Comparison of immunomodulatory properties of mesenchymal stem cells derived from adult human tissues

Mesenchymal stem cells (MSCs), which evoke only minimal immune reactivity, may have anti-inflammatory and immunomodulatory effects. In this study, we conducted a comparative analysis of the immunomodulatory properties of MSCs derived from adult human tissues including bone marrow (BM), adipose tissues (AT), umbilical cord blood (CB), and cord Wharton’s jelly (WJ). Using a multiple cytokine detection assay, we showed that there were no significant differences in levels of secreted factors from non-stimulated MSCs. We compared the immunosuppressive effect of BM-MSCs, AT-MSCs, CB-MSCs, and WJ-MSCs on phytohemagglutinin-induced T-cell proliferation. AT-MSCs, CB-MSCs, and WJ-MSCs effectively suppressed mitogen-induced T-cell proliferation as effectively as did BM-MSCs. Levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α secreted from activated T-cells increased over time, but these levels were significantly reduced when cocultured with each type of MSCs. In addition, the expression of hepatocyte growth factor, IL-10, transforming growth factor-β₁, cyclooxygenase (COX)-1, and COX-2 were unchanged in MSCs treated with IFN-γ and/or TNF-α, while indoleamine 2,3-dioxygenase (IDO) expression increased. IFN-γ and/or TNF-α produced by activated T-cells were correlated with induction of IDO expression by MSCs, which, in turn, suppressed T-cell proliferation. These findings suggest that MSCs derived from AT, CB, or WJ could be substituted for BM-MSCs for treatment of allogeneic conflicts.     

Human mesenchymal stromal cells transiently increase cytokine production by activated T cells before suppressing T-cell proliferation: effect of interferon-γ and tumor necrosis factor-α stimulation

Background aimsMesenchymal stromal cells (MSCs) suppress T-cell proliferation, especially after activation with inflammatory cytokines. We compared the dynamic action of unprimed and interferon (IFN)-γ plus tumor necrosis factor (TNF)-α–pretreated human bone marrow–derived MSCs on resting or activated T cells.MethodsMSCs were co-cultured with allogeneic peripheral blood mononuclear cells (PBMCs) at high MSC-to-PBMC ratios in the absence or presence of concomitant CD3/CD28-induced T-cell activation. The kinetic effects of MSCs on cytokine production and T-cell proliferation, cell cycle and apoptosis were assessed.ResultsUnprimed MSCs increased the early production of IFN-γ and interleukin (IL)-2 by CD3/CD28-activated PBMCs before suppressing T-cell proliferation. In non-activated PBMC co-cultures, low levels of IL-2 and IL-10 synthesis were observed with MSCs in addition to low levels of CD69 expression by T cells and no T-cell proliferation. MSCs also decreased apoptosis in resting and activated T cells and inhibited the transition of these cells into the sub-G0/G1 and the S phases. With inhibition of indoleamine 2,3 dioxygenase, MSCs increased CD3/CD28-induced T-cell proliferation. After priming with IFN-γ plus TNF-α, MSCs were less potent at increasing cytokine production by CD3/CD28-activated PBMCs and more effective at inhibiting T-cell proliferation but had preserved anti-apoptotic functions.ConclusionsUnprimed MSCs induce a transient increase in IFN-γ and IL-2 synthesis by activated T cells. Pre-treatment of MSCs with IFN-γ plus TNF-α may increase their effectiveness and safety in vivo.     

Activated T-cells and pro-inflammatory cytokines differentially regulate prostaglandin E2 secretion by mesenchymal stem cells

