Raman Imaging Providing Insights into Chemical Composition of Lipid Droplets of Different Size and Origin: In Hepatocytes and Endothelium

In this work, 3D linear Raman spectroscopy was used to study lipid droplets (LDs) <i>ex vivo</i> in liver tissue and also <i>in vitro</i> in a single endothelial cell. Spectroscopic measurements combined with fluorescence microscopy and/or histochemical staining gave complex chemical information about LD composition and enabled detailed investigations of the changes occurring in various pathological states. Lipid analysis in fatty liver tissue was performed using a dietary mouse model of liver steatosis, induced by a high fat diet (HFD). HFD is characterized by a high percentage of calories from saturated fat (60%) and reflects closely the detrimental effects of dietary habits responsible for increased morbidity due to obesity and its complications in well-developed Western societies. Such diets lead to obesity, hyperlipidemia, insulin resistance, and steatosis that may also be linked to endothelial dysfunction. In the present work, Raman spectroscopy was applied to characterized chemical composition of lipid droplets in hepatocytes from mice fed HFD and in the endothelium treated with exogenous unsaturated free fatty acid (arachidonic acid). The results demonstrate the usefulness of Raman spectroscopy to characterize intracellular lipid distribution in 2D and 3D images and can be used to determine the degree of saturation. Raman spectroscopy shows the potential to be a valuable tool for studying the role of LDs in physiology and pathology. The method is generally applicable for the determination of LDs of different size, origin, and composition. Moreover, for the first time, the process of LD formation in the endothelium was detected and visualized in 3D

Murine Bone Marrow Lin⁻Sca-1⁺CD45⁻ Very Small Embryonic-Like (VSEL) Cells Are Heterogeneous Population Lacking Oct-4A Expression

<div><p>Murine very small embryonic-like (VSEL) cells, defined by the Lin⁻Sca-1⁺CD45⁻ phenotype and small size, were described as pluripotent cells and proposed to be the most primitive hematopoietic precursors in adult bone marrow. Although their isolation and potential application rely entirely on flow cytometry, the immunophenotype of VSELs has not been extensively characterized. Our aim was to analyze the possible heterogeneity of Lin⁻Sca⁺CD45⁻ population and investigate the extent to which VSELs characteristics may overlap with that of hematopoietic stem cells (HSCs) or endothelial progenitor cells (EPCs). The study evidenced that murine Lin⁻Sca-1⁺CD45⁻ population was heterogeneous in terms of c-Kit and KDR expression. Accordingly, the c-Kit⁺KDR⁻, c-Kit⁻KDR⁺, and c-Kit⁻KDR⁻ subpopulations could be distinguished, while c-Kit⁺KDR⁺ events were very rare. The c-Kit⁺KDR⁻ subset contained almost solely small cells, meeting the size criterion of VSELs, in contrast to relatively bigger c-Kit⁻KDR⁺ cells. The c-Kit⁻KDR⁻FSC^low subset was highly enriched in Annexin V-positive, apoptotic cells, hence omitted from further analysis. Importantly, using qRT-PCR, we evidenced lack of Oct-4A and Oct-4B mRNA expression either in whole adult murine bone marrow or in the sorted of Lin⁻Sca-1⁺CD45⁻FSC^low population, even by single-cell qRT-PCR. We also found that the Lin⁻Sca-1⁺CD45⁻c-Kit⁺ subset did not exhibit hematopoietic potential in a single cell-derived colony <i>in vitro</i> assay, although it comprised the Sca-1⁺c-Kit⁺Lin⁻ (SKL) CD34⁻CD45⁻CD105⁺ cells, expressing particular HSC markers. Co-culture of Lin⁻Sca-1⁺CD45⁻FSC^low with OP9 cells did not induce hematopoietic potential. Further investigation revealed that SKL CD45⁻CD105⁺ subset consisted of early apoptotic cells with fragmented chromatin, and could be contaminated with nuclei expelled from erythroblasts. Concluding, murine bone marrow Lin⁻Sca-1⁺CD45⁻FSC^low cells are heterogeneous population, which do not express the pluripotency marker Oct-4A. Despite expression of some hematopoietic markers by a Lin⁻Sca-1⁺CD45⁻c-Kit⁺KDR⁻ subset of VSELs, they do not display hematopoietic potential in a clonogenic assay and are enriched in early apoptotic cells.</p></div