A procedure for in vitro evaluation of the immunosuppressive effect of mouse mesenchymal stem cells on activated T cell proliferation

Abstract

Background: Mesenchymal stem/stromal cells (MSC) represent adult cells with multipotent capacity. Besides their capacity to differentiate into multiple lineages in vitro and in vivo, increasing evidence points towards the immunomodulatory capacity of these cells, as an important feature for their therapeutic power. Although not included in the minimal criteria established by the International Society for Cellular Therapy as a defining MSC attribute, demonstration of the immunomodulatory capacity of MSC can be useful for the characterization of these cells before being considered MSC.

Methods: Here we present a simple and reliable protocol by which the immunosuppressive effect of mouse bone marrow-derived MSC can be evaluated in vitro. It is based on the measuring of the proliferation of activated T cells cultured in direct contact with irradiated MSC.

Results: Our results showed that mouse MSC have a dose-dependent inhibitory effect on activated T cell proliferation, which can be quantified as a percentage of maximum proliferation. Our data shows that batch-to-batch variability can be determined within one or multiple experiments, by extracting the area under curve of T cell proliferation plotted against the absolute number of MSC in co-culture.

Conclusions: The validation of the immunosupressive capacity of MSC could be added to the characterization of the cells before being used in various MSC-based approaches to treat immunological diseases. Our results showed that mouse MSC have a dose-dependent inhibitory effect on activated T cell proliferation. The immunosuppressive properties of MSC vary between batches, but not between different passages of the same batch.

Keywords: CFSE; Cell culture; Flow cytometry; Immunosuppression; Mesenchymal stem cells; Nylon wool; Proliferation index; Splenocytes; T cells.

Fig. 1

MSC characterization in accordance to International Society for Cellular Therapy. a Adipogenic, osteogenic, and chondrogenic differentiation of MSC, as comparisons between cells cultured in the presence of appropriate differentiation factors and normal culture medium (non-differentiating factors). Exception is chondrogenic differentiation, for which no pellet was formed in the absence of chondrocyte-specific differentiation medium. b–d Representative fields of differentiated MSC by Oil red (b), von Kossa (c), and Alcian Blue (d) staining, demonstrating the capacity of MSC to generate adipocytes (b), osteoblasts (c), and chondroblasts (d), respectively, when cultured in appropriate differentiation media for three weeks. e Phenotypic characterization of MSC by flow cytometry, illustrating the lack of CD45 expression and the expression of Sca-1, CD29, CD105, and CD90

Fig. 2

Schematic design of the main steps for T cell isolation and CFSE labeling. a Isolation of mouse splenocytes and T cell enrichment on nylon wool column. The critical step of CFSE staining of T cells is also shown. b Flow cytometry characterization of splenocytes (upper line), T cells (middle line) and CFSE-labeled T cells (lower line). Note the depletion in B220^pos cells in T cell enriched population, as compared to whole splenocyte population (~ 8% B cells after enrichment, as compared to ~ 60% in the initial population) and the relatively unchanged CD8:CD4 ratio after enrichment. Also note the sharp fluorescent peak of T cell population obtained after CFSE staining

Fig. 3

a The 96-well plate template for experimental design. Thirty wells are required for testing one MSC batch, of which 12 wells serve as controls (the first two lines of the plate). Each additional MSC batch will require another 18 wells, which can be aligned as triplicate columns after the first batch. A total of four different batches can be analyzed in one 96-well plate. b Flow cytometry analysis of the T cell population in the negative and positive controls, at the end of 3-day co-culture experiment. The gating strategy consists of removal of debris, doublets, and dead cells and analysis of the viable cells on the CFSE histogram. Multiple fluorescent peaks are distinguished in the positive control (activated T cells), as compared to the single peak in the negative control (not activated T cells). c Phase-contrast microscopy images of irradiated MSC, resting T cells and activated T cells in culture (in the presence or absence of MSC). Note the integrity of the confluent layer of irradiated MSC after 4 days in culture (MSC only) and the lower number of T cells in the presence of irradiated MSC (MSC +T cells + beads vs. T cells + beads)

Fig. 4

a, b T cell proliferation in the presence of MSC. a Histograms illustrate the CFSE fluorescent peaks of T cell samples activated in the presence of increasing amount of MSC. Note the single fluorescent peak in resting T cells, representing the parent population. Also note the diminishing of the parent population peak with the increasing number of MSC in the culture, concomitant with the increasing in the magnitude of the low-fluorescent peaks, as a result of dye dilution with each cell proliferation. b The diagram shows the decrease of the proliferation index (PI) of activated T cells in the presence of MSC in a dose-dependent manner. The data illustrate mean ± SD of one representative experiment performed in triplicate. c ELISA quantification of IFNγ in the secretome of co-culture. The data represent the mean ± SD of one experiment performed in quadruplicates with two MSC batches

Fig. 5

Dose-dependent inhibition of T cell proliferation by MSC. a The comparative illustration of the effect of actively proliferating and irradiated MSC on T cell proliferation. Note the loss of the dose-dependent effect of proliferative MSC on the inhibition of T cell proliferation. b Comparative analysis of the immunosuppressive effect of a batch of MSC at a low and a higher passage. Note that these MSC properties are not modified with increasing the passage number. The experiment represents one representative experiment from at least 6 experiments performed with different MSC batches. c Comparative analysis of the immunosuppressive properties of 4 batches of MSC measured in separate experiments. The comparison was done by reporting the proliferation as percentage (± SD) of maximum proliferation obtained in individual experiments. By plotting the proliferation against the dose of MSC, the area under curve can be calculated, which makes possible the comparison between the immunosuppressive properties of different batches. The lower the AUC, the higher the immunosuppressive effects