Absence Of Mesenchymal Stromal Cells Research Articles

Intravenous MSC-Treatment Improves Impaired Brain Functions in the R6/2 Mouse Model of Huntington's Disease via Recovered Hepatic Pathological Changes.

Huntington's disease (HD), a congenital neurodegenerative disorder, extends its pathological damages beyond the nervous system. The systematic manifestation of HD has been extensively described in numerous studies, including dysfunction in peripheral organs and peripheral inflammation. Gut dysbiosis and the gut-liver-brain axis have garnered greater emphasis in neurodegenerative research, and increased plasma levels of pro-inflammatory cytokines have been identified in HD patients and various in vivo models, correlating with disease progression. In the present study, we investigated hepatic pathological markers in the liver of R6/2 mice which convey exon 1 of the human mutant huntingtin gene. Furthermore, we evaluated the impact of intravenously administered Mesenchymal Stromal Cells (MSCs) on the liver enzymes, changes in hepatic inflammatory markers, as well as brain pathology and behavioral deficits in R6/2 mice. Our results revealed altered enzyme expression and increased levels of inflammatory mediators in the liver of R6/2 mice, which were significantly attenuated in the MSC-treated R6/2 mice. Remarkably, neuronal pathology and altered motor activities in the MSC-treated R6/2 mice were significantly ameliorated, despite the absence of MSCs in the postmortem brain. Our data highlight the importance of hepatic pathological changes in HD, providing a potential therapeutic approach. Moreover, the data open new perspectives for the search in blood biomarkers correlating with liver pathology in HD.

Human Wharton’s jelly-derived mesenchymal stromal cells promote bone formation in immunodeficient mice when administered into a bone microenvironment

BackgroundWharton’s Jelly (WJ) Mesenchymal Stromal Cells (MSC) have emerged as an attractive allogeneic therapy for a number of indications, except for bone-related conditions requiring new tissue formation. This may be explained by the apparent recalcitrance of MSC,WJ to differentiate into the osteogenic lineage in vitro, as opposed to permissive bone marrow (BM)-derived MSCs (MSC,BM) that readily commit to bone cells. Consequently, the actual osteogenic in vivo capacity of MSC,WJ is under discussion.MethodsWe investigated how physiological bone environments affect the osteogenic commitment of recalcitrant MSCs in vitro and in vivo. To this end, MSC of BM and WJ origin were co-cultured and induced for synchronous osteogenic differentiation in vitro using transwells. For in vivo experiments, immunodeficient mice were injected intratibially with a single dose of human MSC and bone formation was evaluated after six weeks.ResultsCo-culture of MSC,BM and MSC,WJ resulted in efficient osteogenesis in both cell types after three weeks. However, MSC,WJ failed to commit to bone cells in the absence of MSC,BM’s osteogenic stimuli. In vivo studies showed successful bone formation within the medullar cavity of tibias in 62.5% of mice treated with MSC, WJ. By contrast, new formed trabeculae were only observed in 25% of MSC,BM-treated mice. Immunohistochemical staining of human COXIV revealed the persistence of the infused cells at the site of injection. Additionally, cells of human origin were also identified in the brain, heart, spleen, kidney and gonads in some animals treated with engineered MSC,WJ (eMSC,WJ). Importantly, no macroscopic histopathological alterations, ectopic bone formation or any other adverse events were detected in MSC-treated mice.ConclusionsOur findings demonstrate that in physiological bone microenvironment, osteogenic commitment of MSC,WJ is comparable to that of MSC,BM, and support the use of off-the-shelf allogeneic MSC,WJ products in bone repair and bone regeneration applications.

A bone-based 3D scaffold as an in-vitro model of microenvironment-DLBCL lymphoma cell interaction.

