Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

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

1. Splitting and maintenance of H9 on feeder cells
   1. Pipette 2 ml 0.1% gelatin into each 6 cm plate and incubate for 30 min in cell culture incubator (37 °C and 5% CO₂). Aspirate gelatin solution with sterile Pasteur pipette.
   2. Add 2 ml MEF medium containing 0.1 x 10⁶ MEF cells/ml into the two gelatin coated plates and incubate them in cell culture incubator (37 °C and 5% CO₂) O/N.
   3. On next day, remove the H9 cells vial from the liquid nitrogen storage tank using forceps and thaw the vial in a 37 °C water bath using long forceps.
   4. Remove the vial from water bath, bath it with 70% ethanol, air dry in the biosafety cabinet for 15 to 30 sec and transfer the cells to 15 ml falcon tube.
   5. Add 9 ml of H9 culture medium slowly on the inner wall and centrifuge the cells at 200 x g for 5 min.
   6. Aspirate the supernatant and re-suspend the cells in 6 ml culture medium containing ROCK inhibitor (10 µM, Y27632) and gently pipette to mix. Aspirate MEF medium from the 6 cm plate and add 3 ml cell suspension in each plate. Change the medium on day 3 and then every other day. Subculture >80% confluent plate cells with split ratio 1:3.
      Note: Usually in 5 to 7 days plate becomes confluent. Feeders used are obtained from CF1 mice embryo and inactivated by exposure to γ radiation.
2. H9 cell culturing on basement membrane matrix coated plates
   1. Thaw stem cell medium (5x) supplement at RT and add 100 ml into 400 ml basal medium in biosafety cabinet.
   2. Thaw basement membrane matrix on ice. Add suggested volume of basement membrane matrix (refer to certificate of analysis for each batch) in 24 ml chilled DMEM/F-12 basal medium for twelve 6 cm plates. Mix by pipetting up and down.
   3. Add 2 ml in each 6 cm plate. Keep the plate at RT for 1 hr. Remove the medium and add 2 ml of stem cell medium.
   4. Take out four confluent H9 plates on MEFs. Remove the differentiated colonies with 1 ml pipette tip under stereomicroscope kept in biosafety cabinet.
   5. Aspirate the medium and wash the cells with 4 ml PBS and add 2 ml stem cell medium in each plate. Cut the undifferentiated colonies with 26 G needle in to 6 to 9 pieces each.
   6. Gently collect the cells in 50 ml falcon tube. Centrifuge at 200 x g for 5 min.
   7. Aspirate the supernatant and re-suspend in 12 ml stem cell medium. Count the clumps by putting 20 µl on glass slide under the microscope and adjust the volume for 150 clumps per ml. Add 2 ml of suspension in each 6 cm plate.
   8. Move the plates back-and-forth and side-to-side motions for uniform clump distribution and incubate the plates in cell culture incubator (37 °C and 5% CO₂).
   9. Remove the differentiated colonies and give medium change every alternate day.

2. Embryoid Bodies (EBs) Formation

Perform all procedure mentioned below as per aseptic precautions and in the biosafety cabinet.

1. Day 0 — Plating of H9 cells on V bottom plates
   1. Prepare 5% block copolymer such as Pluronic F 127 in PBS and filter through vacuum driven filtration system using 0.22 µm sterile filter.
   2. Coat V bottom 96 well plates with 40 µl of 5% block copolymer per well and incubate at RT for 45 min.
   3. Remove the confluent basement membrane matrix plates with H9 cells from incubator and remove the differentiated colonies with 1 ml pipette tip under stereomicroscope in biosafety cabinet.
   4. Aspirate the medium and wash the cells with 4 ml PBS. Add 2 ml random differentiation medium (H9 culture medium without bFGF, RD medium) in each plate. Use passage tool and cut the H9 cell colonies in clumps of uniform size and shape by observing under stereomicroscope in biosafety cabinet and then gently scrape with the cell scraper.
   5. Collect the clumps in 50 ml falcon tube and centrifuge at 200 x g for 5 min. Aspirate the supernatant and re-suspend the cell in RD medium to get 1,000 clumps per ml.
   6. Aspirate the block copolymer from V bottom plates. Pour the clumps in sterile square plate and with help of multichannel pipette add 100 µl of suspension to each well of V bottom plate.
   7. For the force aggregation of clumps, centrifuge the V bottom plates at 4 °C for 4 min at 400 x g. Incubate plates in cell culture incubator (37 °C and 5% CO₂) for four days.
2. Day 4 — Collection of EBs
   1. Collect the EBs in the sterile square plate from V bottom plates using multichannel pipette and wide bore 200 µl tips.
   2. Collect the EBs from sterile square plate in to 15 ml falcon tube with 10 ml sterile serological pipette. Allow EBs to settle for 2 min. Aspirate the supernatant and wash the EBs with 5 ml PBS.
   3. Allow EBs to settle for 2 min and aspirate the supernatant. Re-suspend EBs in 5 ml RD medium.
   4. Pipette out 10 ml RD medium in 10 cm bacteriological plates. Transfer the EBs in 10 cm bacteriological plates.
   5. Incubate bacteriological plates on horizontal shaker (reciprocation motion 50/min) kept in cell culture incubator (37 °C and 5% CO₂) for required time period. Give medium change (15 ml RD medium) every alternate day.
      Note: Gentle handling is required while culturing hESCs. The size of EBs varies on day 4. Select the uniform size EBs (± 20%) by observing under stereomicroscope for further experiment. Approximately 50% EBs formed with this method are of uniform in size. The transfer of EBs on shaker results in uniform shape.

