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, 22 (12), 1789-801

Clinical-grade Mesenchymal Stromal Cells Produced Under Various Good Manufacturing Practice Processes Differ in Their Immunomodulatory Properties: Standardization of Immune Quality Controls

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Clinical-grade Mesenchymal Stromal Cells Produced Under Various Good Manufacturing Practice Processes Differ in Their Immunomodulatory Properties: Standardization of Immune Quality Controls

Cedric Menard et al. Stem Cells Dev.

Abstract

Clinical-grade mesenchymal stromal cells (MSCs) are usually expanded from bone marrow (BMMSCs) or adipose tissue (ADSCs) using processes mainly differing in the use of fetal calf serum (FCS) or human platelet lysate (PL). We aimed to compare immune modulatory properties of clinical-grade MSCs using a combination of fully standardized in vitro assays. BMMSCs expanded with FCS (BMMSC-FCS) or PL (BMMSC-PL), and ADSC-PL were analyzed in quantitative phenotypic and functional experiments, including their capacity to inhibit the proliferation of T, B, and NK cells. The molecular mechanisms supporting T-cell inhibition were investigated. These parameters were also evaluated after pre-stimulation of MSCs with inflammatory cytokines. BMMSC-FCS, BMMSC-PL, and ADSC-PL displayed significant differences in expression of immunosuppressive and adhesion molecules. Standardized functional assays revealed that resting MSCs inhibited proliferation of T and NK cells, but not B cells. ADSC-PL were the most potent in inhibiting T-cell growth, a property ascribed to interferon-γ-dependent indoleamine 2,3-dioxygenase activity. MSCs did not stimulate allogeneic T cell proliferation but were efficiently lysed by activated NK cells. The systematic use of quantitative and reproducible validation techniques highlights differences in immunological properties of MSCs produced using various clinical-grade processes. ADSC-PL emerge as a promising candidate for future clinical trials.

Figures

FIG. 1.
FIG. 1.
Comparative phenotype of ADSCs expanded with PL (ADSC-PL), BMMSCs expanded with PL (BMMSC-PL), and FCS (BMMSC-FCS). Thawed MSCs were collected at the end of P2 and stained with appropriate antibodies (gray histogram) or isotype-matched controls (black histogram). The ratio of mean fluorescence intensity (rMFI) is indicated on the top right of each panel. One example representative of the 15 MSC batches is shown for commonly expressed markers (A). For differentially expressed markers, data for all MSC batches were shown. Bars: median (B). ADSCs, mesenchymal stromal cells expanded from adipose tissue; BMMSCs, mesenchymal stromal cells expanded from bone marrow; PL, platelet lysate; FCS, fetal calf serum; P2, passage 2.
FIG. 2.
FIG. 2.
Phenotypic modifications induced on MSCs by inflammatory stimuli. (A) Thawed MSCs were stimulated by IFN-γ and TNF-α for 40 h before staining with appropriate antibodies (gray histogram) or isotype-matched controls (black histogram). The ratio of rMFI is indicated on the top right of each panel. One example representative of the 15 MSC batches is shown. (B) The global expression of NK-activating ligands on pMSCs (n=15) was obtained by combining the individual rMFI from MICA/B, ULPB-1, ULBP-2, ULBP-3, CD112, and CD155. This activating profile was then analyzed compared to the level of expression of HLA-ABC, the main NK inhibitory ligand. *p<0.05; **p<0.01. IFN-γ, interferon-γ; TNF-α, tumor necrosis factor-α; pMSCs, primed MSCs.
FIG. 3.
FIG. 3.
Expression of immunosuppressive molecules on resting and pMSCs. Thawed MSCs were stimulated or not by IFN-γ and TNF-α for 40 h, and INDO, PTGS2, NOS2, and TNFAIP6 mRNA expression was measured by RQ-PCR in ADSC-PL (n=5), BMMSC-PL (n=5), and BMMSC-FCS (n=5) and their corresponding primed counterpart. The arbitrary value of 1 was assigned to a pool of PBMCs. INDO and NOS2 expression was undetectable in resting MSC samples. *p<0.05; **p<0.01. RQ-PCR, real-time quantitative polymerase chain reaction; PBMCs, peripheral blood mononuclear cells.
FIG. 4.
FIG. 4.
Immune properties of ADSC-PL, BMMSC-PL, and BMMSC-FCS. (A) Inhibition of T-, B-, and NK cell proliferation by primed and resting ADSC-PL (n=5), BMMSC-PL (n=5), and BMMSC-FCS (n=5) was assessed by the CFSE dilution method. Data are expressed as the percentage of inhibition of immune cell proliferation. (B) Responding T-cells were stimulated with irradiated resting allogeneic MSCs (five ADSC-PL, two BMMSC-PL, and three BMMSC-FCS batches all used at a 1:1 MSC:T ratio), with irradiated autologous PBMCs (auto-MLR) as negative control, and with irradiated allogeneic PBMCs (allo-MLR) and anti-CD3/anti-CD28 antibodies as positive controls. Each experiment was performed in sixplicate culture wells. Proliferation was assessed by the incorporation of tritiated thymidine (3H-TdR). Results represent the mean±SD of the 10 experiments. (C) Lysis of resting versus pMSCs (three ADSC-PL, two BMMSC-PL, and two BMMSC-FCS batches) by activated NK cells was assessed in a standard cytotoxicity assay. Results are represented as the mean for each MSC subtype. *p<0.05, **p<0.01, ***p<0.001. CFSE; carboxyfluorescein succinimidyl ester.
FIG. 5.
FIG. 5.
IDO is involved in the inhibition of T-cell proliferation by MSCs unlike NOS and PGE2. (A) Resting MSCs (n=5) were cocultured at a 10 T:1 MSC ratio with CFSE-labeled purified T-cells stimulated with anti-CD3/anti-CD28 antibodies in the presence or not of l-N-monomethyl arginine (L-NMMA), l-1-methyltryptophan (L-1MT), or NS398 to inhibit iNOS, IDO-1, or Cox-2 activity, respectively, or in the presence of an IFN-γ-blocking antibody. T-cell proliferation was evaluated at day 6, and data are expressed relatively to T-cells alone (assigned to 100%). Results are expressed as mean±SD of the five experiments. NS: not significant; **p<0.01. (B) Representative example of IDO blockade in coculture of activated T-cells with one ADSC-PL and one BMMSC-FCS. The IDO-2 inhibitor D-1MT was used as a negative control. IDO, indoleamine-2,3-dioxygenase; iNOS, inducible nitric oxide synthase; PGE2, prostaglandin-E2.
FIG. 6.
FIG. 6.
Correlation between IDO activity and T-cell inhibition. MSCs (n=15) were stimulated or not by IFN-γ+TNF-α for 40 h. Culture supernatants were collected for IDO activity quantification, and MSCs were cocultured with CFSE-labeled purified T-cells stimulated with anti-CD3/anti-CD28 antibodies for 6 days. (A) The IDO activity of pMSCs was evaluated as the ratio of kynurenine/tryptophan levels as determined by high-performance liquid chromatography. Open symbols correspond to MSC batches that inhibit T-cell proliferation by <50%. (B) The correlation between IDO activity produced by a given pMSCs and the capacity of corresponding resting MSCs to inhibit T cell proliferation was determined by a two-tailed Spearman's test. Each symbol corresponds to a different MSC batch, with circles representing ADSC-PL (n=5); squares, BMMSC-PL (n=5); and triangles, BMMSC-FCS (n=5).

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