Co-culture Of Mesenchymal Stromal Cells Research Articles

Interaction of Tumor Cells with the Hematopoietic Stem and Progenitor Cell Niche

IntroductionThe bone marrow microenvironment regulates the self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs), through a network dependent on cell-cell interaction. This interaction is mediated by morphogens, the extracellular matrix and cell adhesion molecules expressed and secreted by various cell types in the HSPC niche. Mesenchymal stromal cells (MSCs), as the major cellular component, maintain the stemness properties of the niche. The microenvironment thus becomes conducive for HSPCs to remain quiescent, thereby enabling long term self-renewal. Therefore, the safe haven in the bone marrow microenvironment and its constituent cell types can be targeted during tumorigenesis, thus making the niche neoplastic. Dissemination of breast cancer cells into the bone marrow has been described even in the early stages of the disease. The present study focuses on the influence of breast carcinomas on the genetic and functional profile of mesenchymal and hematopoietic progenitor cells of the bone marrow niche.MethodsIn vitro coculture models of breast cancer cell lines- MDA-MB231, MCF-7 and primary MSCs derived from the bone marrow of healthy donors were used in the study. Atomic- force microscopy based single-cell force spectroscopy (AFM-SCFS) and fluorescence based assays were used for cell adhesion experiments. Hydrogel based culture systems were used for 3-dimensional cocultures of breast cancer cells and MSCs. Hypoxic and normoxic culture conditions (0.5% and 20% oxygen respectively) were used for the experiments.ResultsThe breast cancer cell lines caused a significant reduction in HSPC adhesion to MSCs (88% by MDA-MB 231 cells; p<0.005 and 73% by MCF-7 cells; p<0.005). AFM-SCFS studies also indicated a higher binding force between breast cancer cells and MSCs, as compared to HSPCs (MDA-MB231 cells-0.13nN, MCF-7 cells-0.074nN and HSPCs-0.05nN). MDA-MB231and MCF-7 cells express Intercellular adhesion molecule-1(ICAM-1), which has been shown to promote breast cancer metastasis (Hanlon et al, 2002; Rosette et al, 2005; Schröder C. et al, 2011). There was a significant difference in reduction of HSPC adhesion towards MSCs by ICAM-1 knockdown (ICAM-1 KD) tumor cells as compared to MDA-MB231 cells (84.83% by MDA-MB231 cells versus 28.11% by ICAM-1KD tumor cells, p<0.001). AFM-SCFS studies also showed a reduced binding force between ICAM-1 KD tumor cells and MSCs as compared to MDA-MB231cells (MDA-MB231 cells-0.14nN versus ICAM-1-KD tumor cells-0.05nN, p value<0.001). ICAM-1 KD studies thus showed that reduction in HSPC adhesion to MSCs by breast cancer cells was mediated through ICAM-1 signaling. A cytokine array was performed to investigate if breast cancer cell lines affect the cytokine profile of MSCs. The array showed altered expression of growth factors- Basic fibroblast growth factor (bFGF) and Platelet derived growth factor–beta (PDGF-BB) (2.2 fold upregulation and 0.5 fold downregulation in breast cancer cells- MSC cocultures respectively). Based on the array, a bFGF-mediated increase in the proliferation of MSCs and breast cancer cells in coculture was observed. The bFGF upregulation also caused an increased migration of MDA-MB231 cells towards MSCs in a transwell migration assay. An upregulation in the phosphorylation status of Akt was observed in breast cancer cells – MSC cocultures, as a downstream effect of upregulated bFGF levels. The bFGF-mediated increase in the proliferation of breast cancer cells and MSCs in coculture was shown to be dependent on the activation of PI3K-Akt pathway. The bFGF- mediated increase in the migration of MDA-MB231 cells towards MSCs was also inhibited upon addition of the PI3K blocker. Interestingly, the breast cancer cells caused a reduction in osteoblastic differentiation of MSCs by downregulation of PDGF-BB. Studies with 3-dimensional cocultures of breast cancer cells and MSCs also showed a reduction in osteoblastic differentiation of MSCs. Furthermore, long-term cocultures of breast cancer cells, HSPCs and MSCs showed reduced support for primitive HSPCs in the neoplastic niche.ConclusionsThese findings indicate a perturbed HSPC niche upon tumor invasion. The possible role of altered cytokine expression, consecutive downstream signaling in niche activation and bone turnover will be further studied using in vitro and in vivo approaches to recapitulate tumor micrometastases to the HSPC niche. DisclosuresNo relevant conflicts of interest to declare.

Cooperative Targeting of Bcl-2 Family Proteins By ABT-199 (GDC-0199) and Tyrosine Kinase Inhibitors to Eradicate Blast Crisis CML and CML Stem/Progenitor Cells

Bcr-Abl tyrosine kinase supports CML cell survival in part by regulating antiapoptotic Bcl-2 proteins such as Bcl-xL and Mcl-1. Tyrosine kinase inhibition, the front-line therapy for patients with chronic phase CML, is less effective in blast crisis (BC) patients and inactive against quiescent CML stem/progenitor cells. We reported that ABT-737, a dual Bcl-2/Bcl-xL inhibitor, induces apoptosis in BC CML cells including CD34+quiescent CML cells. ABT-199, a potent Bcl-2 specific inhibitor, has entered clinical trials for various hematological malignancies. We hypothesized that cooperative targeting of antiapoptotic Bcl-2 proteins using a combination of ABT-199 and tyrosine kinase inhibitors (TKIs) would exert enhanced activity against BC CML and CML stem/progenitor cells.Cells from patients (n=4) with TKI-resistant BC CML were treated with ABT-199, TKIs, and combinations. Although exerting low activity by itself, ABT-199 in combination with TKIs synergistically induced apoptosis (CI<0.1) in bulk and CD34+38- cells from these patients regardless of their previous clinical responses to TKIs. The combinations had minimal activity against normal CD34+cells (n=3). Mechanistic studies demonstrated that nilotinib inhibited the expression of Bcl-xL and Mcl-1 mRNA and protein, even in cells from TKI (including nilotinib) resistant patients. Individual inhibition of Bcl-xL or Mcl-1, and even more so inhibition of both, by siRNAs increased the sensitivity of cells to ABT-199, suggesting that cooperative inhibition of Bcl-2 by ABT-199 and Bcl-xL/Mcl-1 by TKIs contributes to the synergy.To evaluate the effect of these combinations on TKI-insensitive quiescent stem/progenitor CML cells, BC CML patient cells were stained with the cell division-tracking dye carboxyfluorescein succinimidyl ester (CFSE) and then co-cultured with human bone marrow (BM)-derived mesenchymal stromal cells (MSCs). Once proliferating and quiescent cells were distinguishable by flow cytometry, cells were treated with ABT-199, TKIs, and their combinations for 48 hours with or without MSC co-culture. Apoptosis was measured in proliferating and quiescent progenitor cells, defined as the percentage of annexin V positivity in CD34+CFSEdim and CD34+CFSEbright cells, respectively. ABT-199 as a single agent decreased viability of CML cells cultured alone or co-cultured with MSCs in both proliferating (IC50=191±103nM and 194±64nM, respectively) and quiescent (IC50=221±75nM and 205±123nM, respectively) CD34+ CML cells. Combinations of ABT-199 with TKIs, including imatinib, nilotinib, dasatinib, or ponatinib, synergistically induced death (CI<0.2) and decreased the number of viable cells in proliferating as well as quiescent CD34+progenitor cell populations (n=6). All 6 patients were resistant to TKIs, and 4 had mutations in the BCR-ABL gene, including three with the T315I mutation.To further test the ability of ABT-199 and TKI combinations to eradicate CML stem cells, we used an inducible transgenic CML mouse model in which the BCR-ABL gene is expressed under control of a tet-regulated enhancer of the murine stem cell leukemia (Scl) gene, allowing targeted BCR-ABL expression in stem/progenitor cells. Once BM cells from transgenic Scl-tTa-BCR-ABL/GFP mice were engrafted in wild type recipient mice, the mice were treated with ABT-199, nilotinib, or both. At the end of a 3-week treatment period, each single agent alone, and even more so with the combinations, significantly decreased blood total GFP+ WBC (12.9±1.4, 5.2±0.3, 6.1±0.4, and 1.6±0.3 x106/ml in controls, ABT-199, nilotinib, and combination, respectively) and neutrophils (1.43±0.03, 0.49±0.06, 0.32±0.03, and 0.25±0.05 x106/ml in the respective groups). ABT-199 (P=0.02), and more so with the combination (P<0.01) but not nilotinib alone (P=0.29), significantly decreased BM GFP+ LSK cells (12.0±1.2, 6.8±0.6, 9.5±1.6, and 2.2±0.2 x103 cells in the respective groups). The in vivo experiments are ongoing.Conclusions: ABT-199 and TKIs cooperatively target antiapoptotic Bcl-2 family proteins. This combination is highly effective in killing bulk and CD34+38- CML cells and quiescent CD34+ CML stem/progenitor cells from BC CML patients in vitro and in suppressing leukemia and leukemia stem cells in vivo. This strategy has the potential to eradicate BC CML cells and CML stem/progenitor cells, neither of which are effectively targeted by TKIs alone. DisclosuresCarter:AbbVie, Inc.: Research Funding. Leverson:AbbVie, Inc.: Employment. Konopleva:AbbVie, Inc: clinic trial Other.

