Single-Cell Sorting of Immunophenotyped Mesenchymal Stem Cells from Human Exfoliated Deciduous Teeth

This protocol describes the use of fluorescence-activated cell sorting of human mesenchymal stem cells using the single-cell sorting method. Specifically, the use of single-cell sorting can achieve 99% purity of the immunophenotyped cells from a heterogeneous population when combined with a multiparametric flow cytometry-based approach.

This protocol details a crucial method for accurately characterizing and purifying human mesenchymal stem cells on a single platform, enabling efficient downstream applications like stem cell transplantation. It is particularly significant for clinical labs enhancing molecular and cytogenetic assays through precise single cell sorting of engineered or natural stem cells. The commonly used methods of isolation of purified stem cell populations refer to techniques such as immunomagnetic separation, density gradient, or microfluidic cell sorting.

A technique like index sorting allows it to be linked to single cell RNA profiling that can extract transcriptomic information of each sorted cell. The key challenges involve maintaining sample quality, optimization of cell culture conditions, including cell density and viability during and after sorting is required. Additionally, choosing suitable nozzle sizes for the instrument and ensuring timely access to the sorters would also be considered.

This protocol illustrates a precise staining panel for immunophenotyping and sorting the desired cell population. It ensures standardized sample quality, optimal cell viability density for our cell population sorting and define sorting experimental parameters on the FACSDiva software. This method enables a straightforward selection and immunophenotyping of stem cell population expressing specific biomarkers.

This simple platform allows efficient sorting based on the target population, aiding and obtaining purified samples for subsequent downstream applications. To begin, seed mesenchymal stem cells into T75 flasks, 100 mm dishes and two 48 well plates for all the experiments. Add 200 microliters of 10%MSC culture media to the wells of the 48 well plates.

When the cell monolayer reaches 90%confluency, replace the existing media of the designated control wells with 2%serum media. Add differentiation media of either adipogenic or osteogenic lineage into the last two wells labeled test. On the same day, trypsinize a confluent 100 mm dish and transfer the cell suspension into two labeled 15 milliliter tubes.

Centrifuge the tubes at 300 G for six minutes. Then, pipette two milliliters of the respective media into the control and test tubes. Place the loosely capped tubes in the incubator at 37 degrees Celsius under 5%carbon dioxide for 21 days, with media replacement every three days.

The SHEDs were characterized with standard immunofluorescence assays showing the expression of vimentin, actin filaments, and nuclei stained with DAPI. Short-term cell growth assays showed an increased proliferation rate from day two to day eight. Colony forming unit assay and crystal violet staining confirmed the colony forming ability of SHEDs after 14 days.

To stain the 2D adipocyte lineage, add 200 microliters of permeabilization solution from kit to the plate. Next, incubate the cells in 200 microliters of Oil Red O working solution for 10 minutes. Subsequently, wash the cells five times with 200 microliters of distilled water after removing the stain.

To stain the 2D osteoblast lineage, add 200 microliters of 5%fresh silver nitrate to each well. Then, place the plate under ultraviolet light for an hour. Incubate the cells in 200 microliters of 2.5%sodium thiosulfate for five minutes before a distilled water rinse.

For the 3D cultures, first obtain the cryo sections of the pelleted cultures. To stain the cells of the chondrocyte lineage, add one to two milliliters of washing solution to the air dried sections. Next, incubate them in fixing solution for 30 minutes.

After washing with distilled water, place the cells in one to two milliliters staining solution for 30 minutes. Rinse the cells three times with 0.1 normal hydrochloric acid before rinsing with distilled water. After 21 days, the MSCs under in vitro conditions differentiated into adipocytes, osteoblasts and condregenic lineages.

To begin, centrifuge the trypsinized mesenchymal stem cells at 300 G for six minutes to obtain the cell pellet. Next, resuspend the pellet in one milliliter of MSC media before cell counting. Centrifuge the cell suspension again.

