De novo synthesis of phospholipids and sphingomyelin in multipotent stromal cells - Monitoring studies by mass spectrometry

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Highlights

Abstract

Musculoskeletal diseases are extremely widespread and a significant burden on the health systems of the industrialized countries. The use of mesenchymal stromal cells is a promising approach to cure cartilage and tendon injuries, which often also occur in younger people as consequences of sport accidents. Although particular interest is on the collagen and the glycosaminoglycan composition of the tendon and potential alterations compared to healthy tissue, there is nowadays also increasing evidence that some selected phospholipids (PL) are potential mediators of tissue regeneration. Therefore, PL (and potential changes thereof) attract increasing interest in this field. We have used positive and negative ion matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) to elucidate the lipid compositions of human mesenchymal stromal cells in dependence on the composition of the cell culture medium and the cultivation time. The de novo biosynthesis of PL was monitored by adding 13C labeled glucose or deuterated palmitic acid (d31-PA) to the cells and the incorporation of 13C or 2H into the different PL classes was investigated by electrospray ionization (ESI) mass spectrometry (MS). It is remarkable that all PL classes (for instance, phosphatidylcholine and -inositol) exhibited 13C incorporation - but not the sphingomyelin (SM) which is the most abundant sphingolipid in the majority of human tissues and body fluids. Using suitable internal standards it could be shown, that only 12C-containing SM is de novo generated while no 13C-labeled SM could be monitored - independent of the cultivation time, which was varied between 7 and 28 days. SM impurities stemming from the cell culture medium and the used MALDI matrix compounds (2,5-dihydroxybenzoic acid (DHB) or 9-aminoacridine (9-AA)) could be ruled out. However, incorporation of deuterated palmitic acid (d31-PA) could be observed for multiple PL, including SM. Therefore, it is suggested that there must exist another, so far unknown SM biosynthesis pathway. This pathway does not make use of glucose but relies on the use of other molecules as energy sources. Potential pathways to explain the experimental observations are discussed.

Introduction

Next to abundant MSC (mesenchymal stromal cells)-generated extracellular compounds, such as glycosaminoglycans (GAG) – in particular chondroitin sulfate – or characteristic proteins such as collagens, PL are additional extracellular matrix (ECM) components and known to play an important role in cell augmentation and tissue formation. Sphingolipids, in particular, are nowadays recognized as important second messengers towards signal-transduction pathways that affect cell growth, differentiation, stress responses and programmed death (Khavandgar and Murshed, 2015).

Our main research focus is on the examination of musculoskeletal tissues such as cartilage or tendon and the investigation of how the composition of ECM can be altered and, thus, how the mechanical properties of ECM can be improved under cell culture conditions. In this context we already monitored the de novo biosynthesis of GAG by stable isotope labeling (addition of 13C-labeled glucose) in equine derived multipotent mesenchymal stromal cells by mass spectrometric methods (Roth et al., 2018). MSC were frequently used in recent investigations of tissue regeneration and the so far obtained data are rather promising to cure diseases of the ECM (Uder et al., 2018).

Tendon injuries are often a consequence of sport accidents and affect, thus, particularly younger people. Since the regeneration capacity of tendon is limited, this may massively affect the quality of life of these people. Degenerative processes and mechanical dysfunctions are associated with imbalanced expression patterns in the ECM of cartilage and tendon (Lorenzo et al., 2004; Heijink et al., 2012; Smith et al., 2008; Choi et al., 2016; Riley et al., 1994). The use of MSC from adipose tissue is a promising approach for tissue repair and restoring its natural structure and function (Vangsness et al., 2014; Gaspar et al., 2015). Cell-guided regeneration includes paracrine effects and targeted differentiation leading to cell growth and the production of the extracellular matrix. Cellular mechanisms of action, with regard to the regeneration potential of MSC in, for example, cartilage or tendon have been investigated (Marquass et al., 2011; Burk, 2019). Given the fact that experiments with horses and/or sheep are already underway, a reliable assessment of the de novo synthesis of ECM components by MSC is of particular importance.