3. Cytotoxicity Assay for IC₁₀ Determination

1. Transfer of EBs on optical bottom plates
   1. Thaw 0.1% gelatin in water bath at 37 °C for 15 min and coat optical bottom plates with 50 µl of 0.1% gelatin per well using multichannel pipette. Incubate the plates at RT for 45 min. After incubation aspirate the gelatin from optical bottom plates.
   2. Take out the EBs collected on day 4 in 10 cm bacteriological plate containing RD medium.
   3. Keep optical bottom plates in slanted position in biosafety cabinet. Transfer two uniform size of EBs in 100 µl RD medium per well in optical bottom 96 well plate by observing under stereomicroscope. Keep 12^th column empty.
   4. Incubate plates in cell culture incubator (37 °C and 5% CO₂) for 24 hr.
2. Drug exposure from day 5 to day 14
   1. Weigh the test compound and make highest concentration in known solvent.
   2. Perform half-logarithmic dilution of the test compound serially till 8 dilutions in the solvent containing falcon tubes numbered with A to H, Keep tube no. I as vehicle control, tube no. J as negative control (RD medium) and tube no. K as positive (70% ethanol) control.
   3. Thaw the RD medium in water bath at 37 °C for 15 min. Take out 5 ml RD medium each in 11 sterile falcon tubes labelled from 1 to 11.
   4. Transfer 5 µl of solution from tube A to tube K in to tube 1 to 11 respectively and vortex the tubes. Take out the optical bottom plate from the incubator and carefully remove the media with use of multichannel pipette.
   5. Add 200 µl of media from tube number 1 to 11 into the respective columns of the optical bottom plate. Give medium/drug change every alternate day.
      Note: For half-logarithmic dilutions take 6.48 µl solvent in 7 tubes labeled from 2 to 8. From highest concentration tube no.1 transfer 3 µl to tube no.2, vortex and serially transfer 3 µl to next tube. Keep tube no.9 for vehicle control and tube no.10 for negative control. Tube no.11 is 70% ethanol.
3. Day 14 : Resazurin exposure and fluorescence measurement
   1. Thaw RD medium in water bath at 37 °C for 15 min. Perform all procedure mention below in absence of light in the biosafety cabinet.
   2. Take 10 ml RD medium in 15 ml tube (A) and add recommended volume of resazurin reagent and mix by pipetting. Take out the optical bottom plate from the incubator and carefully remove all medium with multichannel pipette.
   3. Add 100 µl of medium from tube A in each well. Incubate the plate in cell culture incubator (37 °C and 5% CO₂) for 90 min.
   4. Measure the fluorescence using spectrophotometer (560_Ex/590_Em).
4. IC₁₀ value determination
   1. Import the values in graph pad prism after subtracting the blank values. Set x-axis as a dose and y-axis as a fluorescence units.
   2. Normalize the values to obtain percentage on y axis and transform the values (x-axis as log scale). Calculate IC₅₀ value by using sigmoidal-dose response (variable slope) parameter. Calculate log IC₁₀ values by using following equation:
      F = 10 logEC₅₀ = logECF – (1/HillSlope)*log(F/(100-F))
      Y = Bottom + (Top-Bottom)/(1+10^((LogEC₅₀-X)*HillSlope))
   3. Determine the IC₁₀ values to be taken for further studies.

