Flow cytometric analysis of mesenchymal progenitor cells from human osteoarthritic cartilage
No single surface marker protein has yet been found to characterize MPCs. From the accepted markers, we chose seven different cell surface markers and used them in triple combinations for fluorescence-activated cell sorting (FACS) analysis. Immediately after overnight isolation, chondrocytes from OC were directly stained with seven triple combinations of CD9, CD44, CD54, CD90 and CD166 as positive markers, and CD45, CD133/-1 and -2 as negative markers to eliminate haematopoietic and endothelial cells (Table ). The expression of progenitor typical markers varied from nearly no detectable staining to relatively high levels of expression. As shown in Fig. , in the forward/side scatter the fresh isolated OC cells were a heterogeneous population. The majority of the cells stained negative for CD9, CD90 and CD166. The proportion of CD9+/CD90+/CD166+ triple positive cells was only about 5%.
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 (more ...)
CD9+/CD166+ cells could be subdivided in two equivalent populations comprising about 8% of total cells that were either positive or negative for CD90. We analyzed CD9- but CD90+/CD166+, CD90-/CD166+ and CD90-/CD166- cells, and found that these groups comprised 23.0%, 29.7% and 33.7% of cells, respectively. No CD90+/CD9-/CD166- cells were detectable.
The isotype control antibody revealed no specific staining. The distribution of OC cells in forward/side scatter exhibited no difference between isotype and antibody staining. A maximum of 2% positive cells by staining with isotype antibody mouse IG1 or IG2 conjugated with FITC, PE or biotin was allowed and therefore was used to set the markers within the channel display. The distinction to negative assessed cells is presented in Fig. by showing an exemplary FITC-isotype antibody mIgG1 staining.
Comparing total quantities of triple positive cells from OA cartilage (Fig. ), CD9+/CD44+/CD166+ and CD9+/CD54+/CD90+ cells were detected in (mean ± standard deviation) 12.2 ± 10% and 13.3 ± 5.7%, respectively (n = 8). The frequencies of CD9+/CD90+/CD166+ and CD9+/CD44+/CD54+ cells were 8.2 ± 10.4% and 2.5 ± 1.8%, respectively.
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, (more ...)
The combinations CD45+/CD90+/CD166+ and CD9+/CD133(1 or 2)+/CD166+ exhibited less than 1% staining. MPC cultures isolated from bone marrow from different donors served as controls. In these samples 95–98% of all gated cells were triple positive for various combinations of the markers CD9/CD54/CD90/CD166 (data not shown).
Analysis of chondrocytes after adherence and cultivation
The change in cellular morphology and the acquisition of a fibroblastic shape became increasingly apparent as the cells were cultured on plastic. In the primary culture, nonadherent or few loosely adherent small round cells were also present, but these disappeared from the first to the second passage. After an initial lag time of 2–3 days, cells entered a proliferative phase, reaching confluence within 48 hours. An average of one doubling every 3 days was observed upon subsequent passages.
In order to determine the percentage of culture expanded progenitor cells that express antigens recognized by CD9/CD44/CD54/CD90/CD166 monoclonal antibodies, the number of immunoreactive cells was quantified by flow cytometry. Data from adherent, culture expanded cells were collected and the number of triple-positive events was expressed as a percentage of the total cell number. An example for the distribution of cultured OC cells is shown in Fig. . Compared with fresh isolated OC cells, as seen in Fig. , the cultured cells are a homogeneous population on forward/side scatter.
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 (more ...)
In these experiments, the mean frequency of every triple positive staining of cultured OC cells increased markedly compared with native OC cells (Fig. ). The total frequency of the CD9+/CD90+/CD166+ population rose 4.1-fold compared with freshly isolated OC chondrocytes. After cultivation in monolayer, we found 18.8-fold more CD9+/CD44+/CD54+ cells than among native, uncultured OC cells. Analysis of the CD9+/CD44+/CD166+ subpopulation revealed only 1.6-fold expansion, whereas CD9+/CD54+/CD90+ cells expanded 3.4-fold. Within the in vitro expanded cells, CD45+ was still not detectable, whereas the total frequency of CD45-/CD90+/CD166+ population was around 29–45%. These results indicate that cultivation enriches a subpopulation of OC cells that express cell surface markers for MPCs.
