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sc-12-0341_sm_SupplFigure1.pdf77KSupplemental Figure 1. hMSC spheroid-conditioned medium reduces the secretion of TNFα by stimulated macrophages. (A) Images of non-stimulated and LPS-stimulated J774 mouse macrophages after 18 h incubation with hMSC spheroid-conditioned medium. (B) hMSC spheroid-conditioned medium increases the growth of LPS-stimulated J774 mouse macrophages. (C) hMSC spheroid-conditioned medium decreases the secretion of mTNFα by zymosanstimulated mouse macrophages (J774). (D) hMSC spheroid-conditioned medium decreases the secretion of mTNFα by LPS-stimulated mouse macrophages (Raw). (E) hMSC spheroidconditioned medium decreases the secretion of hTNFα by LPS-stimulated human macrophages (U937). Values are mean ± SD (n = 3). ns P ≥ 0.05, ***P < 0.001 compared to control sMΦ in B and vehicle control (CCM) in C, D, and E. Scale bar: 50 μm. Abbreviations: CCM, complete culture medium; CM, conditioned medium; LPS, lipopolysaccharide; MΦ, macrophage; sMΦ, stimulated macrophage; Sph, spheroid hMSC from 3 d hanging drop cultures (25,000 cells/drop).
sc-12-0341_sm_SupplFigure2.tif1080KSupplemental Figure 2. hMSC spheroid-conditioned medium down-regulates the expression of pro-inflammatory genes and up-regulates the expression of antiinflammatory genes in stimulated macrophages. (A-C) hMSC spheroid-conditioned medium down-regulates the expression of Tnf (A), Cxcl2 (B), and Csf2 (C) in LPS-stimulated macrophages. (D-F) hMSC conditioned medium up-regulates the expression of Il10 (D), Il1rn (E), and Tgm2 (F) in LPS-stimulated macrophages. Real-time PCR measurements shown as mean (assay triplicates) relative quantities (RQ) compared to time 0 value of each gene. Abbreviations as in Fig. S1.
sc-12-0341_sm_SupplFigure3.tif1183KSupplemental Figure 3. hMSC spheroid-conditioned medium has an anti-inflammatory effect on stimulated macrophages. (A,B,C, D, E) Comparison of the spheroid and monolayer hMSC conditioned medium effect in production of mCXCL2 (A), mIL6 (B), mIL12p40 (C), mIL23 (D), and mIL1ra (E) by LPS-stimulated macrophages. (F) hMSC spheroid-conditioned medium increases the secretion of mIL10 by LPS-stimulated macrophages in a dose dependent manner. Values are mean ± SD (n = 3). ns P ≥ 0.05, *P < 0.05, **P < 0.01, ***P < 0.001 compared to sMΦ with vehicle control (CCM) in A, B, C, D, and E and compared to corresponding vehicle control (CCM) in F. Abbreviations: hMSC, human mesenchymal stem/stromal cell; Adh VH, adherent monolayer hMSCs plated at very high density (200,000 cells/cm2) and cultured for 3 d. Other abbreviations as in Fig. S1.
sc-12-0341_sm_SupplFigure4.tif814KSupplemental Figure 4. hMSC spheroid-conditioned medium increases the number of CD206 positive macrophages. hMSC spheroid-conditioned medium increases the number of CD206 positive LPS-stimulated macrophages measured with flow cytometry. Histogram overlays of representative samples (A) and combined data (B) are shown. Values are mean ± SD (n = 4). ***P < 0.001 compared to vehicle control (CCM) in B. Abbreviations as in Fig. S1.
sc-12-0341_sm_SupplFigure5.tif928KSupplemental Figure 5. Conditioned medium from adherent hMSC does not have an antiinflammatory effect and the anti-inflammatory effect of hMSC spheroid-conditioned medium is not mediated by nitric oxide. (A,B) Concentrated conditioned medium from adherent hMSC does not lower the mTNFα (A) or increase the mIL10 (B) secretion by LPSstimulated macrophages. (C) Inhibition of nitric oxide synthesis in hMSC spheroids does not prevent the effect of spheroid-conditioned medium on production of mTNFα by LPS-stimulated macrophages. Nitric oxide synthesis inhibitor concentrations are shown in μM. Values are mean ± SD (n = 3). ns P ≥ 0.05, ***P < 0.001 compared to corresponding vehicle control (CCM, or CCM conc) in A and compared to corresponding vehicle control (Wtr) in B. Abbreviations: Adh High, adherent monolayer hMSCs plated at high density (5,000 cells/cm2) and cultured for 3 d; Adh VH, adherent monolayer hMSCs plated at very high density (200,000 cells/cm2) and cultured for 3 d; conc, 28x concentrated conditioned medium; inh, inhibitor; Wtr, water. Other abbreviations as in Fig. S1.