About 30% of patients with diffuse large B-cell lymphoma (DLBCL) relapse or exhibit refractory disease (r/r DLBCL) after first-line immunochemotherapy. Bone marrow (BM) involvement confers a dismal prognosis at diagnosis, likely due to the interaction between neoplastic cells and a complex tumor microenvironment (TME). Therefore, we developed a 3D in-vitro model from human decellularized femoral bone fragments aiming to study the role of mesenchymal stromal cells (MSC) and the extracellular matrix (ECM) in the adaptation, growth, and drug resistance of DLBCL lymphoma cells. The 3D spatial configuration of the model was studied by histological analysis and confocal and multiphoton microscopy which allowed the 3D digital reproduction of the structure. We proved that MSC adapt and expand in the 3D scaffold generating niches in which also other cell types may grow. DLBCL cell lines adhered and grew in the 3D scaffold, both in the presence and absence of MSC, suggesting an active ECM-lymphocyte interaction. We found that the germinal center B-cell (GCB)-derived OCI-LY18 cells were more resistant to doxorubicin-induced apoptosis when growing in the decellularized 3D bone scaffold compared to 2D cultures (49.9% +/- 7.7% Annexin V+ cells in 2D condition compared to 30.7% + 9.2% Annexin V+ 3D adherent cells in the ECM model), thus suggesting a protective role of ECM. The coexistence of MSC in the 3D scaffold did not significantly affect doxorubicin-induced apoptosis of adherent OCI-LY18 cells (27.6% +/- 7.3% Annexin V+ 3D adherent cells in the ECM/MSC model after doxorubicin treatment). On the contrary, ECM did not protect the activated B-cell (ABC)-derived NU-DUL-1 lymphoma cell line from doxorubicin-induced apoptosis but protection was observed when MSC were growing in the bone scaffold (40.6% +/- 5.7% vs. 62.1% +/- 5.3% Annexin V+ 3D adherent cells vs. 2D condition). These data suggest that the interaction of lymphoma cells with the microenvironment may differ according to the DLBCL subtype and that 2D systems may fail to uncover this behavior. The 3D model we proposed may be improved with other cell types or translated to the study of other pathologies with the final goal to provide a tool for patient-specific treatment development.

Bone Marrow Stromal Cells Induce ALDH+ Stem Cell Phenotype and Chemotherapy Resistance in AML Cells through TGF-β1 Mediated Signaling: Reversal By TGFβ Inhibitor Galunisertib

