Molecular tracking of interactions between progenitor and endothelial cells via Raman and FTIR spectroscopy imaging: a proof of concept of a new analytical strategy for in vitro research

Abstract

Circulating endothelial cell progenitors originating from the bone marrow are considered to be a powerful tool in the repair of endothelium damage. Due to their unique properties, endothelial progenitors are now broadly investigated to assess their clinical significance in diseases e.g., associated with brain endothelial dysfunction. However, their distinction in terms of the expression of specific markers remains ambiguous. Additionally, endothelial progenitor cells may change their repertoire of markers depending on the microenvironment of the tissue in which they are currently located. Here, we applied the label-free Raman and FTIR imaging to discriminate mice brain endothelium and endothelial progenitors. Cells cultured separately showed distinctly different spectral signatures extracted from the whole cellular interior as well as the detected intracellular compartments (nucleus, cytoplasm, perinuclear area, and lipid droplets). Then, we used these spectroscopic signals to examine the cells co-cultured for 24Â h. Principal cluster analysis showed their grouping with the progenitor cells and segregation from brain endothelium at a level of the entire cell machinery (in FTIR images) which resulted from biochemical alternations in the cytoplasm and lipid droplets (in Raman images). The models included in partial least square regression indicated that lipid droplets are the key element for the classification of endothelial progenitor-brain endothelial cells interactions.

Keywords: Intercellular interactions; Mouse aorta gonad mesonephros endothelial cells 11.5 (MAgEC11.5); Mouse brain microvascular endothelial cells (MBrMEC); Multivariate analysis; Raman and FTIR imaging.

Fig. 1

Exemplary fluorescence images of Endothelial Progenitor MAgEC11.5 and Brain Endothelial MBrMEC cells stained with a Hoechst 33342 (nuclei), b Dil (lipophilic membranes), and c Phalloidin-Atto 488 (F-actin) dyes. d Merged images. Magnification 60×

Fig. 2

Schematic displaying the workflow of Raman and FTIR spectroscopy imaging of the studied cells

Fig. 3

Bright-field (a) and distribution Raman images (b-d) of single brain endothelial (MBrMEC) and endothelial progenitor (MAgEC11.5) cells collected with a step size of 0.3 µm. The distribution of main macromolecules was calculated for organic matter (integration region: 2800-3030 cm⁻¹), lipids (integration region: 2830-2900 cm⁻¹), and nucleic acids (integration region: 790-810 cm⁻¹). The corresponding false-color KMC maps (e) reveal the presence of the main subcellular compartments: cytoplasm (gray), nucleus (blue), perinuclear area (brown), and lipid droplets (red)

Fig. 4

The scores (left) and loadings (right) plots from principal component analysis performed on Raman spectra of their cellular compartments (3050-500 cm^-1) and FTIR spectra of the whole MAgEC11.5 EPCs and MBrMEC cells (3000-1000 cm^-1): a cytoplasm, b nucleus, c perinuclear area, d lipid droplets, e whole cells. The fingerprint region (below 1800 cm^-1) is shown only for the perinuclear area, because of the lack of high loading vectors in the high-wavenumber region (data not shown). Each point refers to a single cell

Fig. 5

The scores plots (left) and loadings (right) from PCA analysis performed on Raman spectra of cellular compartments of MAgEC11.5 EPCs and MBrMEC cells, and their 24-h co-cultured cells. Each point refers to a single cell

Fig. 6

The PCA scores plots for a 24-h and b 4-h co-cultures of whole MAgEC11.5 EPCs and MBrMEC cells together with c PC-1 loading graphs of the 24-h co-culture and individual cells lines (from Fig. 4e) compared with the difference spectrum (from Fig. S2). Data analysis was performed for the whole cells acquired in FTIR spectroscopy imaging

Fig. 7

Bar chart with the % classification of the 24-h co-cultured cells to whole MAgEC11.5 EPCs and MBrMEC cells based on the a cellular compartment – cytoplasm, cell nucleus, endoplasmic reticulum, and lipid droplets (high-resolution Raman imaging) and b the whole cells (FTIR imaging) compared with negative control