Interactions of human MSC with head and neck squamous cell carcinoma cell line PCI-13 reduce markers of epithelia-mesenchymal transition.

Cancer progression is influenced by tumor microenvironment and communication of stromal cells and tumor cells. Interactions may enhance epithelial-mesenchymal transition (EMT) of tumor cells through signaling proteins such as Wnt/beta-catenin and matrix metalloproteinases (MMP), as well as loss of cellular integrity, which affects invasion, progression, and metastasis of head and neck squamous cell carcinoma (HNSCC). In this study, we are testing the hypothesis that interactions of human mesenchymal stromal cells (MSCs) with HNSCC might influence the expression of markers of EMT and tumor progression by co-culturing human MSC with the PCI-13 HNSCC line. Pooled MSCs were derived from the iliac bone marrow of seven patients and co-cultured in transwell permeable membrane wells with tumor cells of the established HNSCC cell line PCI-13 (UICC: T3, N1, M0). MSCs were characterized through fluorescence-activated cell sorting (FACS) analysis. Expression of Wnt3, E-cadherin, beta-catenin, MMP14, cathepsin b, and ETS1 was assessed by quantitative RT-PCR. We were able to show that co-culture of MSCs and PCI-13 leads to a significantly reduced expression of Wnt3, MMP14, and beta-catenin compared to controls, whereas the expression of cathepsin b and ETS1 was not significantly different between co-cultures and controls. Our results suggest that the interaction between MSCs and PCI-13 may suppress EMT in cancer cells. The influence of MSCs can suppress the onset of EMT in HNSCC, affecting tumor progression and therapy.

Activation Of p53 By MDM2-Inhibition Sensitizes Quiescent Blast Crisis CML Stem/Progenitor Cells To Bcl-2 Inhibitor- and Tyrosine Kinase Inhibitor-Induced Cell Death

The transcriptional activation of pro-apoptotic Bcl-2 family members is a major mechanism of p53-mediated cell death. p53 can also directly contribute to mitochondrial mediated apoptosis by interacting with Bcl-2 family proteins. A number of anti-apoptotic Bcl-2 proteins such as Mcl-1 and Bcl-xL are regulated by the Bcr-Abl tyrosine kinase and confer apoptosis resistance to CML cells. It is therefore not surprising that p53 gene alterations may play a role in the disease progression of CML. The reported p53 mutation rate of 30% suggests potential therapeutic benefit of p53 activation by MDM2 inhibition alone or as sensitization of CML to therapeutic agents.Tyrosine kinase inhibitors (TKIs), the front-line therapy for patients with chronic phase CML, are less effective in patients with blast crisis (BC) CML and inactive against quiescent CML stem/progenitor cells. Here we examine the effects of nutlin3a, which activates p53 by inhibiting MDM2, and its combinations with the Bcl-2 inhibitor ABT-737 and the TKI nilotinib on the viability of proliferating and quiescent CD34+CML stem/progenitor cells obtained from patients with BC CML.Mononuclear cells from patients with BC CML who were resistant to multiple TKIs were stained with the cell division tracking dye carboxyfluorescein succinimidyl ester (CFSE) and then co-cultured with human bone marrow (BM) derived mesenchymal stromal cells (MSCs). Once proliferating and quiescent cells are distinguishable by flow cytometry (within 7 to 12 days), the cells were treated with nutlin3a, ABT-737, nilotinib, nutlin3a plus ABT-737, or nutlin3a plus nilotinib for 48 hours with or without MSC co-culture. The cells were then stained with a CD34 antibody, and apoptosis (annexin V staining) was determined by flow cytometry. Apoptosis in proliferating and quiescent progenitor cells was defined as percentage of annexin V positivity in CD34+CFSEdim and CD34+CFSEbrightcells, respectively.Nutlin3a alone was able to decrease viability of CML cells cultured alone or co-cultured with MSCs in both proliferating (IC50 = 2.50 ± 0.92 μM and 2.54 ± 0.60 μM, respectively) and quiescent (IC50 = 3.70 ± 1.22 μM and 4.27 ± 0.77 μM, respectively) CD34+ CML stem/progenitor cells. Although not very active by itself, ABT-737, when combined with nutlin3a (n = 6), induced apoptosis synergistically in proliferating (CI = 0.24 ± 0.12) and more so in quiescent (CI = 0.09 ± 0.10) CD34+CML stem/progenitor cells, even when cells were co-cultured with MSCs (CI = 0.22 ± 0.09 for proliferating and CI = 0.03 ± 0.05 for quiescent stem/progenitor cells). The combination of nutlin3a with nilotinib (n = 4) was also highly synergistic in proliferating stem/progenitors (CI = 0.02 ± 0.04 without and CI = 0.05 ± 0.04 with co-culture) and in quiescent progenitors (CI = 0.07 ± 0.29 without and CI = 0.03 ± 0.06 with co-culture).Treatment of primary CML blast cells with nultin3a increased p53, and pro-apoptotic Bax and Puma levels in all samples examined, indicating activation of transcriptional activity of p53. In addition, nutlin3a treatment decreased the protein levels of anti-apoptotic Bcl-xL and/or Mcl-1. Nilotinib also decreased the expression, both at the RNA and protein levels, of Mcl-1 and Bcl-xL, even in patients who had not responded to nilotinib treatment clinically. ConclusionsHere we demonstrate that activation of p53 induces apoptosis in proliferating and quiescent BC CML stem/progenitor cells and sensitizes to Bcl-2 inhibitor- and TKI-induced cell death by, at least in part, regulating the expression of Bcl-2 family proteins. Results suggest a potential for utilizing this strategy in the treatment of BC CML, and also for the elimination of quiescent CML stem/progenitor cells. Disclosures:No relevant conflicts of interest to declare.

