Proteomic Analysis of Reprograming of Mesenchymal Stem/Stromal Cells (MSC) Following Interferon Gamma Identifies Pathways That Are Upregulated in Suppression

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 384-384 ◽  
Author(s):  
Qingdong Guan ◽  
Tanner Shpiruk ◽  
Peyman Ezzati ◽  
Oleg Krokhin ◽  
Scott Gilpin ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Proteomic Analysis ◽  
Conflicts Of Interest ◽  
Mass Spectrometry Data ◽  
Paired Samples ◽  
T Lymphocyte Proliferation ◽  
Healthy Donors ◽  
Ifn Γ ◽  
Postdoctoral Fellowship ◽  
Tandem Mass Spectrometry Data

Abstract Assessing potency of mesenchymal stem/stromal cells (MSC) used for immunologic applications such as the treatment of GVHD or other inflammatory disorders has been a challenge. Expression of PD-L1 or production of indoleamine-pyrrole 2,3-dioxygenase (IDO-1) has been proposed as potential potency markers. To screen for other pathways involved with suppression we undertook proteomic analysis of IFN-γ stimulated MSC. MSC isolated and expanded from normal healthy donors to 70-80% confluence were treated overnight with human rIFN-γ (30ng/ml). MSC were harvested using TrypLE Select and then immunologic and proteomic studies were performed. IFN-γ exposure increased a) MSC expression of IDO-1 and PD-L1, b) MSC suppression of 3rd party T lymphocyte proliferation, c) MSC inhibition of development of IFN-γ producing T lymphocytes, and d) MSC promotion of Treg expansion. Cellular proteomic changes that occur with IFN-γ exposures were studied in paired samples of control and IFN-γ treated MSC. Samples were prepared using a modified Filter Aided Sample Prep (FASP) and digests were separated using 2D liquid chromatography and analysed by tandem mass spectrometry. Data was processed with the Global Proteome Machine and only proteins with at least two confident peptides were reported. A total of 7621 proteins were identified of which 5575 were seen in all samples and 232 proteins were significantly upregulated in the IFN-γ treated cells relative to their controls. The proteomic analysis identified constitutive proteins seen in MSC. The upregulated proteins were significantly enriched (p<10-17) for GO processes such as "response to IFN-γ" and "cytokine mediated signaling pathway". Known inhibitory mediators (such as IDO-1, PD-L1, PGE2, galectin-9) were upregulated. Interestingly adhesion molecules (ICAM-1, VCAM-1, and CCL9) were increased. Other proteins with increased expression include Bone Marrow Stromal 2 (BST2). Conclusion: Proteomic analysis of response of MSC to IFN-γ has identified a signature of proteins upregulated with the activation of immune suppressive functions of MSC. Once confirmed these findings will support the development of a potency test for immunosuppressive potential of given MSC preparations - something that is sorely needed in the clinical manufacturing of MSC products. Acknowledgments: Q.D. is holding a postdoctoral fellowship from MS Society of Canada. This research was supported by The Bihlers' Professorship in Stem Cell Research to D.W. Disclosures No relevant conflicts of interest to declare.

Mesenchymal Stromal Cells with Immunosuppressive Potential Can Be Mobilized Into Peripheral Blood by Electro-Acupuncture

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4805-4805
Author(s):  
Lizhen Liu ◽  
Qin Yu ◽  
Shan Fu ◽  
Hui Dong ◽  
Kaimin Hu ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Haematopoietic Stem Cell ◽  
Adherent Cells ◽  
Sprague Dawley ◽  
T Lymphocyte Proliferation ◽  
Immunosuppressive Property ◽  
Tgf Β1