4. Biomarker Study Based on Microarrays

1. Day 0 to day 5:
   1. Embryoid body formation and transfer to 10 cm bacteriological plates — follow the steps mentioned in point 2 for embryoid body formation.
      Note: Use three biological replicates for each study. Divide each biological replicate in to two parts — Drug treatment at IC₁₀ concentration and vehicle control. Prepare drug concentration 10,000 fold above the IC₁₀ concentration in vehicle and from this add 10 µl to 100 ml RD medium with H9 cell clumps in 50 ml Eppendorf tube mix well and seed it on V bottom plates. Follow the same procedure for vehicle control group.
2. For drug exposure on Day 5 to 14, collect the EB´s and transfer them in 10 cm bacteriological plates on day 4 as per the steps mentioned in point 2. Transfer the plates on horizontal shaker (reciprocation motion 50/min) in cell culture incubator (37 °C and 5% CO₂) for 14 days. Give medium change every alternate day.
3. For sample collection, on day 14, collect the EBs from 10 cm plates in to 15 ml falcon tube with sterile serological pipette. Allow EBs to settle for 2 min. Aspirate the supernatant and wash the EBs with 5 ml PBS. Allow EBs to settle for 2 min and aspirate the supernatant. Re-suspend EBs in 1 ml RNAlater solution or TRIzol reagent, vortex and store the sample at -80 °C till further processing.
   Note: Perform all procedure in biosafety cabinet as per good laboratory practices. Rotate the plates in circular motion around the center to bring all EBs in center, aspirate the medium from surrounding with the help of sterile glass pasture pipette, add 15 ml RD medium and then add 15 µl of drug / vehicle for respective group.

5. RNA Isolation and Integrity Testing

1. RNA Isolation:
   Most of the steps mentioned below are to be performed for RNA purification using RNeasy Mini Kit as per the instruction manual. Always use nuclease free tubes, pipette tips and water. While working with TRIzol carry out all procedure in chemical safety hood and wear protective glasses as well as chemical protective gloves.
   1. Thaw the samples on ice. If samples are stored in RNAlater solution, centrifuge the tubes at 12,000 x g for 5 min at 4 °C. Discard the supernatant and add 1 ml TRIzol reagent.
   2. Triturate the samples using 24 G needle and 1 ml syringe. Approximately 15 times trituration is sufficient for disruption of EBs, cell wall and plasma membranes.
   3. Add 200 µl of chloroform in each sample. Vortex to mix the contents uniformly. Centrifuge at 12,000 x g for 15 min at 4 °C. Remove the RNeasy mini spin columns, 1.5 ml tubes and label them properly.
   4. Collect the supernatant in 1.5 ml tubes (While collecting supernatant do not disturb the middle or bottom layer). Add equal volume of chilled 70% ethanol. Mix the contents by gentle shaking.
   5. Apply 700 µl from the tubes to respective mini spin columns and centrifuge them at 12,000 x g for 20 sec at RT. Perform all further steps at RT.
   6. Discard the filtrate and apply remaining solution to the respective columns and centrifuge them at 12,000 x g for 20 sec. Discard the filtrate.
   7. Apply 350 µl of RW1 buffer to the column and centrifuge them at 12,000 x g for 20 sec. Discard the filtrate and apply 10 µl of DNAse and 70 µl RDD buffer to the column.
   8. Incubate at RT for 15 min. Apply 350 µl of RW1 buffer to the column and centrifuge them at 12,000 x g for 20 sec. Discard the filtrate. Apply 500 µl of RPE wash buffer to the column and centrifuge them at 12,000 x g for 20 sec. Discard the filtrate. Again Apply 500 µl of RPE wash buffer to the column and centrifuge them at 12,000 x g for 2 min. Discard the filtrate.
   9. Shift the columns to new 2 ml collection tubes and centrifuge them at 12,000 x g for 1 min. Transfer the columns to labelled 1.5 ml collection tube and apply 22 µl of nuclease free water. Centrifuge the tubes at 12,000 x g for 1 min.
   10. Remove the collection tube and put them on ice. Quantify RNA using automated electrophoresis system.
2. RNA concentration, purity and integrity testing.
   For RNA purity and integrity testing use automated electrophoresis system and respective kit³³.

