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

The overall goal of this procedure is to describe two in vitro developmental toxicity test systems based on human embryonic stem cells and transcriptome studies. This is accomplished by first culturing human embryonic stem cells on mouse embryonic fibroblasts. The second step is to either let the human embryonic stem cells form Embry bodies in which they can generate cells of all three germ layers or differentiate them into neuroectodermal progenitors.

Next, the embryo bodies or neuroectodermal progenitors are treated with compounds of interest at different concentrations during differentiation. The final steps are to assess the viability of the cells and isolate the RNA. Ultimately transcriptome microarray analysis is used to show the changes in RNA expression of cells treated with a compound of interest compared to untreated control cells Based on human neonic stem cells and DNA macro areas, we have established an in vitro methodology allowing monitoring of human relevant developmental toxicity effects.

The methodology can be applied by the pharmaceutical and chemical industry for safety screening of potential drugs and environmental toxicants. This method can help to answer key questions in the field of developmental toxicity, such as the effect of chemicals on the very early neurodevelopment. To begin, thaw the stem cell medium supplement at room temperature and add 100 milliliters of it into 400 milliliters of the basal stem cell medium.

Next, thaw the basement membrane matrix on ice, and then add the supplemented volume of matrix into 24 milliliters of chilled dmm. F 12 basal medium as recommended by the manufacturer, mixed a solution by gently pipetting up and down, and then add two milliliters of the mixture into 12 separate six centimeter plates. Incubate the plates at room temperature for one hour while the matrix coats the plates.

Then remove the medium and add two milliliters of stem cell medium. Next, remove four confluent plates from the incubator containing human embryonic. H nine cells grown on mouse embryonic fiberblast feeder layers that are prepared as described in the accompany intext protocol under a stereoscope, use a one milliliter pipette tip and remove the differentiated colonies from the plate.

Undifferentiated colonies have well-defined borders and cells are compact, whereas differentiated cells start propagating out from the borders and their cells are larger in size. Aspirate the medium and wash the cells with four milliliters of PBS. Then add two milliliters of stem cell medium with the eight of a stereoscope.

Cut the colonies into six to nine pieces each with a 26 gauge needle and scrape the clumps with one milliliter pipette tips. Gently collect the cut colonies in a 50 milliliter falcon tube and pellet them at 200 G for five minutes. Next, aspirate the snat and resuspend the cells in 12 milliliters of stem cell medium.

Place 20 microliters of the cell suspension on a glass slide and count the number of clumps under the microscope. Adjust a concentration of the suspension to 150 clumps per milliliter and then add two milliliters of the suspension into each six centimeter plate. Move the plates back and forth and side to side to distribute the cells uniformly, and then incubate the plates in a cell culture incubator every other day.

Carefully change the medium After removing the differentiated colonies, add 40 microliters of a sterile 5%block copolymer solution to each well of a Vbo 96 well plate and let it incubate at room temperature for 45 minutes. Remove a confluent plate containing colonies from the incubator and pick out any differentiated colonies under a stereo microscope with a one milliliter pipette tip. Then aspirate the medium from the plate and gently wash the cells with four milliliters of PBS.

Next, add two milliliters of random differentiation medium to each plate. Use the passage tool to cut the H nine cell colonies in clumps of uniform size. Scrape the clumps with a cell scraper and collect them gently into a 50 milliliter falcon tube centrifuge.

The colony clumps at 200 G for five minutes and then aspirate the snat and resuspend the cell and random differentiation medium at 1000 clumps per milliliter. Next, aspirate the block copolymer from the V bottom 96 well plate With the help of a multi-channel pipette, add 100 microliters the colony suspension to each well aggregate the clumps to the bottom of the V-shaped wells by centrifuging the plate at four degrees Celsius for four minutes at 400 G.Then place a plate in the incubator for four days using wider bore 200 microliter tips. Remove the embryo bodies from the 96 well plate and place them into a sterile 122 millimeter square plate.

Next, use a 10 milliliter sterile serological pipette to transfer the embryo bodies from the square plate into a 15 milliliter falcon tube. Allow the embryo bodies to settle for two minutes and then aspirate the snat and wash the embryo bodies with five milliliters of PBS. Again, wait for the embryo bodies to settle for two minutes and then aspirate the supernatant.

This time resuspend the embryo bodies in five milliliters of random differentiation medium. Next, transfer the embryo bodies into a 10 centimeter bacteriological plate and add an additional 10 milliliters of random differentiation.Medium. Incubate the plates at 37 degrees Celsius and 5%CO2 on a horizontal shaker.

At 50 RPMs, the representative embryo bodies are shown for day five, day nine, and day 14. Carefully change the medium every other day, replacing all 15 milliliters with fresh random differentiation.Medium. To assess a drug effect on differentiation, begin adding drug to the medium at day five for 10 days as described in the accompanying text protocol.

Using a sterile pipette, collect the embryo bodies from the 10 centimeter plates into a 15 milliliter falcon tube. Allow them to settle for two minutes, then aspirate the supernatant. Wash them with five milliliters of PBS and transfer them into a nuclease free 1.5 milliliter tube.

Allow them to settle and aspirate the snat raise. Suspend the embryo bodies in one milliliter of triol reagent and passed a solution through a 24 gauge needle with a one milliliter syringe approximately 15 times to disrupt the cells. Next, add 200 microliters of chloroform to each sample.

