NF-κB Inhibitor Pyrrolidinedithiocarbamate Can Increase the Infection and Activation of Cytomegalovirus in Mesenchymal Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4948-4948
Author(s):  
Guoqing Wei ◽  
Maofang Lin ◽  
He Huang ◽  
Zhen Cai
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Calf Serum ◽  
Close Relation ◽  
Clinical Work ◽  
Rt Pcr ◽  
Cmv Infection ◽  
Pcr Assay ◽  
Pp65 Antigen

Abstract Objective Because of the central role of the transcription factor nuclear factor-κB (NF-κB) in cell survival and proliferation in many kinds of cancer cells, NF-κB inhibitor may have a potential role in the therapy of cancer. Human cytomegalovirus (CMV) infection is one of the most common complications following stem cells transplantation. Some data report that CMV infection has a very close relation with NF-κB activation. But it is unknown what effect of NF-κB inhibitor pyrrolidinedithiocarbamate (PTDC) on the infection and activation of cytomegalovirus in mesenchymal stem cells(MSCs). Methods MSCs were infected by 1 TCID50 of CMV combined with/without 1μmol/L of PTDC. After 48h of culture with DMEM supplemented with 10% (v/v) fetal calf serum, MSCs shapes were observed. RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene and GAPDH gene. Flow Cytometry was used to detect the CMV PP65 antigen positive cells. Results Shape of some MSCs changed after the infection of 1 TCID50 of CMV. But in MSCs infected by 1 TCID50 of CMV combined with 1μmol/L of PTDC, Cell shape changed more dominantly, almost all cells changed from thin shuttle shape to round and thick ball shape. While in the cells treated only with PDTC, shape of the cells did not changed. RT-PCR assay showed that there was a very bright band of CMV IE mRNA in MSCs infected by 1 TCID50 of CMV combined with 1μmol/L of PTDC compared with the cells infected only with 1 TCID50 of CMV. More CMV pp65 antigen positive cells were found in MSCs infected by 1 TCID50 of CMV combined with 1μmol/L of PTDC with Flow Cytometry. Conclusion NF-κB activation may affect CMV infection of cells. NF-κB inhibitor PTDC can increase the infection and activation of CMV in MSCs and we should pay more attention to CMV infection when we use NF-κB inhibitor in clinical work..

135 Strategies for transfection of bovine mesenchymal stem cells with pBC1-anti-CD3 vector

2020 ◽  
Vol 32 (2) ◽  
pp. 194
Author(s):  
F. B. Duarte ◽  
S. N. Báo ◽  
M. Brígido ◽  
J. M. Araújo ◽  
E. d. O. Melo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Calf Serum ◽  
Genetic Material ◽  
Cell Receptor ◽  
Wharton’S Jelly ◽  
Control Group ◽  
Wharton's Jelly ◽  
Transgenic Cells

Cells from different origins behave differently regarding the incorporation of exogenous genetic material and the formation of transgenic cells. In this context, the objective of this study was to verify the potential of transfection of bovine mesenchymal stem cells from Wharton's jelly and adipose tissue, comparing two transfection protocols, using Lipofectamine LTX and Plus or Xfect reagents, with the integration of humanized anti-CD3. Skin fibroblasts were used as a control group. Humanized anti-CD3 is a monoclonal antibody that interacts with the CD3 molecule of the T-cell receptor, leading to the suppression of T-cells. This antibody is considered an option in the treatment of human autoimmune diseases and against the rejection of transplanted organs. Humanized anti-CD3 was used in this work for the production of bovine transgenic cells that, in the future, will be used in the development of bioreactor animals. In all steps of this study, cell types were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) and antibiotics, in an incubator at 39°C with 5% CO2 in air with saturated humidity. All cells were plated at 5×105 into 24-well culture dishes and co-transfected with vector pBC1-anti-CD3-IRES-FEO and pEF-NEO-GFP using Lipofectamine LTX with reagent Plus or Xfect. Forty-eight hours after transfection, neomycin was added in each treatment and cells were cultured for 2 weeks. Treated cells were submitted to fluorescence microscopy, flow cytometry, and PCR evaluations. Wharton's jelly cells were sensitive to treatments and started necrosis. In the flow cytometry assay, the median fluorescence was higher in adipocytes than in fibroblasts, for both the Xfect reagent (20.057±1.620.7 and 10.601±702.86, respectively, P<0.05) and for LTX (19.590±113.84 and 10.518±442.65 respectively, P<0.05). These results, associated with the evaluation of epifluorescence, demonstrated that adipocytes presented a better response to transfection than did other cells, independent of the kit used. Performing PCR on co-transfected adipocytes and fibroblasts demonstrated the presence of anti-CD3, making this approach feasible in future experiments. Southern blotting analysis is being performed to confirm DNA integration. Financial support was provided by Fundação de Amparo à Pesquisa do Distrito Federal (FAPDF); Embrapa MP1.