6. Microarray Studies

1. Perform transcriptional profiling using commercial available Human array chips. For RNA target preparation, fragmentation, hybridization³⁴ and array chip staining, washing³⁵ use commercial available kits.
2. Perform array chip scanning and quality control check by using standard fluidics station, array scanners and standard operating softwares³⁶. For gene expression analysis import the files generated from scanners to the standard commercial available software³⁷, perform background correction, summarization and normalization with Robust Multi-array Analysis (RMA).
3. For obtaining list of differentially expressed genes (DEG's) perform one way ANOVA analysis. From this list filter out the genes based on the fold change (± 2) and FDR-controlled p-value (< 0.05). Obtain the Principal Component Analysis (PCA), Heat Map, etc. using this software.

Part 2. UKN 1 Test System

1. Maintenance of hESC

1. Seeding of MEFs
   1. For the differentiation use the NSCB#8534 (H9) cell line. Culture cells on mouse embryonic fibroblasts (MEFs) as feeder cells. Coat T25 flask with 4 ml of 0.1% gelatin and incubate for 30 min at 37 °C.
   2. Thaw MEFs in 37 °C water bath and transfer the cells into pre-warmed DMEM/10%FBS.
   3. Spin 3.5 min with 500 x g, remove supernatant and re-suspend cells to obtain 1 x 10⁷ cells/ml. Plate MEFs 4 x 10⁴/cm² in T25 flasks on gelatin. Optionally, use the MEFs for the next two days. Quality of MEF batches are a very critical issue for hESC maintenance. Therefore it is advisable to elucidate the best company and preparation method for the H9 cells. We use PMEF P3.
2. Splitting and maintenance of H9
   1. Add 1 ml pre-warmed dispase per T25 flask H9 and incubate 9 min at 37 °C.
   2. Add 2 ml wash medium to dispase treated cells and pipet 5 times up and down with 5 ml pipet and transfer cell solution to a falcon tube.
   3. Wash the flask with 9 ml wash medium and add cells to the others. Spin 3.5 min with 500 x g, remove supernatant and re-suspend cells in 10 ml hESC medium.
   4. Spin 3.5 min with 500 x g, remove supernatant and re-suspend cells in 4 ml hESC medium. Add 0.5 ml cell suspension and 4.5 ml hESC medium and plate in a new (PBS washed) T25 flask with MEFs. Change entire hESC medium (5 ml) of the flask every day.

2. Differentiation of hESC towards Neuroectodermal Progenitor Cells (NEP)

1. Prepare hESC medium and KCM medium. Coat one 10 cm dish with gelatine (0.1% in PBS) per T25 flask and incubate for 30 min at 37 °C. Remove medium from hESC and add enough accutase to cover the whole bottom of the flask (1 ml per T25 flask) and incubate 25 to 30 min at 37 °C.
2. Prepare basement membrane matrix coated plates during accutase incubation. Add cold DMEM/F12 to frozen basement membrane matrix pellet and resolve it 1:20. Filter basement membrane matrix solution through a 40 µm cell strainer. Add filtered solution to plate, the whole bottom has to be covered (1 ml per 6-well is required) and incubate for 2 hr at RT.
3. After incubation period remove the basement membrane matrix supernatant and seed cells on the coated wells. After accutase step (2.1) stop reaction by addition of 1.5 ml HES medium. Scrape cells from the flask, add 8 ml hESC medium and produce a single cell solution by pipetting with 10 ml pipet thoroughly. Filter cells through a 40 µm cell strainer.
4. Spin cells 3 min with 500 x g, remove supernatant and re-suspend cells in 10 ml of hESC. Spin cells again 3 min with 500 x g, remove supernatant and re-suspend cells in hESC containing ROCK inhibitor Y-27632 at a final concentration of 10 µM.
5. Remove supernatant of gelatin coated dish. Plate cell suspension on gelatin coated dish to remove the MEFs and leave in the incubator for exactly 1 hr.
   NOTE: During this step the MEFs will settle onto the gelatine coated plate, whereas the hESC cannot attach to gelatin. Therefore this is crucial to obtain a feeder-free differentiation. It is a critical step as too long incubation results in hESC clumps and too short incubation in inefficient removal of MEFs. After 45 min of incubation the plate should be investigated for already settled MEFs and hESC clumps.
6. When the MEFs have attached, gently wash non-adherent cells (hESC) off after incubation with the medium already in the plate. If several T25 were used to get more cells, single cells now can be combined. Wash plate once with hESC medium.
7. Spin cells 3 min with 500 x g, remove supernatant and re-suspend cells in approximately 4 ml KCM containing 10 µM ROCK inhibitor Y-27632 and 10 ng/ml FGF-2.
8. Count cells in a hemocytometer using Trypan blue. Plate 18 x 10³ cells/cm² on basement membrane matrix coated plates in KCM containing 10 µM ROCK inhibitor Y-27632 and 10 ng/ml FGF-2 (for 6 well use 1.5 ml per well). It is crucial to plate the cells in the right density to differentiate them successfully into NEPs.
9. After 24 hr, change medium to fresh KCM containing 10 µM ROCK inhibitor Y-27632 and 10 ng/ml FGF-2. After further 24 hr, change medium to fresh KCM containing 10 ng/ml FGF2.
10. 72 hr after seeding the cells, differentiation starts by medium change towards KSR medium. This time point is referred to as day of differentiation 0 (DoD0). The addition of test substances is possible now.
11. On DoD1 and DoD2 the medium is changed exactly as on DoD0. Next medium change is at DoD4 containing 25% N2-S and 75% KSR. At DoD6 the differentiation is stopped and cells are harvested for analysis.

