Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

Summary

The immunomodulatory properties of human mesenchymal stem cells (MSC) appear increasingly relevant for clinical application. Using a co-culture system of MSCs and peripheral blood leukocytes pre-stained with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE), we describe the in vitro assessment of MSC immunomodulation on effector leukocyte proliferation and specific subpopulations.

Abstract

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

Introduction

Human mesenchymal stem cells (MSCs) are somatic progenitors that can differentiate into the paraxial mesodermal lineages of bone, cartilage, and adipose tissue^1-4, as well as to a few extramesodermal lineages⁵. First isolated from the adult bone marrow, these multilineage progenitors have now been found in numerous tissues^6-8 and, unexpectedly, shown to have strong immunomodulatory properties that appear highly amenable to clinical application^9-12. Detailed mechanisms involved in the immunomodulatory effects are actively being investigated for effective application on specific disease entities. One of the most straightforward ways to evaluate immunomodulation is by assessing for the suppression of effector leukocyte proliferation¹³. Most effector leukocytes such as T lymphocytes and monocytes proliferate prodigiously when stimulated or activated. Immunomodulatory function can be assessed when suppression of proliferation is evidenced.

Traditionally, effector leukocyte proliferation has been evaluated by detection of [³H] thymidine incorporation into DNA. However, this method has significant drawbacks due to the concerns of radiation and post-use disposal, as well as the complex equipment needed. While there are non-radioactive assays to assess cell proliferation, the carboxyfluorescein succinimidyl ester (CFSE) assay has other advantages such as allowing for identification of specific cellular populations, which is especially useful in co-culture experiments involving multiple cell types. CFSE is a fluorescent cellular dye which can be assessed by flow cytometric analysis. As cells divide, the intensity of this cellular label is decreased proportionally; this not only enables determination of overall cell proliferation but also allows for the assessment on the number of cell divisions up to 8 divisions before the fluorescence becomes difficult to detect against background signal. Moreover, the stability of the fluorescent CFSE allows for in vivo tracking of labeled cells such that cells can be visualized up to many months¹⁴.

This assay can also be varied to evaluate specific types of effector leukocytes or the immunomodulatory function of specific populations of MSC-induced immunomodulatory leukocytes-such as interleukin-10 (IL-10) producing CD14⁺ monocytes¹⁵ by performing magnetic bead surface marker selection of cell populations of interest prior to or after co-culture as appropriate. Our protocol describes the basic assay of assessing the immunomodulatory effects of MSCs on effector leukocytes (flow chart shown in Figure 1) and a variation on this basic assay for evaluation of MSC-induced leukocyte immunomodulation on allogeneic CD4⁺ effector T lymphocytes (flow chart shown in Figure 4).

Protocol

Patient informed consent as approved by the institutional review board must be obtained for use of human cells.

1. Density Gradient Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs)

1. Add 25 ml heparinized whole blood into a 50 ml tube by a 25 ml pipette.
   1. Dilute cells with 25 ml of phosphate-buffered saline (PBS).
2. Add 15 ml of Ficoll-Paque density gradient into a new 50 ml tube, and while tilting the tube, very slowly and carefully add in 25 ml of the diluted cell suspension over the density gradient so that there is no mixing of the whole blood with the density gradient, i.e., no disturbance of the blood-density gradient interface.
3. Centrifuge the suspension from step 1.2 at 400 × g for 30 to 40 min in a temperature-controlled swinging-bucket rotor without brake at 20 °C.
   Note: Three distinct layers should be apparent after centrifugation: the upper layer being plasma; the bottom clear layer being the Ficoll-Paque density gradient; and a thin, middle cellular layer being the PBMCs
4. Aspirate and discard the upper layer by suction with a Pasteur pipette, with care not to disturb the interphase layer of mononuclear cells (i.e., lymphocytes, monocytes, and platelets).
5. Carefully collect this mononuclear cell layer with a 10 ml pipette to a new 50 ml centrifuge tube.
6. Fill the tube with 20 ml of PBS, mix well, and centrifuge at 300 × g for 10 min at 20°C. Remove the supernatant completely after centrifugation.
7. Resuspend the cell pellet in 20 ml of PBS and centrifuge at 200 × g for 10-15 min at 20 °C. Remove the supernatant completely after centrifugation.
   Note: This step removes the platelets-which are unwanted-within the PBMCs; this step can be repeated to ensure complete removal of the platelets.
8. If no selection of specific populations is desired, resuspend cell pellet in leukocyte complete medium (10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin/streptomycin in RPMI-1640 medium) for culturing after performing a cell count¹⁶ to suspend 1 ml of medium per 10⁷ PBMCs before proceeding to the next step.

2. Magnetic Labeling of Leukocyte Populations (Skip to STEP 4 if no Selection of Specific Populations is Required)

1. Centrifuge PBMC suspension at 300 × g for 10 min and aspirate supernatant completely.
2. Resuspend cell pellet in 80 µl of PBS per 10⁷ total cells.
3. Add 20 µl of specified magnetic beads (i.e., CD14 magnetic beads for isolation of monocytes, CD4 magnetic beads for isolation of T lymphocytes) per 10⁷ total cells.
4. Mix well and incubate for 15 min at 2 to 8 °C in the refrigerator.
5. Add 1-2 ml of PBS per 10⁷ cells to wash, and centrifuge at 300 × g for 10 min.
6. Aspirate supernatant completely and resuspend up to 10⁸ cells in 500 µl of PBS.

