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Mesenchymal stromal cells improve salivary function and reduce lymphocytic infiltrates in mice with Sjögren's-like disease.

Khalili S, Liu Y, Kornete M, Roescher N, Kodama S, Peterson A, Piccirillo CA, Tran SD - PLoS ONE (2012)

Bottom Line: Both MSC and MSC+CFA groups prevented loss of saliva flow and reduced lymphocytic infiltrations in SGs.MSC-therapy alone reduced inflammation (TNF-α, TGF-β), but the combination of MSC+CFA reduced inflammation and increased the regenerative potential of SGs (FGF-2, EGF).The combined use of MSC+CFA was effective in both preventing saliva secretion loss and reducing lymphocytic influx in salivary glands.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Dentistry, McGill University, Montreal, Quebec, Canada.

ABSTRACT

Background: Non-obese diabetic (NOD) mice develop Sjögren's-like disease (SS-like) with loss of saliva flow and increased lymphocytic infiltrates in salivary glands (SGs). There are recent reports using multipotent mesenchymal stromal cells (MSCs) as a therapeutic strategy for autoimmune diseases due to their anti-inflammatory and immunomodulatory capabilities. This paper proposed a combined immuno- and cell-based therapy consisting of: A) an injection of complete Freund's adjuvant (CFA) to eradicate autoreactive T lymphocytes, and B) transplantations of MSCs to reselect lymphocytes. The objective of this was to test the effectiveness of CD45(-)/TER119(-) cells (MSCs) in re-establishing salivary function and in reducing the number of lymphocytic infiltrates (foci) in SGs. The second objective was to study if the mechanisms underlying a decrease in inflammation (focus score) was due to CFA, MSCs, or CFA+MSCs combined.

Methodology/principal findings: Donor MSCs were isolated from bones of male transgenic eGFP mice. Eight week-old female NOD mice received one of the following treatments: insulin, CFA, MSC, or CFA+MSC (combined therapy). Mice were followed for 14 weeks post-therapy. CD45(-)/TER119(-) cells demonstrated characteristics of MSCs as they were positive for Sca-1, CD106, CD105, CD73, CD29, CD44, negative for CD45, TER119, CD11b, had high number of CFU-F, and differentiated into osteocytes, chondrocytes and adipocytes. Both MSC and MSC+CFA groups prevented loss of saliva flow and reduced lymphocytic infiltrations in SGs. Moreover, the influx of T and B cells decreased in all foci in MSC and MSC+CFA groups, while the frequency of Foxp3(+) (T(reg)) cell was increased. MSC-therapy alone reduced inflammation (TNF-α, TGF-β), but the combination of MSC+CFA reduced inflammation and increased the regenerative potential of SGs (FGF-2, EGF).

Conclusions/significance: The combined use of MSC+CFA was effective in both preventing saliva secretion loss and reducing lymphocytic influx in salivary glands.

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Functional assays for multipotent mesenchymal stromal cell (MSCs).Photomicrographs of CD45−/TER119− cells that undergone chondrocytic, osteogenic and adipocytic differentiation when cultured in differentiation media. Lower right photomicrograph represents CD45−/TER119− cells cultured in non-differentiating media (negative control). Collagen II (red) and DAPI (blue nuclei) are used to identify chondrocytes. Von kossa (black) and oil red stainings were used to identify osteoblasts and adipocytes, respectively. Graphs represent the number, size, and frequency of colony-forming unit fibroblasts (CFU-F) of CD45−/TER119− cells versus whole bone marrow cells. All three graphs show that CD45−/TER119− cells had higher CFU-F than whole bone marrow cells (* P<0.05).
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pone-0038615-g007: Functional assays for multipotent mesenchymal stromal cell (MSCs).Photomicrographs of CD45−/TER119− cells that undergone chondrocytic, osteogenic and adipocytic differentiation when cultured in differentiation media. Lower right photomicrograph represents CD45−/TER119− cells cultured in non-differentiating media (negative control). Collagen II (red) and DAPI (blue nuclei) are used to identify chondrocytes. Von kossa (black) and oil red stainings were used to identify osteoblasts and adipocytes, respectively. Graphs represent the number, size, and frequency of colony-forming unit fibroblasts (CFU-F) of CD45−/TER119− cells versus whole bone marrow cells. All three graphs show that CD45−/TER119− cells had higher CFU-F than whole bone marrow cells (* P<0.05).

