This protocol details a method to generate a well-characterized population of human hemogenic endothelial cells. These cells can be utilized to study the molecular regulation and endothelial-to-hematopoietic transition. Begin by culturing human embryonic stem cells for four days to differentiate them into primordial endothelial cells.
On day five, aspirate the medium above the cells. Then gently wash the cells with one milliliter of DMEM/F-12 per well. Add one milliliter of freshly prepared hemogenic endothelial cell differentiation medium per well and incubate the cells for 24 hours at 37 degrees Celsius and 5%carbon dioxide.
On day six and day seven, replace the medium above the cells with one milliliter of freshly prepared homogenic endothelial cell differentiation medium per well, and incubate the cells for another 24 hours. On day eight. after detaching and washing the cells, divide them evenly into microcentrifuge tubes on ice, each containing a minimum of 600 microliters of cells at a density of 1 times 10 to the 5th cells per milliliter.
Add antibodies to the tubes containing cells as appropriate and incubate on ice protected from light for 30 minutes. Pellet the cells by centrifugation at 1000 times G and 4 degrees Celsius for five minutes. Remove the supernatant and resuspend the pellets in 600 microliters of ice-cold sorting buffer.
Strain the samples through the mesh filter cap of five milliliter FACS tubes and store the cells on ice protected from light for immediate cell sorting. To isolate hemogenic endothelial cells by FACS, first gate the CD45-negative cell population. Then within the CD45-negative population, gate the CD31-positive cells.
Next within the CD31-positive population, gate for the VE-cadherin-negative cell population. And then within the VE-cadherin-negative population, gate for the c-Kit-positive cells. From the c-Kit-positive population, gate the CD34-positive cell population.
And finally from the CD34-positive cell population, identify and collect the KDR-positive cells. After seeding hemogenic endothelial cells in a Methylcellulose-based medium formulated for growth of Hematopoietic Progenitor Cells, Colony Forming Unit-Erythroid colonies, and Blast Forming Unit-Erythroid colonies are counted on day eight. Colony-Forming Unit-Granulocyte-Macrophage and Colony-Forming Unit-Granulocyte, Erythroid, Macrophage, and Megacaryocyte.
Multipotent hematopoietic progenitor colonies are counted on day 14. Per 1000 hemogenic endothelial cells plated, approximately 20 colony forming units are generated. Cells with endothelial cell morphology are also visible in the cultures.
These are the hemogenic endothelial cells that give rise to multilineage hematopoietic progenitors on a single-cell level. This protocol is a 2D serum and murine feeder-free culture system that can generate human hemogenic endothelial cells in roughly one week.