In recent years it has become clear that mesenchymal stem or stromal cells (MSCs) are capable of modulating inflammatory and immune responses through interaction with a wide variety of cells. Whereas several studies indicated that PGE2 is one of the chief soluble mediators involved in these processes, here we investigated prostaglandin E2 (PGE2) production of murine bone marrow- (BM-) and adipose tissue- (Ad-) derived MSCs stimulated with pro-inflammatory cytokines TNF-α and IFN-γ, or co-cultured with ConA-induced T-cell blasts. We found that both MSC populations are able to produce high amounts of PGE2 in MSC/activated T-cell co-cultures. This effect was markedly attenuated when direct cell-cell contact was prevented in transwell system, indicating that the elicitation of the PGE2 secretion of MSCs is contact-dependent in this experimental setting. In contrast, when soluble recombinant pro-inflammatory cytokines were added to the MSC cultures, TNF-α and IFN-γ act synergistically to induce PGE2 production, whereas only high amount of TNF-α but not IFN-γ was able to do so alone. Although the PGE2 secretion by MSCs was completely abrogated by addition of indomethacin under all culture conditions tested, L-NMA, a NOS inhibitor could only partially inhibit it when the cells were elicited in the concomitant presence of TNF-α and IFN-γ. These results, combined with others, suggest that NO acts downstream of IFN-γ but upstream of COX2. Taken together, our findings demonstrate that the induction of PGE2 secretion by BM- and Ad-MSCs is not mediated by a single or unique, nonredundant molecular mechanism under different experimental conditions.     

Tolerogenic effect of mesenchymal stromal cells on gliadin-specific T lymphocytes in celiac disease

Background aimsCeliac disease is caused by a dysregulated immune response toward dietary gluten, whose only treatment is a lifelong gluten-free diet. We investigated the effects of mesenchymal stromal cells (MSCs) on gliadin-specific T cells, which are known to induce intestinal lesions, in view of a possible use as new therapy.MethodsBone marrow–derived MSCs and gliadin-specific T-cell lines were obtained from allogeneic donors and mucosal specimens of celiac patients, respectively. The immunosuppressant effect of MSCs was evaluated in terms of proliferative response and interferon (IFN)-γ production upon gliadin stimulation of long-term T-cell lines; the immunomodulant effect was assessed in terms of apoptotic rate, immunophenotype and cytokine profile of short-term T-cell lines generated in the presence of MSCs. Different MSC:T-cell ratios were applied, and statistics were performed as appropriate.ResultsMSCs inhibited both proliferative response and IFN-γ production of long-term T-cell lines in a dose-dependent manner while limiting the expansion of short-term T-cell lines by increasing the apoptotic rate. Moreover, a reduction of the CD4⁺ population and expansion of the regulatory FoxP3⁺ subset were found in T-cell lines cultured with MSCs, in which a significant decrease of interleukin (IL)-21, IFN-γ and IL-10 paralleled by an upregulation of transforming growth factor-β1, IL-6 and IL-8 were observed. Finally, an increase of the indoleamine 2,3-dioxygenase activity was found, possibly playing a key role in mediating these effects.ConclusionsMSCs exert potent immunomodulant effects on gliadin-specific T cells, which may be exploited for future therapeutic application in celiac disease.     

Effects of inflammatory factors on mesenchymal stem cells and their role in the promotion of tumor angiogenesis in colon cancer

Mesenchymal stem cells (MSCs), which are modulated by cytokines present in the tumor microenvironment, play an important role in tumor progression. It is well documented that inflammation is an important part of the tumor microenvironment, so we investigated whether stimulation of MSCs by inflammatory cytokines would contribute to their ability to promote tumor growth. We first showed that MSCs could increase C26 colon cancer growth in mice. This growth-promoting effect was further accelerated when the MSCs were pre-stimulated by inflammatory factors IFN-γ and TNF-α. At the same time, we demonstrated that MSCs pre-stimulated by both inflammatory factors could promote tumor angiogenesis in vivo to a greater degree than untreated MSCs or MSCs pre-stimulated by either IFN-γ or TNF-α alone. A hen egg test-chorioallantoic membrane (HET-CAM) assay showed that treatment of MSC-conditioned medium can promote chorioallantoic membrane angiogenesis in vitro, especially treatment with conditioned medium of MSCs pretreated with IFN-γ and TNF-α together. This mechanism of promoting angiogenesis appears to take place via an increase in the expression of vascular endothelial growth factor (VEGF), which itself takes place through an increase in signaling in the hypoxia-inducible factor 1α (HIF-1α)-dependent pathway. Inhibition of HIF-1α in MSCs by siRNA was found to effectively reduce the ability of MSC to affect the growth of colon cancer in vivo in the inflammatory microenviroment. These results indicate that MSCs stimulated by inflammatory cytokines such as IFN-γ and TNF-α in the tumor microenvironment express higher levels of VEGF via the HIF-1α signaling pathway and that these MSCs then enhance tumor angiogenesis, finally leading to colon cancer growth in mice.     

