Search results for: mesenchymal stromal

Authors: K. Nikovics, D. Riccobono, M. Oger, H. Morin, L. Barbier, T. Poyot, X. Holy, A. Bendahmane, M. Drouet, A. L. Favier

Abstract:

Muscle regeneration after injury (as irradiation) is of great importance. However, the molecular and cellular mechanisms are still unclear. Cytokines are believed to play fundamental role in the different stages of muscle regeneration. They are secreted by many cell populations, but the predominant producers are macrophages and helper T cells. On the other hand, it has been shown that adipose tissue derived stromal/stem cell (ASC) injection could improve muscle regeneration. Stem cells probably induce the coordinated modulations of gene expression in different macrophage cells. Therefore, we investigated the patterns and timing of changes in gene expression of different cytokines occurring upon stem cells loading. Muscle regeneration was studied in an irradiated muscle of minipig animal model in presence or absence of ASC treatment (irradiated and treated with ASCs, IRR+ASC; irradiated not-treated with ASCs, IRR; and non-irradiated no-IRR). We characterized macrophage populations by immunolabeling in the different conditions. In our study, we found mostly M2 and a few M1 macrophages in the IRR+ASC samples. However, only few M2b macrophages were noticed in the IRR muscles. In addition, we found intensive fibrosis in the IRR samples. With in situ hybridization and immunolabeling, we analyzed the cytokine expression of the different macrophages and we showed that M2d macrophage are the most abundant in the IRR+ASC samples. By in situ hybridization, strong expression of the transforming growth factor β (TGF-β) was observed in the IRR+ASC but very week in the IRR samples. But when we analyzed TGF-β level with immunolabeling the expression was very different: many M2 macrophages showed week expression in IRR+ASC and few cells expressing stronger level in IRR muscles. Therefore, we investigated the MMP expressions in the different muscles. Our data showed that the M2 macrophages of the IRR+ASC muscle expressed MMP2 proteins. Our working hypothesis is that MMP2 expression of the M2 macrophages can decrease fibrosis in the IRR+ASC muscle by capturing TGF-β.

Keywords: adipose tissue derived stromal/stem cell, cytokine, macrophage, muscle regeneration

Procedia PDF Downloads 208

Authors: Sandra Pietri, Helene Dubout, Sabrina Ena, Candice Hoste, Enrico Bastianelli

Abstract:

Bone Therapeutics is a bone cell therapy company addressing high unmet medical needs in the field of bone fracture repair, more specifically in non-union and delayed-union fractures where the bone repair process is impaired. The company has developed a unique allogeneic osteoblastic cell product (ALLOB®) derived from bone marrow which is currently tested in humans in the indication of delayed-union fractures. The purpose of our study was to directly compare ALLOB® vs. non-differentiated mesenchymal stem cells (MSC) for their in vitro osteogenic characteristics and their in vivo osteogenic potential in order to determine which cellular type would be the most adapted for bone fracture repair. Methods: Healthy volunteers’ bone marrow aspirates (n=6) were expended (i) into BM-MSCs using a complete MSC culture medium or (ii) into ALLOB® cells according to its manufacturing process. Cells were characterized in vitro by morphology, immunophenotype, gene expression and differentiation potential. Additionally, their osteogenic potential was assessed in vivo in the subperiosteal calvaria bone formation model in nude mice. Results: The in vitro side-by-side comparison studies showed that although ALLOB® and BM-MSC shared some common general characteristics such as the 3 minimal MSC criteria, ALLOB® expressed significantly higher levels of chondro/osteoblastic genes such as BMP2 (fold change (FC) > 100), ALPL (FC > 12), CBFA1 (FC > 3) and differentiated significantly earlier than BM-MSC toward the osteogenic lineage. Moreover the bone formation model in nude mice demonstrated that used at the same cellular concentration, ALLOB® was able to induce significantly more (160% vs.107% for control animals) bone formation than BM-MSC (118% vs. 107 % for control animals) two weeks after administration. Conclusion: Our side-by-side comparison studies demonstrated that in vitro and in vivo, ALLOB® displays superior osteogenic capacity to BM-MScs and is therefore a better candidate for the treatment of bone fractures.

Keywords: gene expression, histomorphometry, mesenchymal stem cells, osteogenic differentiation potential, preclinical

Procedia PDF Downloads 306

Authors: Jienny Lee, In-Soo Cho, Sang-Ho Cha

Abstract:

Adult mesenchymal stem cells (MSCs) have been investigated using preclinical approaches for tissue regeneration. Porcine MSCs (pMSCs) are capable of growing and attaching to plastic with a fibroblast-like morphology and then differentiating into bone, adipose, and cartilage tissues in vitro. This study was conducted to investigate the proliferating abilities, differentiation potentials, and multipotency of miniature pig adipose tissue-derived MSCs (mpAD-MSCs) with or without long-term cryopreservation, considering that cryostorage has the potential for use in clinical applications. After confirming the characteristics of the mpAD-MSCs, we examined the effect of long-term cryopreservation (> 2 years) on expression of cell surface markers (CD34, CD90 and CD105), proliferating abilities (cumulative population doubling level, doubling time, colony-forming unit, and MTT assay) and differentiation potentials into mesodermal cell lineages. As a result, the expression of cell surface markers is similar between thawed and fresh mpAD-MSCs. However, long-term cryopreservation significantly lowered the differentiation potentials (adipogenic, chondrogenic, and osteogenic) of mpAD-MSCs. When compared with fresh mpAD-MSCs, thawed mpAD-MSCs exhibited lower expression of mesodermal cell lineage-related genes such as peroxisome proliferator-activated receptor-g2, lipoprotein lipase, collagen Type II alpha 1, osteonectin, and osteocalcin. Interestingly, long-term cryostoraged mpAD-MSCs exhibited significantly higher cell viability than the fresh mpAD-MSCs. Long-term cryopreservation induced a 30% increase in the cell viability of mpAD-MSCs when compared with the fresh mpAD-MSCs at 5 days after thawing. However, long-term cryopreservation significantly lowered expression of stemness markers such as Oct3/4, Sox2, and Nanog. Furthermore, long-term cryopreservation negatively affected expression of senescence-associated genes such as telomerase reverse transcriptase and heat shock protein 90 of mpAD-MSCs when compared with the fresh mpAD-MSCs. The results from this study might be important for the successful application of MSCs in clinical trials after long-term cryopreservation.

Keywords: mesenchymal stem cells, cryopreservation, stemness, senescence

Procedia PDF Downloads 209

Authors: Sang Chul Lee, Gi-Young Park, Dong Rak Kwon

Abstract:

Objective: The aim of this study was to investigate regenerative effects of ultrasound (US)-guided injection with human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) and/or polydeoxyribonucleotide (PDRN) injection in a chronic traumatic full-thickness rotator cuff tendon tear (FTRCTT) in a rabbit model. Material and Methods: Rabbits (n = 32) were allocated into 4 groups. After a 5-mm sized FTRCTT just proximal to the insertion site on the subscapularis tendon was created by excision, the wound was immediately covered by silicone tube to prevent natural healing. After 6 weeks, 4 injections (0.2 mL normal saline, G1; 0.2 mL PDRN, G2; 0.2 mL UCB-MSCs, G3; and 0.2 mL UCB-MSCs with 0.2ml PDRN, G4) were injected into FTRCTT under US guidance. We evaluated gross morphologic changes on all rabbits after sacrifice. Masson’s trichrome, anti-type 1 collagen antibody, bromodeoxyuridine, proliferating cell nuclear antigen, vascular endothelial growth factor and platelet endothelial cell adhesion molecule stain were performed to evaluate histological changes. Motion analysis was also performed. Results: The gross morphologic mean tendon tear size in G3 and 4 was significantly smaller than that of G1 and 2 (p < .05). However, there were no significant differences in tendon tear size between G3 and 4. In G4, newly regenerated collagen type 1 fibers, proliferating cells activity, angiogenesis, walking distance, fast walking time, and mean walking speed were greater than in the other three groups on histological examination and motion analysis. Conclusion: Co-injection of UCB-MSCs and PDRN was more effective than UCB-MSCs injection alone in histological and motion analysis in a rabbit model of chronic traumatic FTRCTT. However, there was no significant difference in gross morphologic change of tendon tear between UCB-MSCs with/without PDRN injection. The results of this study regarding the combination of UCB-MSCs and PDRN are worth additional investigations.