Vortex the contents briefly, and then centrifuge the samples at 12, 000 G for 15 minutes At four degrees Celsius, collect the supernatant in 1.5 milliliter tubes without disturbing any of the other layers. Then add an equal volume of chilled 70%ethanol to the supernatant and gently mix the contents by shaking. Transfer 700 microliters of the mixture to a mini spin column and follow the manufacturer's instructions to purify the RNA in the final elution step.

Apply 22 microliters of nuclease free water to the spin column and centrifuge the tubes at 12, 000 G for one minute. Then remove the collection tube and put the RNA on ice. Add one milliliter of prewarm disc space per confluent T 25 flask of H nine cells, and incubate the flask for nine minutes at 37 degrees Celsius.

Then add two milliliters of prewarm. Wash medium to the dis displaced treated cells and gently pipette up and down five times with a five milliliter pipette. Transfer the cell solution to a falcon tube.

Next, wash the flask with nine milliliters of wash medium and add it to the tube with the cells. PT the harvest its cells for three and a half minutes at 500 G.Then remove the snat and resuspend the cells in 10 milliliters of wash medium. PT the cells again for three and a half minutes at 500 G and then remove the sup natant and resuspend the cells in four milliliters of human embryonic stem cell medium, add 0.5 milliliters of the cell suspension and 4.5 milliliters of fresh medium into a fresh PBS washed T 25 flask containing mitotically inactivated mouse embryonic fibroblasts.

Change the medium in the flask every day. Coat a 10 centimeter dish with 0.1%gelatine in PBS and incubate for 30 minutes at 37 degrees Celsius. Then harvest a human embryonic stem cells by removing the medium and add one milliliter of acuta to the T 25 flask While the cells are detaching.

Add 6.6 milliliters of cold media to 330 microliters of frozen basement membrane matrix. Filter the solution through a 40 micrometer cell strainer and use one milliliter of the filtered solution to cover each well of a six well plate. The cells will later be plated on the coated plates after the 30 minute incubation of the human embryonic cells with Accutane, stop the reaction by adding 1.5 milliliters of Hess medium.

Scrape the cells from the flask and then add an additional eight milliliters of Hess medium. Produce a single cell solution by gently pipetting up and down with a 10 milliliter pipette filter cells through a 40 micrometer cell strainer, and then centrifuge the cells for three minutes At 500 G, remove the S supernatant and resuspend the cells in 10 milliliters of medium spin cells again for three minutes at 500 G.Then remove the supernatant and reus. Suspend the cells in 10 milliliters of Hess S medium containing rock inhibitor Y 2 7 6 3 2 at a final concentration of 10 micromolar.

Next, remove the supernatant from the gelatin coated dish plate, the cell suspension on the gelatin coated dish and incubate for exactly one hour at 37 degrees Celsius. During this steps, the hypoplast will settle onto the gelatin coated plates where the human embryonic stem cells will remain free. Floating time is crucial as to long incubation will result in clumping of the stem cells and to short incubation will only remove a few of these fibroblasts When the fibroblasts have attached.

Use the medium in the plate to gently wash the non-adherent stem cells off the plate. Wash the plate a second time with 10 milliliters of Hess medium and pool the cells, centrifuge the cells for three minutes at 500 G, remove the resulting S supernatant and resuspend the cells in approximately four milliliters of conditioned medium containing 10 micromolar of rock inhibitor Y 2, 7, 6, 3, 2, and 10 nanograms per milliliter of fiberblast growth factor two. Next, mixed the cells with trian blue and count them in a hemo cytometer plate.

18, 000 cells per square centimeter on the plate before basement. Membrane coated plates cover the cells with 1.5 milliliters of medium per well and incubate at 37 degrees Celsius and 5%carbon dioxide. After 24 hours, refresh the medium After a further 24 hours, change the medium to fresh conditioned medium supplemented with only 10 nanograms per milliliter of fibroblast growth factor 2 72 hours.

After seeding the cells, change the medium to knockout serum replacement medium. This time point is referred to as day zero of differentiation. Refresh the cells with fresh medium each day for the next three days.

When changing the media on the fourth and fifth days, add a consisting of 25%N two supplemented medium and 75%knockout serum replacement. Medium on the sixth day harvest the cells for analysis. The embryo bodies can be used for many different studies.

Here is an example cytotoxicity study showing the effect of varying concentrations of methyl mercury on the viability of the cells. From this graph, it was determined that point 24 micromolar caused 10%cell death and a concentration of 1.61 micromolar caused 50%cell death after exposure for 14 days. RNAs isolated from embryo bodies of three methyl mercury treatment groups were run on a microarray to look at a wide variety of biomarkers.

Those exposed to point 25 micromolar mercury are shown in red. Those exposed to one micromolar methyl mercury are shown in blue, and the control RNA is shown in green. This principle component analysis map shows that the genetic characteristics of each treatment group varies significantly from the other in all more than 276 genes were modified more than twofold when exposed to the one micromolar concentration of methyl mercury.

30 of those genes were also modified by the lower concentration group at point 25 micromolar. On this heat map, one can see that 233 of the genes modified in the one micromolar treatment group were genes that were downregulated and only 43 were upregulated by the treatment. Don't forget that working with triazole or development ants can be extremely hazardous and precautions such as wearing septi goggles, chemical resistant hand gloves, and performing all triol related work in fume hood should always be considered while performing this procedure.

Following this procedure. Other methods like staining, feros formation, which may mix your tube formation can be performed in order to answer additional questions like phenotypic anchoring.