Autophagy inhibition via Becn1 downregulation improves the mesenchymal stem cells antifibrotic potential in experimental liver fibrosis

Liver fibrosis (LF) is the result of a vicious cycle between inflammation-induced chronic hepatocyte injury and persistent activation of hepatic stellate cells (HSCs). Mesenchymal stem cell (MSC)-based therapy may represent a potential remedy for treatment of LF. However, the fate of transplanted MSCs in LF remains largely unknown. In the present study, the fate and antifibrotic effect of MSCs were explored in a LF model induced by CCl₄ in mouse. Additionally, MSCs were stimulated in vitro with LF-associated factors, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and transforming growth factor-β1 (TGF-β1), to mimic the LF microenvironment. We unveiled that MSCs exhibited autophagy in response to the LF microenvironment through Becn1 upregulation both in vivo and in vitro. However, autophagy suppression induced by Becn1 knockdown in MSCs resulted in enhanced antifibrotic effects on LF. The improved antifibrotic potential of MSCs may be attributable to their inhibitory effects on T lymphocyte infiltration, HSCs proliferation, as well as production of TNF-α, IFN-γ, and TGF-β1, which may be partially mediated by elevated paracrine secretion of PTGS2/PGE₂. Thus, autophagy manipulation in MSCs may be a novel strategy to promote their antifibrotic efficacy.     

Inducible indoleamine 2,3-dioxygenase 1 and programmed death ligand 1 expression as the potency marker for mesenchymal stromal cells

Aim

Establishment of a potency assay in the manufacturing of clinical-grade mesenchymal stromal cells (MSCs) has been a challenge due to issues of relevance to function, timeline and variability of responder cells. In this study, we attempted to develop a potency assay for MSCs.

Tryptophan concentration is the main mediator of the capacity of adipose mesenchymal stromal cells to inhibit T-lymphocyte proliferation in vitro

Background aimsMesenchymal stromal cells (MSCs) have immunomodulatory properties that are mediated by cell-to-cell interactions and paracrine effects through soluble factors, among which tryptophan (Trp) conversion into kynurenine (Kyn) through the enzymatic activity of indoleamine 2,3-dioxygenase has been proven to be of special relevance. However, the respective role of Trp depletion and/or Kyn accumulation on the inhibition of T-cell proliferation by MSCs remains unclear.MethodsThe effect of supplementation with increasing concentrations of Trp on the capacity of MSCs to inhibit T-lymphocyte proliferation in vitro was investigated.ResultsWe report that Trp supplementation impairs the capacity of adipose mesenchymal stromal cells (ASCs) to inhibit T-cell proliferation, despite the accumulation of very high concentrations of Kyn in the medium (>200 μmol/L). Moreover, Trp supplementation after 72 h of peripheral blood mononuclear cell:ASC co-culture, once the inhibitory effect of ASCs was established, reverted ASC inhibition and restored T-cell proliferation. Addition to stimulated lymphocytes of Kyn inhibited T proliferation in 3 of 10 peripheral blood mononuclear cell donors, but at different concentrations, suggesting that sensitivity of lymphocytes to Kyn might be donor-dependent.ConclusionsOur results confirm the relevance of Trp metabolism as a key mediator of the immunomodulatory properties of ASCs and clarify the respective roles of the Trp/Kyn balance.     

Indirect co-culture of lung carcinoma cells with hyperthermia-treated mesenchymal stem cells influences tumor spheroid growth in a collagen-based 3-dimensional microfluidic model

BackgroundMesenchymal stem cells (MSCs) have paradoxically been reported to exert either pro- or anti-tumor effects in vitro. Hyperthermia, in combination with chemotherapy, has tumor-inhibiting effects; however, its role, together with MSCs, so far is not well understood. Furthermore, a lot of research is conducted using conventional 2-dimensional in vitro models that do not mimic the actual tumor microenvironment.AimIn light of this fact, an indirect method of co-culturing human amniotic membrane-derived MSCs (AMMSCs) with collagen-encapsulated human lung carcinoma cells (A549) was performed using a 3-dimensional (3D) tumor-on-chip device.MethodsThe conditioned medium of AMMSCs (AMMSC-CM) or heat-treated AMMSCs (heat-AMMSC-CM) was utilized to create indirect co-culture conditions. Tumor spheroid growth characterization, immunocytochemistry and cytotoxicity assays, and anti-cancer peptide (P1) screening were performed to determine the effects of the conditioned medium.ResultsThe A549 cells cultured inside the 3D microfluidic chip developed into multicellular tumor spheroids over five days of culture. The AMMSC-CM, contrary to previous reports claiming its tumor-inhibiting potential, led to significant proliferation of tumor spheroids. Heat-AMMSC-CM led to reductions in both spheroid diameter and cell proliferation. The medium containing the P1 peptide was found to be the least cytotoxic to tumor spheroids in co-culture compared with the monoculture and heat-co-culture groups.ConclusionsHyperthermia, in combination with the anticancer peptide, exhibited highest cytotoxic effects. This study highlights the growing importance of 3D microfluidic tumor models for testing stem-cell-based and other anti-cancer therapies.     

