Identification of subpopulations with characteristics of mesenchymal progenitor cells from human osteoarthritic cartilage using triple staining for cell surface markers

Abstract

We first identified and isolated cellular subpopulations with characteristics of mesenchymal progenitor cells (MPCs) in osteoarthritic cartilage using fluorescence-activated cell sorting (FACS). Cells from osteoarthritic cartilage were enzymatically isolated and analyzed directly or after culture expansion over several passages by FACS using various combinations of surface markers that have been identified on human MPCs (CD9, CD44, CD54, CD90, CD166). Culture expanded cells combined and the subpopulation derived from initially sorted CD9+, CD90+, CD166+ cells were tested for their osteogenic, adipogenic and chondrogenic potential using established differentiation protocols. The differentiation was analyzed by immunohistochemistry and by RT-PCR for the expression of lineage related marker genes. Using FACS analysis we found that various triple combinations of CD9, CD44, CD54, CD90 and CD166 positive cells within osteoarthritic cartilage account for 2-12% of the total population. After adhesion and cultivation their relative amount was markedly higher, with levels between 24% and 48%. Culture expanded cells combined and the initially sorted CD9/CD90/CD166 triple positive subpopulation had multipotency for chondrogenic, osteogenic and adipogenic differentiation. In conclusion, human osteoarthritic cartilage contains cells with characteristics of MPCs. Their relative enrichment during in vitro cultivation and the ability of cell sorting to obtain more homogeneous populations offer interesting perspectives for future studies on the activation of regenerative processes within osteoarthritic joints.

Figure 1

Fluorescence activated cell sorting analysis of fresh isolated chondrocytes from osteoarthritic cartilage. (a) Forward/side scatter. (b) Markers were set in the channel display with a maximum of 2% positive cells by staining with isotype control antibody fluorescein isothiocyanate (FITC)-conjugated mouse IgG₁. (c-e) Triple staining experiments for CD9-FITC/CD90-allophycocyanine (APC)/CD166- phycoerythrin (PE). Panel c shows a histogram of FL1 CD9-FITC. Based on isotype and histogram, cells were divided into positive or negative: panel d, CD9^-, double-stained CD90-APC/CD166-PE; and panel e, CD9⁺, double-stained CD90-APC/CD166-PE.

Figure 2

Flow cytometric analysis of combinations of progenitor markers on freshly isolated and culture expanded chondrocytes from osteoarthritic cartilage. The label 'native' represents the fluorescence-activated cell sorting analysis after tissue digestion, whereas 'cultivated' indicates the analysis after culture expansion. The number of patients used for every analysis is indicated above every box plot. The dot presents the arithmetic mean of all the data in the category.

Figure 3

Fluorescence-activated cell sorting analysis of culture expanded chondrocytes. (a) Forward and side scatter (FCS/SSC) of cultured cells. Histograms of CD9-fluorescein isothiocyanate (FITC) (b) negative and (c) positive stained cells. Dot plots show the expression of triple stained cells: CD9-FITC gated (d) negative and (e) positive double-stained CD90-Biotin/CD166-phycoerythrin (PE).

Figure 4

Reanalysis of triple positive sorted cells. (a) Forward and side scatter characteristics of sorted osteoarthritic cartilage cells. (b-d) CD9-fluorescein isothiocyanate (FITC)/CD166-phycoerythrin (PE), CD9-FITC/CD90-allophycocyanine (APC) or CD90-APC/CD166-PE double positive cells and the fluorescence gate used for sorting. Triple staining: (e) CD9⁺/CD90⁺/CD166⁺ and (d) CD9^-/CD90⁺/CD166⁺.

Figure 5

Chondrogenesis and adipogenesis of culture expanded and progenitor marker sorted osteoarthritic cartilage derived cells. Culture expanded cells were stained as follows: (a) Alzian blue, (c) collagen type II, (e) cartilage oligomeric matrix protein (COMP) and (g) oil-red. The marker sorted cells are shown in (b) Alzian blue, (d) collagen type II, (f) COMP and (h) oil-red.

Figure 6

Polymerase chain reaction analysis of osteogenesis of culture expanded and progenitor marker sorted osteoarthritic cartilage derived chondrocytes. AP, alkaline phosphatase; BSP, bone sialoprotein; COL1, collagen type I; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; M, 100 base-pair size marker; OCN, osteocalcin.