Unraveling The Molecular and Metabolic Basis For Glutamine Addiction In Leukemias

The bone marrow (BM) microenvironment is characterized by hypoxia and the presence of supporting mesenchymal stromal cells (MSC) that promote leukemia cell survival and resistance to therapy, in part by metabolic reprogramming. However mechanisms that couple leukemic cells survival to metabolic processes under different microenvironment conditions have not been elucidated. Glutamine (Gln) provides cells with carbon skeletons to the Krebs cycle (KC) via anaplerosis, sustains cell proliferation, regulates redox homeostasis and modulates activity of signal transduction pathways. Recent data suggests that leukemia cells reduce molecular oxygen utilizing electrons from carbon sources other than pyruvate, and we hypothesize that these electrons could be provided at least in part by glutaminolysis. Our recent studies utilizing gene expression profiling indicate that MSC co-culture under hypoxia promoted glycolytic gene expression in AML cells, as well as genes regulating oxidative phosphorylation (OXPHOS), KC cycle and Gln utilization (GLS1, GOT) (Matre et al., AACR 2013:1887). Here we report studies aimed to unravel metabolic changes in proliferating leukemic cells under hypoxia and upon interaction with MSC and determine the role of Gln as a contributor.First, we performed GC-MS metabolic profiling of OCI-AML3 leukemic cells alone or in co-cultured with MSC under hypoxic or normoxic conditions and observed significant changes in the core metabolic processes. Our data demonstrates that microenvironment promotes glucose-independent OXPHOS to meet bioenergetics needs of leukemic cells. Interaction with MSC propels a glucose-independent oxidative KC through Gln and asparagine catabolism even under conditions where oxygen concentration is limited. Under hypoxia, concentrations of KC intermediates were lower compared to normoxia, however the accumulation of 2-hydroxyglutarate suggests reverse KC activity with glutamate-derived 2-oxoglutarate being converted to citrate via reductive carboxylation pathway. In addition, consumption of glucogenic amino acids was upregulated by MSCs. Glycolytic intermediates accumulated under hypoxia and coculture accompanied by excretion of pyruvate as lactate, suggesting increased availability of carbon skeletons for biomass generation provided, in part, by glutaminolysis.Next, oxygen consumption rates (OCR) and extra-cellular acidification rates (ECAR) in OCI-AML3 and REH cells were assayed using Seahorse Bioscience XF96 EF Analyzer (Billerica, MA). Glutaminase (GLS) inhibition by BPTES or shRNA caused a decrease in basal OCR, reduced ATP production and decreased maximal respiratory capacity of leukemic cells (Fig. 1). Both acute and prolonged exposure to BPTES resulted in a compensatory increase in glycolytic activity as shown by increase in ECAR and confirmed by media lactate levels. [Display omitted] Analysis of a panel of acute leukemia cell lines (n=12) showed that subset of leukemia (75%) markedly dependent on Gln for growth with Gln deprivation causing steep decrease in viable cell number via induction of apoptosis. In addition, in the corresponding subset, inhibition of GLS (GLS1) with BPTES decreased cell growth and increased apoptosis under both normoxia and hypoxia. Notably, MSC co-culture failed to protect firmly attached hypoxic AML cells, which are otherwise resistant to chemotherapy-induced cytotoxicity. Finally, the expression of GLS1 gene splice variants, Glutaminase C (GAC) and kidney glutaminase (KGA), was determined using oligonucleotide microarrays (HG U133 Plus 2.0, Affymetrix) in 288 AML and in 103 normal samples (healthy BM and non-leukemia conditions, Haferlach, JCO 2010). GAC transcript was found to be significantly overexpressed in several AML subtypes, including AML with FLT3 gene mutations and complex cytogenetics. In turn, KGA expression was not different between AML and normal samples.In summary, our results indicate that Gln is a major source of carbon skeletons for KC activity in AML cells, and demonstrate the key role of Gln utilization pathway for the survival of hypoxic BM-resident leukemic cells and “Glutamine-dependent OXPHOS subset” of leukemia. These findings support the notion of targeting microenvironment-fueled leukemia metabolism through pharmacological inhibition of GLS with novel selective GLS1/2 inhibitors entering clinical arena. Disclosures:No relevant conflicts of interest to declare.

EMP1, a Novel Poor Prognostic Factor In Pediatric Leukemia Regulates Prednisolone Resistance, Cellular Proliferation, Migration and Adhesion

Background20% of pediatric precursor B-acute lymphoblastic leukemia (BCP-ALL) and 25% of pediatric T-ALL patients relapse on or after current treatment strategies. Treatment failure has been associated with cellular resistance to prednisolone, the leading drug in multi-drug treatment of ALL. This study aimed to find new druggable targets that may modulate prednisolone resistance. MethodsLeukemic cells from children with newly diagnosed ALL were isolated from bone marrow aspirates, only samples with ≥ 90% leukemic blasts were used. RNA of 178 leukemic patients were hybridized to Affymetrix U133 Plus 2.0 gene expression microarrays and limma analysis was performed. EMP1 knockdown was achieved by lentiviral shEMP1 spin-occulation in four BCP-ALL cell lines and two T-ALL cell lines. Knockdown was confirmed by qRT-PCR and Western blot. Cell proliferation, cell cycle, and cell survival was analyzed by an AnnexinV/PI-staining and by a BrdU/7-AAD-staining. Response to prednisolone was evaluated in an in vitro3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Irradiated human mesenchymal stromal cells (hMSC-TERT) were co-cultured overnight with maroon-labeled leukemic cells, whereafter non-adherent and adherent leukemic cells were counted with the MACSQuant. Maroon-labeled leukemic cells were also inserted in the upper compartment of a 3 µM pore-size transwell system and were allowed to migrate overnight to hMSC-TERT. Migrated leukemic cells were evaluated by trypan blue and the MACSQuant. To assess MSC-mediated prednisolone resistance, leukemic cells alone or in combination with irradiated hMSC-TERT were cultured with or without prednisolone and cell survival was assessed after 72 hours. Cell signaling after shEMP1 knockdown was studied using Milliplex 8-plex Human Src Family kinase kit and the 9-plex Multi-Pathway kit with GAPDH and β-tubuline loading control beads, following the manufacturers' protocol. Cumulative incidence of relapse was calculated as time to relapse with death as a competing event in R using the method of Fine and Gray. Multivariate analysis of event free survival were performed in IBM SPSS Statistics 20 using Cox regression. ResultsGene expression microarray data from 178 primary pediatric ALL patients showed a 3.6-fold increase in epithelial membrane protein 1 (EMP1) expression between in vitro prednisolone-resistant and -sensitive patients (p=0.002). This difference was confirmed with qRT-PCR (p<0.0001). EMP1 is a small hydrophobic four-transmembrane glycoprotein of which the biological function is largely unknown. We show that lentiviral silencing of EMP1 in six different BCP-ALL and T-ALL cell lines increased apoptosis and induced G0/G1 and G2/M arrest leading to 84.1 ± 4.5% reduction in leukemic survival compared to non-silencing control cells (shNSC , p=0.014). Moreover, EMP1 silencing sensitized three out of six cell lines to prednisolone up to 8.8-fold (p<0.001) compared to shNSC. Silencing of EMP1 also abrogated migration and adhesion to mesenchymal stromal cells (MSCs) by 78.3±9.0% and 29.3±4.1% respectively compared to shNSC (p<0.002). Notably, we also discovered that EMP1 contributes to MSC-mediated prednisolone resistance, as prednisolone resistance triggered during leukemic MSC coculture was partially reversed after EMP1 knockdown. Pathway analysis indicated that EMP1 signals through Src kinase family phosphorylation to activate JNK, STAT3, STAT5, CREB and NF-κB. Finally, BCP-ALL patients with high EMP1 mRNA expression showed a higher cumulative incidence of relapse and non-response (p=0.02) and a poorer 5-year event-free survival compared to EMP1-low patients (82%±2 vs. 91%±9 ; p<0.01). Most importantly, Cox regression multivariate analysis with white blood cell count, age, and EMP1 expression as co-variates identified EMP1 as an independent predictor for poor outcome in BCP-ALL (p<0.01, hazard ratio: 2.36 (1.31-4.25) ). ConclusionOur results indicate that EMP1 is a novel poor prognostic factor in pediatric leukemia that regulates prednisolone resistance, cell survival, migration and adhesion. With this study we provide preclinical evidence that EMP1 is an interesting new druggable target to treat pediatric leukemia and sensitize to prednisolone. Disclosures:No relevant conflicts of interest to declare.