Bone marrow (BM) microenvironment in acute myeloid leukemia (AML) consists of various cell types including mesenchymal stromal cells (MSCs), osteoprogenitor cells, osteoblasts, osteocytes, endothelial, immune and perivascular reticular cells that support AML cell growth and protect them from chemotherapy. We have recently reported that AML cells alter the BM microenvironment by inducing osteogenic differentiation in MSCs to gain growth advantage in the bone marrow (Battula et al, JCI Insight, 2017). However the stroma-induced functional alterations in AML cells are not well understood. Stromal cells induce chemo-resistance in AML cells and as chemo-resistance and high tumorigenic potential are the characteristics of leukemia stem cells, we hypothesize that stromal cells can induce stem cell characteristics in AML cells. To test our hypothesis we co-cultured AML cell lines including OCI-AML3 and HL60 cells with BM-derived MSCs for 3 or 5 days and determined aldehyde dehydrogenase (ALDH) enzyme activity (a well-established marker of hematopoietic and leukemia stem cells) and gene expression by RNA sequencing. We found that the percentage of ALDH+ cells in OCI-AML3 increased from 7% to 89.1% in 3 days and from 23% to 92.3% in 5 days of co-culture with MSCs. Similarly, ALDHwas increased in HL60 cells from 7% to 68% in 3 days and from 20.7% to 67.9% in 5 days of co-culture with MSCs suggesting that stromal cells can induce certain stem cell features in AML cells.Next, to better determine the mechanism, OCI-AML3 cells were co-cultured with BM-MSCs (isolated from 3 different donors) for 5 days, the OCI-AML3 cells were FACS sorted and gene expression was analyzed by RNA sequencing. Using R statistics we identified differentially expressed genes in OCI-AML3 cells and found that gene sets associated with stem cell function including ALDH2, Wnt5b, Wnt5a were up-regulated whereas inflammatory and innate immune response genes were down-regulated in OCI-AML3 cells co-cultured with MSCs compared to cells cultured alone. These genes were validated by qRT-PCR assay. Ingenuity pathway analysis® (IPA) revealed a TGFβ1 activated gene signature in OCI-AML3 cells co-cultured with MSCs compared to cells culture alone. To investigate the role of TGFβ1 in the stroma-induced stem cell phenotype in AML cells, OCI- AML3 and HL60 cells were treated with recombinant TGFβ1 (5ng/ml) for 3 or 5 days. Indeed, treatment with TGFβ1 induced ALDH positive cells from 27.7% to 89.5% in 3 days and from 3.4% to 92.5% in 5 days (OCI-AML3). Similarly, in HL60 cells treated with TGFβ1, ALDH+ cells increased from 9.8% to 25.1 in 3 days suggesting that TGFβ1 induces a stem cell phenotype in AML cells. Next, we measured levels of TGFβ1 in cell culture supernatants from MSCs by ELISA and found that MSCs secrete TGFβ1 up to 1ng/ml into the surrounding medium. In addition, to study the role of TGFβ1 in stroma-induced stem cell activity in AML cells, we treated AML cells with or without TGFβ1 type-1 receptor kinase inhibitor Galunisertib (LY2157299 monohydrate from Eli Lilly®) in the presence or absence of MSCs. Strikingly, treatment of galunisertib at 1µM completely inhibited MSCs-induced ALDH activity in OCI-AML3 and HL60 cells in 5 day co-culture setting. To determine if galunisertib sensitizes AML cells to chemotherapy in the presence of MSCs, we treated OCI-AML3 with galunisertib (1µM) or cytarabine (1µM) or in combination for 72hrs in the presence or absence of MSCs. As expected presence of MSCs inhibited cytarabine induced cell killing (positive for annexin-V and DAPI) from 47.1% to 28.3% cells. In turn, the combination of cytarabine and galunisertib negated this affect and induced cell killing in OCI-AML3 cells from 28% to 54% even in the presence of MSCs suggesting complete loss of MSC-mediated chemo-protection in OCI-AML3 cells upon inhibition of TGF-β signaling. In-vivo testing of galunisertib in combination with cytarabine and doxorubicin in AML PDX models in NSG mice is currently ongoing.Collectively, data indicate that stromal cells induce chemo-resistance in AML cell by inducing stem cells phenotype through TGF-beta mediated signaling (Fig 1). TGF-β inhibition by galunisertib, an agent in early clinical trials in solid tumors inhibits stem cell phenotype and BM microenvironment-induced chemo-resistance and facilitates cell killing of AML cells. [Display omitted] DisclosuresBattula:United Therapeutics Corporation: Research Funding.

A Systematic Study of the Effect of Different Molecular Weights of Hyaluronic Acid on Mesenchymal Stromal Cell-Mediated Immunomodulation.

IntroductionOsteoarthritis (OA) is associated with chronic inflammation, and mesenchymal stromal cells (MSCs) have been shown to provide pain relief and reparative effects in clinical investigations. MSCs are often delivered with hyaluronic acid (HA), although the combined mechanism of action is not fully understood; we thus investigated the immunomodulatory effects of combining MSCs with different molecular weights (MW) of HA.MethodsHAs with MWs of 1.6 MDa (hHA), 150 kDa or 7.5 kDa, were added to MSCs alone or MSC-immune cell co-cultures. Gene expression analyses, flow cytometry and cytokine measurements were assessed to determine the effect of HAs on the MSC interactions with immune cells.ResultsMSCs in the presence of HAs, in both normal and lymphocyte-conditioned medium, showed negligible changes in gene expression. While addition of hHA resulted in increased proliferation of activated lymphocytes, both in the presence and absence of MSCs, the overall combined effect was a more regulated, homeostatic one; this was supported by higher ratios of secreted IL10/IFNγ and IL10/IL2, in lymphocyte cultures, than with lower MW HAs or no HA, both in the presence and absence of MSCs. In addition, examination of monocyte-derived macrophages showed an increased M2 macrophage frequency (CD14+CD163+CD206+) in the presence of hHA, both with and without MSCs.ConclusionshHA produces a less pro-inflammatory environment than lower MW HAs. Moreover, combining hHA with MSCs has an additive effect on the MSC-mediated immunomodulation, suggestive of a more potent combination treatment modality for OA.