sc-12-0341_sm_SupplFigure6.tif870KSupplemental Figure 6. hMSC spheroids and spheroid-derived cells produce large amounts of PGE2. The production of PGE2 by hMSC monolayers, spheroids, or spheroidderived cells was determined at time points indicated. Values are mean ± SD (n = 3). ns P ≥ 0.05, **P < 0.01, ***P < 0.001 compared to compared to control (Adh VH) in each time point. Abbreviations: Adh VH, adherent monolayer hMSCs plated at very high density (200,000 cells/cm2) and cultured for 3 d; hMSC, human mesenchymal stem/stromal cell; Sph DC, spheroid-derived cell. Other abbreviations as in Fig. S1.
sc-12-0341_sm_SupplFigure7.tif877KSupplemental Figure 7. Anti-inflammatory effect of hMSC spheroid-conditioned medium on stimulated macrophages is dependent on hMSC COX activity. (A-C) COX inhibition in hMSC spheroids reduces the production of PGE2 (A) and the anti-inflammatory effect of conditioned medium on LPS-stimulated macrophages measured as mTNFα (B) and mIL10 (C) secretion. Indomethacin doses are in μM and dose of 1 μM was used in C. Values are mean ± SD (n = 3). ns P ≥ 0.05, *P < 0.05, **P < 0.01, ***P < 0.001 compared to vehicle control (MetOH) in A, B, and C. Abbreviations: Indo, Indomethacin; MetOH, methanol. Other abbreviations as in Fig. S1.
sc-12-0341_sm_SupplFigure8.tif897KSupplemental Figure 8. COX2 knockdown in spheroid hMSCs reduces the antiinflammatory effect on stimulated macrophages. (A) Real-time PCR to asses the knockdown of COX2 gene, PTGS2, in hMSC spheroids with siRNA. hMSCs plated at a low density and grown for 7 d were used as a baseline. Biological triplicates were pooled and technical triplicates with 95% upper confidence intervals are shown. (B,C) COX2 knockdown in hMSC spheroids reduces the anti-inflammatory effect of conditioned medium on LPS-stimulated macrophages measured as mCXCL2 (B) and mIL1ra (C) secretion. Values are mean ± SD (n = 3). ***P < 0.001 compared to control (Scr) in B and C. Abbreviations: All, combination of COX2 siRNA #1, 2, and 3; Scr, negative control siRNA; Tran, Transfection control. Other abbreviations as in Fig. S1.
sc-12-0341_sm_SupplFigure9.tif778KSupplemental Figure 9. PGE2 can elicit an anti-inflammatory effect on stimulated macrophages. PGE2 reduces the secretion of mCXCL2 (A) and increases the secretion of mIL1ra (B) by LPS-stimulated macrophages in a dose-dependent manner. PGE2 doses are in ng/ml. Values are mean ± SD (n = 3). ns P ≥ 0.05, *P < 0.05, ***P < 0.001 compared to vehicle control (EtOH). Abbreviation: EtOH, ethanol. Other abbreviations as in Fig. S1.
sc-12-0341_sm_SupplFigure10.tif783KSupplemental Figure 10. PGE2 antibody reduces the anti-inflammatory effect of hMSC spheroid-conditioned medium on stimulated macrophages. (A) PGE2 neutralization in hMSC spheroid-conditioned medium reduces the anti-inflammatory effect of the conditioned medium on LPS-stimulated macrophages measured as mIL1ra secretion. PGE2 antibody doses are shown in μg/ml. (B) PGE2 neutralizing antibody inhibits the anti-inflammatory effect of added synthetic PGE2 on LPS-stimulated macrophages. PGE2 was used at 10 ng/ml and PGE2 antibody at 10 μg/ml. Values are mean ± SD (n = 3). ns P ≥ 0.05, ***P < 0.001 compared to vehicle control (PBS) in A and compared to vehicle control (EtOH) and PGE2 with PGE2 Ab in B. Abbreviations: Ab, antibody; EtOH, ethanol; PBS, phosphate buffered saline. Other abbreviations as in Fig. S1.