Activated T cells modulate immunosuppression by embryonic-and bone marrow-derived mesenchymal stromal cells through a feedback mechanism

Background aimsHuman embryonic stem cell (hESC)-derived mesenchymal stromal cells (MSC) (hESC-MSC) are an alternative source of MSC to bone marrow (BM)-derived MSC (BM-MSC), which are being investigated in clinical trials for their immunomodulatory potential. hESC-MSC have the advantage of being consistent because each batch can be generated from hESC under defined conditions. In contrast, BM-MSC have a limited proliferative capacity.MethodsThe ability to suppress the proliferation of anti-CD3/CD28-stimulated CD4 ⁺ T cells by hESC-MSC was compared with adult BM-MSC and neonatal foreskin fibroblast (Fb).ResultshESC-MSC suppress the proliferation of CD4 ⁺ T cells in both contact and transwell systems, although inhibition is less in the transwell system. hESC-MSC are approximately 2-fold less potent (67 cells/100 T cells) than BM-MSC and Fb (37 and 34 cells/100 T cells, respectively) at suppressing T-cell proliferation by 50% in a transwell [inhibitory concentration(IC)₅₀]. The anti-proliferative effect is not contact-dependent but requires the presence of factors such as interferon (IFN)-γ produced by activated T cells. IFN-γ induces the expression of indoleamine-2,3-dioxygenase (IDO) in hESC-MSC, BM-MSC and Fb, contributing to their immunosuppressive property.ConclusionsThe feedback loop between MSC or Fb and activated T cells may limit the immunosuppressive effects of MSC and Fb to sites containing ongoing immunologic or inflammatory responses where activated T cells induce the up-regulation of IDO and immunomodulatory properties of MSC and Fb. These data demonstrate that hESC-MSC may be evaluated further as an allogeneic cell source for therapeutic applications requiring immunosuppression.     

Dimethyloxalylglycine,a small molecule,synergistically increases the homing and angiogenic properties of human mesenchymal stromal cells when cultured as 3D spheroids

Strategies aiming at increasing the survival and paracrine activity of human mesenchymal stromal cells (MSCs) are of utmost importance to achieve the full therapeutic potential of these cells. Herein, we propose both physical and biochemical strategies to enhance the survival, homing, angiogenic, and immunomodulatory properties of MSCs in vitro. To that purpose, we compared the effect of exposing either 2D monolayer or 3D spheroids of MSCs to (i) hypoxia (2% O₂) or to (ii) a hypoxic-mimetic small molecule, dimethyloxalylglycine (DMOG), with cells cultured at 21% O₂. 3D-cultured MSC spheroids evidenced higher survival upon exposure to oxidative stress and expressed higher levels of factors involved in tissue repair processes, namely tumor necrosis factor-stimulated gene-6, matrix metalloproteinase-2, and vascular endothelial growth factor. MSCs cultured as 3D spheroids and further exposed to hypoxia or hypoxic-mimetic conditions provided by DMOG synergistically favored the expression of the cell surface marker C-X-C chemokine receptor type-4, involved in homing processes to injured tissues, and adhesion to extracellular matrix components as fibronectin. These results highlight the role of ex vivo preconditioning approaches, presenting a novel strategy that combine biochemical stimuli with 3D spheroid organization of MSCs to maximize their tissue regeneration potential.     