Quality Controls Of Immune Regulatory Properties Of Ex-Vivo, GMP-Grade Expanded Mesenchymal Stromal Cells For Clinical Use

Aim of CASCADE is to standardize GMP-grade production and clinical use of Mesenchymal Stromal Cells (MSC). Immunological Unit is aimed at setting up and validating a standardized panel of functional assays to fully characterize the immunomodulatory properties of MSC obtained from bone marrow and adipose tissue through different GMP-grade expansion protocols (platelet lysate- vs. fetal calf serum).Immune cells were isolated using indirect immunomagnetic depletion (purity >96%). MSC were expanded in the same medium used for production and harvested at 70% confluence. Primed MSC (pMSC) were obtained after 48h-treatment with rh-IFNγ and rh-TNFα. MSC or pMSC were cocultured with T, B, NK cells for 4 or 6 days, and proliferation was evaluated by CFDA-SE dilution. T cells were stimulated with αCD3 + αCD28 antibodies; B cells were activated with CD40L, its enhancer, IL-2, CpG 2006, and anti-IgM/IgA/IgG; NK cells were activated with 100 U/ml rhIL-2. Cocultures were performed also with specific molecule inhibitors: L-1MT (IDO), snPP (HO-1), NS-398 (COX2), L-NMMA (iNOS) and anti-IFNγ antibody. For MSC immunogenicity assay, allogeneic T cell proliferation was evaluated at day 5 of culture; in addition, NK cells were activated for 2 days with rh-IL2, and MSC and pMSC were used as target cells.Inflammatory milieu significantly upregulated MHC class I and II, CD54, CD106, CD40, CD274, CD112, CD155 expression, and downregulated NKG2D ligands and mesenchymal markers (CD73, CD90, CD105). AT-derived MSC expressed less MHC class II, CD200 and CD106 molecules than BM-MSC. MSC coculture inhibited T and NK cell proliferation without inducing apoptosis, and this effect was greater in presence of primed MSC. Only primed MSC were capable of suppressing B cell proliferation. MSC inhibited apoptosis of resting T, B, and NK cells, while inflammatory priming increased their pro-survival activity. Activation of IDO and HO-1 was the main mechanism involved in MSC immune modulation. MSC never promoted allogeneic T cell proliferation; by contrast, IL-2-activated NK cells could efficiently recognize and kill allogenic unprimed MSC, while primed MSC became insensitive to NK cells. Some differences were observed depending on the origin and culture conditions of clinical-grade MSC.All the experimental protocols to assess MSC inhibitory effects on immune effector cells have been standardized and will be applied for the release of GMP-grade MSC produced inside the CASCADE Consortium. Disclosures:No relevant conflicts of interest to declare.

The influence of bone marrow- and synovium-derived mesenchymal stromal cells from osteoarthritis patients on regulatory T cells in co-culture

There is increasing evidence that inflammation in the synovium plays a major role in the progression of osteoarthritis (OA). However, the immunogenic properties of mesenchymal stromal cells (MSCs), which are considered to regulate immunity in various diseases, remain largely unknown in OA. The purpose of this study was to determine the influence of MSCs from OA patients on regulatory T cells (Tregs ) in an allogeneic co-culture model. Bone marrow (BM) and synovial membrane (SM) were harvested from hip joints of OA patients and co-cultured with lymphocytes enriched in CD4(+) CD25(+) CD127(-) regulatory T cells (Treg (+) LC) from healthy donors. Treg proportions and MSC markers were assessed by flow cytometry. Cytokine levels were assessed after 2 and 5 days of co-cultivation. Additionally, Treg (+) LC cultures were analysed in the presence of interleukin (IL)-6 and MSC-supernatant complemented medium. B-MSCs and S-MSCs were able to retain the Treg proportion compared to lymphocyte monocultures. T cell-MSC co-cultures showed a significant increase of IL-6 compared to MSC cultures. S-MSCs produced higher amounts of IL-6 compared to B-MSCs, both in single and T cell co-cultures. The effect of retaining the Treg percentage could be reproduced partially by IL-6 addition to the medium, but could only be observed fully when using MSC culture supernatants. Our data demonstrate that retaining the Treg phenotype in MSC-T cell co-cultures can be mediated by MSC derived from OA patients. IL-6 plays an important role in mediating these processes. To our knowledge, this study is the first describing the interaction of MSCs from OA patients and Tregs in an allogeneic co-culture model.

Mesenchymal stromal cells: filling the void of immunosuppressive therapy in aplastic anemia?

Mesenchymal stromal cells (MSCs) have been used in the context of hematopoietic stem cell transplantation (HSCT) to treat graft-versus-host disease and stimulate tissue repair. Their relative ease of manufacture, immunomodulatory, differentiation and regenerative properties, weak recognition by host immunity, and favorable risk profile make MSCs attractive in inducing tolerance in the setting of transplantation and autoimmunity (1). Recently, coculture of MSCs with umbilical cord blood cells have led to rapid engraftment comparable to that of an adult donor graft (2). MSC treatments have also been applied in autoimmune disease such as inflammatory bowel disease and multiple sclerosis and in tissue repair in degenerative conditions such as amyotrophic lateral sclerosis (3). In the next few years we can expect the role of MSC in HSCT and autoimmune diseases to be increasingly defined in this rapidly evolving field. Aplastic anemia (AA) is characterized by pancytopenia and a marked reduction in stem and progenitor marrow cells. Evidence from the laboratory, animal models and from the clinic strongly support and autoimmune pathogenesis where effector T cells recognize and destroy marrow elements, resulting in a hypocellular bone marrow (5). More recently, decreased regulatory T (Tregs) and increased TH17 cells have been described in AA patients at presentation, with improvement of this ratio after successful immunosuppressive therapy (4). Anti-thymocyte globulin (ATG) is the primary immunosuppressive agent used in severe AA inducing hematological responses in 60-75% of patients when combined with cyclosporine (5). The mechanism(s) by which ATG restores hematopoiesis in aplastic

Cartilage graft engineering by co-culturing primary human articular chondrocytes with human bone marrow stromal cells

Co-culture of mesenchymal stromal cells (MSCs) with articular chondrocytes (ACs) has been reported to improve the efficiency of utilization of a small number of ACs for the engineering of implantable cartilaginous tissues. However, the use of cells of animal origin and the generation of small-scale micromass tissues limit the clinical relevance of previous studies. Here we investigated the in vitro and in vivo chondrogenic capacities of scaffold-based constructs generated by combining primary human ACs with human bone marrow MSCs (BM-MSCs). The two cell types were cultured in collagen sponges (2 × 6 mm disks) at the BM-MSCs:ACs ratios: 100:0, 95:5, 75:25 and 0:100 for 3 weeks. Scaffolds freshly seeded or further precultured in vitro for 2 weeks were also implanted subcutaneously in nude mice and harvested after 8 or 6 weeks, respectively. Static co-culture of ACs (25%) with BM-MSCs (75%) in scaffolds resulted in up to 1.4-fold higher glycosaminoglycan (GAG) content than what would be expected based on the relative percentages of the different cell types. In vivo GAG induction was drastically enhanced by the in vitro preculture and maximal at the ratio 95:5 (3.8-fold higher). Immunostaining analyses revealed enhanced accumulation of type II collagen and reduced accumulation of type X collagen with increasing ACs percentage. Constructs generated in the perfusion bioreactor system were homogeneously cellularized. In summary, human cartilage grafts were successfully generated, culturing BM-MSCs with a relatively low fraction of non-expanded ACs in porous scaffolds. The proposed co-culture strategy is directly relevant towards a single-stage surgical procedure for cartilage repair.