Sorting and cultivation of progenitor marker positive, fresh isolated osteoarthritic cartilage cells
Findings in fresh isolated chondrocytes suggested that there is a subpopulation in OC that expresses progenitor-associated markers and is capable of osteogenic and chondrogenic differentiation. If a common progenitor cell exists, then it should be found among cells with a CD9+, CD90+ and CD166+ phenotype. Based on phenotypic analysis by FACS, we therefore isolated CD9+/CD90+/CD166+ OC cells from five patients and analyzed the kinetics of cultivation and the potential for differentiation. The data analysis after sorting is exemplarily shown for one patient in Fig. . The gates used for cell sorting are shown in Fig. .
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 (more ...)
In these experiments, the mean frequency of the native CD9+/CD90+/CD166+ population was 32%. Figure shows the forward and side scatter characteristics of sorted OC cells. To confirm the quality of sorting, reanalysis of triple positive sorted cells was performed, and it was found that 99.1% were again triple positive (Fig. ). Serial observations of each culture well that contained triple positive cells were performed after 3, 7, 14, 21 and 28 days. The earliest point at which the growth of triple positive sorted could be detected was after 3 days. Between 7 and 14 days of culture, adherent, fibroblast-like cells scattered in a random pattern across the surface of the culture well. For 21 days of culture, continuous growth of the adherent, fibroblastic cells was observed.
Differentiation of the cultured sorted cells was determined by RT-PCR, histochemistry and immunohistochemistry. The findings confirmed that the CD9/CD90/CD166 triple positive cell population derived from OC was capable of multipotent mesenchymal differentiation.
Osteogenesis, adipogenesis and chondrogenesis of culture expanded osteoarthritic cartilage cells
To study the possible multilineage capacity of some OC derived cells, we differentiated these cell cultures toward the osteogenic, adipogenic and chondrogenic lineages.
Pellet cultures of OC derived cells resulted in the formation of dense nodules consistent with chondrogenic differentiation. These nodules were associated with an Alcian Blue-positive extracellular matrix, which indicates the presence of sulphated proteoglycans within the matrix (Fig. ).
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) (more ...)
Cartilaginous nodules were also observed upon pellet cultures of bone marrow derived MPCs. In addition to the presence of sulphated proteoglycans within the extracellular matrix, transforming growth factor-β3 supplemented OC-derived cells expressed collagen type II and COMP in pellet culture (Fig. ). Overall, these results indicate that a subpopulation of OC-derived cells has the capacity to differentiate toward the chondrogenic lineage. To determine whether OC cells undergo adipogenesis, cells were cultured in medium containing dexamethasone, isobutyl-methylxanthine and indomethacin. About 10–30% of the OC cells were reproducibly induced toward the adipogenic lineage as early as 2 weeks after induction (Fig. ). Using PCR, the expression of peroxisome proliferator-activated receptor-γ demonstrated adipogenic differentiation by progenitor marker sorted and culture-derived OC cells (data not shown).
Differentiation of OC derived cells into osteoblasts was induced in vitro
by treating the cells with ascorbic acid, β-glycerophosphate and dexamethasone [2
]. OC derived cells and bone marrow MPCs formed an extensive network of dense, multilayered nodules that stained positive for alkaline phosphatase.
After 14 days of differentiation, the gene expression profile of osteoblast markers was investigated. For OC cells and bone marrow MPCs we detected a strong signal for expression of genes for all tested osteogenic markers (Fig. ): alkaline phosphatase, bone sialoprotein and osteocalcin.
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 (more ...)