Keywords: mesenchymal stem cell, umbilical cord, polydeoxyribonucleotides, shoulder, rotator cuff, ultrasonography, injections

Procedia PDF Downloads 165

Authors: Almudena Espin Perez, Tyler Risom, Carl Pelz, Isabel English, Robert M. Angelo, Rosalie Sears, Andrew J. Gentles

Abstract:

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly malignancies. The role of the tumor microenvironment (TME) is gaining significant attention in cancer research. Despite ongoing efforts, the nature of the interactions between tumors, immune cells, and stromal cells remains poorly understood. The cell-intrinsic properties that govern cell lineage plasticity in PDAC and extrinsic influences of immune populations require technically challenging approaches due to the inherently heterogeneous nature of PDAC. Understanding the cell lineage plasticity of PDAC will improve the development of novel strategies that could be translated to the clinic. Members of the team have demonstrated that the acquisition of ductal to neuroendocrine lineage plasticity in PDAC confers therapeutic resistance and is a biomarker of poor outcomes in patients. Our approach combines computational methods for deconvolving bulk transcriptomic cancer data using CIBERSORTx and high-throughput single-cell imaging using Multiplexed Ion Beam Imaging (MIBI) to study lineage plasticity in PDAC and its relationship to the infiltrating immune system. The CIBERSORTx algorithm uses signature matrices from immune cells and stroma from sorted and single-cell data in order to 1) infer the fractions of different immune cell types and stromal cells in bulked gene expression data and 2) impute a representative transcriptome profile for each cell type. We studied a unique set of 300 genomically well-characterized primary PDAC samples with rich clinical annotation. We deconvolved the PDAC transcriptome profiles using CIBERSORTx, leveraging publicly available single-cell RNA-seq data from normal pancreatic tissue and PDAC to estimate cell type proportions in PDAC, and digitally reconstruct cell-specific transcriptional profiles from our study dataset. We built signature matrices and optimized by simulations and comparison to ground truth data. We identified cell-type-specific transcriptional programs that contribute to cancer cell lineage plasticity, especially in the ductal compartment. We also studied cell differentiation hierarchies using CytoTRACE and predict cell lineage trajectories for acinar and ductal cells that we believe are pinpointing relevant information on PDAC progression. Collaborators (Angelo lab, Stanford University) has led the development of the Multiplexed Ion Beam Imaging (MIBI) platform for spatial proteomics. We will use in the very near future MIBI from tissue microarray of 40 PDAC samples to understand the spatial relationship between cancer cell lineage plasticity and stromal cells focused on infiltrating immune cells, using the relevant markers of PDAC plasticity identified from the RNA-seq analysis.

Keywords: deconvolution, imaging, microenvironment, PDAC

Procedia PDF Downloads 97

Authors: Sepideh Hassanpour Khodaei

Abstract:

Background/Objectives: The purpose of this study is to evaluate the effect of mummy substances on two issues of proliferation and production of matrix protein synthesis in wound healing. Methods: The methodology used for this aim involves isolating mesenchymal stem cells and human fibroblasts procured at Pastor Institute, Iran. The cells were treated with mummy substances separately and co-cultured between ASCs and WJSCs, and fibroblasts. Proliferation was assessed by Ki67 method in monolayer conditions. Synthesis of components of extracellular matrix (ECM) such as collagen type I, type III, and fibronectin 1 (FN1) was determined by qPCR. Results: The effects of adipocyte stem cells (ASCs), Wharton Jelly Stem Cells (WJSCs), and Mummy material on fibroblast proliferation and migration were evaluated. The present finding underlined the importance of Mummy material, ASCs, and WJSCs in the proliferation and migration of fibroblast cells. Furthermore, the expression of collagen I, III, and FN1 was increased in the presence of the above material and cells. Conclusion: This study presented an effective in vitro method for the healing process. Hence, the prospect of utilizing Mummy material and stem cell-based therapies in wound healing as a therapeutic approach is promising.

Keywords: mummy material, wound healing, adipose tissue, Wharton’s jelly

Procedia PDF Downloads 82

Authors: Isalira Peroba Ramos, Cherley Borba Vieira de Andrade, Grazielle Suhett, Camila Salata, Paulo Cesar Canary, Guilherme Visconde Brasil, Antonio Carlos Campos de Carvalho, Regina Coeli dos Santos Goldenberg

Abstract:

Background: The therapeutic options for patients with cancer now include increasingly complex combinations of medications, radiation therapy (RT), and surgical intervention. Many of these treatments have important potential adverse cardiac effects and are likely to have significant effects on patient outcomes. Cell therapy appears to be promising for the treatment of chronic and degenerative diseases, including cardiomyopathy induced by RT, as the current therapeutic options are insufficient. Aims: To evaluate the potential of bone marrow mesenchymal cells (BMMCs) in radioinduced cardiac damage Methods: Female Wistar rats, 3 months old (Ethics Committee 054/14), were divided into 2 groups, non-treated irradiated group (IR n=15) and irradiated and BMMC treated (IRT n=10). Echocardiography was performed to evaluate heart function. After euthanasia, 3 months post treatment; the left ventricle was removed and prepared for RT-qPCR (VEGF and Pro Collagen I) and histological (picrosirius) analysis. Results: In both groups, 45 days after irradiation, ejection fraction (EF) was in the normal range for these animals (> 70%). However, the BMMC treated group had EF (83.1%±2.6) while the non-treated IR group showed a significant reduction (76.1%±2.6) in relation to the treated group. In addition, we observed an increase in VEGF gene expression and a decrease in Pro Collagen I in IRT when compared to IR group. We also observed by histology that the collagen deposition was reduced in IRT (10.26%±0.83) when compared to IR group (25.29%±0.96). Conclusions: Treatment with BMMCs was able to prevent ejection fraction reduction and collagen deposition in irradiated animals. The increase of VEGF and the decrease of pro collagen I gene expression might explain, at least in part, the cell therapy benefits. All authors disclose no financial or personal relationships with individuals or organizations that could be perceived to bias their work. Sources of funding: FAPERJ, CAPES, CNPq, MCT.