Microenvironmental Protection of CML Stem and Progenitor Cells From Tyrosine Kinase Inhibitors Through N-Cadherin and Wnt Signaling

AbstractAbstract 912BCR-ABL tyrosine kinase inhibitors (TKI) do not eliminate leukemia stem cells (LSC) in chronic myeloid leukemia (CML), which remain a potential source of relapse. TKI treatment effectively inhibits BCR-ABL kinase activity in CML LSC, indicating that additional kinase-independent mechanisms contribute to LSC preservation. We investigated the role of signals from the bone marrow (BM) microenvironment in protecting chronic phase (CP) CML stem/progenitor cells from TKI treatment. Culture with human BM mesenchymal stromal cells (MSC), immortalized by ectopic expression of telomerase, significantly inhibited apoptosis in CP CML CD34+CD38- primitive and CD34+CD38+ commited progenitors treated with imatinib (5μM, p<0.01), nilotinib (5μM, p<0.01), or dasatinib(150nM, p<0.05). MSC coculture reduced TKI-mediated apoptosis in both dividing and non-dividing CD34+CD38- cells, defined using CFSE labeling, and increased the percentage of undivided CD34+CD38- cells (p<0.05). MSC coculture also enhanced colony forming ability of CML CD34+CD38- (p<0.05) and CD34+CD38+ (p<0.05) cells treated with TKI. Importantly MSC coculture, with or without imatinib treatment, significantly enhanced engraftment of CML CD34+ cells in immunodeficient NSG mice, both at 4 weeks (without IM: 27.2±5% human CD45+ cells without stroma, 52.8±8% with stroma, p<0.001; with IM: 4.9±2% without stroma, 10.1±2% with stroma, p<0.05) and at 10 weeks post-transplant (without IM : 1.48±0.2% human CD45+ cells without stroma, 2.37±0.4% with stroma, p=0.07; with IM : 0.74±0.2% without stroma, 1.2±0.3% with stroma, p<0.05). CML progenitors cultured in a transwell insert over MSC showed increased apoptosis following TKI exposure compared to cells cultured in direct contact with MSC (p<0.05). An increased proportion of CML progenitors adhered to MSC after 4 days of TKI treatment (22±4% without IM, 42±5% with IM, p<0.05). Antibody or peptide (NCDH) mediated blockade of the N-Cadherin receptor reduced adhesion of CML progenitors to MSC (p<0.05), and increased apoptosis of TKI-treated CML progenitors cocultured with MSC (p<0.05), indicating an important role for N-Cadherin in MSC-mediated protection of CML progenitors from TKI. Although N-Cadherin expression measured by flow cytometry, Western blot and Q-RT-PCR was lower in CML compared to normal CD34+CD38- and CD34+CD38+ cells, we observed significantly increased N-Cadherin expression in CML cells remaining after 4 days culture with IM and MSC (p<0.05). We observed enhanced β-catenin activity in CML progenitors cocultured with MSC, with and without TKI treatment, as manifested by increased β-catenin protein levels and nuclear localization, enhanced expression of β-catenin target genes, and increased TCF/LEF reporter activity. β-catenin levels and target gene expression were increased primarily in MSC-adherent CML progenitors compared to non-adherent cells, and were significantly reduced by antibody or peptide-mediated inhibition of N-Cadherin-mediated adhesion. Using the Duolink labeling technology, we demonstrated increased protein-protein interactions between N-cadherin and β-catenin in CML progenitors cocultured with MSC, with or without TKI treatment. Finally, we showed that addition of exogenous Wnt1 protected CML progenitors from TKI treatment, whereas inhibition of Wnt receptors by DKK1, or inhibition of β-catenin signaling by the small molecule inhibitor ICG001, enhanced apoptosis of CML progenitors cocultured with MSC, suggesting an important role for exogenous Wnt signaling in MSC-mediated protection of CML progenitors from TKI-induced apoptosis. Microarray analysis of gene expression confirmed enrichment of Wnt/β-catenin and Cadherin related gene sets in CML CD34+ cells cultured on MSC with or without IM, as well as enrichment of genes related to hematopoietic stem cell (HSC) self-renewal, HSC quiescence, and cytokine signaling. In conclusion, our studies reveal an important role for exogenous Wnt-β-catenin signaling, and a close interplay between N-Cadherin and β-catenin, in microenvonment-mediated resistance of CML stem and progenitor cells to TKI treatment. These findings support further development of novel treatment strategies to eradicate residual LSC in TKI-treated CML patients through inhibition of Wnt secretion or blockade of Wnt or N-Cadherin receptors. Disclosures:No relevant conflicts of interest to declare.

Leukemia Microenvironment and Pathologic Hypoxia: Sensitivity to Hypoxia-Activated Cytotoxin TH-302

