Isolation of Human BAMBI^highMFGE8^high Umbilical Cord-Derived Mesenchymal Stromal Cells

Our scope is to isolate specific subgroups of human UC-MSCs and analyze their basic characteristics according to their single-cell transcriptomic factors. We are trying to answer which are the distinct characters and functions for different UC-MSC subpopulations. The most recent developments in our field are the discovery of the heterogeneity of mixed MSC populations, mostly by using single-cell RNA sequencing.

We identified the three sub-populations within human UC-MSCs, including BAMBI high MFGE8 high cells. This subgroup exhibits a distinct phenotype, unique transcriptomic profile, limited adipogenic potential, and reduced immunosuppressive activity in lupus nephritis. These findings advance understanding of UC-MSCs and supports selecting optimal subgroups for treating specific diseases.

We are paving the way for understanding the real functions of not only BAMBI high MFGE8 high UC-MSCs but also the other two subgroups, especially their roles in treating different diseases. We will continue to focus on starting applications of human UC-MSC subgroups on multiple immune-mediated diseases and exploring their molecular mechanisms in treating those diseases. To isolate BAMBI high MFGE8 high UC-MSCs, culture UC-MSC to a density of approximately 5 to 10 times 10 to the power of 6 cells in complete DMEM.

Add 0.5 millimolar EDTA for five minutes to dissociate the cells. Then, add the complete DMEM and transfer the cell suspension to a 15 milliliter conical tube. Pipette the cell suspension up and down several times to prepare a single-Cell suspension.

Take 10 microliters of the cell suspension. Count the cells using a hemocytometer, and calculate the total number of cells harvested. After counting, centrifuge the required number of cells at 300 G for five minutes.

Resuspend the pellet in one milliliter of complete medium to achieve a concentration of 5 to 10 times 10 to the power of 6 cells per milliliter, and place the tube on ice. Next, divide the harvested cells into four 1.5 milliliter microcentrifuge tubes. Add the primary antibodies at a 1 to 100 dilution to MFGE8 and BAMBI tubes.

Mix the contents thoroughly and incubate the cells at room temperature for 15 minutes. After incubation, add PBS to wash the labeled cells. Centrifuge the tubes at 300 G for five minutes and discard the supernatant before resuspending the pellet in one milliliter of complete medium.

Add the conjugated fluorescent secondary antibodies at a 1 to 1, 000 dilution to the resuspended cells. Mix thoroughly and incubate the tubes at room temperature in the dark four 15 minutes. After incubation, add PBS and centrifuge to wash the labeled cells.

Resuspend the pellet in 500 microliters of complete medium and filter them through a 70 micrometer cell strainer to remove clumps and debris. Transfer the filtrate to a 15 milliliter tube for flow cytometry sorting. Run the blank cell tube as a negative control without adding an antibody.

Adjust the forward scatter and side scatter settings on the flow cytometer to establish the gating scale for the unstained population. Then, run the single-antibody-labeled MFGE8 and BAMBI as a gating control to determine where the positivity starts in the plot. Run the experimental sample tubes to sort the BAMBI high MFEG8 high cell population and collect the sorted cells.

Plate the sorted MSCs in a 24-well plate and incubate the cells at 37 degrees Celsius and 5%carbon dioxide. Once the sorted cells have grown through two passages, perform flow cytometry analysis to confirm the purity of the sorted population. Cultured UC-MSCs were strongly positive for CD44, CD73, CD90, and CD105 expression, and negative for CD14, CD34, CD45, CD79, and HLA DR expression.

BAMBI high MFEG8 high MSCs were sorted from cultivated human UC-MSCs, and their elevated BAMBI and MFEG8 expression levels were confirmed to persist through three to four passages. The frequency of BAMBI high MFEG8 high MSCs varied based on the donor and dissociation method.