Keywords: mesenchymal cells, radioation, cardiotoxicity, bone marrow

Procedia PDF Downloads 229

Authors: Elena Filova, Ondrej Kaplan, Marie Markova, Helena Dragounova, Roman Matejka, Eduard Brynda, Lucie Bacakova

Abstract:

Decellularized vessels have been evaluated as small-diameter vascular prostheses. Reseeding autologous cells onto decellularized tissue prior implantation should prolong prostheses function and make them living tissues. Suitable cell types for reseeding are both endothelial cells and bone marrow-derived stem cells, with a capacity for differentiation into smooth muscle cells upon mechanical loading. Endothelial cells assure antithrombogenicity of the vessels and MSCs produce growth factors and, after their differentiation into smooth muscle cells, they are contractile and produce extracellular matrix proteins as well. Fibrin is a natural scaffold, which allows direct cell adhesion based on integrin receptors. It can be prepared autologous. Fibrin can be modified with bound growth factors, such as basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF). These modifications in turn make the scaffold more attractive for cells ingrowth into the biological scaffold. The aim of the study was to prepare thin surface-attached fibrin assemblies with bound FGF-2 and VEGF, and to evaluate growth and differentiation of bone marrow-derived mesenchymal stem cells on the fibrin (Fb) assemblies. Following thin surface-attached fibrin assemblies were prepared: Fb, Fb+VEGF, Fb+FGF2, Fb+heparin, Fb+heparin+VEGF, Fb+heparin+FGF2, Fb+heparin+FGF2+VEGF. Cell culture poly-styrene and glass coverslips were used as controls. Human MSCs (passage 3) were seeded at the density of 8800 cells/1.5 mL alpha-MEM medium with 2.5% FS and 200 U/mL aprotinin per well of a 24-well cell culture. The cells have been cultured on the samples for 6 days. Cell densities on day 1, 3, and 6 were analyzed after staining with LIVE/DEAD cytotoxicity/viability assay kit. The differentiation of MSCs is being analyzed using qPCR. On day 1, the highest density of MSCs was observed on Fb+VEGF and Fb+FGF2. On days 3 and 6, there were similar densities on all samples. On day 1, cell morphology was polygonal and spread on all sample. On day 3 and 6, MSCs growing on Fb assemblies with FGF2 became apparently elongated. The evaluation of expression of genes for von Willebrand factor and CD31 (endothelial cells), for alpha-actin (smooth muscle cells), and for alkaline phosphatase (osteoblasts) is in progress. We prepared fibrin assemblies with bound VEGF and FGF-2 that supported attachment and growth of mesenchymal stem cells. The layers are promising for improving the ingrowth of MSCs into the biological scaffold. Supported by the Technology Agency of the Czech Republic TA04011345, and Ministry of Health NT11270-4/2010, and BIOCEV – Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University” project (CZ.1.05/1.1.00/02.0109), funded by the European Regional Development Fund for their financial supports.

Keywords: fibrin assemblies, FGF-2, mesenchymal stem cells, VEGF

Procedia PDF Downloads 296

Authors: Huseyin Apdik, Aysegul Dogan, Selami Demirci, Ezgi Avsar Apdik, Fikrettin Sahin

Abstract:

Mesenchymal stem cells (MSCs) are crucial cell types for bone maintenance and growth along with resident bone progenitor cells providing bone tissue integrity during osteogenesis and skeletal growth. Any deficiency in this regulation would result in vital bone diseases. Of those, osteoporosis, characterized by a reduction in bone mass and mineral density, is a critical skeletal disease for especially elderly people. The commonly used drugs for the osteoporosis treatment are bisphosphonates (BPs). The most prominent role of BPs is to prevent bone resorption arisen from high osteoclast activity. However, administrations of bisphosphonates may also cause bisphosphonate-induced osteonecrosis of the jaw (BIONJ). Up to the present, the researchers have proposed several circumstances for BIONJ. However, effects of long-term and/or high dose usage of BPs on stem cell’s proliferation, survival, differentiation or maintenance capacity have not been evaluated yet. The present study will be held to; figure out BPs’ effects on MSCs in vitro in the aspect of cell proliferation and toxicity, migration, angiogenic activity, lineage specific gene and protein expression levels, mesenchymal stem cell properties and potential signaling pathways affected by BP treatment. Firstly, mesenchymal stem cell characteristics of Dental Pulp Stem Cells (DPSCs) and Periodontal Ligament Stem Cells (PDLSCs) were proved using flow cytometry analysis. Cell viability analysis was completed to determine the cytotoxic effects of BPs (Zoledronate (Zol), Alendronate (Ale) and Risedronate (Ris)) on DPSCs and PDLSCs by the 3-(4,5-di-methyl-thiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium (MTS) assay. Non-toxic concentrations of BPs were determined at 24 h under growth condition, and at 21 days under osteogenic differentiation condition for both cells. The scratch assay was performed to evaluate their migration capacity under the usage of determined of BPs concentrations at 24 h. The results revealed that while the scratch closure is 70% in the control group for DPSCs, it was 57%, 66% and 66% in Zol, Ale and Ris groups, respectively. For PDLSs, while wound closure is 71% in control group, it was 65%, 66% and 66% in Zol, Ale and Ris groups, respectively. As future experiments, tube formation assay and aortic ring assay will be done to determinate angiogenesis abilities of DPSCs and PDLSCs treated with BPs. Expression levels of osteogenic differentiation marker genes involved in bone development will be determined using real time-polymerase change reaction (RT-PCR) assay and expression profiles of important proteins involved in osteogenesis will be evaluated using western blotting assay for osteogenically differentiated MSCs treated with or without BPs. In addition to these, von Kossa staining will be performed to measure calcium mineralization status of MSCs.

Keywords: bisphosphonates, bisphosphonate-induced osteonecrosis of the jaw, mesenchymal stem cells, osteogenesis

Procedia PDF Downloads 236

Authors: Maryam Farzaneh, Seyyedeh Nafiseh Hassani, Hossein Baharvand

Abstract:

Objective: Chicken gonadal tissue has a two population such primordial germ cells (PGCs) and stromal cells (somatic cells). PGCs and embryonic germ cells (EGCs) that is a pluripotent type of PGCs in long-term culture are suitable sources for the production of chicken pluripotent stem cell lines, transgenic birds, vaccine and recombinant protein production. In general, the effect of growth factors such bFGF and mouse LIF on derivation of PGCs in vitro are important and in this study we could see the unique effect of small molecules such PD032 and SB43 as a chemical, in comparison to growth factors. Materials and Methods: After incubation of fertilized chicken egg up to 6 days and isolation of primary gonadal tissues and culture of mixed cells like PGCs and stromal cells. PGCs proliferate in the present of fetal calf serum (FCS) and small molecules and in another group bFGF, that these factors are important for PGCs culture and derivation. Somatic cells produce a multilayer feeder under the PGCs in primary culture and PGCs make a small cluster under these cells. Results: In present of small molecules and high volume of FCS (15%), the present of EGCs as a pluripotent stem cells were clear four weeks, that they had a positive immune-staining and periodic acid-Schiff staining (PAS), but in present of growth factors like bFGF without any chemicals, the present of PGCs were clear but after 7 until 10 days, there were disappear. Conclusion: Until now we have seen many researches about derivation and maintenance of chicken PGCs, in the hope of understanding the mechanisms that occur during germline development and production of a therapeutic product by transgenic birds. There are still many unknowns in this area and this project will try to have efficient conditions for identification of suitable culture medium for long-term culture of PGCs in vitro without serum and feeder cells.