Then, resuspend the pellet in an appropriate volume of the staining buffer, after discarding the supernatant. For the preparation of compensation controls, label seven FACS tubes as unstained, DAPI, V450, FIT, PE, PerCP Cy5.5, and APC. Next, prepare the single stain tubes for compensation and incubate them in the dark for 30 minutes.

Following incubation, wash the cells in each tube two times and the beads one time with one milliliter of staining buffer. Next, centrifuge the vortexed tubes at 200 G for 10 minutes at room temperature. After discarding the supernatant, resuspend the pellet in 500 microliters of staining buffer.

Incubate the DAPI tube in a 60 degree Celsius water bath for five minutes, followed by a 15 minute incubation on ice to induce the inclusion of the dye. Add five microliters of DAPI to the suspension and incubate until acquisition. Prepare the cell and antibody suspension in five labeled FACS tubes as per the table.

Gently vortex the tubes before incubation. Then, wash each tube two times with one milliliter of the staining buffer. Centrifuge the vortexed tube at 200 G for 10 minutes at room temperature.

Then, resuspend the remaining pellet in 500 microliters of staining buffer. To begin, label two new FACS tubes as mixed tubes one and two. Prepare the cell and antibody suspension as per the given table for flow cytometric analysis.

After incubating the tubes for 30 minutes, wash each tube two times with one milliliter of staining buffer. Centrifuge the vortexed tubes at 200 G for 10 minutes at room temperature. Then, resuspend the pellet in 500 microliters of staining buffer.

Coat the wells of a 48 well plate with 200 to 500 microliters of FBS and keep for one hour. Then pipette 200 to 500 microliters of 10%MSC media after removing the residual FBS. To prepare the samples for single cell sorting, pipette one milliliter of FBS into two new FACS tubes.

Roll the tubes to create an even coat of the FBS on the tube's inner surface. Prepare the cell and antibody suspension in mixed tubes one and two as per the table for the sorting experiment. Then, incubate the tubes in the dark for 30 minutes at room temperature.

After washing with staining buffer, centrifuge the tubes at 200 G for 10 minutes at room temperature. Then, resuspend the pellet in 500 microliters of staining buffer and incubate for 15 minutes in five microliters of DAPI before sorting. To set up the compensation matrix, use the single stain compensation tubes.

Click on experiment from the toolbar in the instrument software. Then, click on compensation setup, followed by create compensation controls. Next, click on unstained tube, followed by parameters in the cytometer dialogue box.

Adjust the voltages by editing the values for each parameter. Click on load in the acquisition dashboard and then click acquire. Drag the P1 gate to the cell population and apply it across all compensation controls to set the voltage and negative gate for each parameter.

Now, load the single stain compensation tubes individually. Record the data and save it. Select the current experiment, right click on it, and select calculate compensation values followed by link and save.

Run the mixed tube one and record 10, 000 cells. Set the gates on the population of interest to be sorted with the proper gating strategy. Next, load a collection plate into the instrument and set a target cell number ranging from 2, 500 to 5, 000 cells per well.

Then, select the single cell sort purity mask. Collect the sorted cell populations in a collection device, keeping them on ice for the duration of the sorting experiment. Finally, shift the plates into a 5%carbon dioxide incubator to maintain the cultures at 37 degrees Celsius.

The multiparametric flow cytometry assays plots showed no expression of the FITC isotype of CD90 FITC antibodies. Positive expression of the markers CD90 and CD73 and negative expression of CD45 are comparable with that of their unstained and FMO controls. The immuno phenotypes showed the marker distribution from one of the early passages with the ISCT recommended markers and CD44, determining the parent populations as MSCs.

The late passage SHEDs were used for single cell sorting of high and dim expressors. The post sorted cells collected in the 48 well plate showed adherence and proliferation like their parent population. The post sorted cells differentiated in the presence of adipogenic growth factors.