Abstract 1523We have recently demonstrated that the leukemic bone marrow (BM) niche is highly hypoxic and that hypoxia promotes resistance of leukemic cells to chemotherapy (Benito et al., PLoS One 2011, e23108). Our preliminary data indicate that AML cells surviving chemotherapy in the human xenograft mouse models of leukemia reside within hypoxic areas of BM microenvironment as documented by staining with the hypoxia marker CAIX. These findings support utility of hypoxia-activated pro-drugs with the goal to eliminate leukemic blasts and leukemic stem cells residing in hypoxic BM microenvironment. TH-302 is a 2-nitroimidazole linked bromo-isophosphoramide mustard cytotoxin that upon hypoxia-dependent activation induces DNA cross-linking. TH-302 exhibited potent hypoxia-selective anti-leukemia activity in pre-B ALL (REH, NALM6), AML (OCI-AML3, MOLM-13, KG-1) and CML cell lines (KBM5), with IC50s at 1% O2 ranging from 0.04μM to 2.3μM and hypoxia cytotoxicity ratio (HCR) ranging from 116 for KBM5 to 11 for REH cells. TH-302 at 5–7.5μM also exhibited hypoxia-dependent anti-leukemia activity in primary ALL and AML samples (N=3; normoxia, 2–8%; hypoxia, 28–65% apoptotic cells).To better recapitulate the multidimensional BM niche we utilized co-cultures of GFP-labeled leukemic cells with bone marrow-derived RFP-labeled mesenchymal stromal cells (MSC) immobilized within Matrigel. MSC and leukemic cells generated three-dimensional (3D) structures- “spheroids”- and co-proliferated over time with colonies of leukemic cells firmly attached to MSC, as monitored by confocal microscopy (Fig. 1). Pimonidazole staining shows that vast hypoxia is present in the MSC/AML spheroids grown at normal oxygen tension, in contrast to what is observed in plastic-based (2-D) stromal co-cultures. Anti-leukemia activity of TH-302 was next determined in 2D vs 3D co-cultures of MSCs plus MOLM-13 or OCI-AML3 cells. In 2D co-cultures, MSC protected MOLM-13 and OCI-AML3 cells from TH-302-induced cytotoxicity, while extensive apoptosis was documented in hypoxic spheroid co-cultures (at 50nM TH-302, reduction in viability 10–15% vs >60%, Fig. 1). These findings suggest that culture conditions faithfully mimicking BM microenvironment promote pathologic hypoxia generated by rapidly proliferating AML cells, which in turn leads to their increased sensitivity to hypoxia-activated cytotoxins. To validate these findings in vivo, we next tested anti-leukemia efficacy of TH-302 in the in vivo model of primary AML established in NSG mice. TH-302 (50 mg/kg IP 3 times a week for three weeks) reduced the number of circulating AML cells (control, 13.2+/−5.7 ×106/ml; TH-302, 2.5+/−2.1 ×106/ml) and prolonged survival of NSG mice engrafted with primary AML cells compared to the vehicle treated mice (median survival time: TH-302=75 days; Control=56 days; P=0.003, n= 8 mice/group). To test the ability of hypoxia-activated prodrug to target leukemia-initiating cells, secondary transplant experiments were performed in which BM cells from control or TH-302 treated mice (collected after two weeks of therapy) were serially diluted and injected into secondary NSG recipient mice at 0.01, 0.005 or 0.0001×106′ cells/mouse (N=5 mice/dilution). Although all mice transplanted with higher cell doses died from leukemia, we observed significantly prolonged survival of animals injected with 0.01×106 cells from TH-302-treated primary recipients compared with vehicle-treated controls (median survival control=68 days; TH-302=79 days; P=0.0031) or with 0.005×106 cells (control=79 days; TH-302=83 days; P=0.0462). In summary, our findings suggest that pathologic hypoxia is a prevalent condition of leukemic BM microenvironment that promotes survival of leukemic blasts and leukemia-initiating cells. The results support targeting hypoxia with hypoxia-activated cytotoxins such as TH-302 to enhance the efficacy of therapeutic regimens in AML. A Phase 1 single agent clinical trial of TH-302 in patients with relapsed/refractory hematologic malignancies is ongoing. [Display omitted] Disclosures:Handisides:Threshold Pharmaceuticals: Employment. Hart:Threshold Pharmaceuticals: Employment. Konopleva:Threshold Pharmaceuticals: Research Funding.

Intercellular cytosolic transfer correlates with mesenchymal stromal cell rescue of umbilical cord blood cell viability during ex vivo expansion

Background aims. Mesenchymal stromal cells (MSC) have been observed to participate in tissue repair and to have growth-promoting effects on ex vivo co-culture with other stem cells. Methods. In order to evaluate the mechanism of MSC support on ex vivo cultures, we performed co-culture of MSC with umbilical cord blood (UCB) mononuclear cells (MNC) (UCB-MNC). Results. Significant enhancement in cell growth correlating with cell viability was noted with MSC co-culture (defined by double-negative staining for Annexin-V and 7-AAD; P<0.01). This was associated with significant enhancement of mitochondrial membrane potential (P<0.01). We postulated that intercellular transfer of cytosolic substances between MSC and UCB-MNC could be one mechanism mediating the support. Using MSC endogenously expressing green fluorescent protein (GFP) or labeled with quantum dots (QD), we performed co-culture of UCB-MNC with these MSC. Transfer of these GFP and QD was observed from MSC to UCB-MNC as early as 24 h post co-culture. Transwell experiments revealed that direct contact between MSC and UCB-MNC was necessary for both transfer and viability support. UCB-MNC tightly adherent to the MSC layer exhibited the most optimal transfer and rescue of cell viability. DNA analysis of the viable, GFP transfer-positive UCB-MNC ruled out MSC transdifferentiation or MSC-UCB fusion. In addition, there was statistical correlation between higher levels of cytosolic transfer and enhanced UCB-MNC viability (P< 0.0001). Conclusions. Collectively, the data suggest that intercellular transfer of cytosolic materials could be one novel mechanism for preventing UCB cell death in MSC co-culture.

Mesenchymal stromal cell neuroprotection of hydrogen peroxide -challenged pheochromocytoma cells through reducing apoptosis and releasing cytokines

Immunoregulation of mesenchymal stromal cells (MSC) is more efficient at restoring biologic function of injured tissue than transdifferentiation during transplantation. However, the exact mechanisms and characteristics of MSC regarding immunomodulation are still unknown, especially in the damaged niche after hypoxic-ischemic insult. We investigated the anti-apoptotic actions of MSC against the neurotoxicity of hydrogen peroxide (H(2)O(2)) on pheochromocytoma (PC12) cells. To mimic hypoxic-ischemic brain injury in vivo, a relatively high H(2)O(2) concentration and short period were used to treat PC12 cells. MSC were co-cultured directly with the injured PC12 cells, and 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazoly)-3-(4-sulfophenyl)tetrazolium, inner salt (MTS), lactose dehydrogenase (LDH) and nitric oxide (NO) assays, intracellular Ca(2+) and resting membrane potential (RMP) were analyzed. Apoptotic-associated genes and cytokine releases were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). After exposure to H(2)O(2), the viability of PC12 cells was significantly decreased, while the levels of LDH and NO increased, resulting in intracellular Ca(2+) accumulation and cell apoptosis. Co-culture of MSC with the H(2)O(2)-treated PC12 cells sustained viability of the PC12 cells, reduced LDH levels and NO release, and improved Ca(2+) influx and cell apoptosis. The injured PC12 cells exhibited lower RMP following co-culture with MSC compared with the injured PC12 cells alone. The mRNA expression levels of Bcl-2 were up-regulated and caspase-3 levels down-regulated in the MSC co-culture groups. The release of interleukin (IL)-10 was gradually reduced by co-cultured MSC, while the release of IL-6 was sharply increased following MSC co-culture. These findings indicate that MSC have neuroprotective effects on suppressing cell apoptosis via regulation of the H(2)O(2)-impaired microenvironment, partly through IL-6 and IL-10 cytokine production.

Systemic Delivery of Human Mesenchymal Stromal Cells Combined with IGF-1 Enhances Muscle Functional Recovery in LAMA2dy/2j Dystrophic Mice

The combination of cell therapy with growth factors could be a useful approach to treat progressive muscular dystrophies. Here, we demonstrate, for the first time, that IGF-1 considerably enhances the myogenesis of human umbilical cord (UC) mesenchymal stromal cells (MSCs) in vitro and that IGF-1 enhances interaction and restoration of dystrophin expression in co-cultures of MSCs and muscle cells from Duchenne patients. In vivo studies showed that human MSCs were able to reach the skeletal muscle of LAMA2(dy/2j) dystrophic mice, through systemic delivery, without immunosuppression. Moreover, we showed, for the first time, that IGF-1 injected systemically together with MSCs markedly reduced muscle inflammation and fibrosis, and significantly improved muscle strength in dystrophic mice. Our results suggest that a combined treatment with IGF-1 and MSCs enhances efficiency of muscle repair and, therefore, should be further considered as a potential therapeutic approach in muscular dystrophies.

Macrophages and mesenchymal stromal cells support survival and proliferation of multiple myeloma cells

Multiple myeloma (MM) is characterized by almost exclusive tropism of malignant cells for the bone marrow (BM) milieu. The survival and proliferation of malignant plasma cells have been shown to rely on interactions with nonmalignant stromal cells, in particular mesenchymal stromal cells (MSCs), in the BM microenvironment. However, the BM microenvironment is composed of a diverse array of cell types. This study examined the role of macrophages, an abundant component of BM stroma, as a potential niche component that supports malignant plasma cells. We investigated the proliferation of MM tumour cell lines when cultured alone or together with MSCs, macrophages, or a combination of MSCs and macrophages, using the carboxyfluorescein succinimidyl ester assay. Consistently, we observed increased proliferation of MM cell lines in the presence of either MSCs or macrophages compared to cell line-only control. Furthermore, the combined co-culture of MSCs plus macrophages induced the greatest degree of proliferation of myeloma cells. In addition to increased proliferation, MSCs and macrophages decreased the rate of apoptosis of myeloma cells. Our in vitro studies provide evidence that highlights the role of macrophages as a key component of the BM microenvironment facilitating the growth of malignant plasma cells in MM.

