In Vitro Characterization of the Human Skeletal Stem Cell-like Properties of Primary Bone-Derived Mesenchymal Stem/Stromal Cells in Patients with Late and Early Hip Osteoarthritis

Abstract

Human skeletal stem cells (hSSCs) were recently identified as podoplanin (PDPN)/CD73/CD164-positive and CD146-negative cells that decline with age, and play a role in the pathogenesis of osteoarthritis (OA). The aim of this study was to identify the hSSC-like properties of bone-derived mesenchymal stem/stromal cells (MSCs) of patients with late and early OA. Methods: First, we performed gene expression profiling for the hSSC markers in 32 patients with late and early OA, and donors without OA. Having identified the low expression of hSSC markers in late OA patients, we further performed trilineage differentiation and immunophenotyping for hSSC makers in the selected subsets from each donor group. Results: Our results show no differences in osteogenesis, chondrogenesis, and adipogenesis between the MSCs from the three groups. However, the immunophenotyping shows lower CD164 in MSCs from early OA patients in comparison with late and no OA subjects (p = 0.002 and p = 0.017). Conclusions: Our study shows that the in vitro hSSC-like properties of bone-derived MSCs are similar in patients with early and late OA, and in donors without OA. However, the lower percentage of CD164-positive MSCs in early OA patients indicates the potential of CD164 as a marker of the onset of OA.

Keywords: CD146; CD164; CD73; bone-derived mesenchymal stem/stromal cells (MSCs); early OA; human skeletal stem cells (hSSCs); immunophenotyping; late osteoarthritis (OA); podoplanin (PDPN); trilineage differentiation.

Figure 1

Study design, subject groups, and analyses. The subjects in our study were divided into 3 donor groups (violet circle), i.e., patients with late OA of the hip, patients with early OA of the hip, and post mortem donors with no degenerative changes of the hip (no OA group). All subjects had their trabecular bone from the femoral head harvested for primary cell isolation. The primary cells from subjects with late OA and without OA were used from our previous study [13]. The current study comprised of two stages. In the first stage (red circle), gene expression profiling for human skeletal stem cell (hSSC) markers was performed on cDNA samples from bone-derived MSCs from 32 subjects. Based on these results, we further selected 15 samples (5 samples per subject group) for the in vitro analyses, such as trilineage differentiation and immunophenotyping, for hSSC markers (green circle).

Figure 2

The results of the expression profiling for the hSSC marker genes. (a) Heat map analysis for hierarchical clustering of hSSC gene expression (columns, as indicated) in the primary cells from the three donor groups (rows). Green, gene expression higher than reference channel; red, gene expression lower than reference channel. Two clusters with high expression of the negative marker CD146, and low expression of the three positive markers are shown (right; boxed in blue), along with the clustering tree analysis (left). The majority of the samples in these two clusters are from late OA patients, i.e., 3 out of 5 in the upper cluster, and 4 out of 4 in the lower cluster. OBV, late OA samples; OKK, early OA samples; SM, samples with no OA. (b) Expression of the hSSC marker genes CD73, CD146, PDPN, and CD164 for each donor group. Individual samples and means are shown. No significant differences in any of the measured gene are found (p > 0.05; general linear model with age as covariate, and a Bonferroni post hoc comparison). OA, osteoarthritis; PDPN, podoplanin.

Figure 3

The results of the osteogenic potential of the primary bone-derived cells. (a) Alizarin red S concentration for each donor group is measured. Individual samples and means are shown. No significant differences are obtained between the three groups of donors (p = 0.380; general linear model with age as covariate, and a Bonferroni post hoc comparison). (b) Gene expression of specific markers of osteogenesis are measured. Individual samples and means are shown. Data are normalized to the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). No significant differences are obtained between the three groups of donors, for any of the genes measured (p > 0.05; general linear model with age as covariate, and a Bonferroni post hoc comparison). (c) Representative images of the wells for each donor group (as indicated) stained with alizarin red S for rate of osteogenesis. Images for all donors are shown in Supplemental Figure S1. Scale bars, 400 µm. OA, osteoarthritis; OC, osteocalcin; COL1A1, collagen type I; ALP, alkaline phosphatase.

Figure 4

The results of the chondrogenic potential of the primary bone-derived cells. (a) Bern score of the toluidine blue-stained chondrogenic pellets for each donor group is determined. No significant differences are obtained between the three donor groups (p = 0.252; general linear model with age as covariate, and a Bonferroni post hoc comparison). Individual samples and means are shown. (b) Representative images of the toluidine blue-stained chondrogenic pellets for each group of donors are shown. Images for all donors are shown in Supplemental Figure S2. (c) von Kossa histology shows no difference in the rate of mineralization between the three donor groups (p >0.05; chi-squared test). Representative images of the von Kossa-stained chondrogenic pellets for each group of donors are shown. Images for all donors are shown in Supplemental Figure S3. (d) Immunofluorescence for the α-1 chain of type II collagen (Col2A1) shows no difference in the presence of the hyaline cartilage between the three donor groups (p > 0.05; chi-squared test). Representative images of the Col2A1-stained chondrogenic pellets for each group of donors are shown. Images for all donors are shown in Supplemental Figure S4. Scale bars, 200 µm. OA, osteoarthritis.

Figure 5

The results of the adipogenic potential of the primary bone-derived cells. (a) The percentage of the oil-red-O-positive adipocytes for each donor group is determined. Individual samples and means are shown. No significant differences are obtained between the three donor groups (p = 0.613; general linear model with age as covariate, and a Bonferroni post hoc comparison). (b) Gene expression of specific markers of adipogenesis is measured. Data are normalized to the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Significant differences are obtained between for fatty acid-binding protein 4 gene (FABP4) between late and early OA, and early and no OA, as indicated (* p < 0.05; general linear model with age as covariate, and a Bonferroni post hoc comparison). (c) Representative images of the oil-red-O-stained adipocytes for each group of donors are shown. Images for all donors are shown in Supplemental Figure S5. Scale bars, 200 µm. OA, osteoarthritis; ADIPOQ, adiponectin; PPARG, peroxisome proliferator-activated receptor γ; FABP4, fatty acid-binding protein 4.

Figure 6

The results of the immunophenotyping for hSSC markers. (a) The percentage of the positive (CD73, PDPN, and CD164) and the negative (CD146) markers within single live CD45/CD235a-negative cells is determined. Significant differences are observed for the positive marker CD164 between all group of patients, and for the negative marker CD146 between early and no OA, as indicated (general linear model with age as covariate, and a Bonferroni post hoc comparison). * p < 0.05 and ** p < 0.01. (b) Representative dot plots for each hSSC marker in each group of donors are shown. OA, osteoarthritis; PDPN, podoplanin.

Figure 6

The results of the immunophenotyping for hSSC markers. (a) The percentage of the positive (CD73, PDPN, and CD164) and the negative (CD146) markers within single live CD45/CD235a-negative cells is determined. Significant differences are observed for the positive marker CD164 between all group of patients, and for the negative marker CD146 between early and no OA, as indicated (general linear model with age as covariate, and a Bonferroni post hoc comparison). * p < 0.05 and ** p < 0.01. (b) Representative dot plots for each hSSC marker in each group of donors are shown. OA, osteoarthritis; PDPN, podoplanin.