sc-12-0341_sm_SupplFigure11.tif753KSupplemental Figure 11. PGE2 addition restores the anti-inflammatory effect of conditioned medium from Indomethacin treated spheroids. Indomethacin doses are in μM and PGE2 doses in ng/ml. Indomethacin was used at 1 μM with PGE2. Values are mean ± SD (n = 3). ns P ≥ 0.05, ***P < 0.001 compared to corresponding vehicle control (MetOH or EtOH). Abbreviations: EtOH, ethanol; Indo, Indomethacin; MetOH, methanol. Other abbreviations as in Fig. S1.
sc-12-0341_sm_SupplFigure12.tif698KSupplemental Figure 12. PGD2 synthase is up-regulated in hMSC spheroids. Relative gene expression levels for PGD2 synthase from microarray data for 2 hMSC donors. Abbreviations: Adh High, adherent monolayer hMSCs plated at high density (5,000 cells/cm2) and cultured for 3 d; Adh Low, adherent monolayer hMSCs plated at low density (100-150 cells/cm2) and cultured for 7 d; D1, donor 1; hMSC, human mesenchymal stem/stromal cell. Other abbreviations as in Fig. S1.
sc-12-0341_sm_SupplFigure13.tif839KSupplemental Figure 13. hMSC spheroid-conditioned medium has a COX2-dependent anti-inflammatory effect on both LPS and zymosan stimulated macrophages. (A) hMSC spheroid-conditioned medium reduces the production of mTNFα by zymosan-stimulated mouse macrophages (J774) in a COX2-dependent manner. (B) hMSC spheroid-conditioned medium reduces the production of mTNFα by LPS-stimulated mouse macrophages (Raw) in a COX2- dependent manner. Indomethacin, COX2 inhibitor, and COX1 inhibitor were used at 1 μM. PGE2 was used at 10 ng/ml. Values are mean ± SD (n = 3). ns P ≥ 0.05, *P < 0.05, ***P < 0.001 compared to corresponding vehicle control (MetOH, Indo EtOH, or DMSO). Abbreviations: DMSO, dimethyl sulfoxide; EtOH, ethanol; Indo, Indomethacin; inh, inhibitor; MetOH, methanol. Other abbreviations as in Fig. S1.
sc-12-0341_sm_SupplFigure14.tif1041KSupplemental Figure 14. Anti-inflammatory effect of hMSC spheroid-conditioned medium on stimulated macrophages is mediated through EP4 receptor. (A) EP4 receptor antagonists inhibit the anti-inflammatory effect of hMSC spheroid-conditioned medium on stimulated macrophages measured as mIL1ra secretion. All EP receptor antagonists were used at 10 μM. (B,C) EP4 receptor antagonists inhibit the anti-inflammatory effect of hMSC spheroidconditioned medium in a dose dependent manner measured as mTNFα (B) and mIL10 (C) secretion. Doses for the two different EP4 receptor antagonists are shown as μM. (D) EP4 receptor antagonist inhibits the anti-inflammatory effect of PGE2 on stimulated macrophages measured as mTNFα secretion. EP4 antagonist was used at 10 μM. Values are mean ± SD (n = 3). ns P ≥ 0.05, ***P < 0.001 compared to vehicle control (DMSO) in A, B, and C and compared to corresponding vehicle control (DMSO, or PGE2 DMSO) in D. Abbreviation: DMSO, dimethyl sulfoxide. Other abbreviations as in Fig. S1.
sc-12-0341_sm_SupplFigure15.tif773KSupplemental Figure 15. EP4 receptor agonist has an anti-inflammatory effect on stimulated macrophages. (A,B) EP4 receptor agonist decreases the production of mCXCL2 (A) and increases the production of mIL1ra (B) by stimulated macrophages in a dose-dependent manner. Doses are shown in μM. Values are mean ± SD (n = 3). ns P ≥ 0.05, *P < 0.05, ***P < 0.001 compared to vehicle control (EtOH). Abbreviation: EtOH, ethanol. Other abbreviations as in Fig. S1.
sc-12-0341_sm_SupplFigure16.pdf907KSupplemental Figure 16. hMSC and hDF spheroids have similar morphology. Microscopy images of hMSC and hDF adherent and hanging drop cultures at 3 days and after 1 day transfer of spheroids. Scale bar 100 μm. Abbreviations: Adh High, adherent monolayer hMSCs plated at high density (5,000 cells/cm2) and cultured for 3 d; Adh VH, adherent monolayer hMSCs plated at very high density (200,000 cells/cm2) and cultured for 3 d.hDF, human adult dermal fibroblast; hMSC, human mesenchymal stem/stromal cell. Other abbreviations as in Fig. S1.
