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. 2019 Apr 30;60(2):78-86.
doi: 10.3325/CroatMedJ_60_0078.

Raman microspectroscopy: toward a better distinction and profiling of different populations of dental stem cells

Jelena Simonović, Boško Toljić, Božidar Rašković, Vladimir Jovanović, Miloš Lazarević, Maja Milošević, Nadja Nikolić, Radmila Panajotović, Jelena Milašin  1
Affiliations

Raman microspectroscopy: toward a better distinction and profiling of different populations of dental stem cells

Jelena Simonović et al. Croat Med J. .

Abstract

Aim: To characterize stem cells originating from different dental tissues (apical papilla [SCAP], dental follicle [DFSC], and pulp [DPSC]) and test the capacity of Raman microspectroscopy to distinguish between the three dental stem cell types.

Methods: SCAP, DFSC, and DPSC cultures were generated from three immature wisdom teeth originating from three patients. Cell stemness was confirmed by inducing neuro-, osteo-, chondro-, and adipo-differentiaton and by mesenchymal marker expression analysis by flow-cytometry and real-time polymerase chain reaction. Cellular components were then evaluated by Raman microspectroscopy.

Results: We found differences between SCAP, DFSC, and DPSC Raman spectra. The ratio between proteins and nucleic acids (748/770), a parameter for discriminating more differentiated from less differentiated cells, showed significant differences between the three cell types. All cells also displayed a fingerprint region in the 600-700 cm-1 range, and characteristic lipid peaks at positions 1440 cm-1 and 1650 cm-1.

Conclusion: Although different dental stem cells exhibited similar Raman spectra, the method enabled us to make subtle distinction between them.

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Figures

Figure 1
Figure 1
Orthopantomograph of the impacted third molar (A) and schematic representation of the three types of tissues used in the analysis: DFSC – dental follicle stem cells; DPSC – dental pulp stem cells; SCAP – apical papila stem cells (B); Ctrl – control.
Figure 2
Figure 2
Representative examples of dental stem cell (apical papila) differentiation into four different lineages (magnification 100 × ). Slender projections indicated neuro-differentiation (A); Alizarin Red S stained extracellular mineral deposits indicated osteogenic differentiation (B); Safranin O stained areas with proteoglycan presence indicated chondrogenic differentiation (C); and positive Oil Red O staining indicated lipid droplets accumulation, ie, adipogenic differentiation (D).
Figure 3
Figure 3
Immunophenotypic profile of mesenchymal stem cells derived from (A) dental pulp, (B) dental follicle, and (C) apical papilla, all strongly positive for CD90, CD73, and CD105 (markers associated with mesenchymal stem cells) and negative for CD45 and CD34 (markers of hematopoietic cells).
Figure 4
Figure 4
Relative gene expression of mesenchymal (CD73 and CD90) and hematopoietic markers (CD45 and CD133) of stem cells isolated from dental pulp (DPSC, black), apical papilla (SCAP, dark gray), and dental follicle (DFSC, light gray).
Figure 5
Figure 5
Raman spectra of different cell types averaged over all three subjects (75 spectra per cell type), offset for clarity. Shaded regions mark the standard deviation of spectra (upper panel); spectra subtracted from each other to emphasize the differences (lower panel).
See this image and copyright information in PMC

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