Metabolism and phospholipid assembly of polyunsaturated fatty acids in human bone marrow mesenchymal stromal cells

Abstract

High arachidonic acid (20:4n-6) and low n-3 PUFA levels impair the capacity of cultured human bone marrow mesenchymal stromal cells (hBMSCs) to modulate immune functions. The capacity of the hBMSCs to modify PUFA structures was found to be limited. Therefore, different PUFA supplements given to the cells resulted in very different glycerophospholipid (GPL) species profiles and substrate availability for phospholipases, which have preferences for polar head group and acyl chains when liberating PUFA precursors for production of lipid mediators. When supplemented with 20:4n-6, the cells increased prostaglandin E2 secretion. However, they elongated 20:4n-6 to the less active precursor, 22:4n-6, and also incorporated it into triacylglycerols, which may have limited the proinflammatory signaling. The n-3 PUFA precursor, 18:3n-3, had little potency to reduce the GPL 20:4n-6 content, while the eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acid supplements efficiently displaced the 20:4n-6 acyls, and created diverse GPL species substrate pools allowing attenuation of inflammatory signaling. The results emphasize the importance of choosing appropriate PUFA supplements for in vitro hBMSC expansion and suggests that for optimal function they require an exogenous fatty acid source providing 20:5n-3 and 22:6n-3 sufficiently, but 20:4n-6 moderately, which calls for specifically designed optimal PUFA supplements for the cultures.

Keywords: arachidonic acid; docosahexaenoic acid; eicosapentaenoic acid; glycerophospholipid; immunomodulation; lipid signaling; mass spectrometry; mesenchymal stromal/stem cell; prostaglandin E2.

Fig. 1.

Fatty acid profiles (mole percent in total fatty acids, mean ± SD) in hBMSCs (SC, in control medium, n = 4) compared with those in different lots of FBS (n = 5) and in clinical samples of human bone marrow (BM, n = 4). A: Total proportions of SFAs, MUFAs, PUFAs, n-6 PUFAs, and n-3 PUFAs The ratio of n-3 to n-6 PUFAs (n-3/n-6) is shown in the insert. B: Individual SFAs and MUFAs. The ratio of MUFA total to SFA total is shown in the insert. C: Individual n-6 PUFAs. D: Individual n-3 PUFAs. As statistics, Kruskal-Wallis nonparametric one-way ANOVA followed by post hoc Mann-Whitney test for the means were used. The means with no common letter differed at the P < 0.05 level. Missing letters on the bars mean that the Kruskal-Wallis test showed no significant differences between the means.

Fig. 2.

Comparison of the fatty acid profiles (mole percent in total fatty acids, mean ± SD) in control (Ctrl) and 18:2n-6- or 18:3n-3-supplemented hBMSCs (SC) and HepG2 (Hep) cells (n = 4). A: Total proportions of SFAs, MUFAs, PUFAs, n-6 PUFAs, and n-3 PUFAs. B: Individual SFAs and MUFAs. C: Individual n-6 PUFAs. D: Individual n-3 PUFAs. Statistics are as in Fig. 1. In the cases where a value exceeded the scale, the value was shown at the top of the bar.

Fig. 3.

Scheme of the metabolism of n-6 and n-3 PUFAs in the hBMSCs and HepG2 cells by desaturase and elongase enzymes. The average levels of different PUFAs in hBMSCs and HepG2 cells on the pathways are visualized with bars and related values (mole percent) of the cells grown in control (Ctrl) media. The measured mRNA levels of the elongases (ELOVL5 and ELOVL2) and desaturases (FADS2, FADS1) involved are marked inside the blue ovals. The values inside the arrows are the average expression levels of the enzymes that were obtained for mesenchymal stem cells (SC) from a public online database, IST, and normalized against hepatocyte (Hep) values of the database. Per β-ox, peroxisomal partial β-oxidation.

Fig. 4.

Fatty acid profiles (mole percent in total fatty acids, mean ± SD) in hBMSCs (n = 4) cultured for 9 days in the control (Ctrl) medium or media supplemented with different n-6 and n-3 PUFAs. A: Profiles of individual SFAs and MUFAs in cells cultured in the Ctrl medium and in the cells the medium of which was supplemented with 18:2n-6, 20:4n-6, 18:3n-3, 20:5n-3, or 22:6n-3. B: Profiles of PUFAs in the cells cultured in the Ctrl medium and in the medium supplemented with 18:2n-6 or 20:4n-6. C: PUFA profiles in the cells cultured in the Ctrl medium and in the medium supplemented with 18:3n-3, 20:5n-3, or 22:6n-3. Statistics are as in Fig. 1. In the cases where the value exceeded the scale, the mean and SD are shown at the top of the bar. D: Scheme of the metabolism of n-6 and n-3 PUFAs in the hBMSCs cultured in control (Ctrl) medium or media supplemented with the different n-6 and n-3 PUFAs. The enzymes metabolizing the PUFAs are indicated (abbreviations explained in Fig. 3). The values on the bars represent mole percent of the PUFAs per total fatty acids.

Fig. 4.