Human Cytomegalovirus Can Induce Alteration of β-Actin mRNA and Microfilaments as Well as Induce Apoptosis in Mesenchymal Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4261-4261
Author(s):  
Guoqing Wei ◽  
Maofang Lin ◽  
Zhen Cai ◽  
He Huang
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Electron Microscope ◽  
Human Cytomegalovirus ◽  
Transmission Electron Microscope ◽  
Cmv Infection ◽  
Transmission Electron ◽  
Stem Cells Transplantation ◽  
Actin Mrna

Abstract Mesenchymal stem cells (MSCs) is a very important cell system in bone marrow stromal and human cytomegalovirus (CMV) infection is one of the most common complications following stem cells transplantation. Our studies were to investigate the cytoskeleton change as well as the apoptosis induction effects of CMV infection on MSCs. In our study, MSCs were infected by 100,10,1 TCID50 of CMV. After 48h of culture with DMEM supplemented with 10% (v/v) fetal calf serum, MSCs shapes were observed with microscope. CMV particles and ultra structure in the cells were detected with transmission electron microscope. RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene,β-actin gene and GAPDH genes. Flow Cytometry was used to detect the apoptotic cells. After the infection of CMV, shape of MSCs changed, turning from thin shuttle shape to round and thick ball shape, even escaping from wall. Cell shape changed more dominantly as the CMV titer increased. With transmission electron microscope, CMV particles and typical cellular apoptotic character as well as the ruptured- Microfilaments could be seen in the cells infected by CMV. MSCs infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time- and titer- dependent manner compared with the uninfected cells whose expression of GAPDH mRNA did not change much. With Flow Cytometry, it was found there were more apoptotic and necrosis cells in the cells infected by CMV. These results indicate that CMV can not only infect MSCs and destroy the cytoskeleton in the cells but also can induce apoptosis of the cells, much more attention of CMV infection on MSCs should be paid after stem cells transplantation.

Human Cytomegalovirus Can Activate and Induce Apoptosis in Mesenchymal Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4947-4947
Author(s):  
Guoqing Wei ◽  
Maofang Lin ◽  
He Huang ◽  
Zhen Cai
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Electron Microscope ◽  
Human Cytomegalovirus ◽  
Transmission Electron Microscope ◽  
Calf Serum ◽  
Cell System ◽  
Cmv Infection ◽  
Gapdh Gene ◽  
Transmission Electron

Abstract Objective Mesenchymal stem cells (MSCs) is a very important cell system in bone marrow stromal and human cytomegalovirus (CMV) infection is one of the most common complications following stem cells transplantation. Our studies were to investigate the CMV infection of MSCs as well as the apoptosis induction effects. Methods MSCs were infected by 100,10,1 TCID50 of CMV. After 48h of culture with DMEM supplemented with 10% (v/v) fetal calf serum, MSCs shapes were observed. CMV particles and ultra structure in the cells were detected with transmission electron microscope. RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene and GAPDH gene. Flow Cytometry was used to detect the apoptotic cells. Results After the infection of CMV, shape of MSCs changed, turning from thin shuttle shape to round and thick ball shape, even escaping from wall. Cell shape changed more dominantly as the CMV titer increased. With transmission electron microscope, CMV particles and typical cellular apoptotic character could be seen in the cells. MSCs infected by CMV could express IE mRNA compare with the uninfected and there were more apoptotic and necrosis cells in the cells infected by CMV. Conclusion CMV can not only infect MSCs and activate in the cells but also can induce apoptosis of the cells.

