Characterization of stem and progenitor cells in the dental pulp of erupted and unerupted murine molars

Abstract

In the past few years there have been significant advances in the identification of putative stem cells also referred to as "mesenchymal stem cells" (MSC) in dental tissues including the dental pulp. It is thought that MSC in dental pulp share certain similarities with MSC isolated from other tissues. However, cells in dental pulp are still poorly characterized. This study focused on the characterization of progenitor and stem cells in dental pulps of erupted and unerupted mice molars. Our study showed that dental pulps from unerupted molars contain a significant number of cells expressing CD90+/CD45-, CD117+/CD45-, Sca-1+/CD45- and little if any CD45+ cells. Our in vitro functional studies showed that dental pulp cells from unerupted molars displayed extensive osteo-dentinogenic potential but were unable to differentiate into chondrocytes and adipocytes. Dental pulps from erupted molars displayed a reduced number of cells, contained a higher percentage of CD45+ and a lower percentage of cells expressing CD90+/CD45-, CD117+/CD45- as compared to unerupted molars. In vitro functional assays demonstrated the ability of a small fraction of cells to differentiate into odontoblasts, osteoblasts, adipocytes and chondrocytes. There was a significant reduction in the osteo-dentinogenic potential of the pulp cells derived from erupted molars compared to unerupted molars. Furthermore, the adipogenic and chondrogenic differentiation of pulp cells from erupted molars was dependent on a long induction period and were infrequent. Based on these findings we propose that the dental pulp of the erupted molars contain a small population of multipotent cells, whereas the dental pulp of the unerupted molars does not contain multipotent cells but is enriched in osteo-dentinogenic progenitors engaged in the formation of coronal and radicular odontoblasts.

Figure 1. Mineralization potential of primary cultures derived from dental pulp, BMSC and calvarial osteoblasts established from P5-7 and P18-21 mice

von Kossa staining of mineralized tissue in primary cultures established from dental pulp (A-D and M-P), BMSC (E-H and Q-T) and calvaria osteoblasts (I-L) of P5-7 (A-L) and P18-21 (M-T) mice at various time points. In cultures derived from dental pulp and BMSC isolated from P5-7, the first sign of mineralization is at day 10 (B, F) and increased at days 14 and 21 (D, H). In cultures derived from calvaria osteoblasts from P5-7 mice, the first sign of mineralization is at day 14 (K). In cultures derived from dental pulp isolated from P18-21 (M-P), the first sign of mineralization is at day 14 (O) with increased at days 14 and 21 (P). Note the reduced amount of mineralization in these cultures as compared to cultures derived from pulps from P5-7 (A-D).

Figure 2. Characterization of mineralized matrix in primary cultures derived from dental pulp, BMSC and COB of P5-7 mice by RT-PCR

In all cultures, the expression of Col1a1 (A) was detected at the highest levels at day 7 with decreases thereafter. In all cultures, expression of BSP (B), OC (C) and DMP1 (D) were not detected at day 7, appeared at day 14 and increased at day 21. Expression of DSPP (E) was detected only in dental pulp cultures. In cultures from dental pulp DSPP expression was detected first at day 14 and increased at day 21. Values represent mean value ± S.E of four independent experiments.

Figure 3. Expression of DSP in mineralized matrix in vitro

Epifluorescence images of 14 days old primary dental pulp cultures stained with Calcein blue (A, D), TO-PRO3 (G) and anti-DSP antibody (B, E, H) C, F, and I represent overlay of the two images generated by AxioVision software. A-C represent the expression of DSP (indicated by white arrows in C) in mineralized nodules stained with Calcein Blue. D-E demonstrate the lack of expression of DSP in another mineralized nodule in the same culture. G-I: Confocal images of 14 days old primary dental pulp cultures showing DSP expression in the cytoplasm and in the extracellular matrix, but not in the nuclei (I). Scale bars in all images = 20 μm.

