Proteome of human stem cells from periodontal ligament and dental pulp

Abstract

Background: Many adult tissues contain a population of stem cells with the ability to regenerate structures similar to the microenvironments from which they are derived in vivo and represent a promising therapy for the regeneration of complex tissues in the clinical disorder. Human adult stem cells (SCs) including bone marrow stem cells (BMSCs), dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) have been characterized for their high proliferative potential, expression of characteristic SC-associated markers and for the plasticity to differentiate in different lineage in vitro.

Methodology/principal findings: The aim of this study is to define the molecular features of stem cells from oral tissue by comparing the proteomic profiles obtained with 2-DE followed by MALDI-TOF/TOF of ex-vivo cultured human PDLSCs, DPSCs and BMSCs. Our results showed qualitative similarities in the proteome profiles among the SCs examined including some significant quantitative differences. To enrich the knowledge of oral SCs proteome we performed an analysis in narrow range pH 4-7 and 6-9, and we found that DPSCs vs PDLSCs express differentially regulated proteins that are potentially related to growth, regulation and genesis of neuronal cells, suggesting that SCs derived from oral tissue source populations may possess the potential ability of neuronal differentiation which is very consistent with their neural crest origin.

Conclusion/significance: This study identifies some differentially expressed proteins by using comparative analysis between DPSCs and PDLSCs and BMSCs and suggests that stem cells from oral tissue could have a different cell lineage potency compared to BMSCs.

Figure 1. Photomicrographs of primary cultures of BMSCs (A) DPSCs (B) and PDLSCs (C) at passage 2 analyzed at morphological and cytofluorimetric levels.

At Scanning (section A, B, and C) and Light microscopy (a1,b1,c1) the culture displays the comparable morphological features consisting of adherent cells having a morphological homogeneous fibroblast-like appearance with a stellate shape and long cytoplasmic processes and numerous filopodia. Nuclei contain one or more nucleoli (Nc). At 28 days of culture in the osteogenic medium BMSCs, DPSCs and PDLSCs (a2,b2,c2) differentiate versus osteoblasts as evidenced by calcium deposition with Alizarin Red staining. Original magnification: 1.000× (scanning microscopy), 10× (light microscopy). The histograms show the cytofluorimetric analysis of the cell culture BMSCs, DPSCs and PDLSCs, surface and intracellular antigens expression profile: CD13, CD14, CD29, CD34, CD44, CD45, CD73, CD90, CD105, CD117 CD133, CD144, CD146, CD166, CD271, OCT3/4, Sox2 and SSEA4. Blue histograms represent cells stained with the expression markers; red histograms show the respective IgG isotype control. These data are representative of four independent biological samples.

Figure 2. Proliferation rate of the primary culture of BMSCs, DPSCs and PDLSCs plated at passage 2 and after 3 days of culture.

The MTT analysis histogram shows that the proliferation in PDLSCs and DPSCs was higher compared to BMSCs. The comparative analysis between the stem cells population demonstrated a difference in terms of cell proliferation to the advantage of stem cells from periodontal ligament. The data represent the mean of four separate experiments carried out in triplicate. Values are reported as mean ±SD (ANOVA test, p≤0.05).

Figure 3. Comparative proteome analysis of BMSCs and oral tissue derived SCs (PDLSCs and DPSCs).

Panel A displays the representative 2D pattern of each stem cell population analyzed. Differences are indicated with numbers. Panel B shows magnification of differentially expressed spot (1–14) and relative % of volume histogram. Panel C summarizes proteins differentially expressed in all cell populations examined.

Figure 4. Functional and cellular compartment distribution of oral tissue SCs proteins.

The assigned proteins were analyzed using Gene Ontology (GO) annotation and grouped in seven categories based on their functions: 1) protein biosynthesis folding and degradation, 2) cellular structure and motility, 3) metabolism, 4) oxidative stress, 5) cell signaling, 6) nucleotide biosynthesis recovery and degradation, 7) transport. (section A) and in terms of cellular localization (section B).

Figure 5. Western Blot analysis of some representative proteins that are differentially expressed between SC populations.

A) 1D western blot data for CLPP, NQ01, SCOT1 and ACTB as loading control. Western blot confirms results obtained by 2D analysis. B) 2D western blot data for DPYL3. BMSCs show a higher level of DPYL3 than oral tissue SCs. The new and more basic DPYL3 isoform is indicated by the arrow.

Figure 6. Acidic and basic oral tissue SCs proteome. DPSCs and PDLSCs narrow range (4–7 and 6–9 pH range) 2D maps.

Differences are indicated with numbers. Magnifications at the right side show differentially expressed protein spots and relative % volume histograms.