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Flow cytometric characterization of freshly isolated and culture expanded human synovial cell populations in patients with chronic arthritis.

Van Landuyt KB, Jones EA, McGonagle D, Luyten FP, Lories RJ - Arthritis Res. Ther. (2010)

Bottom Line: Within the hematopoietic CD45-positive populations, we found no differences in relative amounts of macrophages, T-lymphocytes and B-lymphocytes between patient groups.Culture expanded cells were found to fulfill the multipotent mesenchymal stromal cell marker profile, except for CD34 negativity.Detection of peripheral blood macrophage and B-cell markers was decreased after enzymatic exposure and mechanical forces, respectively, but stromal markers were not affected.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Skeletal Development and Joint Disorders, Division of Rheumatology, Katholieke Universiteit Leuven, Herestraat 49, Leuven, B3000, Belgium. Kristel.Vanlanduyt@med.kuleuven.be

ABSTRACT

Introduction: The synovium is a major target tissue in chronic arthritis and is intensively studied at the cellular and molecular level. The aim of this study was to develop flow cytometry for the quantitative analysis of synovial cell populations pre and post culture and to characterize mesenchymal cell populations residing in the inflammatory synovium.

Methods: Knee synovium biopsies from 39 patients with chronic arthritis and from 15 controls were treated in a short, standardized tissue digestion procedure. Stored, thawed digests were routinely analyzed with flow cytometry including live-dead staining and use of the markers CD45, CD3, CD14, CD20, CD34, CD73, CD105, CD90, CD146, CD163 and HLA-DR to distinguish inflammatory and stromal cells. The influence of the digestion method on the detection of the different surface markers was studied separately. In addition, we studied the presence of a specific cell population hypothesized to be mesenchymal stem cells (MSC) based on the CD271 marker. Cell expansion cultures were set up and a MSC-related surface marker profile in passages 3 and 6 was obtained. Immunohistochemistry for CD34 and von Willebrand factor (vWF) was done to obtain additional data on synovium vascularity.

Results: The cell yield and viability normalized to tissue weight were significantly higher in inflammatory arthritis than in controls. Within the hematopoietic CD45-positive populations, we found no differences in relative amounts of macrophages, T-lymphocytes and B-lymphocytes between patient groups. Within the CD45-negative cells, more CD34-positive cells were seen in controls than in arthritis patients. In arthritis samples, a small CD271 positive population was detected. Culture expanded cells were found to fulfill the multipotent mesenchymal stromal cell marker profile, except for CD34 negativity. Detection of peripheral blood macrophage and B-cell markers was decreased after enzymatic exposure and mechanical forces, respectively, but stromal markers were not affected.

Conclusions: Flow cytometry can distinguish synovial cell populations in tissue digests. The preparation method can influence the detection levels of macrophage and B-cell populations. However, stromal cell markers seem not affected and quantification is possible, supporting flow cytometry tissue analysis as a tool to study these cell populations in arthritis.

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Semi-quantitative assessment of vascularization in synovial tissue by immunohistochemistry. Magnification 100×. (a to c) von Willebrand Factor staining. (a) Positive endothelial cells are detected. (b) Isotype control. (c) Positive (sub)endothelial cells and fibroblasts are detected. (d to f) CD34 staining. (e) Isotype control. (c and f) Scores in different patient groups.
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Figure 6: Semi-quantitative assessment of vascularization in synovial tissue by immunohistochemistry. Magnification 100×. (a to c) von Willebrand Factor staining. (a) Positive endothelial cells are detected. (b) Isotype control. (c) Positive (sub)endothelial cells and fibroblasts are detected. (d to f) CD34 staining. (e) Isotype control. (c and f) Scores in different patient groups.

Mentions: As expected, biopsies from patients with inflammatory arthritis contained a significantly larger proportion of hematopoietic cells than the orthopedic controls (median CD45-positivity 79.6% vs. 30%, P < 0.0001) and a smaller proportion of non-hematopoietic cells than the controls (median CD45-negative cells 16.9% vs. 67%, P < 0.0001; Figure 5). Other results from the quantification are depicted in Table 3. No differences were detected concerning the CD45-positive subpopulations. Within CD45-negative cells, the CD90-positive population was not different between RA and SpA samples. The CD34-positive population contains endothelial cells [24]. CD34-expression was higher in the control samples than in arthritis samples (P = 0.034). CD34-expression was also higher in SpA than in RA, however, not statistically significant (P = 0.089). By immunohistochemistry for CD34 and vWF, median scores for CD34 were two in the SpA group and one in the RA group (P = 0.053). CD34 was positive in (sub)endothelium and also in certain stromal elements (Figure 6). There were no significant correlations between stromal/hematopoietic cell composition of the synovial digests and the inflammatory status of the patient as assessed by C-reactive protein level, erythrocyte sedimentation rate or the white blood cell count of the synovial fluid.