scholarly journals Isolation and characterization of mesenchymal stem cells derived from bovine Wharton's jelly and their potential for use in cloning by nuclear transfer

2016 ◽  
Vol 46 (10) ◽  
pp. 1830-1837 ◽  
Author(s):  
Carolina Gonzales da Silva ◽  
Carlos Frederico Martins ◽  
Tereza Cristina Cardoso ◽  
Elisa Ribeiro da Cunha ◽  
Heidi Christina Bessler ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Nuclear Transfer ◽  
Wharton’S Jelly ◽  
Skin Fibroblasts ◽  
Bovine Embryo ◽  
Rt Pcr ◽  
Wharton's Jelly ◽  
Isolation And Characterization

ABSTRACT: Wharton's jelly is a source of mesenchymal stem cells (MSCs) that had not yet been tested for bovine embryo production by nuclear transfer (NT). Thus, the objective of this study was to isolate, characterize and test MSCs derived from Wharton's jelly for embryo and pregnancy production by NT in cattle. The umbilical cord was collected during calving and cells derived from Wharton's jelly (WJCs) were isolated by explant and cultured in Dulbecco's Modified Eagle Medium. Skin Fibroblasts (FB) were isolated after 6 months of life. Morphological analysis was performed by bright field and scanning electron microscopy (SEM) during cell culture. Phenotypic and genotypic characterization by flow cytometry, immunocytochemistry, RT-PCR and differentiation induction in cell lineages were performed for WJC. In the NT procedure, oocytes at the arrested metaphase II stage were enucleated using micromanipulators, fused with WJCs or FB and later activated artificially. SEM micrographs revealed that WJCs have variable shape under culture. Mesenchymal markers of MSCs (CD29+, CD73+, CD90+ and CD105+) were expressed in bovine-derived WJC cultures, as evidenced by flow cytometry, immunocytochemistry and RT-PCR. When induced, these cells differentiated into osteocytes, chondrocytes and adipocytes. After classification, the WJCs were used in NT. Blastocyst formation rate by NT with WJCs at day 7 was 25.80±0.03%, similar to blatocyst rate with NT using skin fibroblasts (19.00±0.07%). Pregnancies were obtained and showed that WJCs constitute a new cell type for use in animal cloning.

Isolation and Characterization of Mesenchymal Stem Cells from Canine Ovarian Surface Epithelium

2021 ◽  
Author(s):  
Ajeet Kumar Jha ◽  
Anirban Mandal ◽  
Kalyani Ray ◽  
Shyamal Kanti Guha
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stromal Vascular Fraction ◽  
Ovarian Surface Epithelium ◽  
Surface Epithelium ◽  
High Plasticity ◽  
Rt Pcr ◽  
Differentiation Potential ◽  
Ovarian Surface ◽  
Isolation And Characterization

Background: Few studies have confirmed the presence of ovarian tissue stem cells indicating the capacity for differentiation. Based on this fact, it was hypothesized that mesenchymal stem cells (MSC) were found in ovarian surface epithelium (OSE) of canines that could easily be isolated. Methods: Both left and right ovaries were minced and digested using collagenase to obtain a stromal vascular fraction (SVF). MSCs were characterized using RT-PCR. To ascertain the trilineage differentiation potential, MSCs were stained with respective stain for osteocytes, chondrocytes and adipocytes. Result: We observed elongated, spindle-shaped and fibroblast like appearance of cells after 72 h of initial culture. Expression of MSC specific surface markers were observed through RT-PCR. Using Stem Pro® differentiation medium, OSE were differentiated into osteogenic, chondrogenic and adipogenic lineages and were found to be potential source for isolation, characterization and differentiation of MSCs. Canine (OSE) is easily accessible, multipotent and has high plasticity, holding promise for applications in regenerative medicine.