Keywords: chicken gonadal primordial germ cells, pluripotent stem cells, growth factors, small molecules, transgenic birds

Procedia PDF Downloads 407

Authors: Diana Islam, Juan Fang, Vito Fanelli, Bing Han, Julie Khang, Jianfeng Wu, Arthur S. Slutsky, Haibo Zhang

Abstract:

Introduction: MSC delivery in preclinical models of ARDS has demonstrated significant improvements in lung function and recovery from acute injury. However, the role of MSC delivery in ARDS associated pulmonary fibrosis is not well understood. Some animal studies using bleomycin, asbestos, and silica-induced pulmonary fibrosis show that MSC delivery can suppress fibrosis. While other animal studies using radiation induced pulmonary fibrosis, liver, and kidney fibrosis models show that MSC delivery can contribute to fibrosis. Hypothesis: The beneficial and deleterious effects of MSC in ARDS are modulated by the lung microenvironment at the time of MSC delivery. Methods: To induce ARDS a two-hit mouse model of Hydrochloric acid (HCl) aspiration (day 0) and mechanical ventilation (MV) (day 2) was used. HCl and injurious MV generated fibrosis within 14-28 days. 0.5x106 mouse MSCs were delivered (via both intratracheal and intravenous routes) either in the active inflammatory phase (day 2) or during the remodeling phase (day 14) of ARDS (mouse fibroblasts or PBS used as a control). Lung injury accessed using inflammation score and elastance measurement. Pulmonary fibrosis was accessed using histological score, tissue collagen level, and collagen expression. In addition alveolar epithelial (E) and mesenchymal (M) marker expression profile was also measured. All measurements were taken at day 2, 14, and 28. Results: MSC delivery 2 days after HCl exacerbated lung injury and fibrosis compared to HCl alone, while the day 14 delivery showed protective effects. However in the absence of HCl, MSC significantly reduced the injurious MV-induced fibrosis. HCl injury suppressed E markers and up-regulated M markers. MSC delivery 2 days after HCl further amplified M marker expression, indicating their role in myofibroblast proliferation/activation. While with 14-day delivery E marker up-regulation was observed indicating their role in epithelial restoration. Conclusions: Early MSC delivery can be protective of injurious MV. Late MSC delivery during repair phase may also aid in recovery. However, early MSC delivery during the exudative inflammatory phase of HCl-induced ARDS can result in pro-fibrotic profiles. It is critical to understand the interaction between MSC and the lung microenvironment before MSC-based therapies are utilized for ARDS.

Keywords: acute respiratory distress syndrome (ARDS), mesenchymal stem cells (MSC), hydrochloric acid (HCl), mechanical ventilation (MV)

Procedia PDF Downloads 649

Authors: H. M. Imam, H. M. Rezk, A. F. Tohamy

Abstract:

Background: Human umbilical cord blood (HUCB) is considered as a unique source for stem cells. HUCB contain different types of progenitor cells which could differentiate into hepatocytes. Aims: To investigate the potential of rat's liver damage repair using human umbilical cord mesenchymal stem cells (hUCMSCs). We investigated the feasibility for hUCMSCs in recovery from liver damage. Moreover, investigating fibrotic liver repair and using the CCl4-induced model for liver damage in the rat. Methods: Rats were injected with 0.5 ml/kg CCl4 to induce liver damage and progressive liver fibrosis. hUCMSCs were injected into the rats through the tail vein; Stem cells were transplanted at a dose of 1×106 cells/rat after 72 hours of CCl4 injection without receiving any immunosuppressant. After (6 and 8 weeks) of transplantation, blood samples were collected to assess liver functions (ALT, AST, GGT and ALB) and level of Procollagen III as a liver fibrosis marker. In addition, hepatic tissue regeneration was assessed histopathologically and immunohistochemically using antihuman monoclonal antibodies against CD34, CK19 and albumin. Results: Biochemical and histopathological analysis showed significantly increased recovery from liver damage in the transplanted group. In addition, HUCB stem cells transdifferentiated into functional hepatocytes in rats with hepatic injury which results in improving liver structure and function. Conclusion: Our findings suggest that transplantation of hUCMSCs may be a novel therapeutic approach for treating liver fibrosis. Therefore, hUCMSCs are a potential option for treatment of liver cirrhosis.

Keywords: carbon tetra chloride, liver fibrosis, mesenchymal stem cells, rat

Procedia PDF Downloads 313

Authors: Abir O. El Sadik

Abstract:

Introduction: Wound healing involves the interaction of multiple biological processes among different types of cells, intercellular matrix and specific signaling factors producing enhancement of cell proliferation of the epidermis over dermal granulation tissue. Several studies investigated multiple strategies to promote wound healing and to minimize infection and fluid losses. However, burn crisis, and its related morbidity and mortality are still elevated. The aim of the present study was to examine the effects of mesenchymal stem cells (MSCs) in accelerating wound healing and to compare the most efficient route of administration of MSCs, either intradermal or systemic injection, with focusing on the mechanisms producing epidermal and dermal cell regeneration. Material and methods: Forty-two adult male Sprague Dawley albino rats were divided into three equal groups (fourteen rats in each group): control group (group I); full thickness surgical skin wound model, Group II: Wound treated with systemic injection of MSCs and Group III: Wound treated with intradermal injection of MSCs. The healing ulcer was examined on day 2, 6, 10 and 15 for gross morphological evaluation and on day 10 and 15 for fluorescent, histological and immunohistochemical studies. Results: The wounds of the control group did not reach complete closure up to the end of the experiment. In MSCs treated groups, better and faster healing of wounds were detected more than the control group. Moreover, the intradermal route of administration of stem cells increased the rate of healing of the wounds more than the systemic injection. In addition, the wounds were found completely healed by the end of the fifteenth day of the experiment in all rats of the group injected intradermally. Microscopically, the wound areas of group III were hardly distinguished from the adjacent normal skin with complete regeneration of all skin layers; epidermis, dermis, hypodermis and underlying muscle layer. Fully regenerated hair follicles and sebaceous glands in the dermis of the healed areas surrounded by different arrangement of collagen fibers with a significant increase in their area percent were recorded in this group more than in other groups. Conclusion: MSCs accelerate the healing process of wound closure. The route of administration of MSCs has a great influence on wound healing as intradermal injection of MSCs was more effective in enhancement of wound healing than systemic injection.

Keywords: intradermal, mesenchymal stem cells, morphology, skin wound, systemic injection

Procedia PDF Downloads 174

Authors: Iman M. Mansour, Rania A. Zayed, Fadwa S. Abdel-Azim, Lamyaa H. Abdel-Latif

Abstract:

Background and Objectives: The microenvironment of acute myeloid leukemia (AML) is suppressive for immune cells. Regulatory T cells (Tregs) have been recognized to play a role in helping leukemic cells to evade immunesurveillance. The mesenchymal stem cells (MSCs) are essential contributors in immunomodulation of the microenvironment as they can promote differentiation of Tregs via the indoleamine 2,3-dioxygenase (IDO) pathway. The aim of the present work was to evaluate the expression of IDO in bone marrow derived MSCs and to study its correlation to percentage of Tregs. Methods: 37 adult bone marrow samples were cultured in appropriate culture medium to isolate MSCs. Successful harvest of MSCs was determined by plastic adherence, morphology and positive expression of CD271 and CD105; negative expression of CD34 and CD45 using flowcytometry. MSCs were examined for IDO expression by immunocytochemistry using anti-IDO monoclonal antibody. CD4+ CD25+ cells (Tregs) were measured in bone marrow samples by flowcytometry. Results: MSCs were successfully isolated from 20 of the 37 bone marrow samples cultured. MSCs showed higher expression of IDO and Tregs percentage was higher in AML patients compared to control subjects (p=0.002 and p<0.001 respectively). A positive correlation was found between IDO expression and Tregs percentage (p value=0.012, r=0.5). Conclusion: In this study, we revealed an association between high IDO expression in MSCs and elevated levels of Tregs which has an important role in the pathogenesis of AML, providing immunosuppressive microenvironment.

