This protocol can be used to study the molecular and cellular changes that occur during the normal myeloid differentiation of human CD34 positive hematopoietic stem and progenitor cells. The main advantage of this protocol is that it allows the differentiation of CD34 positive cells along all four myeloid lineages including megakaryocytes, erythrocytes, granulocytes, and monocytes. Demonstrating the procedure will be Aditi Bapat, a post-doc from my laboratory.
For mononuclear cell isolation from a human umbilical cord blood sample, sterilize the outer surface of a bag of umbilical cord blood with 70%ethanol before transferring the bag contents into a sterile 250 milliliter plastic bottle within a biological safety cabinet. Dilute the blood with an equal volume of room temperature Hank's buffered saline solution, and add 15 milliliters of mononuclear cell fractionation medium to each of five sterile 50 milliliter conical tubes. After gentle mixing, carefully pour 30 milliliters of the diluted umbilical cord blood onto the mononuclear fractionation medium in each tube, holding the 50 milliliter tubes at an angle to help avoid mixing the layers.
Separate the cells by density gradient centrifugation, and remove the top 15 to 20 milliliters of plasma above the mononuclear cell layer. Use a pipette to carefully transfer the exposed mononuclear cell layer into a new 50 milliliter tube, pooling the mononuclear cells from two to three tubes together, and bring the final volume in each tube up to 50 milliliters with cold PBE buffer. Pellet the mononuclear cell samples by centrifugation, and aspirate the supernatants.
Tap each tube gently to loosen the cell pellets, and dilute the pellets in five milliliters of ice cold PBE buffer. Combine the cells from three to four tubes into one 50 milliliter conical tube, and wash all of the tubes with five milliliters of fresh PBE buffer to collect any remaining cells. After counting, bring the final volume in the tube up to 50 milliliters with fresh ice cold PBE buffer, and collect the cells by centrifugation.
To isolate the CD34 positive mononuclear cells, re-suspend the pellet in 50 milliliters of fresh PBE buffer before collecting the cells with another centrifugation. After discarding the supernatant, tap gently to loosen the pellet, and re-suspend the cells at a one times 10 to the eight mononuclear cells per 300 microliters of ice cold PBE buffer concentration. Block any nonspecific binding with 100 microliters of Fc receptor blocking reagent, mix gently, and then add 100 microliters of CD34 microbeads from a magnetic activated cell-sorting human CD34 microbead kit per one times 10 to the eight cells with gentle mixing, and incubate the cells for 30 minutes at four degrees Celsius.
While the cells are incubating, place an LS column and pre-separation filter in a MACS separator magnetic field. Equilibrate the column with two milliliters of ice cold PBE buffer and the pre-separation filter with one milliliter of buffer. Allow the column to empty into a 15 milliliter conical tube and discard the flow-through.
At the end of the incubation, bring the final volume of the mononuclear cell sample up to 15 milliliters with fresh ice cold PBE buffer and sediment the cells by centrifugation. Re-suspend the pellet in one milliliter of ice cold PBE buffer and load the cells onto the pre-separation filter on the column. Rinse the tube with three milliliters of fresh ice cold PBE buffer and add the buffer to the pre-separation filter.
Wash the column four times with an additional three milliliters of ice cold PBE buffer per wash without the filter, allowing the column to fully drain between each wash. After the final wash, move the column into a new 15 milliliter tube, and plunge five milliliters of fresh ice cold PBE through the column into the tube. To determine the fractions of stem and progenitor populations in the freshly isolated CD34 positive hematopoietic stem and progenitor cells, collect the cells by centrifugation and re-suspend the pellet at a one times 10 to the six cells per 50 microliters of flow cytometry buffer concentration.
Next, transfer the cells in 50 microliter aliquots per five milliliter fluorescence activated cell-sorting tube according to the experimental flow cytometry staining protocol. Add the appropriate antibody cocktail and live/dead staining to the cells, adjusting the volume to 100 total microliters. After 20 minutes in the dark protected from light, wash the cells with one milliliter of fresh flow cytometry buffer per tube, and re-suspend the cells in 500 microliters of fresh flow cytometry buffer per tube.
Then analyze the cells on a flow cytometer according to standard flow cytometric protocols. To differentiate the microbead isolated CD34 positive stem and progenitor cells into myeloid lineage cells, first coat the wells of a sterile, uncoated 48-well plate with 200 microliters per well of recombinant human fibronectin solution in PBS for two hours at room temperature. At the end of the incubation, discard the fibronectin solution from each well and block the wells with 200 microliters of 2%bovine serum albumin.
After 30 minutes at room temperature, wash the wells two times with 500 microliters of sterile PBS per wash. Re-suspend the freshly isolated CD34 positive cells at a five times 10 the five cells per milliliter of warm stimulation medium concentration. Next, seed 200 microliters of cells into each well of the fibronectin fragment coated plate and place the plate in the cell culture incubator for 48 hours.
At the end of the incubation, collect the cells with gentle pipetting, and wash the wells with 500 microliters of warmed Dulbecco's modified Eagle medium, pooling the washes with the cell suspension. After counting, re-suspend the cells at a 2.5 times 10 to the four cells per one milliliter of myeloid differentiation medium concentration. Layer 200 microliters of CD34 positive cells per well in an MS-5 stromal cell seeded 48 well tissue culture plate.
Then place the plate in the cell culture incubator, gently replacing 100 microliters of the supernatant in each well with 100 microliters of fresh 2X myeloid differentiation medium every three to four days. On day 21, harvest the cells for analysis of the myeloid lineage marker expression by flow cytometry as just demonstrated. The typical percentage of total CD34 positive cells isolated from cord blood by microbead separation as demonstrated ranges between 80 to 90%Immunophenotypic analysis reveals that these CD34 positive cells typically consist of an approximately 20%lineage negative CD34 positive CD38 negative hematopoietic stem cell population and a 72%lineage negative CD34 positive CD38 positive multi-potent progenitor cell population.
Within the multi-potent progenitor cell population, about 25%of the cells express common myeloid progenitor markers, about 15%express granulocyte monocyte progenitor markers, and about 56%express megakaryocyte erythroid progenitor markers. Immunophenotyping also demonstrates that the percentage of CD34 positive cells reduces from about 90%at isolation to less than 25%on day 21. Analysis of the CD34 negative population demonstrates a concomitant rise in the percentage of cells expressing mature myeloid lineage markers.
Further, Wright-Giemsa stained cells demonstrate distinct morphological characteristics on days one and 21 with the undifferentiated cultures exhibiting large, round nuclei and very little cytoplasm and the differentiated cultures displaying morphologic characteristics of lineage cells including which are monocytes, granulocytes, and erythroblasts. Remember to pour the diluted cord blood onto the mononuclear cell fractionation medium without mixing the layers, and do not disturb the MS-5 and CD34 positive cells when changing the medium. Following this differentiation protocol, the CD34 positive cell cultures can be analyzed for morphological changes, apoptotic effects, changes in cell cycle patterns, and the degree of differentiation.