Mesenchymal stem cell transplantation increases expression of vascular endothelial growth factor in papain-induced emphysematous lungs and inhibits apoptosis of lung cells

BackgroundPulmonary emphysema is characterized by loss of alveolar structures. We have found that bone marrow (BM) mesenchymal stem cell (MSC) transplantation ameliorates papain-induced pulmonary emphysema. However, the underlying mechanism is not completely understood. It has been shown that blocking the vascular endothelial growth factor (VEGF) signaling pathway leads to apoptosis of lung cells and pulmonary emphysema, and MSC are capable of secreting VEGF. We hypothesized that MSC transplantation may have a protective effect on pulmonary emphysema by increasing VEGF-A expression and inhibiting apoptosis of lung cells.MethodsWe examined the morphology and expression of VEGF-A in rat lung after papain treatment and MSC transplantation. We also used a co-culture system in which MSC and cells prepared from papain-treated lungs or control lungs were cultured together. The levels of VEGF-A in cells and culture medium were determined, and apoptosis of cultured lung cells was evaluated.ResultsVEGF-A expression in rat lungs was decreased after papain treatment, which was partly rescued by MSC transplantation. MSC production of VEGF-A was increased when MSC were co-cultured with cells prepared from papain-treated lungs. Furthermore, the apoptosis of papain-treated lung cells was inhibited when co-cultured with MSC. The induction of MSC production of VEGF-A by papain-treated lung cells was inhibited by adding anti-tumor necrosis factor (TNF)-α antibody to the medium.ConclusionsThe protective effect of MSC transplantation on pulmonary emphysema may be partly mediated by increasing VEGF-A expression and inhibiting the apoptosis of lung cells. TNF-α released from papain-treated lung cells induces MSC to secret VEGF-A.     

Human platelet lysate stimulates high-passage and senescent human multipotent mesenchymal stromal cell growth and rejuvenation in vitro

Background aimsMultipotent mesenchymal stromal cells (MSCs) are clinically useful because of their immunomodulatory and regenerative properties, but MSC therapies are limited by the loss of self-renewal and cell plasticity associated with ex vivo expansion culture and, on transplantation, increased immunogenicity from xenogen exposure during culture. Recently, pooled human platelet lysate (hPL) has been used as a culture supplement to promote MSC growth; however, the effects of hPL on MSCs after fetal bovine serum (FBS) exposure remain unknown.MethodsMSCs were cultured in medium containing FBS or hPL for up to 16 passages, and cell size, doubling time and immunophenotype were determined. MSC senescence was assessed by means of a fluorometric assay for endogenous β-galactosidase expression. MSCs cultured with FBS for different numbers of passages were switched to hPL conditions to evaluate the ability of hPL to “rescue” the proliferative capacity of MSCs.ResultshPL culture resulted in more rapid cell proliferation at earlier passages (passage 5 or earlier) than remove FBS; by day 4, hPL (5%) yielded an MSC doubling time of 1.28 days compared with 1.52 days in 16% FBS. MSCs cultured first in FBS and switched to hPL proliferated more and demonstrated less β-galactosidase production and smaller cell sizes than remove MSCs continuously propagated in FBS.ConclusionshPL enables rapid expansion of MSCs without adversely affecting immunophenotype. hPL culture of aged and senescent MSCs demonstrated cellular rejuvenation, reflected by decreased doubling time and smaller cell size. These results suggest that expansion of MSCs in hPL after FBS exposure can enhance cell phenotype and proliferative capacity.     

Unique characteristics of human mesenchymal stromal/progenitor cells pre-activated in 3-dimensional cultures under different conditions

Background aimsHuman mesenchymal stromal cells (MSCs) are being used in clinical trials, but the best protocol to prepare the cells for administration to patients remains unclear. We previously demonstrated that MSCs could be pre-activated to express therapeutic factors by culturing the cells in 3 dimensions (3D). We compared the activation of MSCs in 3D in fetal bovine serum containing medium and in multiple xeno-free media formulations.MethodsMSC aggregation and sphere formation was studied with the use of hanging drop cultures with medium containing fetal bovine serum or with various commercially available stem cell media with or without human serum albumin (HSA). Activation of MSCs was studied with the use of gene expression and protein secretion measurements and with functional studies with the use of macrophages and cancer cells.ResultsMSCs did not condense into tight spheroids and express a full complement of therapeutic genes in α-minimum essential medium or several commercial stem-cell media. However, we identified a chemically defined xeno-free media, which, when supplemented with HSA from blood or recombinant HSA, resulted in compact spheres with high cell viability, together with high expression of anti-inflammatory (prostaglandin E2, TSG-6 TNF-alpha induced gene/protein 6) and anti-cancer molecules (TRAIL TNF-related apoptosis-inducing ligand, interleukin-24). Furthermore, spheres cultured in this medium showed potent anti-inflammatory effects in a lipopolysaccharide-stimulated macrophage system and suppressed the growth of prostate cancer cells by promoting cell-cycle arrest and cell death.ConclusionsWe demonstrated that cell activation in 3D depends critically on the culture medium. The conditions developed in the present study for 3D culture of MSCs should be useful in further research on MSCs and their potential therapeutic applications.     

