip Hybridisation Oven – 645 | Affymetrix | ||
Genechip Fluidics Station-450 | Affymetrix | ||
Affymetrix Gene-Chip Scanner-3000-7 G | Affymetrix | ||
Spectramax M5 | Molecular Devices | ||
List of softwares | |||
Prism 4 | |||
Affymetrix GCOS | |||
Partek Genomic Suite 6.25 | |||
Online tools for Functional annotation DAVID Onto-tools Intelligent Systems and Bioinformatics Laboratory |
Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.
Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.
Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.