3. Magnetic Selection of Leukocyte Subpopulations

Note: A variety of magnetic bead separators and columns are available for isolation of specific population of leukocytes by cell surface marker from a number of manufacturers. Isolation can be by positive selection based on one or a few positive markers or by negative selection after depletion of unwanted populations. A general approach of performing positive selection using one marker (i.e., CD4 for T helper lymphocytes, or CD14 for monocytes) is outlined below.

1. Prepare and prime magnetic separator including separation column according to manufacturer's instructions.
2. Apply tube containing the sample of labeled PBMCs. Provide two 15 ml centrifuge tubes for collection of the positively labeled and unlabeled cell fractions, following manufacturer's instructions.
3. Count cell number¹⁵ and resuspend the positively selected leukocytes (i.e., CD4⁺ T lymphocytes or CD14⁺ cells) in leukocyte complete medium, using 1 ml of medium per 10⁷ cells.

4. CFSE Staining of Leukocytes for Assessment of Proliferation

Note: CFSE has been widely used in immunological investigations, for both in vivo and in vitro studies. Our protocol has been optimized for in vitro study of MSC/PBMC (or other leukocyte) interactions. While the general steps involved in conducting CFSE in vitro labeling are similar, there may be differences in the specific dose and timing of the protocol¹⁴. This may be due to a number of factors, i.e., the specific cell type used, cell numbers used-which can likely affect the intensity of the CFSE fluorescent signal.

1. Dilute the CFSE stock solution (10 mM) in PBS to the desired working concentration of 10 µM (CFSE-working solution). Prepare 10⁷ cells (i.e., PBMCs or T cells) for labeling with the CFSE-working solution.
2. Centrifuge at 300 × g for 10 min to obtain a cell pellet and aspirate the supernatant.
3. Resuspend the cells gently in 1 ml of pre-warmed (37 °C) CFSE-working solution and incubate the cells for 10 min at 37 °C.
4. To wash off excess CFSE, dilute the cell suspension with 10x (by volume) of precooled (4 °C) RPMI medium containing 10% FBS. Sediment the cells by centrifugation at 300 x g for 5 min and discard the supernatant. Wash the cell pellet in this manner twice more.
5. Re-pellet the cells by centrifugation and count cell number¹⁵. Resuspend 10⁷ cells (either PBMCs or T cells) in 1 ml fresh prewarmed leukocyte complete medium.

5. Co-culture of MSCs with Leukocytes and Activation of Leukocytes

1. Pre-warm MSC complete medium (10% FBS (pretested for optimal MSC growth), 1% L-glutamine, and 1% penicillin/streptomycin in DMEM-low glucose medium) to 37 °C for no more than 30 min.
2. Seed MSCs at 50,000 cells in 1 ml of MSC complete medium in 24-well plates (MSC density: 25,000 cells/cm²) for attachment O/N in a 37 °C incubator, allowing for the stem cells to reach 80% confluence.
3. Aspirate medium, and based on seeded MSC numbers, add CFSE-labeled PBMCs (previously labeled-please refer to protocol Step 4 above) to cultured MSCs (seeded the previous day in 24-well plates) in 1 ml of leukocyte complete medium at a 1:10 (cell ratio) co-culture ratio of MSCs to PBMCs.
4. Add the mitogen phytohemagglutinin (PHA), a non-specific leukocyte activator, to a final concentration of 10 µg/ml in a total volume of 1 ml leukocyte complete medium per well.
   1. Alternatively, to stimulate for the activation of T lymphocytes specifically: use α-CD3/28 microbeads and add to the two-cell co-culture to obtain a bead-to-T lymphocyte ratio of 1:1.
5. For negative control, plate 500,000 PBMCs/well (or a specific effector leukocyte population; cell density: 250,000 leukocytes/cm²) in a 24-well plate with 1 ml leukocyte complete medium only; for positive control, in addition to plating the same number of PBMCs/well, add PHA to a final concentration of 10 µg/ml.
6. On the 3rd and 5th day of the co-culture experiment, assess proliferation of CFSE-labeled leukocytes (placed in round-bottom tubes) by flow cytometric analysis¹⁷ with 488 nm excitation and emission filters appropriate for fluorescein.
   Note: Intracellular cytokine staining for flow cytometric analysis can also be performed to these CFSE-labeled leukocytes at this point to assess for changes in leukocyte cytokine expression profile as modulated by MSCs. Since CFSE is evaluated with a filter appropriate for fluorescein, the antibodies selected to assess various cytokines need to be conjugated to fluorochromes other than fluorescein or similar spectrum (i.e., phycoerythrin, peridinin chlorophyll protein complex (PerCP)).