Flow cytometric characterization of freshly isolated and culture expanded human synovial cell populations in patients with chronic arthritis.

Van Landuyt KB, Jones EA, McGonagle D, Luyten FP, Lories RJ - Arthritis Res. Ther. (2010)

Semi-quantitative assessment of vascularization in synovial tissue by immunohistochemistry. Magnification 100×. (a to c) von Willebrand Factor staining. (a) Positive endothelial cells are detected. (b) Isotype control. (c) Positive (sub)endothelial cells and fibroblasts are detected. (d to f) CD34 staining. (e) Isotype control. (c and f) Scores in different patient groups.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875643&req=5

Figure 6: Semi-quantitative assessment of vascularization in synovial tissue by immunohistochemistry. Magnification 100×. (a to c) von Willebrand Factor staining. (a) Positive endothelial cells are detected. (b) Isotype control. (c) Positive (sub)endothelial cells and fibroblasts are detected. (d to f) CD34 staining. (e) Isotype control. (c and f) Scores in different patient groups.
Mentions: As expected, biopsies from patients with inflammatory arthritis contained a significantly larger proportion of hematopoietic cells than the orthopedic controls (median CD45-positivity 79.6% vs. 30%, P < 0.0001) and a smaller proportion of non-hematopoietic cells than the controls (median CD45-negative cells 16.9% vs. 67%, P < 0.0001; Figure 5). Other results from the quantification are depicted in Table 3. No differences were detected concerning the CD45-positive subpopulations. Within CD45-negative cells, the CD90-positive population was not different between RA and SpA samples. The CD34-positive population contains endothelial cells [24]. CD34-expression was higher in the control samples than in arthritis samples (P = 0.034). CD34-expression was also higher in SpA than in RA, however, not statistically significant (P = 0.089). By immunohistochemistry for CD34 and vWF, median scores for CD34 were two in the SpA group and one in the RA group (P = 0.053). CD34 was positive in (sub)endothelium and also in certain stromal elements (Figure 6). There were no significant correlations between stromal/hematopoietic cell composition of the synovial digests and the inflammatory status of the patient as assessed by C-reactive protein level, erythrocyte sedimentation rate or the white blood cell count of the synovial fluid.

Bottom Line: Within the hematopoietic CD45-positive populations, we found no differences in relative amounts of macrophages, T-lymphocytes and B-lymphocytes between patient groups.Culture expanded cells were found to fulfill the multipotent mesenchymal stromal cell marker profile, except for CD34 negativity.Detection of peripheral blood macrophage and B-cell markers was decreased after enzymatic exposure and mechanical forces, respectively, but stromal markers were not affected.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Skeletal Development and Joint Disorders, Division of Rheumatology, Katholieke Universiteit Leuven, Herestraat 49, Leuven, B3000, Belgium. Kristel.Vanlanduyt@med.kuleuven.be

ABSTRACT

Introduction: The synovium is a major target tissue in chronic arthritis and is intensively studied at the cellular and molecular level. The aim of this study was to develop flow cytometry for the quantitative analysis of synovial cell populations pre and post culture and to characterize mesenchymal cell populations residing in the inflammatory synovium.

Methods: Knee synovium biopsies from 39 patients with chronic arthritis and from 15 controls were treated in a short, standardized tissue digestion procedure. Stored, thawed digests were routinely analyzed with flow cytometry including live-dead staining and use of the markers CD45, CD3, CD14, CD20, CD34, CD73, CD105, CD90, CD146, CD163 and HLA-DR to distinguish inflammatory and stromal cells. The influence of the digestion method on the detection of the different surface markers was studied separately. In addition, we studied the presence of a specific cell population hypothesized to be mesenchymal stem cells (MSC) based on the CD271 marker. Cell expansion cultures were set up and a MSC-related surface marker profile in passages 3 and 6 was obtained. Immunohistochemistry for CD34 and von Willebrand factor (vWF) was done to obtain additional data on synovium vascularity.

Results: The cell yield and viability normalized to tissue weight were significantly higher in inflammatory arthritis than in controls. Within the hematopoietic CD45-positive populations, we found no differences in relative amounts of macrophages, T-lymphocytes and B-lymphocytes between patient groups. Within the CD45-negative cells, more CD34-positive cells were seen in controls than in arthritis patients. In arthritis samples, a small CD271 positive population was detected. Culture expanded cells were found to fulfill the multipotent mesenchymal stromal cell marker profile, except for CD34 negativity. Detection of peripheral blood macrophage and B-cell markers was decreased after enzymatic exposure and mechanical forces, respectively, but stromal markers were not affected.

Conclusions: Flow cytometry can distinguish synovial cell populations in tissue digests. The preparation method can influence the detection levels of macrophage and B-cell populations. However, stromal cell markers seem not affected and quantification is possible, supporting flow cytometry tissue analysis as a tool to study these cell populations in arthritis.

Show MeSH
Related in: MedlinePlus