Comparison of Immunomodulation Properties of Porcine Mesenchymal Stromal/Stem Cells Derived from the Bone Marrow, Adipose Tissue, and Dermal Skin Tissue

Abstract

Mesenchymal stromal/stem cells (MSCs) demonstrate immunomodulation capacity that has been implicated in the reduction of graft-versus-host disease. Accordingly, we herein investigated the capacity of MSCs derived from several tissue sources to modulate both proinflammatory (interferon [IFN] γ and tumor necrosis factor [TNF] α) and immunosuppressive cytokines (transforming growth factor [TGF] β and interleukin [IL] 10) employing xenogeneic human MSC-mixed lymphocyte reaction (MLR) test. Bone marrow-derived MSCs showed higher self-renewal capacity with relatively slow proliferation rate in contrast to adipose-derived MSCs which displayed higher proliferation rate. Except for the lipoprotein gene, there were no marked changes in osteogenesis- and adipogenesis-related genes following in vitro differentiation; however, the histological marker analysis revealed that adipose MSCs could be differentiated into both adipose and bone tissue. TGFβ and IL10 were detected in adipose MSCs and bone marrow MSCs, respectively. However, skin-derived MSCs expressed both IFNγ and IL10, which may render them sensitive to immunomodulation. The xenogeneic human MLR test revealed that MSCs had a partial immunomodulation capacity, as proliferation of activated and resting peripheral blood mononuclear cells was not affected, but this did not differ among MSC sources. MSCs were not tumorigenic when introduced into immunodeficient mice. We concluded that the characteristics of MSCs are tissue source-dependent and their in vivo application requires more in-depth investigation regarding their precise immunomodulation capacities.

Figure 1

Analysis of alkaline phosphatase (AP) and CD marks in mesenchymal stromal/stem cells (MSCs). For analysis of AP activity, each type of passage 1 MSCs was cultured until ca. 80% confluence on 35 mm dishes, fixed in 3.7% formalin, and stained with a BCIP/NBT kit (A). a, b, and c show A-MSCs, BM-MSCs, and DS-MSCs, respectively, derived from a female micropig. MSCs were observed by both macrography (a–c) after AP staining and micrography before (a-1, b-1, and c-1; scale bars = 500 μm) and after (a-2, b-2, and c-2; scale bars = 200 μm) AP staining. The presence of purple/brown color on AP staining was judged to be a positive reaction. (B) MSC-positive (CD90, 105, and 29) and -negative (CD45) CD marks measured in 1 × 10⁵ cells per sample by flow cytometry. Open black histogram represents the isotype-matched control, and green and red open histograms represent positive CD marks.

Figure 2

In vitro differentiation of passage 3 mesenchymal stromal/stem cells (MSCs). MSCs at ca. 80% confluence were subjected to osteogenic or adipogenic conditions, and differentiation was confirmed by staining with alizarin red S or oil red O solution, respectively. (A) MSCs were observed by both macrography before and after differentiation and micrography after differentiation showing A-MSCs, BM-MSCs, and DS-MSCs, respectively, derived from a female micropig. Scale bars = 200 μm. MSCs were analyzed for the expression of osteogenesis- (RUNX2 and BGLAP) and adipogenesis- (PPARG and LPA) related genes before (B) and at 1-week intervals after (C) differentiation. The housekeeping gene GAPDH was used as a control, shown in (B) and (C).

Figure 3

In vitro proliferation capability of passage 3 mesenchymal stromal/stem cells (MSCs). (A) and (B) display growth rate (absolute growth rate and doubling time in a & b, resp.) parameters of MSCs and the cell cycle, respectively. For analysis of the growth rate, MSCs were seeded at 1 × 10³ cells/well in 24-well tissue culture plates, and the number of cells was calculated at 48 h intervals. For cell cycle analysis, MSCs were seeded at a density of 1 × 10⁵ cells per 35 mm dish, cultured until ca. 80% confluence, and stained with propidium iodide (PI). The first red line, the second blue oblique lines, and the third red-filled peak indicate the G0/G1, S, and G2/M phases, respectively. Experiments shown in (A) and (B) were performed in triplicate.

Figure 4

Analysis of OCT3/4 and TERT in passage 3 mesenchymal stromal/stem cells (MSCs). (A) RT-PCR for OCT4 and TERT. (B) Western blot of OCT3/4 and TERT. Porcine testis and MRC5 cells were used as positive control and negative control for both OCT4 and TERT, respectively. F9 and HeLa cells were used as positive controls for OCT3/4 and TERT, respectively. The internal control was GAPDH for RT-PCR and β-actin for western blot. (C) Protein expression of OCT3/4, measured by flow cytometry, was performed in triplicate ((C)a). ^{a, b, c} P < 0.05. ((C)b) black and green open histograms indicate negative and positive immunoreactivity, respectively.

Figure 5

Expression of immunomodulators in passage 3 mesenchymal stromal/stem cells (MSCs). (A) Histograms of the proinflammatory cytokines IFNγ and TNFα and the immunosuppressive cytokines TGFβ1/2 and IL10 (black, isotype-matched control; blue, positive controls). MRC5 cells were used as negative control for all cytokines. This experiment was performed in triplicate. (B) Expression rate (%). (C) Cytokine secretion based on the results of MRC5 cells in (B) displayed as falling, + (expression), or − (no expression) signs. ++ indicates a significant increase relative to MRC5.

Figure 6

Tumor formation in mesenchymal stromal/stem cells (MSCs) transplanted into immunodeficient mice. (A) and (B) Images obtained after elimination of dorsal hair or clothing, respectively, in each mouse. (a–f) Cells (1 × 10⁷) stained with PKH26 were transplanted to both dorsal subcutaneous spaces of ICR mice (a) or NOD/SCID mice (b–f), and the mice were sacrificed after 15 weeks to confirm tumor formation. ((A)a-b) and ((B)a-b) show mice injected with MDA-MB231 as a positive control and c–f show mice injected with PBS + Matrigel (M), A-MSCs + M, BM-MSCs + M, and DS-MSCs + M, respectively. (C) and (D) display tumors isolated from mouse (A)b (Figures 6(A)-6(b)). ((E)a–c) Immunohistochemical staining and ((E)d) H&E staining were performed in tissue sections from tumors. ((E)a) and ((E)b) Red and blue identify the PKH26-stained membrane of cells and counterstaining of nucleic acids, respectively. Arrows indicate the blood vessels. Scale bars = 500 μm.

Figure 7

Xenogeneic human mixed lymphocyte reaction (MLR) test to confirm immunomodulation capacity of mesenchymal stromal/stem cells (MSCs). Tissue-specific MSCs were isolated from 3 pigs. Peripheral blood mononuclear cells (PBMCs) were cocultured with MSCs in 2 ratios (PBMCs : MSCs of 1 : 1 and 10 : 1). (A) and (B) show the result of the MLR test for each MSC type, by MSC source, based on the average value obtained from the 3 pigs. (C) Cell states at 72 h after cell seeding on 96-well plates. Scale bars = 500 μm.