Mesenchymal stromal cell-dependent reprogramming of Kupffer cells is mediated by TNF-α and PGE2 and is crucial for liver transplant tolerance.

The role of mesenchymal stromal cells (MSCs) in the modulation of liver transplant tolerance has attracted significant interest. However, the interaction between MSCs and Kupffer cells (KCs) has received little attention, and the effect of this interaction on liver transplant tolerance remains unclear. KCs were cultured in the presence and absence of MSCs. After 24 h, cells were treated with lipopolysaccharide (LPS), after which the production of cytokines and the expression of surface antigens were measured for cell function identification. Moreover, the effects of the KCs and the prostaglandin E2 (PGE2) levels produced by the MSCs were determined using an experimental rat liver transplantation model. Blood and liver samples were collected at three time points after transplantation for further analysis. After LPS treatment, when compared with the KC single cultures, the expression of pro-inflammatory cytokines (IL-1β, IL-6, MHC-II, CD40, CD80, and CD86) in the coculture system was down-regulated, whereas the expression of anti-inflammatory cytokines (TGF-β, IL-4, PGE2, and IL-10) was markedly increased. These data indicate that MSCs can reprogram the phenotype of KCs. However, KCs treated with miR/TNF-α (tumor necrosis factor) plasmid prior to coculture to inhibit the production of TNF-α resulted in an inhibition of the reprogramming effect of MSCs. Moreover, overexpression of PGE2 in MSCs increased the effect of MSCs on KC reprogramming. After rat liver transplantation, allograft recipients that received MSCs showed better allograft tolerance when compared with rats in which KC function was inhibited. Furthermore, rats treated with MSCs overexpressing PGE2 demonstrated the best liver tolerance of all of the groups tested. MSCs reprogram the phenotype of KCs through TNF-α and PGE2, and this process is crucial for the immunomodulatory function of MSCs in liver transplantation.

Inhibition of CML Stem Cell Growth By Targeting WNT Signaling Using a Porcupine Inhibitor