Abstract Abstract 4805 Background: Mesenchymal stromal cells (MSCs) constitute a population of multipotential cells giving rise to adipocytes, osteoblasts and chondrocytes. Combining with their engraftment promoting capacity and immunosuppressive property, MSCs may be therapeutically useful for haematopoietic stem cell transplantation. A small number of MSCs can be mobilized into circulation by appropriate stimuli, such as hypoxia. However, there is little evidence for clinically useful methods for MSC mobilization. In this study, we used animal model to determine whether MSCs can be mobilized into peripheral blood (PB) by electro-acupuncture (EA), a traditional Chinese medical method. Design and Methods: Adult male Sprague-Dawley rats (200–220g) were randomly divided into there groups: EA-7 days, EA-14 days and control groups. For EA treatment, the rats were immobilized. A pair of stainless needles of 0.35mm diameter was inserted into points ‘Jizhong’ (GV6) and ‘Mingmen’ (DU4) and was then connected with output terminals of an EA apparatus. Alternating strings of dense-sparse frequencies were selected and the intensity was adjusted to induce slight twitch of the skin, with the intensity lasting for 30 min. Electro-acupuncture was applied to rats once a day for 7days or 14days. The control rats were immobilized for the same period without EA. To quantify the number of MSCs and evaluate mobilization efficiency, PB and bone barrow (BM) samples of each group were collected and colony-forming unit fibroblast (CFU-F) assays were performed. Mobilized PB derived MSCs were identified by immunophenotype and trilineage differentiation. Mixed lymphocyte reactions (MLR) were done to evaluate the immunosuppressive potential of mobilized MSCs and the cytokine levels TGF-β1, HGF and IL-10 in the supernatants of MSCs culture were measured by ELISA. Results: We found that MSCs can be mobilized into PB by electro-acupuncture. CFU-F frequency in rat PB was significantly increased after electro-acupuncture for 7days (8.20 ±1.48 vs.1.40 ±0.55 CFU-Fs per 3×106 cells) (p<0.05, n=5). PB CFU-F frequency increased to 12.4±1.82 per 3×106 cells in rats treated with electro-acupuncture for 14 days. However, no significant differences were observed in BM CFU-Fs among varies groups (P>0.05). Mobilized PB derived adherent cells were positive for CD90, CD29 and CD44, but negative for CD34 and CD45. After adipogenic, osteogenic, and chondrogenic induction, adherent cells from mobilized PB were positive for specific stains. In addition, they expressed mRNAs of Lpl and Pparg2 (adipocytic markers), Bglap and Runx2 (osteoblastic markers), and Col2a1 and Col10a1 (chondrocytic markers). These results showed that mobilized PB-derived cells could differentiate into adipocyte, osteoblast, and chondrocyte, which indicated that they are bona fide MSCs. The levels of immunosuppressive cytokines TGF-β1, HGF and IL-10 in the supernatants of PB-MSC culture were similar as BM MSCs. Results of MLR showed that mobilized PB derived MSCs inhibited T lymphocyte proliferation. Conclusion: Taken together, these data revealed, for the first time to the best of our knowledge, that multipotential MSCs can be mobilized by electro-acupuncture. Our study provides a clinical useful method to mobilize MSCs with immunosuppressive potential and highlight a novel insight into the mechanisms of electro-acupuncture therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Endogenous TGF-beta1 Mediated Osteogenic Impairment of Medullar Mesenchymal Stromal Cells in Primary Myelofibrosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1873-1873
Author(s):  
Christophe Martinaud ◽  
Christophe Desterke ◽  
Johanna Konopacki ◽  
Lisa Pieri ◽  
Rachel Golub ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Hematopoietic Cells ◽  
Primary Myelofibrosis ◽  
Osteogenic Potential ◽  
Healthy Donors