Keywords: acute myeloid leukemia, indoleamine 2, 3-dioxygenase, mesenchymal stem cells, T regulatory cells

Procedia PDF Downloads 328

Authors: A. Reum Son, Jin Seon Kwon, Seung Hun Park, Hai Bang Lee, Moon Suk Kim

Abstract:

These days, stem cell therapy is focused on for promising source of treatment in clinical human disease. As a supporter of stem cells, in situ-forming hydrogels with growth factors and cells appear to be a promising approach in tissue engineering. To examine osteogenic differentiation of hTMSCs which is one of mesenchymal stem cells in vivo in an injectable hydrogel, we use a methoxy polyethylene glycol-polycaprolactone blockcopolymer (MPEG-PCL) solution with osteogenic factors. We synthesized MPEG-PCL hydrogel and measured viscosity to check sol-gel transition. In order to demonstrate osteogenic ability of hTMSCs, we conducted in vitro osteogenesis experiment. Then, to confirm the cell cytotoxicity, we performed WST-1 with hTMSCs and MPEG-PCL. As the result of in vitro experiment, we implanted cell and hydrogel mixture into animal model and checked degree of osteogenesis with histological analysis and amount of expression genes. Through these experimental data, MPEG-PCL hydrogel has sol-gel transition in temperature change and is biocompatible with stem cells. In histological analysis and gene expression, hTMSCs are very good source of osteogenesis with hydrogel and will use it to tissue engineering as important treatment method. hTMSCs could be a good adult stem cell source for usability of isolation and high proliferation. When hTMSCs are used as cell therapy method with in situ-formed hydrogel, they may provide various benefits like a noninvasive alternative for bone tissue engineering applications.

Keywords: injectable hydrogel, stem cell, osteogenic differentiation, tissue engineering

Procedia PDF Downloads 421

Authors: Arefeh Jafarian, Mohammad Taghikani, Saied Abroun, Amir Allahverdi, Masoud Soleimani

Abstract:

Introduction: The miRNAs have key roles in control of pancreatic islet development and insulin secretion. In this regards, current study investigated the pancreatic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) by up-regulation of miR-375 and down-regulation of miR-9 by lentiviruses containing miR-375 and anti-miR-9. Findings: After 21 days of induction, islet-like clusters containing insulin producing cells (IPCs) were confirmed by dithizone (DTZ) staining. The IPCs and β cell specific related genes and proteins were detected using qRT-PCR and immunofluorescence on days 7, 14 and 21 of differentiation. Glucose challenge test was performed at different concentrations of glucose as well as extracellular and intracellular insulin and C-peptide were assayed using ELISA kit. In derived IPCs by miR-375 alone are capable to express insulin and other endocrine specific transcription factors, the cells lack the machinery to respond to glucose. The differentiated hMSCs by miR-375 and anti-miR-9 lentiviruses could secrete insulin and c-peptide in a glucose-regulated manner. Conclusion: It was found that over-expression of miR-375 led to a reduction in levels of Mtpn protein in derived IPCs, while treatment with anti-miR-9 following miR-375 over-expression had synergistic effects on MSCs differentiation and insulin secretion in a glucose-regulated manner. The researchers reported that silencing of miR-9 increased OC-2 protein in IPCs that may contribute to the observed glucose-regulated insulin secretion. These findings highlight miRNAs functions in stem cells differentiation and suggest that they could be used as therapeutic tools for gene-based therapy in diabetes mellitus.

Keywords: diabetes, differentiation, MSCs, insulin producing cells, miR-375, miR-9

Procedia PDF Downloads 289

Authors: K. J. Keerthi, Vasundhara Kamineni, A. Ravi Shanker, T. Rammurthy, A. Vijaya Lakshmi, Q. Hasan

Abstract:

Pancreatic β-cells are the predominant insulin-producing cell types within the Islets of Langerhans and insulin is the primary hormone which regulates carbohydrate and fat metabolism. Apoptosis of β-cells or insufficient insulin production leads to Diabetes Mellitus (DM). Current therapy for diabetes includes either medical management or insulin replacement and regular monitoring. Replacement of β- cells is an attractive treatment option for both Type-1 and Type-2 DM in view of the recent paper which indicates that β-cells apoptosis is the common underlying cause for both the Types of DM. With the development of Edmonton protocol, pancreatic β-cells allo-transplantation became possible, but this is still not considered as standard of care due to subsequent requirement of lifelong immunosuppression and the scarcity of suitable healthy organs to retrieve pancreatic β-cell. Fetal pancreatic cells from abortuses were developed as a possible therapeutic option for Diabetes, however, this posed several ethical issues. Hence, in the present study Mesenchymal stem cells (MSCs) were differentiated into insulin producing cells which were isolated from Human Umbilical cord (HUC) tissue. MSCs have already made their mark in the growing field of regenerative medicine, and their therapeutic worth has already been validated for a number of conditions. HUC samples were collected with prior informed consent as approved by the Institutional ethical committee. HUC (n=26) were processed using a combination of both mechanical and enzymatic (collagenase-II, 100 U/ml, Gibco ) methods to obtain MSCs which were cultured in-vitro in L-DMEM (Low glucose Dulbecco's Modified Eagle's Medium, Sigma, 4.5 mM glucose/L), 10% FBS in 5% CO2 incubator at 37°C. After reaching 80-90% confluency, MSCs were characterized with Flowcytometry and Immunocytochemistry for specific cell surface antigens. Cells expressed CD90+, CD73+, CD105+, CD34-, CD45-, HLA-DR-/Low and Vimentin+. These cells were differentiated to β-cells by using H-DMEM (High glucose Dulbecco's Modified Eagle's Medium,25 mM glucose/L, Gibco), β-Mercaptoethanol (0.1mM, Hi-Media), basic Fibroblast growth factor (10 µg /L,Gibco), and Nicotinamide (10 mmol/L, Hi-Media). Pancreatic β-cells were confirmed by positive Dithizone staining and were found to be functionally active as they released 8 IU/ml insulin on glucose stimulation. Isolating MSCs from usually discarded, abundantly available HUC tissue, expanding and differentiating to β-cells may be the most feasible cell therapy option for the millions of people suffering from DM globally.

Keywords: diabetes mellitus, human umbilical cord, mesenchymal stem cells, differentiation

Procedia PDF Downloads 234

Authors: Bhawna Chandravanshi, Ramesh Bhonde

Abstract:

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 48h 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 facilitate the 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. 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, oxidative stress, and inflammatory cytokines by inhibiting p38 MAPK protein.

Keywords: hypoxia, islet-like cell aggregates, inflammatory cytokines, oxidative stress

Procedia PDF Downloads 235

Authors: Reva Sharan Thakur, Mrinalini Tiwari, Jyoti das

Abstract:

Cerebral malaria-associated over expression of pro-inflammatory cytokines and chemokines ultimately results in the up-regulation of adhesion molecules in the brain endothelium leading to sequestration of mature parasitized RBCs in the brain. The high-parasitic load subsequently results in increased mortality or development of neurological symptoms within a week of infection. Studies in the human and experimental cerebral malaria have implicated the breakdown of the integrity of blood-brain barrier during the lethal course of infection, cerebral dysfunction, and fatal organ pathologies that result in multi-organ failure. In the present study, using Plasmodium berghei Anka as a mouse model and in vitro conditions, we have investigated the effect of MSCs to attenuate cerebral malaria pathogenesis by diminishing the effect of inflammation altered organ morphology, reduced parasitemia, and increased survival of the mice. MSCs are also validated for their role in preventing BBB dysfunction and reducing malarial toxins. It was observed that administration of MSCs significantly reduced parasitemia and increased survival in Pb A infected mice. It was further demonstrated that MSCs play a significant role in reversing neurological complexities associated with cerebral malaria. Infusion of MSCs in infected mice decreased hemozoin deposition; oedema, and haemorrhagic lesions in vascular organs. MSCs administration also preserved the integrity of the blood-brain barrier and reduced neural inflammation. Taken together, our results demonstrate the potential of MSCs as an emerging anti-malarial candidate.

