Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell (CB-SC)-Derived Exosomes

The Stem Cell Educator therapy was developed to treat type I diabetes and the other autoimmune disease. This method was developed to explore the molecule mechanism underlying immune moderation of Stem Cell Educator therapy through CB-SC released exosomes. This protocol provide guidance on how to purify exosomes from a human cord blood stem cells cultures and determine their immune modulation of monocytes.

Demonstrating the procedure will be Wei Hu, a PhD student, from my lab. To begin, centrifuge the CB-SC-derived conditioned medium three times as described in the text manuscript, collecting the supernatant into a new 50 milliliter conical tube after each centrifugation. After the third centrifugation, filter the obtained supernatant with a 0.22 microliter filter and transfer 15 milliliters of media to each 10 kilodalton centrifugal filter unit.

Centrifuges at 4, 000 x g for 30 minutes to isolate the concentrated exosome media. Transfer the concentrated exosomes to an ultracentrifuge tube. Then pellet the exosomes at 100, 000 x g for 80 minutes at four degrees Celsius.

Then discard the supernatant and resuspend the pelleted exosomes in 10 milliliters of PBS. Perform a final ultracentrifugation to collect the exome pellet and resuspend it in 200 microliters of PBS. Add 30 million human PBMCs to a 15 milliliter tube and centrifuge at 300 x g for 10 minutes at four degrees Celsius.

Resuspend the cells in 300 microliters of cold PBS and add 60 microliters of CD14 microbeads. Mix well and incubate the cells on ice for 15 minutes. Add six more milliliters of cold PBS and centrifuge at 300 x g for 10 minutes at four degrees Celsius.

Then resuspend the pelleted cells in 500 microliters of cold running buffer. Place the separation column in the magnet separator and wash it three times with two milliliters of cold running buffer. Transfer the resuspended cells into the separation column and let them pass.

Again, wash the column three times with two milliliters of running buffer per wash. Then lift the column from the magnet separator and place it over a 15 milliliter centrifuge tube on ice. Elute the CD14 positive cells with two milliliters of cold running buffer and centrifuged at 300 x g for 10 minutes at four degrees Celsius to pellet the cells.

Resuspend the pellet in two milliliters of cold chemical defined serum medium and transfer 50 microliters of it into a 1.5 milliliter tube. Stain the obtained cells with 10 microliters of Krome Orange-conjugated anti-human CD14 monoclonal antibodies for 20 minutes. Add one milliliter of PBS and centrifuge at 300 x g for 10 minutes to pellet the cells.

Resuspend the cell pellet in 200 microliters of PBS and transfer it into a five milliliter tube. Determine the purity of CD14+monocytes by flow cytometry. Seed one million of the obtained purified monocytes in a tissue culture treated six-well plate and incubate for two hours at 37 degrees Celsius under 5%carbon dioxide.

After incubation discard the supernatant and gently add two milliliters of 37 degree pre-warmed chemical defined serum-free culture medium, then add 80 micrograms of the CB-SC-derived exosomes to the monocyte culture in the six-well plate with a total volume of two milliliters. Incubate the plate at 37 degrees Celsius under 5%carbon dioxide for three to four days. Then image the cell morphology using an inverted microscope at 200X magnification.

Add one milliliter of PBS-based cell dissociation buffer to detach the cells by pipetting up and down and harvest the remaining cells with a cell scraper. Collect the cells by centrifuging at 1, 690 x g for five minutes and resuspend them in 200 microliters of PBS. Add five microliters of FC blocker to block the non-specific binding, then add the antibodies.

Incubate the cells at room temperature for 30 minutes. Add one milliliter of PBS to the incubated cells and centrifuge them at 300 x g for 10 minutes. Resuspend the pellet in 200 microliters of PBS and add five microliters of propidium iodide per sample.

Transfer the cells to a new five milliliter flow tube and perform flow cytometry to evaluate the levels of CD14, CD80, CD86, CD163, CD206, and CD209 expression. The phenotype and purity of CB-SCs were examined by flow cytometry with CB-SC associated markers. The analysis showed high levels of CD45, OCT3/4, SOX2, CD270 and galectin 9 expression but no expression of CD34 was observed.

Flow cytometry analysis also confirm the expression of exome specific markers, CD9, CD 81, and CD63 on CB-SC-derived exosomes. The size morphology of exosomes was characterized by TEM and the size distribution was determined by DLS. Western blot further prove the expression of the exome associated marker Alix without expression of the ER associated marker, calnexin.

The direct interaction of Dio-labeled CB-SC-Exo with PBMC was observed using fluorescence microscopy. To better define which cell population interacted with the dial labeled CB-SC-Exo, different cell compartments were gated with cell specific markers, such as CD3 for T cells, CD11C for myeloid dendritic cells, CD14 for monocytes, CD19 for B cells, and CD56 for NK cells. Monocytes were primarily targeted by the CB-SC-derived exosomes as they exhibited higher median fluorescence intensities than those of other immune cells.

The exosome treated monocytes successfully differentiated into spindle-like morphologies. There was an upregulation in the level of M2 associated markers, such as CD163, CD206, and CD209 after the treatment with CB-SC-derived exosomes. Phenotypic comparison between conventional M2 macrophages and the CB-SC exome-induced M2 macrophages showed no significant differences.

The key step in this protocol is adding CB-SC-derived exosomes to the monocyte of culture in six-well plate and incubate it for three to four days, which needed to the cell's differentiation to other type 2 macrophages with spindle-like morphology. Next, we are going to explore the molecule components inside of CB-SC-derived exosomes that contributed to the induction of differentiation of monocyte into type 2 macrophages.