Abstract Primary myelofibrosis (PMF) is myeloproliferative neoplasm characterized by clonal myeloproliferation, dysmegakaryopoiesis, extramedullary hematopoiesis associated with myelofibrosis and altered stroma in bone marrow and spleen. Mesenchymal stromal cells (MSCs) are reported to play a pivotal role in fibrosis and stromal changes are considered as a reactive counterpart of the cytokine production by clonal hematopoietic cells. The present study shows that MSCs from patients demonstrate functional abnormalities that are unexpectedly maintained ex-vivo, in culture. Material and Methods: we studied MSCs and bone marrow sections from PMF patients (n=12) as compared to healthy donors (HDs) (n=6). We tested their proliferation, immunophenotype, hematopoiesis supporting capacities, differentiation abilities, in-vivo osteogenic assays, and performed secretome and transcriptome analysis. Results: We found that PMF-MSCs exhibit similar proliferative capacity and long-term hematopoiesis supporting abilities as compare to healthy donors. They overproduce interleukin 6, VEGF, RANTES, PDGF, BMP-2 and surprisingly TGF-beta1. MSCs from fibrotic PMF patients express high levels of glycosaminoglycans. Adipocytes and chondrocytes differentiation abilities were not different as compared to HDs but PMF-MSCs exhibit an increased in vitro potential. Implementation on scaffold in nude mice confirmed, in vivo, this increased osteogenic potential. We then looked into gene expression and discovered that PMF-MSCs show an original transcriptome signature related to osteogenic lineage and TGF-beta1. Indeed, osteogenic genes such as Runx2, Dlx5, Twist1, Noggin, Sclerostin, GDF5 and Serpine1 are deregulated and suggest a potential osteoprogenitor priming of PMF-MSCs. These molecular results also advocated for a TGF-beta1 impregnation that prompted us to study its impact on PMF-MSCs osteogenic differentiation. First, we then showed that Smad2 is intrinsically over-activated in PMF-MSC and that stimulation by TGF-beta1 is associated with an increase phospho-Smad2 level and an enhancement of bone master gene regulator Runx2 expression. Then, we inhibited TGF-beta1 pathway by by SB-431542 and evidenced a specific behavior of osteogenic MSCs differentiation in patients, suggesting involvement of TGF-beta1 in osteogenic impairment. Conclusion: Altogether, our results identify a signature of PMF-MSCs and suggest that they participate in PMF osteogenic dysregulation independently from in vivo local stimulation by clonal hematopoietic cells Disclosures No relevant conflicts of interest to declare.

scholarly journals Serum IL-35 is decreased in overweight patients with rheumatoid arthritis: its correlation with Th1/Th2/Th17-related cytokines

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yuxuan Li ◽  
Yang Jie ◽  
Xiaofei Wang ◽  
Jing Lu
Keyword(s):  
Rheumatoid Arthritis ◽  
Medical Records ◽  
Clinical Information ◽  
Serum Cytokines ◽  
Healthy Donors ◽  
Potential Biomarker ◽  
Tnf Α ◽  
Ifn Γ ◽  
Quantitative Changes ◽  
Anti Inflammatory Cytokine

Abstract Background Obesity is correlated with worse drug responses and high disease activity in patients with rheumatoid arthritis (RA). Interleukin (IL)-35 is a novel anti-inflammatory cytokine that mainly produced by regulatory T (Treg). This study was performed to analyze whether IL-35 was correlated with obesity in RA and investigate the correlation between other Th1/Th2/Th17-related cytokines and obesity in RA. Results The serum IL-35 level was analyzed in RA (n = 81) and healthy donors (n = 53) by ELISA assay, and was compared between three groups (body mass index (BMI) < 18.5,≥18.5 to 25, > 25). Serum cytokines including IL-2, IL-4, IL-10, IL-17, INF-γ, TNF-α levels were measured using Flowcytometry assay. Clinical information was extracted from medical records. Serum IL-35 level in overweight patients were significantly decreased than those in lean patients. Furthermore, Th1/Th2/Th17-related cytokines from overweight patients with RA showed the characteristic immunological features. Serum IL-6, IL-17 and TNF-α levels were positively correlated with BMI. However, serum IL-2, IL-4, IL-10 and IFN-γ concentrations were not correlated with BMI. Conclusions Quantitative changes in serum IL-35 level were characteristic in overweight patients with RA. These findings indicate that IL-35 plays an important role in the development of RA and may prove to be a potential biomarker of active RA.

scholarly journals The therapeutic efficacy of mesenchymal stromal cells on experimental colitis was improved by the IFN-γ and poly(I:C) priming through promoting the expression of indoleamine 2,3-dioxygenase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ji-Young Lim ◽  
Byung-Su Kim ◽  
Da-Bin Ryu ◽  
Tae Woo Kim ◽  
Gyeongsin Park ◽  
...  
Keyword(s):  
Weight Loss ◽  
Disease Activity ◽  
Stromal Cells ◽  
Therapeutic Efficacy ◽  
Activity Index ◽  
Disease Activity Index ◽  
Experimental Colitis ◽  
Poly I:C ◽  
Colon Tissue ◽  
Ifn Γ