Lysis and phenotypic modulation of mesenchymal stromal cells upon blood contact triggers anti-inflammatory skewing of the peripheral innate immune repertoire

Background aimsMesenchymal stromal cells (MSCs) are used to treat immune-related disorders, including graft-versus-host disease. Upon intravenous infusion, MSCs trigger the instant blood-mediated inflammatory response, resulting in activation of both complement and coagulation cascades, and are rapidly cleared from circulation. Despite no/minimal engraftment, long-term immunoregulatory properties are evident. The aim of this study was to establish the effects of blood exposure on MSC viability and immunomodulatory functions.MethodsHuman, bone marrow derived MSCs were exposed to human plasma +/– heat inactivation or whole blood. MSC number, viability and cellular damage was assessed using the JC-1 mitochondrial depolarization assay and annexin V staining. C3c binding and expression of the inhibitory receptors CD46, CD55 and CD59 and complement receptors C3aR and C5aR were evaluated by flow cytometry. MSCs pre-exposed to plasma were cultured with peripheral blood mononuclear cells (PBMCs) and monocyte subsets characterized by flow cytometry. The PBMC and MSC secretome was assessed using enzyme-linked immunosorbent assays against tumor necrosis factor alpha, interleukin (IL)-6 and IL-10. Monocyte recruitment towards the MSC secretome was evaluated using Boyden chambers and screened for chemotactic factors including monocyte chemoattractant protein (MCP)-1. MSC effects on the peripheral immune repertoire was also evaluated in whole blood by flow cytometry.ResultsPlasma induced rapid lysis of 57% of MSCs, which reduced to 1% lysis with heat inactivation plasma. Of those cells that were not lysed, C3c could be seen bound to the surface of the cells, with a significant swelling of the MSCs and induction of cell death. The MSC secretome reduced monocyte recruitment, in part due to a reduction in MCP-1, and downregulated PBMC tumor necrosis factor alpha secretion while increasing IL-6 levels in the co-culture supernatant. A significant decrease in CD14⁺ monocytes was evident after MSC addition to whole blood alongside a significant increase in IL-6 levels, with those remaining monocytes demonstrating an increase in classical and decrease in non-classical subsets. This was accompanied by a significant increase in both mononuclear and polymorphonuclear myeloid-derived suppressor cells.ConclusionsThis study demonstrates that a significant number of MSCs are rapidly lysed upon contact with blood, with those surviving demonstrating a shift in their phenotype, including a reduction in the secretion of monocyte recruitment factors and an enhanced ability to skew the phenotype of monocytes. Shifts in the innate immune repertoire, towards an immunosuppressive profile, were also evident within whole blood after MSC addition. These findings suggest that exposure to blood components can promote peripheral immunomodulation via multiple mechanisms that persists within the system long after the infused MSCs have been cleared.     

Uncarinic acid C plus IFN-γ generates monocyte-derived dendritic cells and induces a potent Th1 polarization with capacity to migrate