It is assumed that (particularly) during healing processes (e.g. after surgery) phospholipids (in combination with e.g. hyaluronic acid or lubricin) play important roles in the prevention of adhesion (Sun et al., 2008), which leads (next to scarring) to functional impairment of tendons in their tendon sheath and impaired mobility (Khanna et al., 2009). Thus, PL possess a significant impact on the biomechanical properties of the tendons.

Mass spectrometry (MS) is presumably the most powerful technique to study the compositions of lipid mixtures in body fluids or tissues (Wang et al., 2019). This particularly applies because the lipids of interest are normally only available in small amounts. This prevents the application of spectroscopic techniques, such as nuclear magnetic resonance (NMR) spectroscopy, due to their poor sensitivities (Li et al., 2017). MS is much more sensitive and particularly enables the differentiation of acyl residues, which differ in the number of carbon atoms. However, all “soft” ionization MS techniques, such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) suffer from the effect of ion suppression (Gross, 2011), i.e. the individual lipids within a mixture are not detected according to their concentration but according to their tendency to generate ions. This is the reason why phosphatidylcholines and sphingomyelins (both contain positively charged quaternary ammonium groups) are nearly exclusively detected if lipid mixtures are investigated by positive ion MS and suppress the detection of other lipid classes (Petkovic et al., 2001). Fortunately, this problem can be largely overcome by comparing the positive and negative ion mass spectra and at least the most abundant lipid classes can be detected using this approach.

Section snippets

Chemicals

If not otherwise specified, all chemicals (salts, phospholipid standards (synthesized by AVANTI Polar Lipids), buffer components, and solvents) were purchased at highest commercially available purity from Merck KGaA (Darmstadt, Germany) and were used without further purification.

In Dulbeccos Modified Eagles Medium (DMEM)

Equine MSCs (eMSC) were isolated from equine adipose tissue by collagenase I digestion as essentially described in Burk et al., 2013. The isolated cells were washed twice in phosphate buffered saline (PBS), suspended in

Lipid extraction and mass spectrometry (MS)

The lipid extraction was performed according to the procedure by Bligh and Dyer (1959). First of all, cell pellets were suspended in 100 μL H2O and afterwards mixed with chloroform/methanol (1:1, v/v). Complete phase separation was achieved after a 5 min centrifugation step at 1000 × g. The lower chloroform phase was isolated with a glass syringe (Hamilton Germany GmbH). The lipid extracts were immediately analyzed by mass spectrometry or stored at −20 °C until analysis was possible.

Results and discussion

First, MSC were cultured in DMEM, supplemented either with 13C-labeled d-glucose or with standard (12C) d-glucose, to obtain evidence for the de novo biosynthesis of phospholipids, i.e. for augmentation of cells and to investigate whether 12C or 13C induce major differences on the cellular level (Fig. 2).

There were no major differences - independent if the cultivation was performed in the presence of standard d-glucose or 13C-labeled glucose. This was expected because no radioactive labeling,

Conclusion and outlook

These new results suggest that MSC-derived SM, is not synthesized through a biosynthesis pathway which is reliant on the use of the supplemented glucose. Since palmitic acid is incorporated into SM during de novo biosynthesis, we were able to establish a clear difference between PC and SM synthesis. Further investigations could shine a light on a novel biosynthesis pathway of de novo SM production. Details of this pathway are largely unknown so far.

Ethics

The consent of the donors for sampling and the approval of the local ethics committee are available; AZ 053−13-11032013.

Funding

This work was supported by the German Research Council (DFG NI 1396/3-2 as well as Project 59307082 – TRR67/A8 & Z3 as well as SCHI 476/16−1).

Acknowledgements

The authors thank Dr. Maria Fedorova for support with the LC–MS measurements.

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