Discrimination of functional hepatocytes derived from mesenchymal stem cells using FTIR microspectroscopy

Abstract

Functional hepatocytes differentiated in vitro from mesenchymal stem cells (MSCs) need to be fully characterized before they could be applied as a therapy to treat liver disease. Here, we employed Fourier Transform Infrared (FTIR) microspectroscopy to investigate the characteristics of hepatocyte-like cells derived from rat bone marrow mesenchymal stem cells (rBM-MSCs) by detecting changes in macromolecular composition occurring during the hepatogenesis process. Partial Least Squares Discriminant Analysis (PLS-DA) enabled us to discriminate undifferentiated rBM-MSCs, and early, mid-stage and late stage rBM-MSCs derived hepatocytes by their characteristic FTIR "spectroscopic signatures". The predominant spectroscopic changes responsible for this discrimination were changes in FTIR absorbance bands at: 3012 cm(-1) (cis C[double bond, length as m-dash]C stretch from unsaturated lipids), 2952 cm(-1) (ν(as)CH(3) from lipids), 2854 cm(-1) (ν(s)CH(2) from lipids) and 1722 cm(-1) (C[double bond, length as m-dash]O stretching from lipids), which were associated with triglyceride and unsaturated fatty acid accumulation in the hepatocyte-like cells occurring during differentiation. Based on these findings, rBM-MSCs derived hepatocytes are characterized by high lipid content which facilitates a means of identifying hepatocytes from their stem cells progenitors by using FTIR microspectroscopy. Other complex changes in spectral bands assigned to proteins and nucleic acids were observed during hepatocyte differentiation indicating that mRNA translation was taking place producing proteins related to the formation of the new hepatocyte-like phenotype, which was corroborated by immunohistochemistry. The results show FTIR microspectroscopy combined with bioinformatic modeling constitutes a powerful new phenotypic-based methodology for monitoring and characterization of the process of stem cell differentiation leading to the formation of hepatocytes, providing complementary information to existing methodologies such as immunohistochemistry and gene analysis, but having advantages of being reagent-free and non-destructive of the sample.

Fourier transform infrared microspectroscopy reveals biochemical changes associated with glioma stem cell differentiation

“…Spectral changes related to proteins clearly suggested that differentiated glioma stem cells express a different set of genes/proteins compared to control stem cells, although the specific proteins remain unidentified. Similar conclusions were drawn by Ye et al [25], where they observed several changes in protein secondary structure at different levels of differentiation of mesenchymal stem cells. In addition, both PC4 loading and the difference spectrum showed a decrease of the spectral band centered at 965 cm −1 , assigned to the symmetric stretching mode of di-anionic phosphate mono-esters of phosphorylated proteins [26] and therefore pointing to a decrease in the level of phosphorylated proteins in the differentiated glioma cells.…”

The Characterisation of Pluripotent and Multipotent Stem Cells Using Fourier Transform Infrared Microspectroscopy

“…[27] aligned with non-spectroscopic studies which report that hepatocytes routinely synthesise lipoproteins and synthesise and store triglycerides as a result of activities from carbohydrate metabolic pathways. Moreover, a significant increase in unsaturated fatty acid levels was seen in the late stage hepatocytes, indicated by an increase in the intensity of C–H stretching band associated with the cis double bond C=C at 3010 cm −1 .…”

“…The spectroscopic technique has also been adopted for studies of rat bone marrow mesenchymal stem cells (rBM-MSCs) differentiated to hepatocytes [27]. The main findings from the work were that the late stage differentiated cells possessed significantly higher levels of lipid compared to the stem cell progenitors and early stage differentiated cells, with notable differences in the FTIR absorbance bands at: 3012 cm −1 (cis C=C stretch from unsaturated lipids), 2952 cm −1 (ν as CH 3 from lipids), 2854 cm −1 (ν s CH 2 from lipids) and 1722 cm −1 (C=O stretching from lipids).…”

“…The correlation coefficients ( R 2 > 0.98 for all calibration sets) indicated that all the datasets were well modelled. PLS-DA correctly classified and discriminated all of the validation spectra [27]. …”

Opportunities for Live Cell FT-Infrared Imaging: Macromolecule Identification with 2D and 3D Localization

“…Most characterization approaches rely on sample manipulation to gain information beyond morphology and are incapable of analyzing intact 3D cell aggregates that are known to be crucial for the maintenance of stem cell state [82]. Recently, 2D vibrational spectroscopy has been applied to study stem cells and several reports have noted a significant spectroscopic change in bands associated pluripotent versus multipotent cells [83,84] and between multipotent cells and derived hepatocytes [85]. The utility of vibrational spectroscopy in the future would benefit greatly from the 3D spectral microtomography technique and associated preservation of crucial cell–cell and cell–extracellular matrix interactions.…”