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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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Related in: MedlinePlus

Flow cytometry analysis of CD45−/Ter119− cells.After 3 passages, CD45−/Ter119− cells were stained with the following surface markers: CD45, Ter119, CD11B, Sca-1, CD106, CD105, CD73, CD29 and CD44. Data are representative of at least three separate experiments. This experiment shows 97.8% CD45−, 99.7% TER119−, 99.6% CD11b− and 84.4% Sca1+, 86.5% CD106+, 64.1% CD105+, 19.3% CD73+, 86.4% CD29+, 81.2% CD44+ cells.
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pone-0038615-g006: Flow cytometry analysis of CD45−/Ter119− cells.After 3 passages, CD45−/Ter119− cells were stained with the following surface markers: CD45, Ter119, CD11B, Sca-1, CD106, CD105, CD73, CD29 and CD44. Data are representative of at least three separate experiments. This experiment shows 97.8% CD45−, 99.7% TER119−, 99.6% CD11b− and 84.4% Sca1+, 86.5% CD106+, 64.1% CD105+, 19.3% CD73+, 86.4% CD29+, 81.2% CD44+ cells.

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)

Flow cytometry analysis of CD45−/Ter119− cells.After 3 passages, CD45−/Ter119− cells were stained with the following surface markers: CD45, Ter119, CD11B, Sca-1, CD106, CD105, CD73, CD29 and CD44. Data are representative of at least three separate experiments. This experiment shows 97.8% CD45−, 99.7% TER119−, 99.6% CD11b− and 84.4% Sca1+, 86.5% CD106+, 64.1% CD105+, 19.3% CD73+, 86.4% CD29+, 81.2% CD44+ cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038615-g006: Flow cytometry analysis of CD45−/Ter119− cells.After 3 passages, CD45−/Ter119− cells were stained with the following surface markers: CD45, Ter119, CD11B, Sca-1, CD106, CD105, CD73, CD29 and CD44. Data are representative of at least three separate experiments. This experiment shows 97.8% CD45−, 99.7% TER119−, 99.6% CD11b− and 84.4% Sca1+, 86.5% CD106+, 64.1% CD105+, 19.3% CD73+, 86.4% CD29+, 81.2% CD44+ cells.
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