3. Chromatin Immunoprecipitation (ChIP) of hESC and NEP

1. Preparation of nuclei
   1. Add 500 µl accutase to each 6 well which should be analyzed and incubate for 25 to 30 min. Count cells in a Neubauer chamber using Trypan blue.
   2. Resuspend cells in 1% formaldehyde in DMEM/F12 for crosslink. Add Tris pH 7.5 to a final concentration of 125 mM after 10 min to stop the crosslink.
   3. Spin cells 3 min with 500 x g at 4 °C, remove supernatant and re-suspend cells in cold PBS.
   4. Spin cells 3 min with 500 x g at 4 °C, remove supernatant and re-suspend cells in 1 ml L1- buffer / 1 x 10⁶ cells.
   5. Incubate for 5 min on ice. Spin 5 min with 800 x g at 4 °C, remove supernatant and re-suspend nuclei in 1 ml L2- buffer / 2 x 10⁶ cells.
2. Sonication and quality control
   1. Sonicate so that DNA fragments of 300–700 bp length are generated. Spin 1 min with 10,000 x g at 4°C. Transfer supernatant to a new tube. The fragments need to have the correct size, otherwise the immunoprecipitation will be inefficient as well as the followed qPCR.
   2. Remove 50 µl and mix with 50 µl L2 buffer to check efficiency of sonication by running an agarose gel.
   3. Reverse crosslink by incubation at 65 °C for 4 hr and 500 rpm. Load samples 1:5 with Orange G loading dye on a 1.5% agarose gel and run 45 min at 110 V in 1x TBE buffer. Control fragment size (should be between 300–700 bp).
3. Chromatin Immunoprecipitation
   1. Dilute samples 1:5 in dilution buffer and aliquot 1 ml per IP in siliconized tubes.
   2. Remove 5% (volume) from diluted chromatin sample (step 3.3.1) and store at 4 °C as “input”.
   3. Incubate samples with antibodies of your choice and with unspecific IgG O/N at 4 °C on a rotator.
   4. Add 50 µl Protein-A/G Sepharose beads to each sample after immunoprecipitation. Incubate samples 3 hr at 4 °C on a rotator. Spin 1 min with 1,500 x g at 4 °C and remove supernatant.
   5. Wash with 1 ml washing beads. Spin 1 min with 1,500 x g at 4 °C and remove supernatant. Repeat step g to h. Wash with 1 ml final washing buffer. During the washing steps you should not lose any of the beads, because this alters the amount of eluate directly.
   6. Centrifuge 1 min with 1,500 x g at 4 °C and remove supernatant. Add 125 µl elution buffer and incubate 15 min with 65 °C at 1,000 rpm on a shaker.
   7. Spin 1 min with 1,500 x g and transfer supernatant to a new tube Repeat step k and l. Add 200 µl elution buffer to input (3.3.2). Add Proteinase K and RNase to each sample and incubate 30 min with 37 °C at 500 rpm on a shaker and afterwards 4 hr with 65 °C at 500 rpm on a shaker.
      NOTE: For DNA extraction use commercial available ChIP DNA Clean and Concentrator Kit³⁸.