Figure 4. Quantification of the dentinogenic potential in the primary dental pulp culture

Cultures from dental pulp (A-F) and BMSC (G-I) of P5-7 mice were stained with Calcein Blue (A, D, G) and anti-DSP antibody (B, E, H). Note that the expression of DSP in mineralized nodules in cultures from dental pulp (B, E), but not in BMSC (H). C, F and I represent overlay of the two images generated by AxioVision software. DSP expression is not detected in all the mineralized areas of the dental pulp cultures. Values in figures A and D represent mean ± SE of the total area of cultures stained with Calcein Blue. Values in B and E represent mean ± SE of the total area of cultures stained with anti-DSP antibody. Values in C and F represents the ratio of DSP+ area to Calcein Blue+ areas. Values are mean ± S.E from at least three independent experiments. Scale bar in all images = 1mm.

Figure 5. Characterization of the expression of cell surface markers

Flow cytometry analysis of the expression of CD45 (y axis), CD90, CD117 and Sca-1 (X-axes) cell surface markers in freshly isolated and cultured dental pulp cells and freshly isolated bone marrow from P5-7 mice. Note that freshly isolated dental pulp cells from unerupted molars contained high number of cells expressing markers of MSC (CD90, CD117 and Sca-1), and very low number of cells expressing hematopoietic stem cell marker CD45. On the other hand, cells isolated from bone marrow contained high number of CD45+, and moderate number of cells expressing CD90, CD117 and Sca-1. During the 7 days in culture there were increases in the number of Sca-1+ and decreases in the number of CD90+ and CD117+ cells.

Figure 6. Adipogenic differentiation by cultures derived from dental pulp

A-C represent images of representative cultures after 6 weeks from BMSC (A) and dental pulp from unerupted (P5-7) (B) and erupted (P18-21) molars (C) stained with Oil Red O. A’-C’ represent bright field images of the areas of cultures indicated in boxes showing lipid vacuoles stained with ORO. Scale bars in A’-C’= 100 μm. (D) RT-PCR analysis of expression of FABP4 and PPARγ2, markers of adipogenesis in these cultures. Note the high levels of PPARγ2 and FABP4 expression in the BMSC cultures. Dental pulp cells derived from erupted (P18-P21) molars display low, but detectable levels of PPARγ2 and FABP4 expression. The expression of PPARγ2 and FABP4 is not detected in dental pulp cells derived from unerupted (P5-7) molars.

Figure 7. Chondrogenic differentiation by cultures derived from dental pulp

A-D are representative images from the whole micromass cultures stained with Alcian Blue. Micromass cultures were established from mesenchyme obtained from E11.5 mandibles (A), BMSC (B) and dental pulp obtained from unerupted (P5-7) (C) and erupted (P18-P21) (D) molars. Cultures were grown under chondrogenic condition for 10 days (A), 14 days (B) and 4 weeks (C, D). A’-D’ represent bright field images of the same cultures. Micromass cultures derived from E11.5 mandibular mesenchyme display extensive chondrogenesis indicated by cartilage nodules stained with Alcian Blue (A, A’). Limited number of Alcian Blue stained nodules is detected in micromass cultures derived from BMSC (arrows in B, B’) and dental pulp from erupted (P18-P21) molars (arrows in D, D’). Chondrogenesis and Alcian Blue stained nodules were not detected in micromass cultures derived from dental pulps from unerupted (P5-7) molars (C, C’). Scale bars in A’-D’= 100 μm. (E) RT-PCR analysis of expression of marker of chondrogenesis in micromass cultures Note the high levels of Col2a1 and Sox9 expression in micromass cultures established from E11.5 mandibular mesenchyme. Levels of Col2a1 expression in the micromass cultures from the erupted (P18-P21) molars and BMSC are low but detectable. Also note the lack of Col2a1 expression in the micromass cultures from the dental pulps from unerupted (P5-7) molars. Sox9 expression was detected in low but detectable levels in all micromass cultures.

Figure 8. Vascularization in dental pulp

Cross-sections of molars from P5-7 (A, B) and P18-21 (C, D) mice processed for Orcein/Giemsa staining (A, C) and CD31 immunohistochemistry (B, D) for detection of erythrocytes (stained red) and endothelial cells in the vessel walls (stained brown) respectively. Note the increases in the number of blood vessels containing red stained erythrocytes and CD31+ endothelial cells (examples indicated by arrows) in pulps from P18-21 (C) as compared to P5-7 mice. Inset in C represent higher magnification of a blood vessel indicated by asterisk showing CD31+ in the endothelial lining of a large blood vessel in the pulp from P18-21 mice. Scale bar = 100μm