Desenvolvimento de um ensaio In Vitro para dosar o Progenitor e Hematopoietic Stem Cells (HSPCs) no desenvolvimento de embriões de peixe-zebra

The overall goal of this assay is to enumerate and examine the differentiation of hematopoietic progenitor cells in the developing zebrafish embryo. This method can help answer some really important questions in the hematopoietic field, such as, how many stem and progenitor cells are actually in a developing zebrafish embryo. The main advantage of this technique, is that it’s quick, it’s relatively inexpensive and it’s easy to perform.

Though this method can provide insight into the number of progenitors present in the developing zebrafish, it also allows researchers to measure the functional effect that a drug or genetic mutation has on blood progenitor formation. We had the idea for this method, when we were knocking down genes that we hypothesized were crucial for blood development. We predicted that we would be able to measure the difference between normally developing embryos and those that were morphants with this assay.

To bleach zebrafish embryos, use plastic containers to create three wash stations, with one containing one milliliter of five percent bleach per one liter of E3 and two stations containing sterile E3.With a transfer pipette, place embryos into a tea strainer and submerge them in wash station number one for five minutes. Then transfer the tea strainer with the embryos into wash station number two for five minutes. Repeat for wash station number three for a final five minutes.

After the last wash, remove the embryos from the tea strainer by turning it upside down over a clean 10 centimeter petri dish and use a transfer pipette and sterile E3 to gently rinse the embryos off the strainer. To dechorionate the embryos, use a transfer pipette to remove and discard as much E3 as possible from the petri dish. Then add 500 microliters of dechorionation prodeas to the embryos and incubate them at room temperature for five minutes.

Next, gently tap the side of the petri dish to completely remove the chorions. Then, with a serological pipette, add 20 milliliters of sterile E3 to dilute the prodeas. Allow embryos to settle and use a pipette to remove the E3.Repeat this wash step three times to remove all traces of the dechorionation prodeas.

To prepare embryo samples, use a pipette to place 10 embryos into a single sterile 1.5 milliliter microcentrifuge tube. Then, carefully pipette out the E3 and discard it. Transfer samples to a laminar flow hood, and wash the embryos by adding one milliliter of sterile E3, allowing embryos to settle to the bottom of the tube.

Then, use a P1000 pipette to remove the supernatant. Repeat for a total of three washes. Following the last wash, remove the E3 and discard it.

Then add one milliliter of 10 millimolar DTT in E3 to remove the mucus coating and any spores, yeasts or bacteria that may be trapped in it surrounding the embryos. Now, lay the microcentrifuge tube horizontally and incubate it at room temperature for 25 minutes. To dissociate the embryos, use one milliliter of sterile DPBS to wash the sample three times.

After the last wash, add 500 microliters of DPBS with calcium and magnesium and five microliters of five milligram per milliliter dissociation prodeas. Incubate the samples at 37 degrees Celsius on a horizontal orbital shaker at 180 rpm for 60 minutes. Then, place the samples and the laminar flow hood and use a P1000 to triturate the embryos until the samples are fully dissociated.

Use caution and check embryos periodically to determine when they are dissociated. You want some tissue to be present. It is possible to over digest the embryos which will destroy the progenitor cells.

Pipette the 10 dissociated embryos onto the top reservoir of a five milliliter poly styrene round bottom tube with a 35 micrometer cell strainer cap. With a pipette and sterile PBS, rinse the embryo tube and transfer the solution to the five milliliter poly styrene tube containing filtered cells. Repeat the rinse until four milliliters of liquid is present in the tube.

Centrifuge the tubes at 300 times the gravity and four degrees Celsius for five minutes to palette the homogenized cell sample. With a pipette, remove and discard the supernatant from the round bottom tube, taking care to not disrupt the cells pelleted at the bottom. Then use 100 microliters of PBS to re-suspend the cells.

Prepare one X complete methylcellulose stock solution and use three milliliter syringes and 16 gage needles to add 2.5 milliliters of sterile round bottom 14 milliliter tubes. Use one tube for each condition tested. For myeloid differentiation, add one percent carp serum and 0.3 milligram per milliliter of recombinant zebrafish granulocyte colony-stimulating factor or GCSF to methyl cellulose medium.

For erythroid differentiation, add one percent carp serum and 0.1 milligram per milliliter of recombinant zebrafish erythropoietin or EPO to methyl cellulose medium. To examine multi-lineage progenitors, add EPO and GCSF. Using a pipette, add 100 microliters of the 10 dissociated embryos to the surface of the prepared methyl cellulose with the recombinant cytokines in carp serum.

Cap the lid and gently vortex to fully homogenize the sample. Each tube should now contain the dissociated tissue of 10 embryos. Using three milliliter syringes and 16 gage needles, aliquot one milliliter of sample into two separate 35 millimeter petri dishes.

Make sure that the sample is fully dispersed throughout the petri dish. Repeat for each sample. Place the 35 millimeter petri dishes with cells and methyl cellulose into a 15 centimeter petri dish.

To each large plate, add one 35 millimeter dish with five milliliters of sterile water to humidify the samples. Incubate the dishes at 32 degrees Celsius and five percent carbon dioxide for seven to 10 days. Finally, visualize the colony forming units according to the text protocol.

After seven days in culture with hematopoietic supportive factors, progenitors from 48 HPF embryonic zebrafish were counted and imaged. Importantly, the quantity of CF used directly correlates with the number of embryos plated. Plating either four or eight embryos per milliliter, which correlates to starting with 10 or 20 embryos, at the beginning of the experiment, yields approximately 1200 or 2400 CFUs respectively.

Once mastered, this technique can be done in two and a half hours if performed properly. While attempting this procedure, it’s important to remember not to over digest the embryos as this will destroy the progenitor cells. With this procedure, you can compare the number of progenitors present at different time points.

You can compare mutant animals with wild type animals, as well as test the effect of drugs on blood development. After watching this video, you should have a good understanding of how to perform a methyl cellulose assay to enumerate zebrafish hematopoietic progenitors. You can alter this procedure to fit your specific experimental needs, such as testing specific cytokines.

Don’t forget, that working with vertebrate blood and needles can be extremely hazardous and care should always be taken when performing this assay.