We offer produce blood cells from human pluripotent stem cells. The main advantage of this technique is that we get consistent and precise yields of hemogenic endothelial cells. We expect that our platform will enable broad applications in disease monitoring and screening for therapeutic compounds.
This technique is particularly beneficial for hematology and immunology research. The method can be applied to genetic intervention or gene editing of human blood cells. It allows for easy induction of vectors in hemogenic endothelial cells.
The most crucial point is LM511-E8 coating for typing PSC colonies. We do not recommend skipping the coating or substituting with another matrix. Visual demonstrations is important because we want to show how to rinse the cells with a dissociation buffer and how to coat plates with LM511-E8.
Grow human pluripotent stem cells or hPSCs to between 70 and 80%confluency on an LM511-E8-coated six-well plate in mTeSR1 medium. Four days prior to hemogenic induction, aspirate the medium and wash twice with PBS. Rinse the cells with dissociation solution, and incubate at 37 degrees Celsius for 15 minutes.
Resuspend the cells in PSC maintenance medium and transfer the suspension to a 1.5 milliliter microcentrifuge tube. Centrifuge the cells at 200 times G for three minutes. Aspirate the supernatant, and resuspend the cells in PSC plating medium.
Plate the PSC suspension according to the manuscript directions onto a microfabricated plastic vessel, and incubate overnight. Three days prior to hemogenic induction, gently pipette the PSC spheroids into a 15 milliliter conical tube and leave them for two minutes at room temperature to precipitate by gravity. Aspirate the supernatant and resuspend the spheroids in spheroid plating medium.
Distribute the suspension into an LM511-E8 coated 10 centimeter culture dish at a density of four spheroids per square centimeter and incubate for three days. After three days, aspirate the medium, add day zero differentiation medium, and culture the cells for two days in a hypoxic incubator. Then, aspirate the day zero medium, add day two differentiation medium, and culture for another two days.
Two days later, aspirate the medium and wash the cells twice with PBS. Rinse the cells with dissociation solution and incubate for 30 minutes at 37 degrees Celsius with 5%carbon dioxide. After incubation, dissociate the cells by gently resuspending them in one millimolar EDTA.
Transfer the suspension into a 50 milliliter conical tube, and centrifuge the cells at 200 times G for three minutes. Aspirate the supernatant and resuspend the cell pellet with 300 microliters of magnetic separation buffer. Add 100 microliters of CD34-positive microbeads to the cells, and gently mix by pipetting.
Incubate at room temperature for 30 minutes, then use a 40 micrometer cell strainer to strain the suspension and separate the CD34-positive cells using a magnetic separator. Dispense 0.5 milliliters of 5 microgram per milliliter fibronectin coating solution into each well of a 24 well culture plate, and incubate the plate at room temperature for 30 minutes. Meanwhile, centrifuge the sorted CD34-positive cells at 200 times G for three minutes, and resuspend the pellet in one milliliter of EHT medium.
Determine the viable cell density with a cell counter, and adjust the cell density to 200, 000 cells per milliliter by adding EHT medium. Aspirate and discard the coating solution from the fibronectin coated wells, and dispense 0.5 milliliters of CD34-positive cell suspension into each well. Incubate the cells in the hypoxic incubator for one week.
To collect the floating cells, transfer the culture medium to a 15 milliliter conical tube, and centrifuge at 200 times G for three minutes. Then aspirate the supernatant. To collect the adherent cells, wash them twice with 0.5 milliliters of PBS, and rinse them with dissociating buffer.
Aspirate the surplus liquid and incubate the plate at 37 degree Celsius for five minutes. Resuspend the cells in one milliliter of Facs buffer, and mix the floating and adherent cells. Centrifuge the mixed cells at 200 times G for three minutes, then aspirate the supernatant and resuspend the cells in 50 microliters of Facs buffer.
Dilute anti-CD34 and anti-CD45 antibodies in 50 microliters of Facs buffer;add them to the cells and incubate the cells for one hour at room temperature in the dark. Then, wash the cells twice with PBS, centrifuging at 200 times G for three minutes after each wash. Aspirate the supernatant and resuspend the cells in 0.5 milliliters of Facs buffer with 0.5 micrograms per milliliter dappy.
Measure CD34 and CD45 expression by flow cytometry. To collect the hematopoietic cells, transfer the culture medium to a 15 milliliter conical tube. Collect the remaining cells by gently rinsing the well twice with PBS.
Centrifuge the cells at 200 times G for three minutes then aspirate the supernatant and resuspend in one milliliter of EHT medium. After counting the cells, add 10, 000 cells to three milliliters of methylcellulose spaced media, supplemented with antibiotics, and use a 16 gauge syringe to mix five times. Dispense the whole media suspension into each well of a six-well plate.
To keep the cells moist, add water or PBS to the empty wells on the plate. Incubate the cells for two weeks, making sure not to disturb the dish as colonies are motion sensitive. After two weeks, count the colonies under a microscope.
Cell morphologically change from endothelial to hematopoietic cells upon stimulation with the hematopoietic cocktail. Hematopoietic cell colonies emerge in the culture. Hematopoietic progenitor cells express CD34 and CD45, so flow cytometry is used to confirm the presence of hematopoietic cell colonies by analyzing expression of CD34 and CD45.
A colony forming unit assay demonstrated the generation of granulocytes or macrophage colonies from the CD34-positive and CD45-positive hematopoietic progenitor cells. The colony number was estimated to be 38 colony forming units per 10, 000 cells. The most crucial steps are tallying PSC spheroids.
We found that the surface metric is the most deterministic factor in efficiency of this protocol. This procedure can be followed by off-the-shelf production of inergical cells such as naturally occurring cells and macrophages. This platform allows researchers to explore key deterministic factors in human hematopoietic development.
