Abstract
Multipotent mesenchymal stromal cells (MSC) have emerged as therapeutic tools for a wide range of pathological conditions. Yet, the still existing deficits regarding MSC phenotype characterization and the resulting heterogeneity of MSC used in different preclinical and clinical studies hamper the translational success. In search for novel MSC characterization approaches to complement the traditional trilineage differentiation and immunophenotyping assays reliably across species and culture conditions, this study explored the applicability of lipid phenotyping for MSC characterization and discrimination. Human peripheral blood mononuclear cells (PBMC), human fibroblasts, and human and equine adipose-derived MSC were used to compare different mesodermal cell types and MSC from different species. For MSC, cells cultured in different conditions, including medium supplementation with either fetal bovine serum or platelet lysate as well as culture on collagen-coated dishes, were additionally investigated. After cell harvest, lipids were extracted by chloroform/methanol according to Bligh and Dyer. The lipid profiles were analysed by an untargeted approach using liquid chromatography coupled to mass spectrometry (LC-MS) with a reversed phase column and an ion trap mass spectrometer. In all samples, phospholipids and sphingomyelins were found, while other lipids were not detected with the current approach. The phospholipids included different species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) in all cell types, whereas phosphatidylglycerol (PG) species were only present in MSC. MSC from both species showed a higher phospholipid species diversity than PBMC and fibroblasts. Few differences were found between MSC from different culture conditions, except that human MSC cultured with platelet lysate exhibited a unique phenotype in that they exclusively featured PE O-40:4, PG 38:6 and PG 40:6. In search for specific and inclusive candidate MSC lipid markers, we identified PE O-36:3 and PG 40:7 as potentially suitable markers across culture conditions, at which PE O-36:3 might even be used across species. On that basis, phospholipid phenotyping is a highly promising approach for MSC characterization, which might condone some heterogeneity within the MSC while still achieving a clear discrimination even from fibroblasts. Particularly the presence or absence of PG might emerge as a decisive criterion for future MSC characterization.
Highlights
Multipotent mesenchymal stromal cells (MSC) are being explored as a therapeutic agent for a wide range of pathological conditions, in human as well as in veterinary medicine
For human MSC derived from bone marrow, this study revealed that their plasma membranes contained high proportions of fully saturated and polyunsaturated fatty acids but less diunsaturated lipids, and nearly no phosphatidylserine headgroups, which appeared to be distinct from other cell types
Less lipid diversity was observed in the other mesodermal cell types, human buffy coat PBMC (hPBMC) and hFibroblasts
Summary
Multipotent mesenchymal stromal cells (MSC) are being explored as a therapeutic agent for a wide range of pathological conditions, in human as well as in veterinary medicine. This results from the versatility of MSC characteristics depending on the MSC origin tissue (Hass et al, 2011; Burk et al, 2013; Fabre et al, 2019) and species (Schwarz et al, 2012; Hillmann et al, 2016), as well as on the culture conditions. Variations in the latter include culture media and supplements used (Schubert et al, 2018; Czapla et al, 2019; Hagen et al, 2021), 2D vs 3D culture and possible scaffold materials (Rashedi et al, 2017; Yang et al, 2018), or static vs dynamic culture and oxygen tension (Frith et al, 2010; Yeatts et al, 2013). The ISCT has recommended to complement this traditional characterization with functional assays (Galipeau et al, 2016), the choice of which depends on the therapeutic target and aim, this cannot be universally applied
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