6. Variation: Effector Suppression Assay Magnetic Bead-selected, MSC-induced Immunomodulatory Leukocytes on Activated CFSE-labeled Effector CD4 ⁺ T Cells

1. Seed MSCs at 250,000 cells in 3 ml of MSC complete medium in 6-well plates (MSC density: 25,000 cells/cm²) for attachment O/N in a 37 °C incubator, allowing for the stem cells to reach 80% confluence.  At least 3 6-well plates are necessary to ensure enough MSC-cocultured PBMCs for subpopulation selection. 
2. Aspirate medium, and add PBMCs separated as per Step 1 but in 6-well plates at 2.5 x 10⁶ cells/well (cell density: 250,000 leukocytes/cm²) with 3 ml of leukocyte complete medium. Co-culture for 48-72 hr in a 37 °C incubator.
3. Magnetic bead-select specific population of MSC-induced immunomodulatory leukocytes (i.e., CD14⁺ cells) as per Sections 2-3.
   Notes: Intracellular cytokine staining for flow cytometric analysis can be performed at this point to assess for changes in leukocyte cytokine expression profile, i.e., expression of interleukin-10-as modulated by MSCs.
4. Add CFSE-labeled allogeneic CD4⁺ T cells generated as per Sections 2-4 in 24-well plates in 1 ml of leukocyte complete medium (T cell density: 250,000 cells/cm²) to bead-selected MSC-induced leukocytes at various ratios, i.e., 1:10, 1:5, 1:2, and 1:1 (cell to cell) ratios.
5. To stimulate CD4⁺ T cells, add α-CD3/28 conjugated microbeads to obtain a bead-to-cell ratio of 1:1.
6. For a negative control, plate 500,000 CD4⁺ T cells/well in a 24-well plate (T cell density: 250,000 cells/cm²) with 1 ml leukocyte complete medium only; for positive control, in addition to plating the same number of CD4⁺ T cells/well, add α-CD3/28 conjugated microbeads to obtain a bead-to-cell ratio of 1:1.
7. On the 3^rd day of co-culture, assess proliferation of CFSE-labeled CD4⁺ T cells (placed in round-bottom tubes) by flow cytometric analysis¹⁶ with 488 nm excitation and emission filters appropriate for fluorescein.

Results

Figure 1 denotes the overall schema of the experiment, and Figure 2 demonstrates the appearance of the various cell culture conditions as visualized by phase-contrast inverted microscopy. MSCs are adherent cells with a fibroblastic, spindle-shaped morphology, whereas PBMCs and leukocytes are small round non-adherent cells. These two morphologically different cell types can be clearly seen in the co-culture. At the end of the assay, when the PBMCs (or leuckotyes) are aspirated for flow cytometric analyses, ev...

Discussion

Increasingly, the immunomodulatory properties of MSCs are being translated into clinical use more rapidly than the multilineage capacity of these stem cells^18-20. Thus, co-culture techniques of MSCs with leukocytes and assays to evaluate immune function are important to further delineate the specific mechanisms involved in these properties for optimizing effective therapeutic application.

One of the most critical technical aspects for success in these assays is having adequate PBMC...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ficoll-Paque PLUS	GE Healthcare	71-7167-00 AG	Density grandient for isolation of peripheral blood mononuclear cells (PBMCs)
Vibrant CFDA-SE Cell Tracer Kit (CFSE)	Life Technologies	V12883	Cellular label for detection of cell division
Phytoagglutinin (PHA)	Sigma	L8902	Activation of human PBMCs
Dynabeads Human T-Activator CD3/28	Life Technologies	111.32D	Activation of human T lymphocytes, e.g. CD4+ T cells, CD8+ T cells, etc.
autoMACS™ Separator	Miltenyi Biotec	autoMACS™ Separator	Magnetic based cell separator
autoMACS® Columns	Miltenyi Biotec	130-021-101	separation columns
CD14 microbeads, human	Miltenyi Biotec	130-050-201	For positive selection of CD14+ human monocytes and macrophages from PBMCs
CD4 microbeads, human	Miltenyi Biotec	130-045-101	For positive selection of CD4+ human T lymphocytes from PBMCs
RPMI 1640 Medium	Life Technologies	11875	Human PBMC/leukocyte culture medium
DMEM, Low glucose, pyruvate	Life Technologies	11885	Human mesenchymal stem cell (MSC) culture medium
L-glutamine	Life Technologies	25030-081	Supplementation for MSC complete medium
Penicillin/Streptomycin	Life Technologies	15070-063	Supplementation for PBMC/leukocyte and MSC complete medium
Fetal bovine serum (FBS)	1) Hyclone, for MSC culture                   2) Life Technologies, for all other cells (i.e. PBMCs, specific leukocyte populations)	1) SH30070.03M 2) 10091-148	Pre-test lots for support of MSC in vitro culture
24-well cell culture plate	Corning	COR3524	Co-culture plate
50 ml centrifuge tube	Corning	430291	Isolation PBMCs from whole blood by Ficoll-Paque PLUS
15 ml centrifuge tube	Corning	430766	Collection of the labeled and unlabeled cell fractions
Round-bottom tubes	BD Falcon	352008	Collection of cells for flow cytometric analysis