sc-12-0341_sm_SupplFigure17.tif901KSupplemental Figure 17. Expression of genes involved in PGE2 synthesis are lower in hDF spheroids than in hMSC spheroids. (A-C) Real-time PCR expression data for PTGES (A), PLA2G4A (B), and PLA2G4C (C) in hMSC and hDF monolayers and spheroids. hMSCs plated at a low density and grown for 7 d were used as a baseline. Values are mean ± SD (n = 3). ns P ≥ 0.05, *P < 0.05, ***P < 0.001 compared to hMSC Adh Low. Abbreviations: Adh High, adherent monolayer hMSCs plated at high density (5,000 cells/cm2) and cultured for 3 d; Adh Low, adherent monolayer hMSCs plated at low density (100-150 cells/cm2) and cultured for 7 d; Adh VH, adherent monolayer hMSCs plated at very high density (200,000 cells/cm2) and cultured for 3 d; hDF, human adult dermal fibroblast; hMSC, human mesenchymal stem/stromal cell; RQ, relative quantity. Other abbreviations as in Fig. S1.
sc-12-0341_sm_SupplFigure18.tif823KSupplemental Figure 18. hDF spheroid-conditioned medium does not have an antiinflammatory effect on stimulated macrophages. (A,B) hDF spheroid-conditioned medium does not reduce the secretion of mCXCL2 (A) or increase the secretion of mIL1ra (B) by LPSstimulated mouse macrophages. Values are mean ± SD (n = 3). ns P ≥ 0.05, *P < 0.05, ***P < 0.001 compared to vehicle control (CCM). Abbreviations: Adh High, adherent monolayer hMSCs/hDFs plated at high density (5,000 cells/cm2) and cultured for 3 d; Adh VH, adherent monolayer hMSCs/hDFs plated at very high density (200,000 cells/cm2) and cultured for 3 d; hDF, human adult dermal fibroblast; hMSC, human mesenchymal stem/stromal cell. Other abbreviations as in Fig. S1.
sc-12-0341_sm_SupplFigure19.tif711KSupplemental Figure 19. hMSC but not hDF spheroids produce large quantities of PGE2. Spheroids from 5 hMSC donors (D1-5) produced similar high levels of PGE2 whereas spheroids from 4 hDF donors (D6-9) produced only very small amounts of PGE2. Values are mean ± SD (n = 4). Abbreviations: Adh VH, adherent monolayer hMSCs/hDFs plated at very high density (200,000 cells/cm2) and cultured for 3 d; D1, donor 1; hDF, human adult dermal fibroblast; hMSC, human mesenchymal stem/stromal cell. Other abbreviations as in Fig. S1.
sc-12-0341_sm_SupplFigure20.tif910KSupplemental Figure 20. Caspase inhibition in hMSC spheroids reduces the antiinflammatory effect of the conditioned medium on stimulated macrophages. (A-C) Broadspectrum caspase inhibition reduces the production of PGE2 (A) and the anti-inflammatory effect of conditioned medium on LPS-stimulated macrophages measured as mTNFα (B) and mIL10 (C) secretion. Caspase inhibitor doses are in μM. Values are mean ± SD (n = 3). ns P ≥ 0.05, **P < 0.01, ***P < 0.001 compared to vehicle control (DMSO). Abbreviations: DMSO, dimethyl sulfoxide, inh, inhibitor. Other abbreviations as in Fig. S1.
sc-12-0341_sm_SupplFigure21.tif849KSupplemental Figure 21. NFκB inhibition in hMSC spheroids reduces the antiinflammatory effect of the conditioned medium on stimulated macrophages. (A-C) Inhibitor of NFκB transcriptional activation in hMSC spheroids reduces the production of PGE2 (A) and the anti-inflammatory effect of conditioned medium on LPS-stimulated macrophages measured as mTNFα (B) and mIL10 (C) secretion. NFκB inhibitor was used at 1 μM. Values are mean ± SD (n = 3). ***P < 0.001 compared to vehicle control (DMSO). Abbreviations: DMSO, dimethyl sulfoxide; inh, inhibitor. Other abbreviations as in Fig. S1.
sc-12-0341_sm_SupplTable1.pdf55KSupplemental Table 1.
sc-12-0341_sm_SupplTable2.pdf54KSupplemental Table 2.
sc-12-0341_sm_SupplTable3.pdf9KSupplemental Table 3.
sc-12-0341_sm_SupplTable4.pdf24KSupplemental Table 4.

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