BCR-ABL tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of chronic myeloid leukemia (CML). However, TKIs are unable to eradicate the leukemia stem cells (LSCs) from which CML arises, despite effective inhibition of BCR-ABL kinase activity in this population. There is evidence that extrinsic signals from the bone marrow (BM) microenvironment play an important role in the resistance of CML LSC to TKI treatment. Our previous studies have shown a role for Wnt signaling in protecting CML LSC from TKI treatment (Blood. 2013; 121(10):1824-38). Palmitoylation of Wnts by the Porcupine acyltransferase (PORCN) is required for their secretion and activity. LGK974, a potent PORCN inhibitor, has recently been shown to inhibit Wnt signaling and shows in vivo efficacy against several Wnt-dependent tumors (PNAS. 2013; 110(50):20224-9). In this study, we investigated whether inhibition of Wnt secretion using LGK974 could sensitize CML stem/progenitor cells to TKI treatment. CML and normal CD34+ cells and purified CD34+CD38- stem cells and CD34+CD38+ committed progenitors were treated with LGK974 with or without addition of the BCR-ABL TKI (nilotinib), and in the presence and absence of human BM mesenchymal stromal cells (MSC). CML CD34+ cells showed enhanced Wnt signaling following exposure to recombinant Wnt3a (200ng/ml) compared to normal CD34+ cells as evidenced by increased phospho-LRP6 (ser1490) and β-catenin protein levels. Analysis of all 10 frizzled receptors expression by qRT-PCR revealed a significant upregulation of FZD4 (p=0.026, n=10), and FZD5 (p=0.003, n=10), but downregulation of FZD8 (p=0.009, n=10), in CML compared to normal CD34+ cells. Co-culture with MSC was associated with increased Wnt signaling in CML CD34+ cells as detected by increased expression and nuclear translocation of β-catenin protein, and increased expression of Wnt target genes including c-Myc, Cyclin-D1, PPARδ, and Axin2. Treatment with LGK974 reduced Wnt secretion from MSC measured in a TCF/LEF reporter assay (LGK 0.5μM, 0.74 fold reduction compared to control, p=0.016, n=5, LGK 1μM, 0.62 fold reduction, p=0.007, n=5) and potently inhibited Wnt signaling in CML CD34+ cells both in the absence and presence of MSC. Treatment with LGK974 (1μM) in combination with nilotinib (1μM) resulted in significantly increased inhibition of CML CFC growth compared to nilotinib alone, both in the presence and absence of MSC (34+38- cells LGK alone 82.75±6.38% inhibition of control, Nil alone 43.12±15.32%, combination 23.17±6.48%, p<0.05, n=4; 34+38+ cells LGK alone 87.65±6.13% inhibition of control, Nil alone 26.99±4.54%, combination 16.04±0.48%, p<0.05, n=4) Importantly, LGK974 inhibited CML CFC growth to a significantly greater extent than the normal CFC growth ( normal 34+38- cells LGK alone 88.09±6.85% inhibition of control, Nil alone 89.79±10.09%, combination 70.95±9.39%, p=ns, n=4; 34+38+ cells LGK alone 86.42±8.41% inhibition of control, Nil alone 87.43±4.66%, combination 73.39±6.91%, p=ns, n=4). However, LGK974 treatment did not induce apoptosis in CML or normal progenitors. These results suggest that the effects of LGK974 on clonogenic growth could be related to the long-term inhibition of LSC self-renewal. Therefore, studies designed to evaluate the effect of in vivo administration of LGK974 in a transgenic BCR-ABL mouse model of CML are in progress. Finally, our preliminary studies indicate that in vivo treatment of control healthy mice with LGK974 (5mg/kg bid p.o.), nilotinib (50mg/kg qd p.o.), or the combination for 4 weeks (n=7 per cohort) did not result in clinical toxicity or alter the short-term and long-term hematopoietic stem cell populations. In conclusion, our results indicate that treatment with the PORCN inhibitor LGK974 effectively inhibits both autocrine and paracrine WNT signaling in CML stem cells and inhibits their clonogenic capacity in vitro, and support further evaluation of this approach to selectively target CML stem cells. DisclosuresWang:Novartis Institute for Biomedical Research: Employment. McLaughlin:Novartis Institute for Biomedical Research: Employment.

MDS-Derived Stromal Cells Exhibit Altered Gene Expression and Support The Engraftment Of lin-CD34+CD38- Disease-Initiating Stem Cells In a Xenograft Model Of Lower Risk MDS

