In vitro migration and proliferation ("wound healing") potential of mesenchymal stromal cells generated from human CD271(+) bone marrow mononuclear cells.

BACKGROUND: Emerging evidence indicates that mesenchymal stromal cells (MSCs) isolated from different tissue sources may be used in vivo as tissue restorative agents. To date, there is no evidence, however, on migration and proliferation ("wound healing") potential of different subsets of MSCs. The main goal of this study was therefore to compare the in vitro "wound healing" capacity of MSCs generated from positively selected CD271(+) bone marrow mononuclear cells (CD271-MSCs) and MSCs generated by plastic adherence (PA-MSCs). METHODS: The in vitro model of wound healing (CytoSelect™ 24-Well Wound Healing Assay) was used in order to compare the migration and proliferation potential of CD271-MSCs and PA-MSCs of passage 2 and 4 cultured in presence or absence of growth factors or cytokines. RESULTS: CD271-MSCs of both passages when compared to PA-MSCs demonstrated a significantly higher potential to close the wound 12 and 24 h after initiation of the wound healing assay (P < 0.003 and P < 0.002, respectively). Noteworthy, the migration capacity of PA-MSCs of second passage was significantly improved after stimulation with FGF-2 (P < 0.02), PDGF-BB (P < 0.006), MCP-1 (P < 0.002) and IL-6 (P < 0.03), whereas only TGF-ß enhanced significantly migration process of PA-MSCs of P4 12 h after the treatment (P < 0.02). Interestingly, treatment of CD271-MSCs of both passages with growth factors or cytokines did not affect their migratory potential. CONCLUSIONS: Our in vitro data provide the first evidence that CD271-MSCs are significantly more potent in "wound healing" than their counterparts PA-MSCs.

Cytochrome P4502S1: a novel monocyte/macrophage fatty acid epoxygenase in human atherosclerotic plaques.

Cytochrome P450 (CYP) epoxygenases metabolize endogenous polyunsaturated fatty acids to their corresponding epoxides, generating bioactive lipid mediators. The latter play an important role in vascular homeostasis, angiogenesis, and inflammation. As little is known about the functional importance of extra-vascular sources of lipid epoxides, we focused on determining whether lipid epoxide-generating CYP isoforms are expressed in human monocytes/macrophages. Epoxides were generated by freshly isolated human monocytes and production increased markedly during differentiation to macrophages. Mass spectrometric analysis identified CYP2S1 as a novel macrophage CYP and CYP2S1-containing microsomes generated epoxides of arachidonic, linoleic and eicosapentaenoic acid. Macrophage CYP2S1 expression was increased by LPS and IFN-γ (classically activated), and oxidized LDL but not IL-4 and IL-13 (alternatively activated), and was colocalised with CD68 in inflamed human tonsils but not in breast cancer metastases. Prostaglandin (PG) E(2) is an immune modulator factor that promotes phagocytosis and CYP2S1 can metabolize its immediate precursors PGG(2) and PGH(2) to 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT). We found that CYP inhibition and siRNA-mediated downregulation of CYP2S1 increased macrophage phagocytosis and that the latter effect correlated with decreased 12-HHT formation. Although no Cyp2s1 protein was detected in aortae from wild-type mice it was expressed in aortae and macrophage foam cells from ApoE(-/-) mice. Consistent with these observations CYP2S1 was colocalised with the monocyte marker CD68 in human atherosclerotic lesions. Thus, CYP2S1 generates 12-HHT and is a novel regulator of macrophage function that is expressed in classical inflammatory macrophages, and can be found in murine and human atherosclerotic plaques.

Mesenchymal stromal cells derived from CD271(+) bone marrow mononuclear cells exert potent allosuppressive properties.

BACKGROUND AIMS. Because data on the immunosuppressive effect of different subsets of mesenchymal stromal cells (MSC) are sparse, we investigated the molecular and cellular mechanisms underlying the allosuppressive effect of MSC generated from bone marrow CD271(+) cells (CD271-MSC) and asked whether this potential is comparable with that of MSC generated through plastic adherence (PA-MSC). METHODS. The immunosuppressive effect of CD271-MSC on the allogeneic reaction was investigated by mixed lymphocyte reaction (MLR). RESULTS. CD271-MSC significantly suppressed the alloantigen-induced proliferation of mononuclear cells (MNC) of two HLA-disparate donors at all MSC:MNC ratios, 1:1, 1:2 and 1:10. They also demonstrated a significantly higher allosuppression than PA-MSC at an MSC:MNC ratio of 1:1. This inhibitory effect was associated with significantly elevated levels of prostaglandin E2 (PGE2) at ratios of 1:1 and 1:2 (about 4-fold), but not at a ratio of 1:10. Indomethacin, and inhibitor of cyclooxygenase-1 and 2 necessary for the biosynthesis of PGE2, mitigated suppressive effects of CD271-MSC only at a ratio of 1:1, indicating that PGE2 is not involved in MSC-mediated inhibition when allogeneic MNC are in excess. The increase of PGE2 was associated with a significant decrease of pro-inflammatory cytokine levels (interferon-gamma and tumor necrosis-alpha), while no changes in levels of interleukin-10, soluble HLA-G and nitric oxide were observed. In addition, CD271-MSC induced an expansion of highly suppressive naive CD4(+)CD25(high)CD45RA(+)CD62L(+) T-regulatory cells, which may extend their allosuppressive effect. CONCLUSIONS. Our data suggest that CD271-MSC exert potent allosuppressive properties and therefore can be used as a reasonable alternative to PA-MSC for the treatment of patients with graft-versus-host disease.

Comparison of fibrotic response in the human lens capsular bag after femtosecond laser-assisted cataract surgery and conventional phacoemulsification.

PURPOSE: To compare the effect of different laser pulse energy settings in femtosecond laser-assisted cataract surgery with that of standard phacoemulsification and no energy at all used on posterior capsule opacification (PCO) in vitro. SETTING: Cell and Molecular Biology Research Laboratory, Department of Ophthalmology, Ludwig-Maximilians-University Munich, Real Eyes, Ophthalmology Center, Munich, and Institute for Clinical Pathology, Goethe University Frankfurt, Frankfurt, Germany. DESIGN: Experimental study. METHODS: Fifteen cadaver capsular bags were cultivated from 8 human donors under standard cell culture conditions. For preparation of the capsular bag, 4 groups were established as follows: femtosecond laser-assisted cataract surgery standard energy (n = 3), femtosecond laser-assisted cataract surgery high energy (n = 3), phacoemulsification (n = 6), and hydrodissection without energy (extracapsular cataract extraction) (n = 3). Growth of lens epithelial cells was observed and photodocumented. The days until full cell coverage of the posterior capsule were documented. Capsular bags were stained for fibronectin, α-smooth muscle actin, and collagen type 1. RESULTS: Cell growth patterns in all treatment groups were comparable, with no statistically significant differences detected at any timepoint measured (P = .81, Kruskal-Wallis). The markers for fibrosis were equally distributed in all groups, indicating an equal fibrotic reaction in all groups. CONCLUSION: Femtosecond laser-assisted cataract surgery did not increase different cellular responses in PCO formation comparison with phacoemulsification in vitro, even when higher laser pulse energy levels were used.