The overall goal of this procedure is to use human cord blood samples to isolate pure CD34-positive cells to study the effects of specific drugs of interest on megakaryocyte differentiation, proplatelet formation, and platelet production. This method can help answer key questions on the megakaryocyte and platelet field, such as what are the effects of a specific treatments on megakaryocyte differentiation and platelet production and function. The main advantage of this technique is that it provides a method for the consistent isolation of viable CD34-positive cells from fresh umbilical cord blood.
Through this method can provide insight into megakaryopoiesis, other cell types such as lymphocytes, erythrocytes, and macrophages, can also be studied. Begin by adding 10 milliliters of separation buffer into 150 milliliter conical tube per 10 milliliters of cord blood. Using an 18 gauge blunt needle mounted on a 10 milliliter syringe, transfer 10 milliliters of blood into each tube, followed by the addition of 15 milliliters of lymphocyte separation medium to the bottom of each tube.
Separate the cells by density gradient centrifugation, then at the end of the spin, carefully collect five to 10 milliliters of mononuclear cell containing buffy coat layer into one new tube per sample. Bring the total volume in each tube up to 50 milliliters with a fresh separation buffer and centrifuge the cells again. Re-suspend the pellets in five to 10 milliliters of fresh separation buffer and pool the cell samples.
Adjust the volume to 50 milliliters with fresh separation buffer for counting and set aside 100 microliters of cells to determine the percentage of CD34 positive cells by flow cytometry. Centrifuge the cells again and re-suspend the pellet in 300 microliters of separation buffer, 100 microliters of FC receptor human IgG blocking reagent, and 100 microliters of CD34 magnetic beads per one times 10 to the eighth cells. Mix the cells gently before placing them at four degrees Celsius for a 30 to 40 minute incubation.
During the incubation, place an appropriately sized magnetic bead separation column into the corresponding magnetic holder and wash the column with three milliliters of separation buffer. At the end of the incubation, remove the unbound beads by centrifugation in five to 10 milliliters of separation buffer and re-suspend the magnetic bead and cell pellet in 1.5 milliliters of fresh separation buffer. Load the labeled cells onto the column and collect the unlabeled flow through in a 15 milliliter collection tube.
Load the effluent back onto the column and collect the second round of flow through into the same collection tube. Then wash the column three times with three milliliters of separation buffer and remove the column from the magnet. Using the column plunger, gradually flush two milliliters of separation buffer through the column into a new 15 milliliter tube and return the column to the magnet.
Load the flushed cells back onto the column. Collect the flow through and wash the column with two milliliters of separation buffer as just demonstrated. After removing the column from the magnet, flush the column with two milliliters of fresh separation buffer to collect the CD34-positive cell fraction as just demonstrated, and collect the CD34-positive cells by centrifugation.
Then resuspend the pellet in serum-free medium for CD34-positive cells. For megakaryocyte differentiation, seed five x 10 to the fifth CD34-positive cells per milliliter in 2 milliliters of serum-free medium, supplemented with recombinant human thrombopoietin per well in a 12-well plate for their incubation at 37 degrees Celsius, and 5%carbon dioxide, in a humidified atmosphere. At the appropriate differentiation monitoring time points, harvest the cells from two to three wells per time point without disturbing the cells in the other wells, and stain the collected cells with anti-CD41, and anti-CD42a antibodies to determine the percentage of mature megakaryocytes within the cultures.
For microscopic visualization at the cell surface markers, wash the harvested cells in 1 milliliter of separation buffer, and resuspend the pellet in 100 microliters of fresh buffer. Use a cytospin to seed the cells onto a glass slide, and fix the cells to the slide with a 30-second submersion in methanol. After air drying, label the cells with 20 microliters of DAPI-supplemented mounting medium, and cover the cells with a cover slip for visualization by fluorescent microscopy.
To count the proplatelets, harvest the cells at day eight or nine of differentiation, and seed the collected cells at one x 10 to the fourth cells per 200 microliters of fresh serum-free medium, supplemented with recombinant human thrombopoietin per well in a 48-well plate. After five days at 37 degrees Celsius and 5%carbon dioxide, count the number of proplatelet-bearing megakaryocytes per well on an inverted light microscope, using the 10 or 20 times objective. To analyze the platelet activation, on day 14 or 15 of culture, gently mix the cells with a Pasteur pipette, and collect 100 microliters of cells.
Add 1 milliliter of Tyrode's buffer to the cells and pellet them by centrifugation. Collect the supernatant for centrifugation to pellet the platelet-sized particles, and resuspend the particles in 100 microliters of fresh Tyrode's buffer, then label the particles with anti-PAK1 antibody, activate them with the adenosine diphosphate for 20 minutes at room temperature, and analyze the percentage of PAK1 positive events by flow cytometry. The typical percentage of CD34-positive cells in cord blood is approximately 1.3%and the purity of the CD34-positive CD45-positive cells after isolation ranges from 90 to 99%CD41-positive megakaryocytes are observed early in serum-free CD34-positive cell cultures, with the percentage of mature CD41-positive CD42a-positive megakaryocytes reaching 30 to 40%by day 7, and the highest levels of double positive cells observed between days 10 and 12 of differentiation.
Cultured megakaryocytes appear as large, usually multi-nucleated cells that express CD42b Von Willebrand factor, and P-selectin in their cytoplasm. At least three classes of ploidy are also commonly observed in these cell cultures. The percentage of megakaryocytes that bear proplatelets, long beaded fibers that extend from the megakaryocyte body, is typically around 1.3%Most platelets in the cell culture supernatant fall within the analytical gate of peripheral blood platelets, are positive for the CD41 platelet marker, can be activated by platelet agonists like adenosine diphosphate, as demonstrated by an increased binding of the activation specific PAK1 monoclonal antibody.
After watching this video, you shall have a good understanding of how to isolate CD34-positive cells from cord blood. You shall also be able to set up megakaryocyte differentiation cultures, count proplatelets, and analyze platelets and megakaryocytes produced in vitro. Don't forget that working with human samples can be hazardous, and precautions, such as working in a class II biological safety cabinet should always be taken while performing this procedure.