Mentions: Multiparameter flow cytometry was used to characterize CD45−/TER119− cells freshly isolated from the bones of eGFP mice (before cell culture; Figure S3), and then again after three passages in culture (before cell transplantation procedures; Figure 6). An antibody panel to identify mouse MSCs consisting of positive markers for Sca-1, CD106, CD105, CD73, CD29, CD44 and negative markers for CD45, TER119 and CD11b was used. Flow cytometry results (based on 3 experiments; mean± SD) of freshly isolated CD45−/TER119− cells demonstrated following frequencies; 70.75%±11.52 CD45−, 98.45%±0.63 TER119−, 97.50%±0.56 CD11b− and 46.85%±3.04 Sca1+, 62.75%±2.75 CD106+, 55.0%±3.67 CD105+, 47.7%±0.56 CD73+, 87.7%±2.82 CD29+, 40.9%±4.52 CD44+ (Figure S3). After three passages in culture, these adherent CD45−/TER119− cells were enriched to 96.1%±5.09 CD45−, 97.93%±2.80 TER119−, 99.00%±0.84 CD11b−, and 83.45%±1.34 Sca1+, 73.72%±13.34 CD106+, 60.44%±9.91 CD105+, 23.92%±6.55 CD73+, 87.85%±2.05 CD29+, 84.45%±4.59 CD44+ (Figure 6). The number, size and frequency of colony-forming unit fibroblasts (CFU-F) were statistically higher in CD45−/TER119− cells than unfractionated bone marrow cells (Figure 7; P<0.05). CD45−/TER119− cells underwent osteogenic, chodrocytic and adipocytic differentiation when cultured in differentiating media (Figure 7). All these characteristics indicated that transplanted CD45−/TER119− cells were multipotent mesenchymal stromal cells (MSCs).


Mesenchymal stromal cells improve salivary function and reduce lymphocytic infiltrates in mice with Sjögren's-like disease.

Khalili S, Liu Y, Kornete M, Roescher N, Kodama S, Peterson A, Piccirillo CA, Tran SD - PLoS ONE (2012)

Functional assays for multipotent mesenchymal stromal cell (MSCs).Photomicrographs of CD45−/TER119− cells that undergone chondrocytic, osteogenic and adipocytic differentiation when cultured in differentiation media. Lower right photomicrograph represents CD45−/TER119− cells cultured in non-differentiating media (negative control). Collagen II (red) and DAPI (blue nuclei) are used to identify chondrocytes. Von kossa (black) and oil red stainings were used to identify osteoblasts and adipocytes, respectively. Graphs represent the number, size, and frequency of colony-forming unit fibroblasts (CFU-F) of CD45−/TER119− cells versus whole bone marrow cells. All three graphs show that CD45−/TER119− cells had higher CFU-F than whole bone marrow cells (* P<0.05).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369846&req=5