IntroductionMyelodysplastic syndromes (MDS) are clonal hematologic disorders characterized by ineffective hematopoiesis, dysplasia and increased risk of progression to acute myeloid leukemia. The development of targeted therapies for MDS has been lagging behind and remains a key clinical challenge that has been hampered, at least in part, by difficulties to establish in vivo model systems that recapitulate disease heterogeneity and complexity. Attempts to generate a xenograft model of lower risk MDS have only achieved low and often transient levels of engraftment. Recent evidence from mouse studies suggests that MDS is a disease in which both the hematopoietic system and the bone marrow microenvironment might be involved. Thus, we hypothesized that a specific MDS microenvironment might be required for the successful modeling of low risk MDS in mice, proposing a dependency of the “disease propagating cells“ on their corresponding niche cells in human MDS. MethodsOur study is based on xenotransplantation of material from 19 MDS patients classified as follows: IPSS low risk (n=6), intermediate-1 risk (n=13), WHO 2008 classification: MDS 5q- (n=7), MDS RCMD (n=7), MDS RAEB I (n=3), MDS-U (n=1), MDS RARS (n=1). MDS CD34+ cells were co-injected with patient-derived mesenchymal stromal cells (MSCs) directly in the bone marrow cavity (i.f) of NOD.Cg-Prkdscid Il2rgtm1Wjl/Szj (NSG) or NSGS (NSG mice expressing human SCF, IL3 and GM-CSF) mice. Molecular tracking of MDS cells was carried out by copy number analysis (Affymetrix SNP 6.0 Arrays), metaphase cytogenetics, interphase FISH, Roche 454 deep sequencing and pyrosequencing of known mutations. Mice were analyzed after a minimum of 16 weeks post transplantation. ResultsWe show that co-injection of MDS CD34+ cells with their corresponding MSCs leads to significant and long-term engraftment of over 77% of the MDS patients analyzed, both in NSG (10/13 patients, range hCD45+= 1-18%) and NSGS mice (7/8 patients, range hCD45+=2.2-74%). In contrast, absence of MSCs or co-injection of healthy age-matched MSCs only gave rise to limited engraftment in NSG mice (2/7 patients (hCD45+=1-3.8%) and 1/2 patients (hCD45+=2%), respectively). Transplanted samples exhibited a clear myeloid bias and significant engraftment of cells with progenitor (CD34+CD38+) and stem cell phenotype (CD34+CD38-) that could be serially transplanted. In addition, presence of morphologically dysplastic cells was readily detectable in NSGS mice. Importantly, molecular analysis of the engrafted cells confirmed their “diseased” origin as they carried identical lesions to the ones present in the original MDS patient. Furthermore, we could demonstrate that disease-propagating stem cells in lower risk MDS exclusively reside within the lin-CD34+CD38- stem cell fraction. Finally, RNA sequencing analysis comparing MDS and age-matched healthy control MSCs revealed altered expression of key genes involved in cellular adhesion, extra-cellular matrix (ECM) remodeling and cellular cross-talk in diseased MSCs, strongly supporting the notion of a complex interplay between MDS hematopoietic cells and their corresponding stroma. In addition, patient MSCs exhibited clear molecular features of fibrosis, a clinical feature often associated with MDS. ConclusionIn this study we have identified patient-derived MSCs as a critical functional component of lower risk MDS. Together with MDS stem cells, these patient MSCs form a functional stem cell-niche unit, which allows the propagation of the disease in a xenograft recipient. The striking changed expression in diseased MSCs of genes involved in processes like cytokine-cytokine receptor interaction, cellular adhesion, ECM remodeling as well as hypoxia further suggests that diseased MDS cells might alter the function of the normal HSC niche into one that can support the requirement of MDS cells. Studying the interaction of MDS stem cells and MSCs at the cellular and molecular level will provide a platform for unraveling the molecular basis of clonal dominance in MDS as well as allow the design of targeted strategies aimed to disrupt the MDS stem cell-MSC niche interactions. Disclosures:No relevant conflicts of interest to declare.

Mesenchymal stromal cells impair the differentiation of CD14++ CD16− CD64+ classical monocytes into CD14++ CD16+ CD64++ activate monocytes

Mesenchymal stromal cells (MSC) possess immunomodulatory activity both in vitro and in vivo. However, little information is available regarding their function during the initiation of immunologic responses through their interactions with monocytes. While many studies have shown that MSC impair the differentiation of monocytes into dendritic cells and macrophages, there are few articles showing the interaction between MSC and monocytes and none of them has addressed the question of monocyte subset modulation. To understand better the mechanism behind the benefit of MSC infusion for graft-versus-host treatment through monocyte involvement, we performed mixed leucocyte reactions (MLR) in the presence and absence of MSC. After 3 and 7 days, cultures were analyzed by flow cytometry using different approaches. MSC induced changes in monocyte phenotype in an MLR. This alteration was accompanied by an increase in monocyte counting and CD14 expression. MSC induced monocyte alterations even without contact, although the parameters above were more pronounced with cell-cell contact. Moreover, the presence of MSC impaired major histocompatibility complex (MHC) I and II, CD11c and CCR5 expression and induced CD14 and CD64 expression on monocytes. These alterations were accompanied by a decrease in interleukin (IL)-1β and IL-6 production by these monocytes, but no change was observed taking into account the phagocytosis capacity of these monocytes. Our results suggest that MSC impair the differentiation of CD14(++) CD16(-) CD64(+) classical monocytes into CD14(++) CD16(+) CD64(++) activated monocytes, having an even earlier role than the differentiation of monocytes into dendritic cells and macrophages.