Cord Blood Hematopoietic Progenitor Cell in Co-Culture with Bone Marrow Mesenchymal Stromal Cells: A Synergic Effect

Abstract 4811The number of hematopoietic stem and progenitor cells (HPCs) in cord blood units are limited and this can result in delayed engraftment. In vitro expansion of HPCs provides a perspective to overcome these limitations. Different combinations of cytokines as well as mesenchymal stromal cells (MSC) have been shown to separately support HPCs ex vivo expansion, but the combining effects are under evaluation. Data derived from ex vivo co-culture systems using MSC as a feeder layer suggest that cellular contacts could have a significant impact on expansion. We have evaluated the expansion rate of thawed cord blood samples (n=6) in a medium containing SCF (100 ng/ml) and G-CSF (100 ng/ml) plated over a pre-established bone marrow derived MSC layer in comparison to the absence of either MSC layer or cytokines. After 7 days cultures were demi-depopulated. At 14 days of culture adherent and non-adherent cells were harvested, counted and evaluated for antigens expression and clonogenic capacity. Immunophenotypic analysis was performed using CD34-PE, CD38-FITC, CD45-PE-Cy7, CD133-APC. Clonogenic assay was performed in semisolid methylcellulose culture medium (MethoCult, Stem Cell Technologies), CFU frequencies and total CFU numbers per cord blood sample were determined. After 14 days of culture, in the presence of MSC layer, an 11.2-fold increase (range 4.4–18.4) in total number of cells was observed, in comparison to a 4.8-fold increase (range1.1-10.35) in the absence of MSC layer. The presence of MSC layer generated a 4.3-fold increase (range 1.5–7.2) in the number of CD34 positive cells, compared to a 3.3-fold increase (range 0.9–5.7) in the absence of MSC; when considering the more immature CD34+/CD38− subpopulation the corresponding increase were 26.9-fold vs 2.85-fold, respectively. Moreover, the percentage of the CD34+/CD38− subpopulation was higher in the adherent compared to the non-adherent fraction (76% vs 15%). The selection effect given by the MSC layer was confirmed by the presence of hematopoiesis foci growing onto the MSC layer. Our data show that cord blood HPCs can be expanded in vitro, moreover the co-culture on a MSC layer shows a synergistic effect on TCN, CD34+ cells and on more primitive CD34+/CD38− cells. Therefore, a clinical protocol of cord blood HPCs and MSC co-culture could represent a promising approach for improving engraftment kinetics in cord blood transplant recipients. Disclosures:No relevant conflicts of interest to declare.

In Vitro Propagation of Mesenchymal Stromal Cells From Marrow Aspirates of Patients with Chronic Lymphocytic Leukemia Is Dependent Upon Physiologic Oxygen Tension

Abstract 2839The tumor microenvironment may play an important role in the growth and/or survival of leukemia cells of patients (pts) with chronic lymphocytic leukemia (CLL). Studies on the interaction of CLL cells with the microenvironment have been facilitated by our capacity to culture accessory cells in vitro. However, the conditions for culturing such cells in ambient oxygen(O2) at 21% are different than those present in lymphoid tissues which have O2 concentrations ranging from 1–7%. The difference between in vivo and in vitro O2 tensions might influence the biology of leukemia accessory cells.To examine this, we studied the effect(s) of O2 tension on our ability to propagate mesenchymal stromal cells (MSCs) from marrow aspirates of pts with CLL. Equal numbers of fresh or viably frozen marrow mononuclear cells were seeded in DMEM with 10% FBS into separate flasks for culture at 37° C in incubators at atmospheric O2 (Atmos-O2) or at 5% O2 (physiologic; Phys-O2), both with 5% CO2. The cells were monitored for viability and growth over time. We found that only Phys-O2 tension allowed for the generation and long-term expansion of MSCs. Out of the 6 pts tested, 3 developed virtually no MSCs (<10 cells), and 3 generated less than 6×104 MSCs (ranging from 2±1×104 to 6±1×104) in Atmos-O2 after 47±7 days in vitro. In contrast, high numbers of MSCs developed in Phys-O2 for all 6 pts, ranging from 45±8×104 to 80×104 cells, resulting in highly significant differences in yields between the 2 culture conditions (p<0.01). Moreover, the MSCs generated in Phys-O2 continued to proliferate over time, whereas MSCs in Atmos-O2 did not. Under Phys-O2, MSCs were successfully expanded from marrow aspirates of 16 out of 18 CLL pts. The morphology and phenotype of the MSCs generated were similar to that of healthy MSCs, expressing CD29, CD44, CD105 and D7-FIB, and lacking expression of CD14, CD31, CD34, or CD45.We next examined whether the differences in cell yields between the two culture conditions could be the result of compromised MSC proliferation in Atmos-O2. To address this, MSCs generated in Phys-O2 were seeded into separate flasks and exposed to Atmos-O2 or Phys-O2 and proliferation was monitored by BrdU incorporation and viable cell counts. We found that MSCs seeded in Atmos-O2 proliferated significantly less well than MSCs in Phys-O2 (n=3). However, MSC viability was not significantly affected by the change in O2 tension, suggesting that replicative senescence could be induced in MSCs exposed to Atmos-O2. To test this hypothesis, MSCs generated in Phys-O2 were seeded separately under Atmos-O2 or Phys-O2 and stained for the senescence-associated beta-galactosidase (SA-B-Gal) marker. We found a significant increase in the fraction SA-B-gal+ MSCs exposed to Atmos-O2 compared to Phys-O2 (70±18% vs. 13±5%; p<0.0001). MSC morphology in Atmos-O2 was also consistent with senescence, marked by a wide-spread cytoplasm and enlarged nucleus. The cell cycle regulator p16INK4 also was distinctively induced in MSCs exposed to Atmos-O2 compared to Phys-O2 (n=2), consistent with its role in inhibiting cell cycle progression and mediating senescence.We next ask if a disruption of the redox balance plays a role in MSC biology modulated by O2, using the free radical scavenger beta-mercaptoethanol (BME). MSCs generated in Phys-O2 were seeded separately under Phys-O2 or Atmos-O2 +/− BME. We observed that MSC proliferation in Atmos-O2 was restored by the addition of BME to the levels observed in Phys- O2 using BrdU incorporation (n=3). However, in a similar context, BME did not restore MSC expansion in Atmos-O2 measured by viable cell counts, or in the generation of MSCs in Atmos-O2 when added at culture initiation (n=2), suggesting that redox balance disruption is not the main mechanism by which high O2 tension affects MSC biology.We finally evaluated whether co-cultures of CLL cells and MSCs in Phys-O2 and Atmos-O2 tensions equally support CLL cell survival. We found that CLL cell survival was significantly enhanced when co-cultured in Phys-O2 compared to Atmos-O2 after 17 days (87±15% vs 44±17% viable cells; p<0.0001). These results suggest that Phys-O2 tension is not only critical to generate MSCs in vitro, but it also has a profound impact on the biology of these accessory cells, which in turn affects the survival of the leukemic cells. Studies conducted under Phys-O2 tension might further our understanding of the mechanisms governing CLL cell survival in vivo. Disclosures:No relevant conflicts of interest to declare.

