A Single-Cell Raman Spectroscopy Analysis of Bone Marrow Mesenchymal Stem/Stromal Cells to Identify Inter-Individual Diversity

Abstract

The heterogeneity of stem cells represents the main challenge in regenerative medicine development. This issue is particularly pronounced when it comes to the use of primary mesenchymal stem/stromal cells (MSCs) due to a lack of identification markers. Considering the need for additional approaches in MSCs characterization, we applied Raman spectroscopy to investigate inter-individual differences between bone marrow MSCs (BM-MSCs). Based on standard biological tests, BM-MSCs of analyzed donors fulfill all conditions for their characterization, while no donor-related specifics were observed in terms of BM-MSCs morphology, phenotype, multilineage differentiation potential, colony-forming capacity, expression of pluripotency-associated markers or proliferative capacity. However, examination of BM-MSCs at a single-cell level by Raman spectroscopy revealed that despite similar biochemical background, fine differences in the Raman spectra of BM-MSCs of each donor can be detected. After extensive principal component analysis (PCA) of Raman spectra, our study revealed the possibility of this method to diversify BM-MSCs populations, whereby the grouping of cell populations was most prominent when cell populations were analyzed in pairs. These results indicate that Raman spectroscopy, as a label-free assay, could have a huge potential in understanding stem cell heterogeneity and sorting cell populations with a similar biochemical background that can be significant for the development of personalized therapy approaches.

Keywords: Raman spectroscopy; human bone marrow mesenchymal stem/stromal cells (BM-MSCs); inter-individual heterogeneity; single cell.

Figure 1

Morphology and immunophenotype of BM-MSCs derived from pediatric donors. (A) Adherent BM-MSCs from five donors (D1–D5) with fibroblast-like shape grown in GM under standard conditions for 3 days (scale bars: 50 µM); Florescent images of TRITC-conjugated phalloidin labeled F-actin (red) merged with DAPI (4′,6-diamidino-2-phenylindole) nuclear staining (blue) (scale bars: 20 µM). (B) Immunophenotypic characteristics of BM-MSCs estimated by follow cytometry. Representative histograms for each donor presenting percentages of cells positive (empty peaks) for mesenchymal markers (CD29, CD73, CD90, CD105) and hematopoietic markers (CD45, HLA-DR) in comparison to isotype control (shaded peaks).

Figure 2

Multilineage differentiation potential of BM-MSCs. Representative images of cells cultivated in GM or differentiation medium (DM) are shown. Osteogenic differentiation detected after 7 days of cultivation by staining for alkaline phosphatase (ALP) activity, and after 21 days for calcium depositions by Alizarin red staining (Scale bar: 50 µM). Chondrogenic differentiation detected with Safranin O staining of proteoglycans after 21 days cultivation (scale bar: 50 µM). Adipogenic differentiation determined based on the presence of intracellular lipid droplets by Oil Red O staining after 21 days (scale bar: 20 µM).

Figure 3

Clonogenic capacity and expression of pluripotency-associated markers in BM-MSCs. (A). Representative images of colony-forming unit–fibroblast (CFU-F) stained by crystal violet are shown. CFU-F number and efficiency (number of colonies relative to number of seeded cells) of BM-MSCs are presented as mean ± SEM of three independent experiments. (B) Expression of pluripotency-associated transcription factors (OCT4, SOX-2 and NANOG) determined by indirect immunofluorescence labeling with FITC-conjugated corresponding secondary antibodies. Cell nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). Representative images are shown (scale bars: 50 µm).

Figure 4

Growth characteristics of BM-MSCs. (A) Metabolic activity of cells isolated from 5 donors during 24 h and 48 h estimated by MTT test. (B) Population doubling time (PDT) of BM-MSCs. Cells were cultivated in standard conditions, passaged at 90% confluency, and enumerated at each passage (up to 6th passage). For PDTs calculation, the formula described in Material and Methods was applied. Results on the graph are presented as ± SEM of independent experiments. (C) Representative images of BM-MSCs stained for β-galactosidase expression after one day of cultivation under standard conditions (scale bar: 50 µM). (D) Expression of proliferation-associated marker Ki67 and p53 detected by indirect immunofluorescence labeling with corresponding FITC-conjugated or AlexaFlour555-conjugated secondary antibodies. Cell nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). Representative images are shown (scale bars: 10 µm).

Figure 5

Comparative representation of BM-MSCs Raman spectra, derived from five donors. BM-MSCs were seeded on rounded CaF2 slides (Raman grade quality) and cultivated under standard conditions during 24 h. Before Raman scattering experiments, cells were washed with saline buffer and fixed with methanol for 10 min at the room temperature. On average, 50 to 100 cells per each cell population were analyzed. (A) Averaged Raman spectra of BM-MSCs for each donor are presented with purple (D1), green (D2), blue (D3), orange (D4), and yellow (D5) lines. (B) 3D PCA score plots (PC1–PC2 and PC1–PC3) are presented with purple (D1), green (D2), blue (D3), orange (D4), and yellow (D5) dots.

Figure 6

Comparative representation of BM-MSCs Raman spectra presented in pairs. BM-MSCs were seeded on rounded CaF2 slides (Raman grade quality) and cultivated under standard conditions during 24 h. Before Raman scattering experiments, cells were washed with saline buffer and fixed with methanol for 10 min at the room temperature. On average, 50 to 100 cells per each cell population were analyzed. A comparative display of the averaged Raman spectra (red and blue lines) per cell populations: (A) D1–D2, (B) D1–D3, (C) D1–D4, (D) D1–D5, and (E) D2–D3. Principal component analysis (PCA) score plots are represented with red and blue dots.

Figure 7

Comparative representation of BM-MSCs Raman spectra presented in pairs. BM-MSCs were seeded on rounded CaF2 slides (Raman grade quality) and cultivated under standard conditions for 24 h. Prior Raman scattering experiments cells were washed with saline buffer and fixed with methanol for 10 min at the room temperature. On average, 50 to 100 cells per each cell population were analyzed. A comparative display of the averaged Raman spectra (red and blue lines) per cell populations: (A) D2–D4, (B) D2–D5, (C) D3–D4, (D) D3–D5, and (E) D4–D5. PCA score plots are represented with red and blue dots.