Keywords: cerebral malaria, mesenchymal stem cells, erythropoesis, cell death

Procedia PDF Downloads 78

Authors: Carmela Nardelli, Laura Iaffaldano, Valentina Capobianco, Antonietta Tafuto, Maddalena Ferrigno, Angela Capone, Giuseppe Maria Maruotti, Maddalena Raia, Rosa Di Noto, Luigi Del Vecchio, Pasquale Martinelli, Lucio Pastore, Lucia Sacchetti

Abstract:

Maternal obesity and nutrient excess in utero increase the risk of future metabolic diseases in the adult life. The mechanisms underlying this process are probably based on genetic, epigenetic alterations and changes in foetal nutrient supply. In mammals, the placenta is the main interface between foetus and mother, it regulates intrauterine development, modulates adaptive responses to sub optimal in uterus conditions and it is also an important source of human amniotic mesenchymal stem cells (hA-MSCs). We previously highlighted a specific microRNA (miRNA) profiling in amnion from obese (Ob) pregnant women, here we compared the miRNA expression profile of hA-MSCs isolated from (Ob) and control (Co) women, aimed to search for any alterations in metabolic pathways that could predispose the new-born to the obese phenotype. Methods: We isolated, at delivery, hA-MSCs from amnion of 16 Ob- and 7 Co-women with pre-pregnancy body mass index (mean/SEM) 40.3/1.8 and 22.4/1.0 kg/m2, respectively. hA-MSCs were phenotyped by flow cytometry. Globally, 384 miRNAs were evaluated by the TaqMan Array Human MicroRNA Panel v 1.0 (Applied Biosystems). By the TargetScan program we selected the target genes of the miRNAs differently expressed in Ob- vs Co-hA-MSCs; further, by KEGG database, we selected the statistical significant biological pathways. Results: The immunophenotype characterization confirmed the mesenchymal origin of the isolated hA-MSCs. A large percentage of the tested miRNAs, about 61.4% (232/378), was expressed in hA-MSCs, whereas 38.6% (146/378) was not. Most of the expressed miRNAs (89.2%, 207/232) did not differ between Ob- and Co-hA-MSCs and were not further investigated. Conversely, 4.8% of miRNAs (11/232) was higher and 6.0% (14/232) was lower in Ob- vs Co-hA-MSCs. Interestingly, 7/232 miRNAs were obesity-specific, being expressed only in hA-MSCs isolated from obese women. Bioinformatics showed that these miRNAs significantly regulated (P<0.001) genes belonging to several metabolic pathways, i.e. MAPK signalling, actin cytoskeleton, focal adhesion, axon guidance, insulin signaling, etc. Conclusions: Our preliminary data highlight an altered miRNA profile in Ob- vs Co-hA-MSCs and suggest that an epigenetic miRNA-based mechanism of gene regulation could affect pathways involved in placental growth and function, thereby potentially increasing the newborn’s risk of metabolic diseases in the adult life.

Keywords: hA-MSCs, obesity, miRNA, biosystem

Procedia PDF Downloads 493

Authors: M. Karzhauov, А. Mukhambetova, M. Sarsenova, E. Raimagambetov, V. Ogay

Abstract:

Regeneration of injured articular cartilage remains one of the most difficult and unsolved problems in traumatology and orthopedics. Currently, for the treatment of cartilage defects surgical techniques for stimulation of the regeneration of cartilage in damaged joints such as multiple microperforation, mosaic chondroplasty, abrasion and microfractures is used. However, as shown by clinical practice, they can not provide a full and sustainable recovery of articular hyaline cartilage. In this regard, the current high hopes in the regeneration of cartilage defects reasonably are associated with the use of tissue engineering approaches to restore the structural and functional characteristics of damaged joints using stem cells, growth factors and biopolymers or scaffolds. The purpose of the present study was to investigate the effects of chondroinductive growth factors and synovium-derived mesenchymal stem cells (SD-MSCs) on the regeneration of cartilage defects in rabbits. SD-MSCs were isolated from the synovium membrane of Flemish giant rabbits, and expanded in complete culture medium α-MEM. Rabbit SD-MSCs were characterized by CFU-assay and by their ability to differentiate into osteoblasts, chondrocytes and adipocytes. The effects of growth factors (TGF-β1, BMP-2, BMP-4 and IGF-I) on MSC chondrogenesis were examined in micromass pellet cultures using histological and biochemical analysis. Articular cartilage defect (4mm in diameter) in the intercondylar groove of the patellofemoral joint was performed with a kit for the mosaic chondroplasty. The defect was made until subchondral bone plate. Delivery of SD-MSCs and growth factors was conducted in combination with hyaloronic acid (HA). SD-MSCs, growth factors and control groups were compared macroscopically and histologically at 10, 30, 60 and 90 days aftrer intra-articular injection. Our in vitro comparative study revealed that TGF-β1 and BMP-4 are key chondroinductive factors for both the growth and chondrogenesis of SD-MSCs. The highest effect on MSC chondrogenesis was observed with the synergistic interaction of TGF-β1 and BMP-4. In addition, biochemical analysis of the chondrogenic micromass pellets also revealed that the levels of glycosaminoglycans and DNA after combined treatment with TGF-β1 and BMP-4 was significantly higher in comparison to individual application of these factors. In vivo study showed that for complete regeneration of cartilage defects with intra-articular injection of SD-MSCs with HA takes time 90 days. However, single injection of SD-MSCs in combiantion with TGF-β1, BMP-4 and HA significantly promoted regeneration rate of the cartilage defects in rabbits. In this case, complete regeneration of cartilage defects was observed in 30 days after intra-articular injection. Thus, our in vitro and in vivo study demonstrated that combined application of rabbit SD-MSC with chondroinductive growth factors and HA results in strong synergistic effect on the chondrogenesis significantly enhancing regeneration of the damaged cartilage.

Keywords: Mesenchymal stem cells, synovium, chondroinductive factors, TGF-β1, BMP-2, BMP-4, IGF-I

Procedia PDF Downloads 275

Authors: Hai-Ying Liu, Yi-Hao Chen, Shu-Yin Pang, Feng-Hua Wang, Xiao-Fang Peng, Li-Yuan Yang, Zheng-Rong Chen, Yi Chen, Bing Zhu

Abstract:

Objective: Biliary atresia (BA) is characterized by progressive liver fibrosis. Epithelial-mesenchymal transition (EMT) has been implicated as a key mechanism in the pathogenesis of organ fibrosis. MiR-200a has been shown to repress EMT. We aim to explore the role of miR-200a in the fibrogenesis of BA. Methods: We obtained the plasma samples and liver samples from patients with BA or controls to examine the role of miR-200a. Histological liver fibrosis was assessed using the Ishak fibrosis scores. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to detect the expression of miR-200a in plasma. We also evaluated the expression of miR-200a in liver tissues using tyramide signal amplification fluorescence in situ hybridization (TSA-FISH). The expression of EMT related proteins zinc finger E-box-binding homeobox 1 (ZEB1), E-cadherin and α-smooth muscle actin (α-SMA) in the liver sections were detected by immunohistochemical staining. Results: We found that the expression of miR-200a was both elevated in the plasma and liver tissues from BA patients compared with the controls. The hepatic expression of ZEB1 and α-SMA were markedly increased in the liver sections from BA patients compared to the controls, whereas E-cadherin was downregulated in the BA group. Simultaneously, we noted that the hepatic expression of miR-200a, E-cadherin and α-SMA were upregulated with the progression of liver fibrosis in the BA group, while ZEB1 was downregulated with the progression of liver fibrosis in BA patients. Conclusion: These findings suggest EMT has a critical effect on the fibrotic process of BA, and the interaction between miR-200a and ZEB1 may regulate EMT and eventually influence liver fibrogenesis of BA.

