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. 2023 Oct 7:24:528-535.
doi: 10.1016/j.reth.2023.09.016. eCollection 2023 Dec.

Raman spectroscopy to assess the differentiation of bone marrow mesenchymal stem cells into a glial phenotype

Sulei Bautista-González  1 , Nidia Jannette Carrillo González  1 , Tania Campos-Ordoñez  1 , Mónica Alessandra Acosta Elías  2 , Martín Rafael Pedroza-Montero  2 , Carlos Beas-Zárate  1 , Graciela Gudiño-Cabrera  1
Affiliations

Raman spectroscopy to assess the differentiation of bone marrow mesenchymal stem cells into a glial phenotype

Sulei Bautista-González et al. Regen Ther. .

Abstract

Background: Mesenchymal stem cells (MSCs) are multipotent precursor cells with the ability to self-renew and differentiate into multiple cell linage, including the Schwann-like fate that promotes regeneration after lesion. Raman spectroscopy provides a precise characterization of the osteogenic, adipogenic, hepatogenic and myogenic differentiation of MSCs. However, the differentiation of bone marrow mesenchymal stem cells (BMSCs) towards a glial phenotype (Schwann-like cells) has not been characterized before using Raman spectroscopy.

Method: We evaluated three conditions: 1) cell culture from rat bone marrow undifferentiated (uBMSCs), and two conditions of differentiation; 2) cells exposed to olfactory ensheathing cells-conditioned medium (dBMSCs) and 3) cells obtained from olfactory bulb (OECs). uBMSCs phenotyping was confirmed by morphology, immunocytochemistry and flow cytometry using antibodies of cell surface: CD90 and CD73. Glial phenotype of dBMSCs and OECs were verified by morphology and immunocytochemistry using markers of Schwann-like cells and OECs such as GFAP, p75 NTR and O4. Then, the Principal Component Analysis (PCA) of Raman spectroscopy was performed to discriminate components from the high wavenumber region between undifferentiated and glial-differentiated cells. Raman bands at the fingerprint region also were used to analyze the differentiation between conditions.

Results: Differences between Raman spectra from uBMSC and glial phenotype groups were noted at multiple Raman shift values. A significant decrease in the concentration of all major cellular components, including nucleic acids, proteins, and lipids were found in the glial phenotype groups. PCA analysis confirmed that the highest spectral variations between groups came from the high wavenumber region observed in undifferentiated cells and contributed with the discrimination between glial phenotype groups.

Conclusion: These findings support the use of Raman spectroscopy for the characterization of uBMSCs and its differentiation in the glial phenotype.

Keywords: Cell differentiation; Glial phenotype; Mesenchymal stem cells; Raman spectroscopy; conditioned medium.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Immunophenotypic characterization of rat bone marrow mesenchymal stem cells (BMSCs). (A) uBMSCs showed a fibroblast-like morphology (white arrows) and were positive for CD90 and CD73; and a scarce stain of CD45 and Alexa fluor 488 (AF488) were observed by immunocytochemistry, Bar: 50 μm. (B) Representative flow cytometry analysis of cell population was selected by prior elimination of duplicated events. Expression of CD73, CD45 and CD90 is shown in stained cells (C) and unstained cells (D). (E) uBMSCs show a high expression of CD73 and CD90 and a very low expression of CD45. BMSCs: bone marrow mesenchymal stem cells. FSC: forward scatter, SSC: side scatter.
Fig. 2
Fig. 2
BMSCs in vitro differentiation. MSCs culture evolution in three conditions is shown: 1) 10% FBS medium showed fibroblast shaped cells proliferated (black arrow), morphology was maintained during cell culture time, 2) B-27 medium showed similar fibroblast shaped morphology (black arrows), and 3) In OECs-CM medium a decrease in cell body size and development of elongated and thin cell projections were observed (blue arrows). Phase contrast microscopy, 10× magnification, Bar: 100 μm. OECs-CM: Olfactory Ensheathing Cell Conditioned Medium.
Fig. 3
Fig. 3
Differentiated cell were identified by immunocytochemistry of p75NTR+ and GFAP+ antibodies. (A) uBMSCs showed a fibroblast-like morphology (yellow arrow) and positive expression to CD90+ and GFAP+. (B) dBMSCs showed elongated spindle-shape and small cell body morphology (white arrow) with positive expression to p75NTR+ and GFAP+. (C) OECs also showed elongated spindle-shape and small cell body morphology (white arrow) and positive expression to p75NTR+ and GFAP+. These data suggest that dBMSCs had a Schwann-like phenotype after the exposure to OECs-CM. Bar 50 μm.
Fig. 4
Fig. 4
BMSCs glial differentiation by Raman spectroscopy. Spectra from each group represents the mean of 5 measures from five different cells. Raman shift includes the fingerprint region (600–1800 cm−1) and high wavenumber region (2500–3200 cm−1). Values from 2000 to 2800 cm−1 were cut for illustrative means. Raman spectra of BMSCs before and after cell differentiation exhibited great modifications in the fingerprint region and the high wavenumber region. In contrast, differentiated groups (OECs and BMSCs exposed to OECs-CM) showed a similar Raman spectra with differences in peak intensities and a prominent Raman peak at 1610 cm−1, which is maintained after cell differentiation. Intensity values are represented in arbitrary units (a.u.). OECs: Olfactory Ensheathing Cells. BMSCs: Bone Marrow Mesenchymal Stem Cells.
Fig. 5
Fig. 5
Principal Component Analysis of glial differentiation of BMSCs. PCA showed different distribution and scores between undifferentiated BMSCs, OECs and uBMSCs after 72 h of treatment. BMSCs: Bone Marrow Mesenchymal Stem Cells. OECs: Olfactory Ensheathing Cells.
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