Identification, quantification and isolation of mesenchymal progenitor cells from osteoarthritic synovium by fluorescence automated cell sorting S Fickert,

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Identification, quantification and isolation of mesenchymal progenitor cells from osteoarthritic synovium by fluorescence automated cell sorting S Fickert, M.D., J Fiedler, Ph.D., R.E Brenner, M.D. Osteoarthritis and Cartilage Volume 11, Issue 11, Pages 790-800 (November 2003) DOI: 10.1016/S1063-4584(03)00167-5

Fig. 1 FACS analysis of freshly isolated SM cells and isotype control. Distribution of fresh isolated SM cells in forward/side scatter (A). Markers were set in the channel display with a maximum of 2% positive cells by staining with isotype control antibody FITC-conjugated mouse IgG1(B). The triple staining experiments were shown for CD9-FITC/CD90-APC/CD166-PE (C–E). According to histogram of FL1 CD9-FITC (C) cells were divided into CD9 negative and positive stained. CD9 negative, double-stained CD90-APC/CD166-PE cells are shown in (D) and CD9 positive, double-stained CD90-APC/CD166-PE fresh isolated SM cells are shown in (E). Osteoarthritis and Cartilage 2003 11, 790-800DOI: (10.1016/S1063-4584(03)00167-5)

Fig. 2 FACS analysis of freshly isolated SM cells of CD45-FITC/CD90-APC and CD166-PE. FSC/SSC used the same gate for all experiments (A). Histogram of cells stained with isotype control antibody FITC-conjugated mouse IgG1(B) and CD45-FITC (C). CD45 negative, double-stained CD90-APC/CD166-PE cells are shown in (D) and CD45 positive, double-stained CD90-APC/CD166-PE fresh isolated SM cells are shown in (E). Osteoarthritis and Cartilage 2003 11, 790-800DOI: (10.1016/S1063-4584(03)00167-5)

Fig. 3 Flow cytometric analysis of combinations of progenitor markers on freshly isolated SM cells and culture expanded SM cells. Data presented as means±SD. All native marked SM cells are from freshly isolated SM from eight donors and the amount of total triple positive cells of five donors after cultivation as described in Materials and methods. Osteoarthritis and Cartilage 2003 11, 790-800DOI: (10.1016/S1063-4584(03)00167-5)

Fig. 4 FACS analysis of culture expanded SM cells from the patient with the highest relative amount of triple positive cells after cultivation. FSC/SSC of analyzed cultured cells (A). Histogram of FITC-mIgG1(B) and CD9-FITC (C) stained cells. Dot plots are showing the expression of triple stained cells: FITC-mIgG1positive gated PE-mIgG1/biotin-mIgG1(D), CD9-FITC gated positive, double-stained CD90-biotin/CD166-PE (E). Osteoarthritis and Cartilage 2003 11, 790-800DOI: (10.1016/S1063-4584(03)00167-5)

Fig. 5 Reanalysis of triple positive sorted cells. Forward and side scatter characteristics of sorted SM cells (A). CD9-FITC/CD166-PE, CD9-FITC/CD90-APC or CD90-APC/CD166-PE double positive cells and the fluorescence gate used for previous cell sorting (B–D). Triple staining of CD9 positive, CD90/CD166 (E) and CD9 negative, CD90/CD166 (F). Osteoarthritis and Cartilage 2003 11, 790-800DOI: (10.1016/S1063-4584(03)00167-5)

Fig. 6 Differentiation of culture expanded SM cells and CD9/CD90/CD166 sorted SM cells. Chondrogenic differentiation of culture expanded (A,C,E) and sorted SM cells (B,D,F) was proved by Alcian blue staining (A,B), COMP (C,D) and collagen type II (E,F) staining of micromass cultures. In addition, von Kossa staining of native SM cells after osteogenic differentiation in monolayer is shown (G, H). Magnification: A–F 100×; G 200×; H 400×. Osteoarthritis and Cartilage 2003 11, 790-800DOI: (10.1016/S1063-4584(03)00167-5)

Fig. 7 Osteogenesis of culture expanded arthrotic SM cells and progenitor marker sorted SM cells. The expression of GAPDH, collagen type I (COL1), alkaline phosphatase (AP), osteocalcin (OC) and bone sialoprotein (BSP) in native SM is presented in A and B. Osteogenic differentiation was shown by presence of alkaline phosphatase, osteocalcin and bone sialoprotein for culture expanded SM cells (C) and progenitor marker sorted SM cells (D). Osteoarthritis and Cartilage 2003 11, 790-800DOI: (10.1016/S1063-4584(03)00167-5)