Keywords: biliary atresia, liver fibrosis, MicroRNA, epithelial-mesenchymal transition, zinc finger E-box-binding homeobox 1

Procedia PDF Downloads 332

Authors: Ainur Mukhambetova, Miras Karzhauov, Vyacheslav Ogay

Abstract:

Introduction: Mesenchymal stem cells (MSCs) have the capacity to be differentiated into several cell lineages and are a promising source for cell therapy and tissue engineering. However, currently most MSCs culturing protocols use media supplemented with fetal bovine serum (FBS), which limits their application in clinic due to the possibility of zoonotic infections, contamination and immunological reactions. Consequently, formulating effective serum free culture medium becomes one of the important problems in contemporary cell biotechnology. Objectives: The aim of this study was to define an optimal serum-free medium for culturing of periosteum derived MSCs. Materials and methods: The MSCs were extracted from human periosteum and transferred to the culture flasks pretreated with CELLstart™. Immunophenotypic characterization, proliferation and in vitro differentiation of cells grown on STEM PRO® MSC SFM were compared to the cells cultured in the standard FBS containing media. Chromosome analysis and flow cytometry were also performed. Results: We have shown that cells were grown on STEM PRO® MSC SFM retained all the morphological, immunophenotypic (CD73, CD90, CD105, vimentin and Stro-1) and cell differentiation characteristics specific to MSCs. Chromosome analysis indicated no anomalies in the chromosome structure. Flow cytometry showed a high expression of cell adhesion molecules CD44 (98,8%), CD90 (97,4%), CD105 (99,1%). In addition, we have shown that cell is grown on STEM PRO® MSC SFM have higher proliferation capacity compared to cell expanded on standard FBS containing the medium. Conclusion: We have shown that STEM PRO® MSC SFM is optimal for culturing periosteum derived human MSCs which subsequently can be safely used in cell therapy.

Keywords: cell technologies, periosteum-derived MSCs, regenerative medicine, serum-free medium

Procedia PDF Downloads 273

Authors: Bing–Jing Zhao, Chang-Kui Liu, Hong Wang, Min Hu

Abstract:

Electron beam rapid manufacturing (EBRM) or Selective laser melting is an additive manufacturing process that uses 3D CAD data as a digital information source and energy in the form of a high-power laser beam or electron beam to create three-dimensional metal parts by fusing fine metallic powders together.Object:The present study was conducted to evaluate the mechanical properties ,the phase transformation,the corrosivity and the biocompatibility of Ti-6Al-4V by EBRM,SLM and forging technique.Method: Ti-6Al-4V alloy standard test pieces were manufactured by EBRM, SLM and forging technique according to AMS4999,GB/T228 and ISO 10993.The mechanical properties were analyzed by universal test machine. The phase transformation was analyzed by X-ray diffraction and scanning electron microscopy. The corrosivity was analyzed by electrochemical method. The biocompatibility was analyzed by co-culturing with mesenchymal stem cell and analyzed by scanning electron microscopy (SEM) and alkaline phosphatase assay (ALP) to evaluate cell adhesion and differentiation, respectively. Results: The mechanical properties, the phase transformation, the corrosivity and the biocompatibility of Ti-6Al-4V by EBRM、SLM were similar to forging and meet the mechanical property requirements of AMS4999 standard. a­phase microstructure for the EBM production contrast to the a’­phase microstructure of the SLM product. Mesenchymal stem cell adhesion and differentiation were well. Conclusion: The property of the Ti-6Al-4V alloy manufactured by EBRM and SLM technique can meet the medical standard from this study. But some further study should be proceeded in order to applying well in clinical practice.

Keywords: 3D printing, Electron Beam Rapid Manufacturing (EBRM), Selective Laser Melting (SLM), Computer Aided Design (CAD)

Procedia PDF Downloads 431

Authors: S. AliReza Mirghasemi, Zameer Hussain, Mohammad Saleh Sadeghi, Narges Rahimi Gabaran, Mohamadreza Baghaban Eslaminejad

Abstract:

Background: Despite promising results have shown by osteogenic cell-based demineralized bone matrix composites, they need to be optimized for grafts that act as structural frameworks in load-bearing defects. The purpose of this experiment is to determine the effect of bone-marrow-mesenchymal-stem-cells seeding on partially demineralized laser-perforated structural allografts that have been implanted in critical femoral defects. Materials and Methods: P3 stem cells were used for graft seeding. Laser perforation in four rows of three holes was achieved. Cell-seeded grafts were incubated for one hour until they were planted into the defect. We used four types of grafts: partially demineralized only (Donly), partially demineralized stem cell seeded (DST), partially demineralized laser-perforated (DLP), and partially demineralized laser-perforated stem cell seeded (DLPST). histologic and histomorphometric analysis were performed at 12 weeks. Results: Partially demineralized laser-perforated had the highest woven bone formation within graft limits, stem cell seeded demineralized laser-perforated remained intact, and the difference between partially demineralized only and partially demineralized stem cell seeded was insignificant. At interface, partially demineralized laser-perforated and partially demineralized only had comparable osteogenesis, but partially demineralized stem cell seeded was inferior. The interface in stem cell seeded demineralized laser-perforated was almost replaced by distinct endochondral osteogenesis with higher angiogenesis in the vicinity. Partially demineralized stem cell seeded and stem cell seeded demineralized laser-perforated graft surfaces had extra vessel-ingrowth-like porosities, a sign of delayed resorption. Conclusion: This demonstrates that simple cell-based composites are not optimal and necessitates the supplementation of synergistic stipulations and surface changes.

Keywords: structural bone allograft, partial demineralization, laser perforation, mesenchymal stem cell

Procedia PDF Downloads 391

Authors: ZhenlinJu, Christopher P. Vellano, RehanAkbani, Yiling Lu, Gordon B. Mills

Abstract:

The epithelial–mesenchymal transition (EMT) is a process by which epithelial cells acquire mesenchymal characteristics, such as profound disruption of cell-cell junctions, loss of apical-basolateral polarity, and extensive reorganization of the actin cytoskeleton to induce cell motility and invasion. A hallmark of EMT is its capacity to promote metastasis, which is due in part to activation of several transcription factors and subsequent downregulation of E-cadherin. Unfortunately, current approaches have yet to uncover robust protein marker sets that can classify tumors as possessing strong EMT signatures. In this study, we utilize reverse phase protein array (RPPA) data and consensus clustering methods to successfully classify a subset of cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) tumors into an EMT protein signaling group (EMT group). The overall survival (OS) of patients in the EMT group is significantly worse than those in the other Hormone and PI3K/AKT signaling groups. In addition to a shrinkage and selection method for linear regression (LASSO), we applied training/test set and Monte Carlo resampling approaches to identify a set of protein markers that predicts the EMT status of CESC tumors. We fit a logistic model to these protein markers and developed a classifier, which was fixed in the training set and validated in the testing set. The classifier robustly predicted the EMT status of the testing set with an area under the curve (AUC) of 0.975 by Receiver Operating Characteristic (ROC) analysis. This method not only identifies a core set of proteins underlying an EMT signature in cervical cancer patients, but also provides a tool to examine protein predictors that drive molecular subtypes in other diseases.

