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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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Detection and quantification of CD271 and CD34 in digests and cultured cells. (a) Plots of two representative samples. The CD45-negative fraction is gated in G1. Within this fraction, a portion of cells is positive for CD271 or CD34. Double positive cells are sometimes well clustered (lower plot). To be uniform, the quantification is performed using quadrant analysis. (b) Data points representing the expression levels in percentage positivity on the cell surface detected in the fresh digests. (c) Plots of cultured cells after staining. Cells are gated according to scatter properties (G1), viability (G2) and the signal for the markers is compared with the isotype controls. No double positive cells are recognized here. 7-AAD = 7-aminoactinomycin; APC = allophycocyanin; FITC = fluoro-isothiocyanate; FSC = forward scatter; PE = phycoerythrin; SSC = side scatter.
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Figure 7: Detection and quantification of CD271 and CD34 in digests and cultured cells. (a) Plots of two representative samples. The CD45-negative fraction is gated in G1. Within this fraction, a portion of cells is positive for CD271 or CD34. Double positive cells are sometimes well clustered (lower plot). To be uniform, the quantification is performed using quadrant analysis. (b) Data points representing the expression levels in percentage positivity on the cell surface detected in the fresh digests. (c) Plots of cultured cells after staining. Cells are gated according to scatter properties (G1), viability (G2) and the signal for the markers is compared with the isotype controls. No double positive cells are recognized here. 7-AAD = 7-aminoactinomycin; APC = allophycocyanin; FITC = fluoro-isothiocyanate; FSC = forward scatter; PE = phycoerythrin; SSC = side scatter.

Mentions: CD271 has been associated with bone marrow-derived mesenchymal progenitor cells but does not appear to be present on expanded mesenchymal progenitor cells from the synovium [14,16,17,25]. We therefore used this molecule as an example to see whether CD271-positive cells are present in the synovium. In a set of eight patients, staining for CD34 was combined with CD271. The plots are illustrated in Figure 7a. CD271 was expressed in most of the samples (Figure 7b), with a median value of 4.7% of the stromal compartment, and 3.1% of the stromal cells were both CD271 and CD34 positive. In contrast, expression in synovium-derived fibroblastic cells cultured for one month was tested in three patients of whom digest data were also available. A certain expression of CD271 was maintained but cells positive for both CD271 and CD34 were not longer detected (Table 5).


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)

Detection and quantification of CD271 and CD34 in digests and cultured cells. (a) Plots of two representative samples. The CD45-negative fraction is gated in G1. Within this fraction, a portion of cells is positive for CD271 or CD34. Double positive cells are sometimes well clustered (lower plot). To be uniform, the quantification is performed using quadrant analysis. (b) Data points representing the expression levels in percentage positivity on the cell surface detected in the fresh digests. (c) Plots of cultured cells after staining. Cells are gated according to scatter properties (G1), viability (G2) and the signal for the markers is compared with the isotype controls. No double positive cells are recognized here. 7-AAD = 7-aminoactinomycin; APC = allophycocyanin; FITC = fluoro-isothiocyanate; FSC = forward scatter; PE = phycoerythrin; SSC = side scatter.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Detection and quantification of CD271 and CD34 in digests and cultured cells. (a) Plots of two representative samples. The CD45-negative fraction is gated in G1. Within this fraction, a portion of cells is positive for CD271 or CD34. Double positive cells are sometimes well clustered (lower plot). To be uniform, the quantification is performed using quadrant analysis. (b) Data points representing the expression levels in percentage positivity on the cell surface detected in the fresh digests. (c) Plots of cultured cells after staining. Cells are gated according to scatter properties (G1), viability (G2) and the signal for the markers is compared with the isotype controls. No double positive cells are recognized here. 7-AAD = 7-aminoactinomycin; APC = allophycocyanin; FITC = fluoro-isothiocyanate; FSC = forward scatter; PE = phycoerythrin; SSC = side scatter.
Mentions: CD271 has been associated with bone marrow-derived mesenchymal progenitor cells but does not appear to be present on expanded mesenchymal progenitor cells from the synovium [14,16,17,25]. We therefore used this molecule as an example to see whether CD271-positive cells are present in the synovium. In a set of eight patients, staining for CD34 was combined with CD271. The plots are illustrated in Figure 7a. CD271 was expressed in most of the samples (Figure 7b), with a median value of 4.7% of the stromal compartment, and 3.1% of the stromal cells were both CD271 and CD34 positive. In contrast, expression in synovium-derived fibroblastic cells cultured for one month was tested in three patients of whom digest data were also available. A certain expression of CD271 was maintained but cells positive for both CD271 and CD34 were not longer detected (Table 5).

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