Uncarinic acid C (URC) is triterpene isolated from Uncaria rhynchophylla and modulates human DC function in a fashion that favors Th1 cell polarization depending on TLR4 signaling. The induction of dendritic cells (DC) is critical for the induction of Ag-specific T lymphocyte responses and may be essential for the development of human vaccines relying on T cell immunity. Monocyte-derived DC used as adjuvant cells in cancer immunotherapy and have shown promising results. We studied the effect of interferon’s (IFN-α and IFN-γ) and TNF-α on phenotypic and functional maturation, and cytokine production of URC-primed DC in vitro. Human monocytes were exposed to either URC alone, or in combination with TNF-α, IFN-α or IFN-γ, and thereafter co-cultured with naïve T cells. We found that the expression levels of CD1a, CD83 and HLA-DR on URC-primed DC were influenced by IFN-γ and IFN-γ augmented the T cell stimulatory capacity in allo MLR to URC-primed DC. Moreover, the production of IL-12p70 by URC-primed DC was enhanced by IFN-γ. IL-12p70 production by URC-primed DC alone was influenced following treatment with anti-TLR4 mAb, but not DC differentiated with URC plus IFN-γ. URC plus IFN-γ-primed DC induced a substantial increase in the secretion of IFN-γ by T cells, which is dependent on IL-12 secretion. DC maturated with URC plus IFN-γ had an intermediate migratory capacity towards CCL19 and CCL21. In addition, the expression levels of CCR7 on URC-primed DC were enhanced by IFN-γ. In contrast, surface molecule up-regulation and function of URC-primed DC were slightly enhanced by TNF-α, and IFN-α. These results suggest that the enhancement of Th1 cells polarization to URC-primed DC induced by IFN-γ depends on the activation of IL-12p70 and independent on TLR4. DC differentiated with URC in combination with IFN-γ might be used on DC-based vaccine for cancer immunotherapy.     

Functional potentials of human hematopoietic progenitor cells are maintained by mesenchymal stromal cells and not impaired by plerixafor

Background aimsMesenchymal stromal cells (MSCs) resemble an essential component of the bone marrow niche for maintenance of stemness of hematopoietic progenitor cells (HPCs). Perturbation of the C-X-C chemokine receptor type 4 (CXCR4)/stromal cell-derived factor-1α (SDF-1α) axis by plerixafor (AMD3100) mobilizes HPCs from their niche; however, little is known about how plerixafor affects interaction of HPCs and MSCs in vitro.MethodsWe monitored cell division kinetics, surface expression of CD34 and CXCR4, migration behavior and colony-forming frequency of HPCs on co-culture with MSCs either with or without exposure to plerixafor.ResultsCo-culture with MSCs significantly accelerated cell division kinetics of HPCs. Despite this, the proportion of CD34⁺ cells was significantly increased on co-culture, whereas the expression of CXCR4 was reduced. In addition, co-culture with MSCs led to significantly higher colony-forming capacity and enhanced migration rate of HPCs compared with mono-culture conditions. The composition of MSC sub-populations—and conversely their hematopoiesis supportive functions—may be influenced by culture conditions. We compared the stromal function of MSCs isolated with three different culture media. Overall, the supporting potentials of these MSC preparations were quite similar. Perturbation by the CXCR4-antagonist plerixafor reduced the cell division kinetics of HPCs on co-culture with MSCs. However, the progenitor cell potential of the HPCs as reflected by colony-forming capacity was not affected by plerixafor.ConclusionsThese results support the notion that the CXCR4/SDF-1α axis is critical for HPC-MSC interaction with regard to migration, adhesion and regulation of proliferation but not for maintenance of primitive progenitor cells.     

Effect of 1,25 dihydroxyvitamin D3 on intracellular IFN-γ and TNF-α positive T cell subsets in pulmonary tuberculosis

We studied the immunomodulatory effect of 1,25(OH)₂D₃ on single cell expression of IFN-γ and TNF-α cytokines in T cell subsets of pulmonary tuberculosis (PTB) patients (n = 22) and normal healthy subjects (n = 22). Peripheral blood mononuclear cells (PBMCs) were cultured with live Mycobacterium tuberculosis (MTB) with or without 1,25(OH)₂D₃ (10^?7 M) for 48 h. T cell subsets positive for IFN-γ and TNF-α were enumerated by flow cytometry and the culture supernatants were assayed for both the cytokines using ELISA. In both NHS and PTB patients, a significantly reduced percentage of IFN-γ and TNF-α expressing CD3+, CD3+CD4+ and CD3+CD8+ T cells were observed in cultures stimulated with live MTB and treated with 1,25(OH)₂D₃ compared to cultures without 1,25(OH)₂D₃ (NHS; CD3+ IFN-γ+: p < 0.0001; CD3+TNF-α +: p = 0.0292 and PTB; CD3+ IFN-γ+: p = 0.0292; CD3+ TNF-α +: p = 0.0028). The levels of IFN-γ and TNF-α in the culture supernatants of 1,25(OH)₂D₃ treated cultures were also found to be significantly decreased in both groups (NHS; IFN-γ: p = 0.0001; TNF-α: p < 0.0001) and (PTB; IFN-γ: p < 0.0001; TNF-α: p < 0.0001). A positive correlation was observed between IFN-γ and TNF-α expressing CD3+CD8+ T cells in MTB stimulated cultures treated with or without 1,25(OH)₂D₃ in NHS (p = 0.0001; p = 0.001, respectively) and PTB patients (p = 0.002; p = 0.005, respectively). The present study revealed the suppressive effect of 1,25(OH)₂D₃ on single cell expression of IFN-γ and TNF-α by CD3+CD4+ and CD3+CD8+ T cells in pulmonary tuberculosis. This suppressive effect of 1,25(OH)₂D₃ on proinflammatory and Th1 cytokine positive cells might have a role in reducing inflammation at the site of infection.     