Keywords: consensus clustering, TCGA CESC, Silhouette, Monte Carlo LASSO

Procedia PDF Downloads 437

Authors: Brian M. Mehling, Louis Quartararo, Marine Manvelyan, Paul Wang, Dong-Cheng Wu

Abstract:

Numerous investigations suggest that Mesenchymal Stem Cells (MSCs) in general represent a valuable tool for therapy of symptoms related to chronic inflammatory diseases. Blue Horizon Stem Cell Therapy Program is a leading provider of adult and children’s stem cell therapies. Uniquely we have safely and efficiently treated more than 600 patients with documenting each procedure. The purpose of our study is primarily to monitor the immune response in order to validate the safety of intravenous infusion of human umbilical cord blood derived MSCs (UC-MSCs), and secondly, to evaluate effects on biomarkers associated with chronic inflammation. Nine patients were treated for conditions associated with chronic inflammation and for the purpose of anti-aging. They have been given one intravenous infusion of UC-MSCs. Our study of blood test markers of 9 patients with chronic inflammation before and within three months after MSCs treatment demonstrates that there is no significant changes and MSCs treatment was safe for the patients. Analysis of different indicators of chronic inflammation and aging included in initial, 24-hours, two weeks and three months protocols showed that stem cell treatment was safe for the patients; there were no adverse reactions. Moreover data from follow up protocols demonstrates significant improvement in energy level, hair, nails growth and skin conditions. Intravenously administered UC-MSCs were safe and effective in the improvement of symptoms related to chronic inflammation. Further close monitoring and inclusion of more patients are necessary to fully characterize the advantages of UC-MSCs application in treatment of symptoms related to chronic inflammation.

Keywords: chronic inflammatory diseases, intravenous infusion, stem cell therapy, umbilical cord blood derived mesenchymal stem cells (UC-MSCs)

Procedia PDF Downloads 403

Authors: Afzaal Bashir, Sunaina Afzaal

Abstract:

Background: Facial contour deformities associated with pigmentary changes are of major concern for plastic surgeons, both being important and difficult in treating such issues. No definite ideal treatment option is available to simultaneously address both the contour defect as well as related pigmentation. Objectives: The aim of the current study is to compare the long-term effects of conventional adipose tissue grafting and ex-vivo expanded Mesenchymal Stem Cells enriched adipose tissue grafting for the treatment of contour deformities with pigmentary changes on the face. Material and Methods: In this study, eighty (80) patients with contour deformities of the face with hyperpigmentation were recruited after informed consent. Two techniques i.e., conventional fat grafting (C-FG) and fat grafts enriched with expanded adipose stem cells (FG-ASCs), were used to address the pigmentation. Both techniques were explained to patients, and enrolled patients were divided into two groups i.e., C-FG and FG-ASCS, per patients’ choice and satisfaction. Patients of the FG-ASCs group were treated with fat grafts enriched with expanded adipose stem cells, while patients of the C-FGs group were treated with conventional fat grafting. Patients were followed for 12 months, and improvement in face pigmentation was assessed clinically as well as measured objectively. Patient satisfaction was also documented as highly satisfied, satisfied, and unsatisfied. Results: Mean age of patients was 24.42(±4.49), and 66 patients were females. The forehead was involved in 61.20% of cases, the cheek in 21.20% of cases, the chin in 11.20% of cases, and the nose in 6.20% of cases. In the GF-ASCs group, the integrated color density (ICD) was decreased (1.08×10⁶ ±4.64×10⁵) as compared to the C-FG group (2.80×10⁵±1.69×10⁵). Patients treated with fat grafts enriched with expanded adipose stem cells were significantly more satisfied as compared to patients treated with conventional fat grafting only. Conclusion: Mesenchymal stem cell-enriched autologous fat grafting is a preferred option for improving the contour deformities related to increased pigmentation of face skin.

Keywords: hyperpigmentation, color density, enriched adipose tissue graft, fat grafting, contour deformities, Image J

Procedia PDF Downloads 70

Authors: A. Bajek, J. Skopinska-Wisniewska, A. Rynkiewicz, A. Jundzill, M. Bodnar, A. Marszalek, T. Drewa

Abstract:

Traumatic injury and age-related degenerative diseases associated with cartilage are major health problems worldwide. The articular cartilage is comprised of a relatively small number of cells, which have a relatively slow rate of turnover. Therefore, damaged articular cartilage has a limited capacity for self-repair. New clinical methods have been designed to achieve better repair of injured cartilage. However, there is no treatment that enables full restoration of it. The aim of this study was to evaluate how collagen gel with bone marrow mesenchymal stem cells (MSCs) and collagen gel alone will influence on the hip cartilage repair after injury. Collagen type I was isolated from rats’ tails and cross-linked with N-hydroxysuccinimide in 24-hour process. MSCs were isolated from rats’ bone marrow. The experiments were conducted according to the guidelines for animal experiments of Ethics Committee. Fifteen 8-week-old Wistar rats were used in this study. All animals received hip joint surgery with a total of 30 created cartilage defects. Then, animals were randomly divided into three groups and filled, respectively, with collagen gel (group 1), collagen gel cultured with MSCs (group II) or left untreated as a control (control group). Immunohistochemy and radiological evaluation was carried out 11 weeks post implantation. It has been proved that the surface of the matrix is non-toxic, and its porosity promotes cell adhesion and growth. However, the in vivo regeneration process was poor. We observed the low integration rate of biomaterial. Immunohistochemical evaluation of cartilage after 11 weeks of treatment showed low II and high X collagen expression in two tested groups in comparison to the control one, in which we observed the high II collagen expression. What is more, after radiological analysis, we observed the best regeneration process in control group. The biomaterial construct and mesenchymal stem cells, as well as the use of the biomaterial itself was not sufficient to regenerate the hip cartilage surfaces. These results suggest that the collagen gel based biomaterials, even with MSCs, are not satisfactory in repar of hip cartilage defect. However, additional evaluation is needed to confirm these results.

Keywords: collafen gel, MSCs, cartilage repair, hip cartilage

Procedia PDF Downloads 432

Authors: Pelin Balcik Ercin

Abstract:

Introduction: ZEB transcription factor family member ZEB2, has a role in epithelial to mesenchymal transition during development and metastasis. The altered circulating extracellular miRNAs expression is observed in diseases, and extracellular miRNAs have an important role in cancer cell microenvironment. In ChIP-Seq study, the expression of miR-2110 was found to be regulated by ZEB2. In this study, the effects of miR2110 on cell proliferation and migration of hepatocellular carcinoma (HCC) cells were examined. Material and Methods: SNU398 cells transfected with mimic miR2110 (20nM) (HMI0375, Sigma-Aldrich) and negative control miR (HMC0002, Sigma-Aldrich). MicroRNA isolation was accomplished with miRVANA isolation kit according to manufacturer instructions. cDNA synthesis was performed expression, respectively, and calibrated with Ct of controls. The real-time quantitative PCR (RT-qPCR) reaction was performed using the TaqMan Fast Advanced Master Mix (Thermo Sci.). Ct values of miR2110 were normalized to miR-186-5p and miR16-5p for the intracellular gene. Cell proliferation analysis was analyzed with the xCELLigence RTCA System. Wound healing assay was analyzed with the ImageJ program and relative fold change calculated. Results: The mimic-miR-2110 transfected SNU398 cells nearly nine-fold (log2) more miR-2110 expressed compared to negative control transfected cells. The mimic-miR-2110 transfected HCC cell proliferation significantly inhibited compared to the negative control cells. Furthermore, miR-2110-SNU398 cell migration capacity was relatively four-fold decreased compared to negative control-miR-SNU398 cells. Conclusion: Our results suggest the miR-2110 inhibited cell proliferation and also miR-2110 negatively affect cell migration compared to control groups in HCC cells. These data suggest the complexity of microRNA EMT transcription factors regulation. These initial results are pointed out the predictive biomarker capacity of miR-2110 in HCC.

Keywords: epithelial to mesenchymal transition, EMT, hepatocellular carcinoma cells, micro-RNA-2110, ZEB2

Procedia PDF Downloads 91