Donor-matched comparison of dental pulp stem cells and bone marrow-derived mesenchymal stem cells in a rat model

Abstract

Dental pulp stem cells (DPSCs) have drawn much interest for the regeneration of mineralized tissues, and several studies have compared DPSCs to bone marrow-derived mesenchymal stem cells (BMMSCs). However, conflicting results, possibly due to donor-associated variability, have been published and the regenerative potential of DPSCs is currently unclear. In the present study we have sought to address this problem using a donor-matched experimental design to robustly compare the biological properties of DPSCs and BMMSCs. All experiments were performed using cells isolated from a single adult Sprague-Dawley rat. Our results show that DPSCs and BMMSCs had similar morphologies and flow cytometry profiles, were capable of forming colonies in vitro and were capable of osteogenic, chondrogenic and adipogenic differentiation. However, quantitative comparisons revealed that DPSCs had a faster population doubling time and a higher percentage of stem/progenitor cells in the population, as determined by clonogenic assays. Furthermore, while both cell populations formed mineral in vitro, DPSCs had significantly higher alkaline phosphatase activity than BMMSCs after 3 weeks in osteogenic medium. These data show several key differences between DPSCs and BMMSCs and support the possibility of using DPSCs for mineralized tissue regeneration.

Figure 1. Appearance of early DPSC and BMMSC cultures

Representative photographs (40× magnification) of DPSC and BMMSC cultures at days 2 and 5 show that both cell types appeared healthy, were proliferating, and exhibited a fibroblastic morphology typical of MSC.

Figure 2. DPSC and BMMSC express similar cell surface marker profiles

Early passage DPSC and BMMSC were found to possess a similar profile of cell surface markers (positive for adhesion molecules CD29, CD59, CD90, and CD106; negative for hematologic markers CD45 and CD11b). The open histograms signify staining with isotype controls, and the filled histograms represent staining with the specified surface marker antibody.

Figure 3. DPSC have a significantly shorter population doubling time than BMMSC

DPSC and BMMSC were plated at 10,000 and 20,000 cells per well in 6 well plates and cultured for 96 hours. DPSC were found to have a doubling time of 39.6 ± 2.5 hours, which was significantly shorter than the 61.7 ± 4.1 hours observed for BMMSC (N = 4; P = 0.0192).

Figure 4. DPSC generate significantly more colonies and contain significantly more clonogenic potential than BMMSC

(A) DPSC and BMMSC were plated at 2,000 and 5,000 cells per dish in 15 cm dishes, and after 14–21 days in culture colonies ≥1 mm in diameter per dish were scored. DPSC had significantly more colonies, with 151.5 ± 52.3 colonies per 1,000 cells plated compared to 63.8 ± 18.1 colonies per 1,000 cells plated for BMMSC (N = 4; P = 0.023). (B) DPSC and BMMSC were plated at an average of one cell per well in 96 well plates. After 21 days DPSC had formed 58.1 ± 4 clones per plate, which was significantly greater than the 11.1 ± 2.6 clones per plate formed by BMMSC (P < 0.0001). (C) Thirty-six clones of each cell type were transferred to 24 well plates and evaluated on the basis expansion potential. While few BMMSC retained their ability to proliferate after replating (4/36), a significantly larger fraction of DPSC clones (29/36) proliferated (N = 8 plates from two independent experiments; P < 0.0001).

Figure 5. DPSC and BMMSC both undergo trilineage differentiation

DPSC and BMMSC were cultured in osteogenic (left), chondrogenic (middle), or adipogenic (right) differentiation medium for three weeks and stained as described in the Materials and Methods. Representative macroscopic views show positive staining for osteogenic and chondrogenic differentiation. Adipogenic differentiation was indicated by the visualization of positively stained lipid vacuoles (40X magnification). RT-PCR results show that expression of COL II (40 amplification cycles), PPAR-γ (35 amplification cycles), osteopontin (20 amplification cycles), and RUNX-2 (35 amplification cycles) was upregulated in both DPSC and BMMSC, which confirmed differentiation.

Figure 6. DPSC exhibit significantly greater alkaline phosphatase activity than BMMSC

DPSC and BMMSC were cultured in osteogenic differentiation medium for three weeks. Positive staining with alizarin red showed that both DPSC (A) and BMMSC (B) formed mineralized nodules (20X magnification). (C) DPSC had a significantly higher alkaline phosphatase activity of 121.3 ± 31.3 nmole/mg*min, compared to 67.7 ± 5.4 nmole/mg*min for BMMSC (N = 3; P = 0.0102). Results shown are from one of two independent experiments performed in triplicate.