Abstract Background Inflammatory bowel disease is a chronic and excessive inflammation of the colon and small intestine. We previously reported that priming of mesenchymal stromal cells (MSCs) with poly(I:C) induced them to express indoleamine 2,3-dioxygenase (IDO). We tried to find out whether the IFN-γ and poly(I:C)-primed MSCs have better therapeutic efficacy on the experimental colitis in the IDO1-dependent manner. Methods To compare the therapeutic effects between the unstimulated MSCs and primed MSCs on murine colitis, mice (C57BL6) were administered with 2.5% dextran sodium sulfate (DSS) in drinking water for 5 days and injected with MSCs intraperitoneally on days 1 and 3 following DSS ingestion. The disease activity index score and body weight loss were assessed daily until day 9. Results Mice receiving the IFN-γ and poly(I:C)-primed MSCs showed a reduced disease activity index and less weight loss. Colon tissue from the same mice presented attenuated pathological damage, increased Paneth cells, increased IDO1-expressing cells, and better proliferation of enterocytes. The primed MSC treatment upregulated the mRNA expression of intestinal stem cell markers (Lgr5, Olfm4, and Bmi1), enterocyte differentiation markers (Muc2, Alpi, Chga, and occludin), and regulatory T (Treg) cells (Foxp3). The same treatment decreased inflammatory cell infiltration to lymphoid organs and the level of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6, and MCP-1) in colon tissue. Notably, in vivo pharmacologic inhibition of the IDO1 activity blocked the Foxp3 upregulation in colon tissue and diminished the protective effects of the primed MSC. Conclusions The priming of MSCs with the IFN-γ and poly(I:C) is a promising new strategy to improve the therapeutic efficacy of MSC and is worth further research.

scholarly journals Label-free proteomic analysis of serum exosomes from paroxysmal atrial fibrillation patients

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Hanwen Ni ◽  
Wenqi Pan ◽  
Qi Jin ◽  
Yucai Xie ◽  
Ning Zhang ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Protein Folding ◽  
Protein Content ◽  
Complement System ◽  
Proteomic Analysis ◽  
Surface Composition ◽  
Label Free ◽  
Healthy Donors ◽  
Variable Surface ◽  
Serum Exosomes

Abstract Background Atrial fibrillation (AF) is the most common cardiac heterogeneous rhythm disorder. It represents a major cause of mortality and morbidity, mainly related to embolic events and heart failure. Mechanisms of AF are complex and remain incompletely understood. Recent evidence suggests exosomes are membrane-coated objects released by many cell-types. Their presence in body fluids and the variable surface composition and content render them attractive as a mechanism for potential biomarkers. However, the content of serum exosomes of AF patients has not been fully delineated. Methods In this work, the serum exosomes from AF patients and healthy donors were used to compare changes in the exosome protein content. Exosomes were isolated from serum of AF patients and healthy donors and their purity was confirmed by Western blotting assays and transmission electron microscopy (TEM). Label-free LC–MS/MS quantitative proteomic analysis was applied to analyze protein content of serum exosomes. Results A total of 440 exosomal protein groups were identified, differentially expressed proteins were filtrated with fold change ≥ 2.0 (AF/controls protein abundance ratio ≥ 2 or ≤ 0.5) and p value less than 0.05 (p < 0.05), significantly changed in abundance group contains 39 elevated proteins and 18 reduced proteins, while consistent presence/absence expression profile group contains 40 elevated proteins and 75 reduced proteins. Bioinformatic analysis of differential exosomal proteins confirmed the significant enrichment of components involved in the anticoagulation, complement system and protein folding. Parallel-Reaction Monitoring Relative Quantitative Analysis (PRM) further suggested that AF related to complement system and protein folding. Conclusions These results revealed the composition and potential function of AF serum exosomes, thus providing a new perspective on the complement system and protein folding to AF.