Stromal Interaction of Lymphoma Cells Regulates Survival and Chemoresistance

Abstract 5195High-grade lymphomas are aggressive but largely curable, whereas low-grade lymphomas are indolent, but frequently recur to be incurable, paradoxically. Mesenchymal stromal cells (MSCs) have been known to participate for reconstituting microenvironment. Studies show that signalings between normal lymphoid cells and stromal cells were frequently altered in high grade lymphomas, but relatively conserved in low grade lymphomas. However, which cell and mechanism is responsible for lymphoma recurrence remains unclear. Here we hypothesized that the interaction with stroma may play a role for lymphoma cell growth and survival. For this, we investigated the effect of MSCs on lymphoma cell growth and chemo-resistance by using a coculture system with MSCs (derived from tonsil), as a stromal microenvironment for lymphoma cells. First, coculture of lymphoma cell line (Pfeiffer) and primary lymphoma cells with/without MSCs for 3 days showed that the MSC-cocultured cells grew more rapidly (1.4 times and 1.8 times for Pfeiffer and primary cells, respectively) than MSC-free lymphoma cells. To further investigate the underlying mechanism of promoting growth, lymphoma cells were cocultured with MSCs in the presence or absence of transwell filter. At day 4, the proliferation of lymphoma cells in the absence of transwell was 3.5 times higher than in the presence of transwell. Interestingly, the lymphoma cells cocultured with MSCs showed increased expression of CXCR4 compared with lymphoma cells cultured alone. When the cyto-protective effect of MSCs was examined by doxorubicin treatment (1ug/ml) in the presence or absence of MSCs, 91% of MSC-cocultured cells survived, whereas only 28% of stroma-free cultured cells survived. These results demonstrate that the direct contact interaction with stroma might be important for lymphoma cell growth and survival.In conclusion, our data suggest that the interaction with stromal microenvironment is an important factor for survival of lymphoma cells which cells might be responsible for chemoresistance, and raise the possibility that the stromal interaction could be a potential target for lymphoma treatment. Disclosures:No relevant conflicts of interest to declare.This research was supported by a grant (10172KFDA993) from Korea Food & Drug Administration in 2011. Disclosures:No relevant conflicts of interest to declare.

Human Bone Marrow Stromal Cells and Skin Fibroblasts Inhibit Natural Killer Cell Proliferation and Cytotoxic Activity

Mesenchymal stromal cells (MSCs) are potent immunomodulators that have successfully been used to circumvent various types of inflammations, including steroid-resistant graft-versus-host disease. Although initially believed to be restricted to multipotent MSCs, this immunoregulatory function is shared with differentiated cells from the mesenchymal lineage such as skin fibroblasts (SFs). Mesenchymal cell-induced immunoregulation is so potent that it may allow the reactivation of dormant malignancies, a fact that would preclude using such cells as therapeutic agents. Because NK cells are pivotal effectors controlling tumor cell containment we investigated the effect of allogenic MSCs and SFs on NK cell function in vitro. When NK cells were incubated with IL-15 and MSCs or SFs for 6 days, their proliferation and cytotoxic activity were significantly decreased compared to NK cells cultured with IL-15 alone or with human venous endothelial cells. Cytotoxic activity inhibition reached 86% when assayed on MHC-I(+) allogenic primary hematopoietic blasts, and was associated with a significant decrease in cytolytic granule exocytosis and in perforin release. Stromal cell-mediated inhibition was effective only if cell-cell proximity was long lasting: when NK cells were activated with IL-15 in the absence of MSCs and assayed for cytotoxicity in their presence no inhibition occurred. MSC inhibition was ultimately mediated by a soluble factor generated upon incubation with NK cells activated by IL-15 or IL-2. The indoleamine 2,3 dioxygenase was activated in MSCs and SFs because L-kynurenine was detected in inhibitory supernatants, but its blockade did not restore NK cell functions. The profound inhibition of cytotoxic activity directed against allogenic hematopoietic blasts exerted by MSCs and SFs on NK cells may be a concern. Should this occur in vivo it may induce the inability of NK cells to control residual or dormant malignant diseases after infusion of therapeutic MSCs.

