Ex vivo expansion of hematopoietic stem cell from umbilical cord blood unit hold great promise for HSC application in regenerative medicine and transplantation therapy. This protocol use a cytokine cocktail combined with valproic acid treatment to rapidly expand a great number of functional HSCs with characteristics that closely resemble primary primitive HSCs. Due to the prompt effects of valproic acid treatment, this method also has the potential to overcome the loss of HSCs associated with gene editing.
This method might be beneficial for generation of a higher number of genetically-modified cells within a period of time that is relevant for currently-used gene modification protocols. The valproic acid ex vivo expansion protocol is relatively simple and reliable. Purification of CD34-positive cells with the cell separator device is highly reproducible and rapid, allowing for fast recovery of highly purified CD34-positive cells.
24 hours prior to the isolation of CD-34 positive cells from umbilical cord blood, prepare the separation buffer by adding two milliliters of 0.5 molar EDTA and 33 milliliters of 7.5%BSA to 465 milliliters of 1X PBS. Mix the buffer gently and maintain it overnight at four degrees Celsius. On the day of purification, warm the media to room temperature.
Dispense the whole umbilical cord blood unit into a 75-square-centimeter flask. Dilute the blood with an equal volume of PBS at room temperature and mix gently. Next, determine the number of tubes required for processing the whole umbilical cord blood unit.
Add 15 milliliters of density gradient media to each tube. Dispense the diluted blood very slowly on top of the density gradient while holding the tube at a 45-degree angle to create two layers. Centrifuge the tube at 400 g for 30 minutes with a low acceleration and deceleration rate.
After the centrifugation, the mononuclear cells will be located in the white layer between the plasma and density gradient media layers. Carefully transfer the buffy coat layer into a new, 50-milliliter tube. Collect all of the mononuclear cells from the same umbilical cord blood unit into a 50-milliliter tube until reaching 25 milliliters.
Add 25 milliliters of cold PBS to the tube containing 25 milliliters of mononuclear cells and mix well. Centrifuge at 400 g and at four degrees Celsius for 10 minutes. Carefully aspirate the supernatant and resuspend the cell pellet in 50 milliliters of cold PBS.
After this, remove 20 microliters of the cell suspension for counting. Use an automated cell counter to count the number of cells stained with either acridine orange or propidium iodide and calculate the total number of mononuclear cells. Then, centrifuge the cells at 400 g and at four degrees Celsius for 15 minutes.
First, mix 300 microliters of separation buffer with 100 microliters of human FCR human IgG and 100 microliters of CD34 magnetic beads to isolate the CD34-positive cells from 10 million mononuclear cells. Next, carefully remove the supernatant from a tube of previously-centrifuged mononuclear cells. Resuspend the pellet in the magnetic bead solution using 500 microliters per 10 million cells.
Mix gently and incubate at four degrees Celsius for 30 minutes. During the incubation, prepare the cell separator device by replacing the storage solution with the working buffer and running a washing program followed by a rinsing program. Then, add cold cell separator running buffer to the tube that contains the cell mixture until the tube is completely filled.
Centrifuge the tube at 400 g and at four degrees Celsius for 15 minutes. Aspirate the supernatant and resuspend the cell pellet in cold separator running buffer using two milliliters per 10 million cells. Transfer the two-milliliter cell suspension mixture into 15-milliliter tubes.
Load three sets of the tubes into the cell separator rack, which has been pre-chilled to four degrees Celsius. One set of tubes contains MMCs, the second set of tubes will be used to collect the negative fraction, and a third set of tubes will be used to collect purified CD34-positive cells. Load the rack into the cell separator device and run the preset program.
After this, centrifuge the tubes containing the positive fraction at 400 g at and four degrees Celsius for 15 minutes. Aspirate the supernatant and resuspend the purified CD34-positive cells in one milliliter of serum-free media. Then, use an automated cell counter to count the cells stained with acridine orange or propidium iodide.
First, prepare a sufficient volume of media to plate the purified CD34-positive cells at a density of 33, 000 cells per milliliter. Plate the purified CD34-positive cells in a 12-well plate at a cell density of 50, 000 cells in 1.5 milliliters of media per well. Incubate at 37 degrees Celsius in a 5%carbon dioxide humidified incubator for 16 hours.
Add valproic acid to the cultures such that the final concentration is one millimolar. Continue culturing the cells at the same conditions for an additional seven days. In this ex vivo protocol, the number of primitive hematopoietic stem cells generated from CD34-positive cells isolated from umbilical cord blood is increased.
The total number of nucleated cells is significantly higher in cultures treated with the cytokine cocktail alone. Despite the higher number of total nucleated cells, the number of hematopoietic stem cells in the cultures receiving the cytokine cocktail alone remains low during the entire expansion period, compared to cultures treated with cytokine cocktail and valproic acid. The greatest number of hematopoietic stem cells is generated in cultures treated with a combination of the cytokine cocktail and valproic acid.
In particular, the greatest expansion is reached after five to seven days following treatment with valproic acid. This increased number of hematopoietic stem cells correlates with a prompt increase in the percentage of hematopoietic stem cells, which is notable within 24 hours of treatment with valproic acid. While the increase in the percentage is maintained during the first four days of ex vivo culture, it declines progressively after five to seven days of treatment.
However, this decrease is inversely correlated with an increase in the absolute number of hematopoietic stem cells. The rapid increase in the percentage of hematopoietic stem cells in valproic-acid-treated cultures is due to the acquisition of the CD90 phenotype. CD34-positive cells expressing the CD90 phenotypic marker reach almost 40 to 50%of cells expanded in ex vivo cultures treated with the cytokine cocktail and valproic acid for four days.
The acquisition of the CD90 phenotype is then confirmed by ex vivo expansion of the highly-purified CD34-positive cells that lack expression of the CD90 marker. Within 24 hours of treatment with valproic acid, almost 75%of the ex vivo expanded cells express CD90. This phenotype is highly retained during the first four days in these cultures.
The diluted cord blood should be dispensed very slowly on top of the density gradient while holding the tube at a 45-degree angle to create two distinct layers. Following this ex vivo expansion procedure, the expanded cells can be injected into the myeloablated immune-deficient NSG mice to test their functionality and engraftment potential. The expanded cells can be used to test a variety of questions regarding the biology of both human and mouse HSC and the role of different genes or critical player on the functional integrity of HSCs.
The functionality of ex vivo expanded HSC from human umbilical cord blood unit, using this technique, will be soon tested in a clinical trial in patient with hematological malignancies who require allogenic bone marrow transplantation. This technique has also allowed us to investigate the mechanism underlying the ex vivo expansion with VPA treatment and get insights into the biology of primitive HSCs.