scholarly journals Akt Inhibitors Induce Apoptosis in Chronic Lymphocytic Leukemia Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1731-1731
Author(s):  
Mercè de Frias ◽  
Daniel Iglesias-Serret ◽  
Ana M Cosialls ◽  
Llorenç Coll-Mulet ◽  
Antonio F Santidrián ◽  
...  
Keyword(s):  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
Therapeutic Option ◽  
Mrna Level ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Healthy Donors ◽  
Induced Apoptosis

Abstract Abstract 1731 Poster Board I-757 Phosphatidylinositol-3-kinase (PI3K)/Akt pathway has been described to be critical in the survival of chronic lymphocytic leukemia (CLL) cells. Here, we have analyzed the effect of two selective chemical inhibitors of Akt (Akti-1/2 and A-443654) in the survival of CLL cells. We studied by cytometric analysis the cytotoxic effects of Akt inhibitors on peripheral B and T lymphocytes from patients with CLL and from healthy donors. Both inhibitors induced apoptosis in CLL cells in a dose-dependent manner. Moreover, B cells from CLL samples were more sensitive to Akt inhibitors than T cells from CLL samples, and B or T cells from healthy donors. Survival factors for CLL cells, such as IL-4 and SDF-1a, were not able to block the apoptosis induced by both Akt inhibitors. We studied the changes induced by Akti-1/2 and A-443654 at mRNA level by performing reverse transcriptase multiplex ligation–dependent probe amplification (RT-MLPA). Akti-1/2 did not induce any change in the mRNA expression profile of genes involved in apoptosis, while A-443654 induced some changes, including an increase in NOXA and PUMA mRNA levels, suggesting the existence of additional targets for A-443654. We also studied the changes induced by both Akt inhibitors in some BCL-2 protein family members on CLL cells by Western blot. Both inhibitors induced an increase in PUMA and NOXA protein levels, and a decrease in MCL-1 protein level. Moreover, Akti-1/2 and A-443654 induced apoptosis irrespective of TP53 status. These results demonstrate that Akt inhibitors induce apoptosis of CLL cells and might be a new therapeutic option for the treatment of CLL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Effects of Interferon-γ on Proliferation and Ability of Secretion of Human Amniotic Mesenchymal Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5012-5012
Author(s):  
Ya Gao ◽  
Ying Xu ◽  
Weiru Li ◽  
Yintian Zhang ◽  
Baohong Ping ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Conflicts Of Interest ◽  
Interferon Γ ◽  
Cell Counting ◽  
Inflammatory Factors ◽  
Complete Medium ◽  
Amniotic Mesenchymal Stem Cells ◽  
Soluble Hla ◽  
Ifn Γ

Objective:The immunoregulatory properties and proliferation of mesenchymal stem cells (MSCs) could be affected by inflammatory factors. However, there have been few studies about human amniotic MSCs (hAMSCs). We investigated the effects of interferon (IFN)-γ on the proliferation and apoptosis of hAMSCs, and measured the level of inflammatory factors secreted by hAMSCs. Result:hAMSCs were cultured with complete medium with different concentrations of IFN-γ. We detected the proliferation of hAMSCs by Cell Counting Kit-8 assays, analysed apoptosis by flow cytometry (FCM) at 48 h, and mesasured the level of inflammatory factors such as solube HLA-G and prostaglandin E2 (PGE2) in the supernatant at 48 h by ELISA. The level of kynurenine (KYN) was measured by ultraviolet spectrophotometry. As culture time increased, the proliferation of hAMSCs with different concentrations of IFN-γ increased rapidly from day 1 to day 4, and then the growth rate slowed. FCM indicated that there was no significant apoptosis in the 100 ng/ml IFN-γ group compared with cells without IFN-γ. The level of PGE2 and soluble HLA-G in cells with IFN-γ was higher compared with those without IFN-γ. The level of KYN increased significantly in the cells with IFN-γ. Conclusion:IFN-γ did not affect the growth and proliferation of hAMSCs, and promoted secretion of PGE2 and soluble HLA-G, and enhanced activity of indoleamine 2,3-dioxygenase (IDO), providing a theoretical basis for hAMSCs to prevent and treat graft-versus-host disease. Disclosures No relevant conflicts of interest to declare.

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