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

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.

Nano alterations of membrane structure on both γ-irradiated and stored human erythrocytes.

PURPOSE: Storage and ionizing radiation of human red blood cells (RBC) produce alterations on RBC membranes and modify their normal shape and functionality. We investigated early morphological and biochemical changes in RBC due to those stressing agents at the nanoscale level and their impact on blood quality. MATERIALS AND METHODS: Whole blood samples from healthy donors were γ-irradiated with 15, 25, 35, and 50 Gy. Non-irradiated and non-stored RBC were used as control samples. Irradiated blood samples were stored separately at 4 °C and analyzed immediately and after 5 and 13 d. Atomic force microscopy (AFM), osmotic fragility and Raman spectroscopy were used to detect morphological and biochemical changes. RESULTS: RBC function is challenged by both irradiation and storage. The storage procedure caused nanometric variations over the surface of RBC membrane for both irradiated and non-irradiated cells. The membrane of RBC became more fragile, while the biochemical fingerprint of hemoglobin (Hb) remained unaltered. CONCLUSIONS: Our work shows that the irradiation procedure leads to an increase in the number and size of nanovesicles along with the dose. The functionality of RBC can be affected from changes in the roughness, becoming more fragile and susceptible to breakage.