Effect of priming of multipotent mesenchymal stromal cells with interferon γ on their immunomodulating properties

Multipotent mesenchymal stromal cells (MSCs) are widely used for cell therapy, in particular for prophylaxis and treatment of graft-versus-host disease. Due to their immunomodulatory properties, MSCs affect the composition of lymphocyte subpopulations, which depends on the immunological state of the organism and can change in different diseases and during treatment. Administration of MSCs is not always effective. Treatment of MSCs with different cytokines (in particular IFN-γ) leads to enhancement of their immunomodulatory properties. The aim of this study was to investigate sub-populational alterations and activation markers in lymphocytes (activated and non-activated) after interaction with MSCs and MSCs pretreated with IFN-γ (γMSCs) in vitro. Lymphocytes were co-cultured with MSCs or γMSCs for 4 days. The proportion of CD4⁺ and CD8⁺ expressing CD25, CD38, CD69, HLA-DR, and PD-1 and distribution of memory and effector subsets were measured by flow cytometry after co-cultivation of lymphocytes with MSCs or γMSCs. The distribution of lymphocyte subpopulations changes during culturing. In non-activated lymphocytes cultured without MSCs, decrease in the proportion of naïve cells and increase in the number of effector cells was observed. That could be explained as activation of lymphocytes in the presence of serum in culturing medium. Co-culturing of lymphocytes with MSCs and γMSCs leads to retention of their non-activated state. Activation of lymphocytes with phytohemagglutinin increases the number of central memory cells and activates marker expression. Interaction with MSCs and γMSCs prevents activation of lymphocytes and keeps their naïve state. Priming with IFN-γ did not induce MSCs inhibitory effect on activation of lymphocytes.     

Evaluation of methods for cultivating limbal mesenchymal stromal cells

Background aimsMesenchymal stromal cells (MSC) with similar properties to bone marrow-derived mesenchymal stromal cells (BM-MSC) have recently been grown from the limbus of the human cornea. We have evaluated methods for culturing human limbal MSC (L-MSC).MethodsFour basic strategies were compared: serum-supplemented medium (10% fetal bovine serum; FBS), standard serum-free medium supplemented with B-27, epidermal growth factor and fibroblast growth factor 2, or one of two commercial serum-free media, defined keratinocyte serum-free medium (Invitrogen) and MesenCult-XF® (Stem Cell Technologies). The resulting cultures were examined using photography, flow cytometry (for CD34, CD45, CD73, CD90, CD105, CD141 and CD271), immunocytochemistry (alpha-smooth muscle actin; α-sma), differentiation assays (osteogenesis, adipogenesis and chrondrogenesis) and co-culture experiments with human limbal epithelial (HLE) cells.ResultsWhile all techniques supported the establishment of cultures to varying degrees, sustained growth and serial propagation were only achieved in 10% FBS medium or MesenCult-XF medium. Cultures established in 10% FBS medium were 70–80% CD34^? CD45^? CD90⁺ CD73⁺ CD105⁺, approximately 25% α-sma⁺ and displayed multipotency. Cultures established in MesenCult-XF were > 95% CD34^? CD45^? CD90⁺ CD73⁺ CD105⁺, 40% CD141⁺, rarely expressed α-sma, and displayed multipotency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of MesenCult-XF-grown L-MSC. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker ?Np63, along with the corneal differentiation marker cytokeratin 3.ConclusionsMesenCult-XF is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells.