Presence Of MSCs Research Articles

The role of mesenchymal cells in cholangiocarcinoma.

The tumour microenvironment (TME) significantly influences tumour formation and progression through dynamic interactions. Cholangiocarcinoma (CCA), a highly desmoplastic tumour, lacks early diagnostic biomarkers and has limited effective treatments due to an incomplete understanding of its molecular pathogenesis. Investigating the TME's role in CCA progression could lead to better therapies. RNA sequencing was performed on seven CCA PDXs and their corresponding patient samples. Differential expression analysis was conducted, and Qiagen Ingenuity Pathway Analysis (IPA) was used to predict dysregulated pathways and upstream regulators. PDX and cell line-derived spheroids, with and without immortalised mesenchymal stem cells, were grown and analysed for morphology, growth, and viability. Histological analysis confirmed biliary phenotypes. RNA sequencing indicated upregulation of ECM-receptor interaction and PI3K-Akt pathways in the presence of MSCs, with several genes linked to poor survival. MSCs restored the activity of inhibited cancer-associated kinases (ICAKs). This study shows that adding MSCs to CCA spheroid models restores key paracrine signalling pathways lost in PDXs, enhancing tumour growth and viability. These findings highlight the importance of including stromal components in cancer models to improve pre-clinical studies.

The Role of Mesenchymal Stem Cells in the Treatment of a Chronic Rhinosinusitis-An In Vivo Mouse Model.

Objectives/Hypothesis: It is acknowledged that the treatment of chronic rhinosinusitis (CRS) represents an important challenge for rhinology and for social and economic life. At present, one of the most common treatments for CRS is represented by local corticosteroids followed by endoscopic sinus surgery (ESS). Starting from the example of the mesenchymal stem cell’s (MSC) capacity to migrate and to modulate a real response in the nasal mucosa of an allergic rhinitis mouse model, we try to obtain a response in a CRS mouse model, using MSC derived by adipose tissue. The aim of this study is to demonstrate that the MSC can be used in CRS treatment and could change its priorities. Methods: Seventy female mice (6 MSC donor mice) were randomized in two stages of study, 32 Aspergillus fumigatus (Af) exposure mice (20 for histological comparison to 1st control mice and 12 for MSC administration, to CRS/MCS model) and 32 control mice (20 for histological comparison to CRS model and 12 for MSC administration and histological control to MSC model); in the first stage, the Aspergillus fumigatus (Af) CRS mouse model was targeted, in this section were included 64 (n = 32) mice (treated and control group). In order to assess the inflammation level (histological analysis), the animals were euthanized; in the second stage MSCs (1 × 106/animal) were administered intravenously to a total of 24 (n = 24) mice (12 mice from the exposed group and 12 mice from the second control group). Results: After 12 weeks of Af intranasal instillation, the inflammation parameters evaluated indicated a severe diffuse chronic inflammation, associated with diffuse severe hyperplasia and mature diffuse squamous metaplasia. The MSCs’ injection via the ophthalmic vein induced important histopathological changes in the CRS experimental group, starting with the presence of MSCs in all samples and continuing with the important degenerative character of inflammation. Conclusions: MSC administration demonstrated a real improvement of CRS evolution on the CRS mouse model.

Umbilical cord mesenchymal stem cells as well as their released exosomes suppress proliferation of activated PBMCs in multiple sclerosis.

Multiple sclerosis (MS) is a central nervous system (CNS) degenerative disorder which is caused by a targeted autoimmune-mediated attack on myelin proteins. Previously, mesenchymal stem cells were considered as a novel and successful treatment of MS. One of the underlying mechanisms behind their immunomodulatory function is the release of extracellular vesicles, particularly exosomes. In this study, we aimed to evaluate the suppressive efficacy of MSCs and their exosomes on the proliferation of peripheral mononuclear blood cells (PBMC) in relapsing-remitting MS (RRMS) patients and healthy subjects. To do, mesenchymal stem cells were derived from human umbilical cord tissues and used for exosome isolation through ultracentrifugation. Suppressive function of MSCs and MSC-derived exosomes was examined in a coculture with CFSE-labelled PBMCs in vitro. PBMC proliferation of the patients and healthy individuals was measured using flow cytometry. We first demonstrated that proliferation of PBMCs decreased in the presence of MSCs and suppression was more efficient by MSC-derived exosomes, with a minimum alloreaction rate. However, suppression capacity of MSCs and their exosomes significantly decreased during extensive sub-culturing. The present study showed that MSC-derived exosomes as an effective cell-free therapy could prevent proliferation of PBMCs. However, further evaluations are need to move towards a functional approach that can be translated to the clinic.

Epidural fat mesenchymal stem cells: Important microenvironmental regulators in health, disease, and regeneration: Do EF-MSCs play a role in dural homeostasis/maintenance?

Mesenchymal stem cells (MSCs) are present in fat tissues throughout the body, yet little is known regarding their biological role within epidural fat. We hypothesize that debridement of epidural fat and/or subsequent loss of MSCs within this tissue, disrupts homeostasis in the vertebral environment resulting in increased inflammation, fibrosis, and decreased neovascularization leading to poorer functional outcomes post-injury/operatively. Clinically, epidural fat is commonly considered a space-filling tissue with limited functionality and therefore typically discarded during surgery. However, the presence of MSCs within epidural fat suggests that itis more biologically active than historically believed and may contribute to the regulation of homeostasis and regeneration in the dural environment. While the current literature supports our hypothesis, it will require additional experimentation to determine if epidural fat is an endogenous driver of repair and regeneration and if so, this tissue should be minimally perturbed from its original location in the spinal canal. Also see the video abstract here https://youtu.be/MIol_IWK1os.

7-T MRI tracking of mesenchymal stromal cells after lung injection in a rat model

BackgroundMesenchymal stromal cells (MSCs) are able to migrate and engraft at sites of inflammation, injuries, and tumours, but little is known about their fate after local injection. The purpose of this study is to perform MSC tracking, combining in vivo 7-T magnetic resonance imaging (MRI) and histological assessment, following lung injection in a rat model.MethodsFive lungs were injected with ferumoxide-labelled MSCs and five with perfluorocarbon-labelled MSCs and underwent 7-T MRI. MRI acquisitions were recorded immediately (T0), at 24 h (T24) and/or 48 h (T48) after injection. For each rat, labelled cells were assessed in the main organs by MRI. Target organs were harvested under sterile conditions from rats sacrificed 0, 24, or 48 h after injection and fixed for histological analysis via confocal and structured illumination microscopy.ResultsFerumoxide-labelled MSCs were not detectable in the lungs, whereas they were not visible in the distant sites. Perfluorocarbon-labelled MSCs were seen in 5/5 injected lungs at T0, in 1/2 at T24, and in 1/3 at T48. The fluorine signal in the liver was seen in 3/5 at T0, in 1/2 at T24, and in 2/3 at T48. Post-mortem histology confirmed the presence of MSCs in the injected lung.ConclusionsFerumoxide-labelled cells were not seen at distant sites; a linear decay of injected perfluorocarbon-labelled MSCs was observed at T0, T24, and T48 in the lung. In more than half of the experiments, perfluorocarbon-labelled MSCs scattering to the liver was observed, with a similar decay over time as observed in the lung.

Systemic oncolytic adenovirus delivered in mesenchymal carrier cells modulate tumor infiltrating immune cells and tumor microenvironment in mice with neuroblastoma.

Celyvir (autologous mesenchymal cells -MSCs- that carry an oncolytic adenovirus) is a new therapeutic strategy for metastatic tumors developed by our research group over the last decade. There are limitations for studying the immune effects of human oncolytic adenoviruses in murine models since these viruses do not replicate naturally in these animals. The use of xenografts in immunodeficient mice prevent assessing important clinical aspects of this therapy such as the antiadenoviral immune response or the possible intratumoral immune changes, both of tumor infiltrating leukocytes and of the microenvironment. In our strategy, the presence of MSCs in the medicinal product adds an extra level of complexity. We present here a murine model that overcomes many of these limitations. We found that carrier cells outcompeted intravenous administration of naked particles in delivering the oncolytic virus into the tumor masses. The protection that MSCs could provide to the oncolytic adenovirus did not preclude the development of an antiadenoviral immune response. However, the presence of circulating antiadenoviral antibodies did not prevent changes detected at the tumor masses: increased infiltration and changes in the quality of immune cells per unit of tumor volume, and a less protumoral and more inflammatory profile of the tumor microenvironment. We believe that the model described here will enable the study of crucial events related to the immune responses affecting both the medicinal product and the tumor.

Mesenchymal Stem Cell Therapy in Osteoarthritis and Regenerative Medicine.

Mesenchymal Stem Cell Therapy in Osteoarthritis and Regenerative Medicine.

Xenogenic Implantation of Equine Synovial Fluid-Derived Mesenchymal Stem Cells Leads to Articular Cartilage Regeneration.

Horses are widely used as large animal preclinical models for cartilage repair studies, and hence, there is an interest in using equine synovial fluid-derived mesenchymal stem cells (SFMSCs) in research and clinical applications. Since, we have previously reported that similar to bone marrow-derived MSCs (BMMSCs), SFMSCs may also exhibit donor-to-donor variations in their stem cell properties; the current study was carried out as a proof-of-concept study, to compare the in vivo potential of equine BMMSCs and SFMSCs in articular cartilage repair. MSCs from these two sources were isolated from the same equine donor. In vitro analyses confirmed a significant increase in COMP expression in SFMSCs at day 14. The cells were then encapsulated in neutral agarose scaffold constructs and were implanted into two mm diameter full-thickness articular cartilage defect in trochlear grooves of the rat femur. MSCs were fluorescently labeled, and one week after treatment, the knee joints were evaluated for the presence of MSCs to the injured site and at 12 weeks were evaluated macroscopically, histologically, and then by immunofluorescence for healing of the defect. The macroscopic and histological evaluations showed better healing of the articular cartilage in the MSCs' treated knee than in the control. Interestingly, SFMSC-treated knees showed a significantly higher Col II expression, suggesting the presence of hyaline cartilage in the healed defect. Data suggests that equine SFMSCs may be a viable option for treating osteochondral defects; however, their stem cell properties require prior testing before application.

Isolation, culture, characterization, and osteogenic differentiation of canine endometrial mesenchymal stem cell.

Aim:In this study, the canine endometrium tissue is characterized for its stem cell properties such as adherence to tissue culture plate (plasticity), short population doubling time, serial clonal passaging, long-term culturing properties, stem cell marker expression, and multilineage differentiation potential.Materials and Methods:The present work describes a novel isolation protocol for obtaining mesenchymal stem cells from the uterine endometrium and is compared with cells derived from umbilical cord matrix as a positive control. These cells are clonogenic, can undergo several population doublings in vitro, and can be differentiated to the osteocytes in mature mesenchymal tissues when grown in osteogenic differentiation media as detected by Alizarin Red-S staining.Results:It is reported for the first time that the cells derived from the canine endometrium (e-multipotent stem cells [MSCs]) were able to differentiate into a heterologous cell type: Osteocytes, thus demonstrating the presence of MSCs. Thus, the endometrium may be told as a potential source of MSCs which can be used for various therapeutic purposes.Conclusion:The endometrium can be used as a potential source of MSCs, which can be used for various therapeutic purposes.

Interferon-γ mediates the immunosuppression of bone marrow mesenchymal stem cells on T-lymphocytes in vitro

ABSTRACTObjectives: In the present study, we first confirmed the suppressive function of MSCs in allogeneic T cell proliferation and then examined the underlying mechanisms for MSCs’ immunomodulation and the role of the pro-inflammatory cytokine interferon (IFN)-γ.Methods: Human MSCs were cultured in the presence or absence of IFN-γ. The expression level of prostaglandin E2 (PGE2), hepatocyte growth factor (HGF), transforming growth factor (TGF)-β1 and indoleamine 2,3-dioxygenase (IDO) by MSCs were measured. T lymphocytes were isolated from peripheral blood of healthy donors and then induced to proliferate under the stimulation of anti-human CD3 mAb and anti-human CD28 mAb. In the presence of MSCs, T cell proliferation was examined by BrdU incorporation. In addition, PGE2, HGF, TGF-β1, Kynurenine, recombinant human IFN-γ and anti-IFN-γ mAb were added and cell proliferation was examined.Results: Compared to the controls (MSCs alone), MSCs cocultured with IFN-γ expressed significantly higher concentrations of PGE2, HGF and TGF-β1. The mRNA level of IDO was remarkably increased. Human bone marrow-derived MSCs alone notably suppressed T lymphocytes proliferation in vitro. Addition of exogenous IFN-γ did not ablate the immunosuppressive effects of MSCs. Addition of anti-IFN-γ mAb partially restored suppression of T cell proliferation by MSCs.Conclusions: Human MSCs constitutively expressed immunosuppressive levels of PGE2, HGF and TGF-β1. The proinflammatory cytokine IFN-γ exhibited synergistic effects with MSCs on immunosuppression, possibly by up-regulating PGE2, HGF and TGF-β1 in MSCs and inducting MSCs expression of IDO, involved in tryptophan catabolism.

Shielding Engineered Islets With Mesenchymal Stem Cells Enhance Survival Under Hypoxia.

In the present study we focused on the improvisation of islet survival in hypoxia.The Islet like cell aggregates (ICAs) derived from wharton's jelly mesenchymal stem cells (WJ MSC) were cultured with and without WJ MSC for 48 h in hypoxia and normoxia and tested for their direct trophic effect on β cell survival. The WJ MSCs themselves secreted insulin upon glucose challenge and expressed the pancreatic markers at both transcription and translational level (C-peptide, Insulin, Glucagon, and Glut 2). Direct contact of MSCs with ICAs facilitated highest viability under hypoxia as evidenced by fluorescein diacetate/propidium iodide and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cytokine analysis of the co-cultured ICAs revealed amplification of anti-inflammatory cytokine like TGFβ and TNFα accompanied by depletion of pro-inflammatory cytokines. The increment in VEGF and PDGFa was also seen showing their ability to vascularize upon transplantation. This was further accompanied by reduction in total reactive oxygen species, nitric oxide, and super oxide ions and down regulation of Caspase3, Caspase8, p53, and up regulation of Bcl2 confirming prevention of apoptosis in ICAs. The western blot analysis confirmed the cytoprotective effect of WJ MSC on ICAs as they enhanced the anti-apoptotic marker BCL2 and reduced the expression of apoptotic markers, Annexin 5 and Caspase 3. There was a significant reduction in the expression of p38 protein in the presence of MSCs making the ICAs responsive to glucose. Taken together our data demonstrate for the first time that the WJ MSC expressed pancreatic markers and their supplementation protected engineered islets against hypoxia and oxidative stress. J. Cell. Biochem. 118: 2672-2683, 2017. © 2017 Wiley Periodicals, Inc.

TNF-α modulates the immunosuppressive effects of MSCs on dendritic cells and T cells

Mesenchymal stem cells are progenitor cells that have capabilities to differentiate different cell types. Also, MSCs possess immune suppressive effects on DC differentiation and T cell activation through a wide range of soluble factors and receptors. The properties of MSCs change through activation of cytokines particularly IFN-γ and TNF-α. The DC phenotypes and functions including the expression of co-stimulatory and co-inhibitory molecules and capabilities of DCs to induce allogeneic activation of CFSE-labeled splenocytes as well as cytokine production when they were differentiated in the presence of MSCs, TNF-α activated MSCs, IFN-γ activated MSCs and IFN-γ & TNF-α activated MSCs were analyzed. Treg population and T cell polarization were investigated using flowcytometry and real-time PCR respectively. Here, we showed that IFN-γ slightly enhances immunosuppressive effects of MSCs on immune system through induction tolerogenic DCs with elevated expression of IDO and increasing Treg population. Conversely, TNF-α decreases immunomodulation properties of MSCs on immune cells through the enhancement of co-stimulatory molecules such as ICOSL and HLA-DR, reduction of PDL-1 and PDL-2 expression and decrease of TGF-β and IL-10 in DCs as well as inhibition of T cell polarization into TH2 and Treg. Taken together, these data showed crucial effects of microenvironments on MSC behaviors indicating that functions of MSCs differentially altered in the presence of different cytokines.

Abstract 3595: Mesenchymal stem cells play crucial roles in the generation of osteogenic prostate to bone metastases

Abstract The skeleton is a common target for prostate metastases. In bone, prostate cancer cells induce extensive bone formation/osteogenesis by promoting osteoblast differentiation. This results in painful bony lesions that are incurable. The precise mechanisms through which prostate cancer cells induce bone formation are not well understood. Analysis of human and mouse specimens of prostate to bone metastasis demonstrate the presence of MSCs (smooth muscle actin positive). Given that MSCs are precursors of osteoblasts, we hypothesizes that MSC recruitment to the bone microenvironment would contribute to the formation of osteoblastic lesions and enhance tumor progression. Methodology: Using in-vitro and in-vivo studies we aim to elucidate the effects of MSC's on prostate tumor progression in bone. To determine the effect of the MSCs on the growth of the prostate cancer cells, mouse MSCs were directly co-cultured at various ratios with luciferase expressing PAIII prostate cancer cells for 24-96 hours. In-vivo, mice were intratibially inoculated with either PAIIIs, PAIIIs/MSCs (1:1), or MSCs (n=8/group, 104 total cells in 20µl). Tumor growth was measured over 15 days using luminescence imaging. Subsequently the tibias were collected for x-ray, micro CT, and histological analysis. Results: In-vitro MSCs inhibited the growth of the PAIII cells from 24 to 96 hours. At a 1:1 ratio, MSCs inhibited PAIII growth by up to 55% at 96 hours (p<0.05). A cleaved caspase 3 western blot of the lysates of PAIIIs treated with MSC CM provides evidence that soluble factors from the MSCs are inducing caspase dependent apoptosis. Consistent with our in vitro observations, we observed in vivo that at day 11, the average tumor size in the PaIII/MSC (1:1 ratio) group was 57.7% significantly smaller than the PAIII group alone (p<0.05). However by day 15 the tumors were growing at comparable rates. Tumor bearing tibias were X-rayed and an overall decrease in tumor-induced osteolysis was observed between the PAIII/MSC and PAIII groups. μCT scans of tumor bearing bones show an increase in bone volume in bones injected with MSCs alone and PaIII/MSCs when compared to PAIIIs, a finding that was consistent with X-ray and hisotmorphometrical data. Conclusions: In conclusion we have found that in-vitro and in-vivo MSCs have an early inhibitory effect on the growth of prostate tumor cells. This impact on growth is coupled to an apparent decrease in tumor-induced osteolysis and increase in osteogenesis. Currently, we are examining 1) the impact of the MSCs on tumor-induced osteogenesis and 2) identifying the MSC derived factors that are inhibiting prostate cancer growth. Understanding the role of MSCs in the prostate tumor-bone microenvironment will lead to the identification of new therapeutic targets to treat the disease. Citation Format: Jeremy Jonathan Mcguire, Gemma Shay, Leah Cook, Jeremy Frieling, Conor Lynch. Mesenchymal stem cells play crucial roles in the generation of osteogenic prostate to bone metastases. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3595. doi:10.1158/1538-7445.AM2014-3595

TGF-β Promotes Immune Responses in the Presence of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) possess potent immunosuppression capacity and could exert strong therapeutic effects in many diseases, especially inflammatory disorders, in animal models and clinical settings. Although inflammatory cytokines are critical in inducing the immune modulatory properties of MSCs, detailed molecular mechanisms are yet to be fully understood. TGF-β is a well-known anti-inflammatory cytokine and exists in various inflammatory processes; therefore, we investigated whether it could synergize with MSCs in suppressing immune responses. To our surprise, we found that TGF-β actually reversed the immunosuppressive effect of MSCs on anti-CD3 activated splenocytes. Using TGF-β unresponsive MSCs, we demonstrated that the TGF-β directly acted on MSCs. Furthermore, we showed that the effect of TGF-β is exerted through inhibiting inflammatory cytokines induced inducible NO synthase (iNOS) expression in a SMAD3-dependent manner. Interestingly, we found that TGF-β produced by MSCs could act in an autocrine manner to reduce inflammatory cytokine-induced inducible NO synthase expression by MSCs themselves. Therefore, our study revealed a previously unrecognized property of TGF-β in promoting immune responses in the presence of MSCs.

Molecular Mechanisms Of Adipocyte-Leukemia Interactions Revealed By The Quantitative Proteomics Technology: Pro-Survival Function Of Adipocyte-Derived Free Fatty Acids On Acute Monoblastic Leukemia Mitochondrial Biogenesis

Acute monocytic leukemia (AMoL) is a distinct subtype of AML with an average 3-year overall survival of 31% (Tallman, J Clin Oncol 2004), and the majority of patients die from disease progression after relapse. Adipocytes represent an essential component of the adults bone marrow (BM) microenvironment (Battula, Blood 2013, Tabe, Blood 2004, Konopleva, Blood 1999), and promote survival of monoblastic leukemia cells (Tabe, ASH, 2012).In this study, we employed a proteomic approach based on isobaric tags for relative and absolute quantification (iTRAQ, Applied Biosystems) to examine the molecular mechanism of pro-survival properties of BM adipocytes in co-culture with AMoL. The specific pathway alterations were identified by Metacore (GeneGo, St. Joseph, MI). We first confirmed the more prominent protective role of BM-derived adipocytes as compared to BM stromal cells (MSC) on AMoL cells. Co-culture with adipocytes derived from differentiated MSCs significantly protected U937 and primary AMoL cells (n = 5) from serum starvation-induced apoptosis compared to MSCs (U937 p = 0.029, primary samples p = 0.034). Gene expression analysis demonstrated striking upregulation of mRNA expression levels of CD36 (4.1+0.4-fold),FABP4 (569.3+40.1-fold), and PPARG (2.1+0.2-fold) in U937 cells co-cultured with adipocytes compared to the MSC co-cultured cells. Notably, Bcl-2 mRNA expression in U937 cells was significantly upregulated after adipocyte co-culture compared to MSCs (5.4+0.3-fold). We previously reported that fatty acids (FAs) promote leukemic cell survival via leukemia cells metabolic shifting from pyruvate oxidation to fatty acid oxidation for glycolysis, which links to the Bcl-2 anti-apoptotic machinery (Samudio, J Clin Invest. 2010). Mature adipocytes are capable of releasing abundant free fatty acids (Fas), the essential ligands of a nuclear receptor PPARgamma (PPARG), which are internalized by leukemic cells via scavenger receptor CD36 and ligate PPARG through fatty acid binding protein 4 (FABP4). Enhanced transcriptional activity of FA-ligated PPARG may in turn stimulate glucose metabolism, regulate fatty acids storage, maintain mitochondrial membrane potential, and prevents apoptosis by upregulating anti-apoptotic Bcl-2 resulting in further increase of its downstream target genes including CD36 and FABP4. To gain further insights into metabolic alterations induced in adipocyte-leukemia co-cultures, we performed iTRAQ proteomic analysis of U937 cells cultured in the presence of MSCs or adipocytes. A total of 1,610 proteins were detected by this technology, with 106 proteins differentially expressed between co-culture conditions; 50 genes were up-regulated and 56 genes were down-regulated by adipocytes co-culture compared to MSCs. Among the upregulated proteins, FA synthase (p = 0.007) ATP-citrate synthase responsible for cytosolic acetyl-CoA synthesis (p = 0.019); and glyceraldehyde-3-phosphate dehydrogenase (p = 0.028) involved in the activation of glycolysis and gluconeogenesis pathways (p< 0.0001) were identified. Of note, 20% of the upregulated proteins (10/50) were ribosomal proteins, accompanied by an increase in six DNA elongation or replication related proteins. Anti-apoptotic chaperone proteins known to assist ribosome biogenesis, HSP70, HSP90alpha and HSP90beta were also increased by adipocyte co-culture (p<0.01). The mass spectrometry further revealed more prominent repression of Cytochrome c (p=0.01) as well as the proline metabolism pathway (p=0.0011) under adipocyte co-culture conditions, consistent with inhibition of mitochondrial apoptosis of U937 cells. Summaryour results indicate that abundant FAs produced by BM adipocytes support AMoL cell survival via stimulation of PPARG transcription which directly increases the FA metabolism through uptake and translocation of FAs by CD36 and FABP4 in AMoL cells. FA metabolism in turn facilitates supply of Acetyl-CoA via oxidative phosphorylation, which drives the TCA cycle; and enhances ribosomal biogenesis of AMoL cells, resulting in pro-survival signaling in AMoL cells. It is conceivable that increased adipocyte content of the BM in adult AML patients may promote leukemogenesis and negatively affect the responsiveness to chemotherapy. Disclosures:No relevant conflicts of interest to declare.

Histological characterization of abdominal aorta post endovascular autologous transplantation of Mesenchymal Stem cells (MSCs) in Porcine AAA Model

IntroductionMSCs transplantation is being investigated in porcine AAA models for their potential to repair degenerated aorta. This study aims to characterize histological changes in the tissues of porcine AAA models post MSC transplant to build reliable methods of tracking the cells.Methods and ResultsMSCs were isolated from pig's bone marrow and expanded in vitro. Lenti Virus + GFP transfected to MSCs for 48 hrs. The cells are prepared with mixture of 10% sucrose, puramatrix and PBS. The AAA is induced and MSCs labeled with GFP are injected. The pig is sacrificed 2 wks post surgery and the tissues are collected and formalin fixed for histological staining such as H&amp;E, Trichrome, Pearl, and Picrosirius red(PSR). IHC is performed to localize GFP positive cells. Figure 1A: IHC staining for anti GFP showing bright green signal ‐positive spindle cells at the border of Media and adventitia layer of the tissue. Cluster of MSCs showing two different morphologies, round and spindle shapes indicating differentiation of MSCs within the damaged region. Figure 1B: PSR stains showing distinct color gradient between new collagen fibers in green Vs. old collagen fibers. Figure 2A: H&amp;E stain shows the presence of MSCs. Figure 2B: Trichrome confirms figure 1A findings.ConclusionThe histology slides clearly make it easy to visualize MSCs in the aorta post transplant confirming that it is possible to localize and track MSCs.

Tu1235 Multicentre Follow-up Study of Phagocyte-Derived S100A12 as a Surrogate Marker of Intestinal Inflammation in Inflammatory Bowel Disease

demonstration of MSc markers in granulomas of ITB and evaluate whether the distribution of MSc markers could differentiate between granulomas of CD & ITB. Materials & methods: We retrospectively enrolled 17 patients with confirmed ITB(8)or CD(9) and granulomas on histopathology. The 8 ITB cases were culture +ve for myco.tb and mTB PCR +ve. The 9 CD cases were identified according to WHO criteria for definite CD. Paraffinembedded tissue blocks were sectioned to determineMScmarkers CD29, CD90 and CD73 expression by immunofluorescence (Bioimager , BD BioSciences,USA). Conjugated monoclonal antibodies against CD29 (mouse, 313001; BIolegend,USA.), CD73 (mouse, ab54217) & CD90 (rabbit, ab92574) (Abcam) were used for identification of the MScs. Double staining was carried to confirm presence of MScs. Results: CD29 and CD90 positive cells were noted around both ITB and CD granulomas. However CD73, a marker for permeability barrier was expressed around the granulomas of ITB alone and was completely absent in the granulomas of CD (Fig 1). Double staining showed that CD73 Cells were also expressing CD90 & CD29 confirming MSc. Discussion: The differential CD73 expression around ITB granuloma indicates that Mtb evades host immunity by recruiting MScs with high CD73. The study also demonstrates CD73 expression in MScs as a marker for differentiation between granulomas of ITB and CD. These features postulate and support the loss of permeability barrier (reduced CD73 expression) in CD but present in ITB due to presence of the bacterium. MSC therapy has shown benefit in CD2. Conclusion: Granulomas of ITB and CD can be differentiated by CD73 MSc surface marker expression.MSCs with increased CD73 expression could be the future for therapeutic intervention in Crohns. Ref: 1. Raghuvanshi S et al Proc Natl Acad Sci USA. 2010Dec 14; 107(50):21653-8 2. Ciccocioppo R et al Gut 2011; 60:788-98

Isolation of G-CSF Mobilized Human Mesenchymal Stem Cells by Elutriation.

AbstractAbstract 1183Mesenchymal stem cells (MSCs) are a population of adult stem cells that constitute a promising source for therapeutic applications. MSCs exist in various adult tissues, such as bone marrow and umbilical cord blood. The most common method for MSC isolation relies on plastic adherence, however, this isolation procedure is hampered by the unpredictable influence of other co-cultured adhering cells. Phenotypic characterization of MSCs is usually carried out by FACS analysis of cell surface marker expression, but no unique marker has yet been identified. To circumvent these limitations, using granulocyte-colony stimulating factor (G-CSF) mobilized adult human peripheral blood (PB), we explored the use of Elutra® Cell Separation System (Caridian BCT) to isolate MSC enriched fractions based on their size and density.A group of 28 donors were studied after mobilization for two consecutive day of G-CSF injection (480μg/day subcutaneously). Total nucleated cells (TNC) were collected by apheresis on the third day. An average of 6-fold increase in MSCs in the post-mobilized sample was observed. At least 1 million TNC in pre- and post-mobilized PB were analyzed by FACS for the presence of MSCs by cell surface staining with antibodies to CD105, CD90, CD73, CD31, CD45 and CD34. Circa 3 × 1010 TNC were loaded onto the Elutra to fractionate the apheresis product into several distinct cell fractions. Typically, the MSCs were enriched in 2 fractions, and yielded about 5–10% of the initial loaded cell number. Following culturing and passage, cells from these 2 fractions were grown on plastic with low contamination by other cell types. The cultured cells showed spindle-shaped morphology. Cells were further characterized by FACS analysis and shown to stained positive for mesenchymal specific markers CD90 and CD105; and negative for hematopoietic markers of CD45 and CD34. Furthermore, after cells from all fractions were plated for colony forming unit-fibroblast assay (CFU-F), no colony formation were observed from other than the 2 fractions.In summary, we demonstrate that MSCs are mobilized into PB by G-CSF and can be collected by apheresis. Size-based Elutra fractionation of apheresis product in a closed system could provide a novel strategy for the large-scale isolation and expansion of human MSCs for autologous cell therapy *Dr. Marasco is a paid consultant for NeoStem and an equity holder. Disclosures:No relevant conflicts of interest to declare.

Mesenchymal Stem Cells Inhibit Generation and Function of Both CD34+-Derived and Monocyte-Derived Dendritic Cells

Mesenchymal stem cells (MSCs) are not only able to evade the immune system, but they have also been demonstrated to exert profound immunosuppressive properties on T cell proliferation. However, their effect on the initiators of the immune response, the dendritic cells (DCs), are relatively unknown. In the present study, the effects of human MSCs on the differentiation and function of both CD34+ -derived DCs and monocyte-derived DCs were investigated. The presence of MSCs during differentiation blocked the differentiation of CD14+CD1a- precursors into dermal/interstitial DCs, without affecting the generation of CD1a+ Langerhans cells. In line with these observations, MSCs also completely prevented the generation of immature DCs from monocytes. The inhibitory effect of MSCs on DC differentiation was dose dependent and resulted in both phenotypical and functional modifications, as demonstrated by a reduced expression of costimulatory molecules and hampered capacity to stimulate naive T cell proliferation. The inhibitory effect of MSCs was mediated via soluble factors. Taken together, these data demonstrate that MSCs, next to the antiproliferative effect on T cells, have a profound inhibitory effect on the generation and function of both CD34+ -derived and monocyte-derived DCs, indicating that MSCs are able to modulate immune responses at multiple levels.