pone-0038615-g007: Functional assays for multipotent mesenchymal stromal cell (MSCs).Photomicrographs of CD45−/TER119− cells that undergone chondrocytic, osteogenic and adipocytic differentiation when cultured in differentiation media. Lower right photomicrograph represents CD45−/TER119− cells cultured in non-differentiating media (negative control). Collagen II (red) and DAPI (blue nuclei) are used to identify chondrocytes. Von kossa (black) and oil red stainings were used to identify osteoblasts and adipocytes, respectively. Graphs represent the number, size, and frequency of colony-forming unit fibroblasts (CFU-F) of CD45−/TER119− cells versus whole bone marrow cells. All three graphs show that CD45−/TER119− cells had higher CFU-F than whole bone marrow cells (* P<0.05).
Mentions: Multiparameter flow cytometry was used to characterize CD45−/TER119− cells freshly isolated from the bones of eGFP mice (before cell culture; Figure S3), and then again after three passages in culture (before cell transplantation procedures; Figure 6). An antibody panel to identify mouse MSCs consisting of positive markers for Sca-1, CD106, CD105, CD73, CD29, CD44 and negative markers for CD45, TER119 and CD11b was used. Flow cytometry results (based on 3 experiments; mean± SD) of freshly isolated CD45−/TER119− cells demonstrated following frequencies; 70.75%±11.52 CD45−, 98.45%±0.63 TER119−, 97.50%±0.56 CD11b− and 46.85%±3.04 Sca1+, 62.75%±2.75 CD106+, 55.0%±3.67 CD105+, 47.7%±0.56 CD73+, 87.7%±2.82 CD29+, 40.9%±4.52 CD44+ (Figure S3). After three passages in culture, these adherent CD45−/TER119− cells were enriched to 96.1%±5.09 CD45−, 97.93%±2.80 TER119−, 99.00%±0.84 CD11b−, and 83.45%±1.34 Sca1+, 73.72%±13.34 CD106+, 60.44%±9.91 CD105+, 23.92%±6.55 CD73+, 87.85%±2.05 CD29+, 84.45%±4.59 CD44+ (Figure 6). The number, size and frequency of colony-forming unit fibroblasts (CFU-F) were statistically higher in CD45−/TER119− cells than unfractionated bone marrow cells (Figure 7; P<0.05). CD45−/TER119− cells underwent osteogenic, chodrocytic and adipocytic differentiation when cultured in differentiating media (Figure 7). All these characteristics indicated that transplanted CD45−/TER119− cells were multipotent mesenchymal stromal cells (MSCs).

Bottom Line: Both MSC and MSC+CFA groups prevented loss of saliva flow and reduced lymphocytic infiltrations in SGs.MSC-therapy alone reduced inflammation (TNF-α, TGF-β), but the combination of MSC+CFA reduced inflammation and increased the regenerative potential of SGs (FGF-2, EGF).The combined use of MSC+CFA was effective in both preventing saliva secretion loss and reducing lymphocytic influx in salivary glands.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Dentistry, McGill University, Montreal, Quebec, Canada.

ABSTRACT

Background: Non-obese diabetic (NOD) mice develop Sjögren's-like disease (SS-like) with loss of saliva flow and increased lymphocytic infiltrates in salivary glands (SGs). There are recent reports using multipotent mesenchymal stromal cells (MSCs) as a therapeutic strategy for autoimmune diseases due to their anti-inflammatory and immunomodulatory capabilities. This paper proposed a combined immuno- and cell-based therapy consisting of: A) an injection of complete Freund's adjuvant (CFA) to eradicate autoreactive T lymphocytes, and B) transplantations of MSCs to reselect lymphocytes. The objective of this was to test the effectiveness of CD45(-)/TER119(-) cells (MSCs) in re-establishing salivary function and in reducing the number of lymphocytic infiltrates (foci) in SGs. The second objective was to study if the mechanisms underlying a decrease in inflammation (focus score) was due to CFA, MSCs, or CFA+MSCs combined.

Methodology/principal findings: Donor MSCs were isolated from bones of male transgenic eGFP mice. Eight week-old female NOD mice received one of the following treatments: insulin, CFA, MSC, or CFA+MSC (combined therapy). Mice were followed for 14 weeks post-therapy. CD45(-)/TER119(-) cells demonstrated characteristics of MSCs as they were positive for Sca-1, CD106, CD105, CD73, CD29, CD44, negative for CD45, TER119, CD11b, had high number of CFU-F, and differentiated into osteocytes, chondrocytes and adipocytes. Both MSC and MSC+CFA groups prevented loss of saliva flow and reduced lymphocytic infiltrations in SGs. Moreover, the influx of T and B cells decreased in all foci in MSC and MSC+CFA groups, while the frequency of Foxp3(+) (T(reg)) cell was increased. MSC-therapy alone reduced inflammation (TNF-α, TGF-β), but the combination of MSC+CFA reduced inflammation and increased the regenerative potential of SGs (FGF-2, EGF).

Conclusions/significance: The combined use of MSC+CFA was effective in both preventing saliva secretion loss and reducing lymphocytic influx in salivary glands.

Show MeSH
Related in: MedlinePlus