Microporous Biphasic Calcium Phosphate Granules (MBCP®) Retain Immunological Properties of Bone Marrow-Derived Mesenchymal Stromal Cells and Promote Osteoblastic Differentiation

Abstract 1924Bone is among the most frequently transplanted tissue with about 1 million procedures annually in Europe. Allografts and autografts account for more than 80% of total graft volume, despite their considerable disadvantages, including the risk of disease transfer and immunologic rejection, limited supply of bone, costs and complications. Significant growth opportunities exist for synthetic bone grafts in association with mesenchymal stromal cells (MSC) from autologous or allogeneic sources as alternatives to biological bone grafts in orthopaedic and maxillofacial surgery. The objective of REBORNE is to perform clinical trials using advanced biomaterials and cells triggering bone healing in patients. To reach this goal, five phase I clinical studies with 20 patients have been planned in 12 clinical Centers spread in 8 European countries.Aim of the Immunological Unit of Reborne is to assess the MSC immunomodulatory properties in presence of the biomaterial used as scaffold for MSC delivery. All the functional experiments were performed in parallel, by comparing the effects of standard culture conditions and three-dimensional culture setting using MBCP (Biomatlante).Material and methods: Bone marrow MSC were provided from REBORNE Consortium Centers. To perform proliferation assays, different immune effector cells (T, B and NK cells) were stained with CFSE according to manufacturer's protocol. Active caspase-3 cell staining was used for survival quantification of immune effector cells after co-culture experiments. Differentiation potential was evaluated by culturing MSC with two different media containing either bone morphogenetic protein 4 (BMP4) or dexamethasone. After three weeks, osteogenic differentiation was quantified by qRT-PCR, alkaline phosphatase activity and alizarin red staining. Results:We found that primed MSC, pre-treated with the inflammatory cytokines IFNg and TNFa, displayed upregulation of HLA-ABC, CD54, CD106 and de novo expression of HLA-DR, both in standard culture conditions and in association with MBCP. Immune effector cells could be cultured and collected even in presence of MBCP and no significant differences were found between standard- (MSC + effector cells) and 3D-coculture conditions (MSC + MBCP + effector cells), in terms of immune effector cell proliferation. In both experimental conditions MSC suppressed T and NK cell proliferation (% suppression: MSC + T = 68.4; MSC + MBCP + T = 62.4; MSC + NK = 17.5; MSC + MBCP + NK = 20.2) and increased B cell proliferation (MSC + B = +13%; MSC + MBCP + B = +12.3%). In addition, immune effector cells viability was not affected by MBCP and MSC co-culture increased their survival even in presence of MBCP; in fact, in each culture condition the percentage of inhibition of T, B and NK cell apoptosis was higher than 20% in comparison to immune effector cells cultured without MSC.Dexamethasone and BMP4 were capable of inducing MSC differentiation into osteoblast-like cells, as confirmed by qRT-PCR analysis. We demonstrated that BMP4-based medium led to fully differentiated osteoblasts (Osterix+, RUNX2+, DLX5+ and alkaline-phosphatase+). Moreover, MBCP was more efficient in increasing osteoblastic differention as compared to standard culture conditions, as shown by the higher expression of Osteocalcin and Osterix.These data show that the association of MBCP and MSC does not affect MSC properties and suggest that it could be a treatment of choice of bone defects instead of allograft and autograft transplantation. Disclosures:No relevant conflicts of interest to declare.

Quality Controls of Immune Regulatory Properties of Ex-Vivo, GMP-Grade Expanded Mesenchymal Stromal Cells for Clinical Use (European multicenter study CASCADE),

Abstract 4049Aim of the European Consortium CASCADE is to standardize GMP-grade production and clinical use of Mesenchymal Stromal Cells (MSC) to treat skin and corneal wounds. MSC possess immunogenicity and immunomodulatory properties that must be carefully addressed before clinical use. CASCADE Immunological Unit is aimed to set up and validate a wide panel of functional assays to fully characterize in a standardized and reproducible manner the immunomodulatory properties of MSC obtained inside CASCADE Units from bone marrow, adipose tissue, cord blood, and amniotic membrane (BM, AT, CB, AM) through different GMP-grade expansion protocols including platelet lysate- and fetal calf serum-based culture conditions.Immune cells were isolated using indirect immunomagnetic depletion; samples with less than 96% of purity were discarded. For the experiments, MSC were expanded in the same medium used for production and harvested at 70% confluence. Primed MSC were obtained by 48h-treatment with 10 ng/ml of rh-INFγ and 15 ng/ml of rh-TNFα.Cocultures were set up by plating primed or unprimed MSC in 96 or 48 flat bottomed – well plates; CFSE-stained T, B, NK cells were seeded at different effector cell:MSC ratios. Cells were harvested after 4 or 6 days of coculture for proliferation evaluation by FACS analysis. T cells were stimulated with mitogenic αCD3 plus αCD28 antibodies at 0.5 μg/ml each; B cells were activated with CD40L at 50 ng/ml, its enhancer at 5 g/ml, IL-2 20 UI/ml, CpG 2006 2.5 μg/mL, and F(ab’)2 anti-IgM/IgA/IgG 2 μg/mL; NK cells were activated with 100 U/ml rh-IL2. To identify the molecular mechanisms involved in immunomodulatory properties of MSC, coculture of immune effector cells and MSC were performed in the presence of specific inhibitors, after identifying their non-toxic and effective concentrations: 1 mM for L-1MT (IDO inhibitor), 2 μM for snPP (HO-1 inhibitor), 5 μM for NS-398 (COX2 inhibitor), 1 mM for L-NMMA (iNOS inhibitor) and 10 μg/ml for anti-IFNγ neutralizing antibody.We also studied the capacity of resting and primed MSC to sustain the survival of unstimulated T, B, and NK cells through the evaluation of the percentage of caspase-3negCD45pos viable immune cells after 4 to 6 days in culture with or without MSC.For MSC immunogenicity assay, the proliferation of allogeneic T was evaluated at day 5 of culture by incorporation of 3H-Thymidine; in addition, NK cells were activated for 2 days with 100 U/ml of rh-IL2 whereas resting or primed MSC were loaded with non radioactive fluorophore (BaTDA) or with Cr51 and used as target cells.Inflammatory milieu significantly upregulated MHC class I and II, CD54, CD106, CD40, CD274, CD112, CD155 expression, and downregulated NKG2D ligands (ULBP 1–3, MICA/B) and mesenchymal markers (CD73, CD90, CD105). AT-derived MSC expressed less MHC class II, CD200 and CD106 molecules than BM-MSC. MSC coculture inhibited T and NK cell proliferation without inducing apoptosis, and this effect was greater in presence of primed MSC. On the contrary, only primed MSC were capable of suppressing B cell proliferation. In addition, MSC inhibited apoptosis of resting T, B, and NK cells, while inflammatory priming increased their pro-survival activity. T cell/MSC coculture showed that activation of IDO and HO-1 was the main mechanism involved in MSC immune modulation, as the addition of specific inhibitors (L-1-MT and snPP) significantly reverted the phenomenon. MSC never promoted allogeneic T cell proliferation; by contrast, IL-2-activated NK cells could efficiently recognize and kill allogenic unprimed MSC. However, MSC became insensitive to NK cells once primed with inflammatory cytokines. Some differences were observed depending on the origin and culture conditions of clinical-grade MSC.All the experimental protocols to assess MSC inhibitory effects on immune effector cells have been standardized and will be applied for the release of GMP-grade MSC produced inside the CASCADE Consortium. Disclosures:No relevant conflicts of interest to declare.