Fatty acid profiles (mole percent in total fatty acids, mean ± SD) in hBMSCs (n = 4) cultured for 9 days in the control (Ctrl) medium or media supplemented with different n-6 and n-3 PUFAs. A: Profiles of individual SFAs and MUFAs in cells cultured in the Ctrl medium and in the cells the medium of which was supplemented with 18:2n-6, 20:4n-6, 18:3n-3, 20:5n-3, or 22:6n-3. B: Profiles of PUFAs in the cells cultured in the Ctrl medium and in the medium supplemented with 18:2n-6 or 20:4n-6. C: PUFA profiles in the cells cultured in the Ctrl medium and in the medium supplemented with 18:3n-3, 20:5n-3, or 22:6n-3. Statistics are as in Fig. 1. In the cases where the value exceeded the scale, the mean and SD are shown at the top of the bar. D: Scheme of the metabolism of n-6 and n-3 PUFAs in the hBMSCs cultured in control (Ctrl) medium or media supplemented with the different n-6 and n-3 PUFAs. The enzymes metabolizing the PUFAs are indicated (abbreviations explained in Fig. 3). The values on the bars represent mole percent of the PUFAs per total fatty acids.

Fig. 5.

PC species profiles in hBMSCs cultured for 9 days in the control (Ctrl) medium or medium supplemented with 18:2n-6 or 20:4n-6 (mole percent per total PC, mean ± SD). For the isobaric species (marked horizontally) having more than one quantitatively important species (acyl chain combination marked vertically), the two most important are listed (in the reading order, the first one being the main species). When the double bond positions of the acyl chains were not marked, several isomers (revealed by GC-MS of fatty acids) were present in the cells for that particular chain. Statistics are as in Fig. 1.

Fig. 6.

PE (A) and PEp (B) species profiles in hBMSCs cultured for 9 days in the control (Ctrl) medium or medium supplemented with 18:2n-6 or 20:4n-6 (mole percent per total PE or PEp, mean ± SD). For the isobaric species (marked horizontally) having more than one quantitatively important species (acyl and alkenyl chain combination marked vertically), the two most important are listed (in the reading order, the first one being the main species). When the double bond positions of the acyl chains were not marked, several isomers (revealed by GC-MS of fatty acids) were present in the cells for that particular chain. Statistics are as in Fig. 1.

Fig. 7.

PS species profiles in hBMSCs cultured for 9 days in the control (Ctrl) medium or medium supplemented with 18:2n-6 or 20:4n-6 (mole percent per total PS, mean ± SD). For the isobaric species (marked horizontally) having more than one quantitatively important species (acyl chain combination marked vertically), the two most important were listed (in the reading order the first one being the main species). Quantitatively important PI species are shown in the insert. When the double bond positions of the acyl chains were not marked, several isomers (revealed by GC-MS of fatty acids) were present in the cells for that particular chain. Statistics are as in Fig. 1.

Fig. 8.

PC species profiles in hBMSCs cultured for 9 days in the control (Ctrl) medium or medium supplemented with 18:3n-3, 20:5n-3, or 22:6n-3 (mole percent per total PC, mean ± SD). For the isobaric species (marked horizontally) having more than one quantitatively important species (acyl chain combination marked vertically) the two most important are listed (in the reading order, the first one being the main species). When the double bond positions of the acyl chains were not marked, several isomers (revealed by GC-MS of fatty acids) were present in the cells for that particular chain. Statistics as in Fig. 1.

Fig. 9.

PE (A) and PEp (B) species profiles in hBMSCs cultured for 9 days in the control (Ctrl) medium or medium supplemented with 18:3n-3, 20:5n-3, or 22:6n-3 (mole percent per total PE or PEp, mean ± SD). For the isobaric species (marked horizontally) having more than one quantitatively important species (acyl and alkenyl chain combination marked vertically), the two most important are listed (in the reading order, the first one being the main species). When the double bond positions of the acyl chains were not marked, several isomers (revealed by GC-MS of fatty acids) were present in the cells for that particular chain. Statistics are as in Fig. 1.

Fig. 10.

PS species profiles in hBMSCs cultured for 9 days in the control (Ctrl) medium or medium supplemented with 18:3n-3, 20:5n-3, or 22:6n-3 (mole percent per total PS, mean ± SD). For the isobaric species (marked horizontally) having more than one quantitatively important species (acyl chain combination marked vertically), the two most important are listed (in the reading order, the first one being the main species). Quantitatively important PI species are shown in the insert. When the double bond positions of the acyl chains were not marked, several isomers (revealed by GC-MS of fatty acids) were present in the cells for that particular chain. Statistics are as in Fig. 1.

Fig. 11.

Distribution of lipid species carrying important PUFA precursors of signaling molecules between different GPL classes in hBMSCs cultured for 9 days in the control (Ctrl) medium or medium supplemented with 18:2n-6, 20:4n-6, 18:3n-3, 20:5n-3, or 22:6n-3 (mole percent sums of all species with the specific PUFA in each GPL class, mean ± SD). A: Distribution of species carrying 20:4n-6 between the GPL classes. B: Distribution of species carrying 20:5n-3 between the GPL classes. C: Distribution of species carrying 22:6n-3 between the GPL classes. Statistics are as in Fig. 1.