Mesenchymal Stromal Cells Promote a Sustained Increase of the CD69 Marker on Activated CD3+ Lymphocytes: Potential Immunomodulatory Role

Mesenchymal stromal cells (MSCs) exert an immune regulatory function and suppress T-cell proliferation in vitro and in vivo. One of the ways by which MSCs may modulate immune responses is by the induction of CD4+CD25+FOXP3+ regulatory T cells (Treg), which suppress alloreactivity in vitro and prevent or attenuate GVHD in animal models. TGF-b (TGFB1 gene), one of the factors secreted by MSC, is known to induce the expression of FOXP3 and to drive the generation of Tregs from CD4+CD25− T cells. T cell activation is characterized by the expression of many surface molecules, and CD69 is one of the earliest markers, transiently expressed following activation. CD69 is selectively expressed at sites of chronic inflammation and recent in vivo and in vitro results indicate that this receptor may modulate the inflammatory response, by inducing TGF-b production. Despite the importance of secreted factors, cell to cell contacts promote increased lymphocyte immune modulation. Interestingly, TGF-b is known to induce the expression of b-IG-H3 (TGFBI gene), a secreted extracellular matrix adaptor protein, whose expression is higher on hematopoietic stem cells adherent to MSC. Given the potential role of CD69 as a regulatory molecule, we explored the effects of MSC on the expression of CD69, TGFB1, FOXP3 and TGFBI on co-cultured T-cells. Peripheral blood mononuclear cells (PBMC) from 6 individuals were co-activated by anti-CD3/CD28 beads and cultured either in the presence or in the absence of MSC (5:1) adhered to the bottom of culture wells. IL2 (20U/ml) was added in the 3rd day for full activation. Cultured PBMC were collected 1, 3 and 5 days after activation. Percentage of CD69+ cells and proliferation of activated lymphocytes (cell division tracking by CFSE) were evaluated by flow cytometry on gated CD3+ lymphocytes. Transcripts levels of TGFB1, TGFBI, FOXP3 and IL10 were determined by real time PCR and normalized using ACTB as an endogenous control. Relative expression levels were calculated in comparison with activated PBMC cultivated alone at the 5th day. Proliferation of lymphocytes co-cultured with MSC was significantly inhibited. As expected, activation of lymphocytes cultured alone was evidenced by the expression of CD69 in 5% of cells in the first day (mean percentage), followed by a decrease in the subsequent days (4% and 3%, respectively). Interestingly, lymphocytes co-cultured with MSC displayed a completely different pattern, with a similar initial activation (7%) which was followed by significant increase in the 3rd day (16%), maintained in the 5th day (14%). Furthermore, TGFBI and IL10 were both expressed at significantly higher levels on PBMC co-cultured with MSC, compared to PBMC alone, in all days evaluated. In addition, their transcript levels decreased faster on PBMC cultured alone. In the other hand, TGFB1 and FOXP3 levels in PBMC cultured alone or in the presence of MSC were similarly higher in the 1st day, but decreased till the 5th day, when their levels were slightly, but significantly higher on PBMC co-cultured with MSC, compared to PBMC cultured alone. We demonstrate for the first time that co-culture with MSC causes a sustained increase of the CD69 marker on CD3+ lymphocytes, which is accompanied by increased levels of TGFB1, TGFBI, FOXP3 and IL10 on total PBMC. Our results are in line with the proposed immunoregulatory role of CD69. In addition, higher TGFBI levels on PBMC may increase lymphocyte adherence to MSC, thus favoring immune modulation. This work was supported by FINEP, CNPq and FAPESP.