scholarly journals Effect of Placental Extract on Omentum Mesenchymal Stem Cells Differentiation in NMRI Mice

2017 ◽  
Vol 7 (1) ◽  
pp. 176
Author(s):  
Maryam Sadat Nezhadfazel ◽  
Kazem Parivar ◽  
Nasim Hayati Roodbari ◽  
Mitra Heydari Nasrabadi
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Flow Cytometry ◽  
Endothelial Cells ◽  
Mesenchymal Stem Cells ◽  
Control Group ◽  
Fat Cell ◽  
Nmri Mice ◽  
Differentiated Cells ◽  
Placental Extract

Omentum mesenchymal stem cells (OMSCs) could be induced to differentiate into cell varieties under certain conditions. We studied differentiation of OMSCs induced by using placenta extract in NMRI mice. Mesenchymal stem cells (MSCs) were isolated from omentum and cultured with mice placenta extract. MSCs, were assessed after three passages by flow cytometry for CD90, CD44, CD73, CD105, CD34 markers and were recognized their ability to differentiate into bone and fat cell lines. Placenta extract dose was determined with IC50 test then OMSCs were cultured in DMEM and 20% placenta extract.The cell cycle was checked. OMSCs were assayed on 21 days after culture and differentiated cells were determined by flow cytometry and again processed for flow cytometry. CD90, CD44, CD73, CD105 markers were not expressed, only CD34 was their marker. OMSCs were morphologically observed. Differentiated cells are similar to the endothelial cells. Therefore, to identify differentiated cells, CD31 and FLK1 expression were measured. This was confirmed by its expression. G1 phase of the cell cycle shows that OMSCs compared to the control group, were in the differentiation phase. The reason for the differentiation of MSCs into endothelial cells was the sign of presence of VEGF factor in the medium too high value of as a VEGF secreting source.

scholarly journals Characterization of neuronal precursors obtained from human adipose-derived mesenchymal stem cells

2021 ◽  
Vol 31 (Supplement_2) ◽  
Author(s):  
N B Oliveira ◽  
A C Irioda ◽  
P E F Stricker ◽  
B F Mogharbel ◽  
N N Rosa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Neural Cells ◽  
High Plasticity ◽  
Rt Pcr ◽  
Isolated Cells ◽  
Neural Precursors ◽  
Hla Dr ◽  
Neuronal Precursors ◽  
Neural Growth

Abstract Background Mesenchymal stem cells (MSCs) can be isolated from any tissue derived from the mesoderm and have as main characteristics: high plasticity, the ability to originate mesodermal and non-mesodermal tissues, acting in the modulation of the inflammatory response, and the tissue repair. When grown in microenvironments with elasticity comparable to the human brain, these cells can differentiate efficiently in neural cells due to the mechanism related to the YAP protein, which can mediate responses to substrate stiffness in mesenchymal stem cells. Methods Human adipose-derived MSCs were isolated*, then it was done the trilineage test into adipocytes, osteocytes and, chondrocytes. Besides that, differentiation to neural precursor cells was through neurospheres after seeding the cells over a natural biopolymer matrix as NFBX. Those cells were analyzed using flow cytometry for the surface markers CD13, CD34, CD45, CD73, CD90, CD105, HLA-DR, HLA-ABC, immunocytochemistry for the proteins Nestina, ß-tubulin III, YAP and AMOT and RT-PCR for the NEFM and TUBB3 genes. Results Isolated cells demonstrated characteristics of MSCs. Those cells were differentiated in neural precursors, expressing the proteins Nestina and ß-tubulin III on immunocytochemistry and, the NEFM and TUBB3 genes in RT-PCR. Regarding the YAP and AMOT proteins, it was possible to observe the translocation of the YAP protein in response to the regulation of AMOT out of the cell nucleus, proving neurodifferentiation. Conclusions Human adipose-derived MSCs seeded in a natural biopolymer matrix were able to differentiate into neural precursors expressing characteristic neural markers without adding any neural growth factors or genetic induction.

scholarly journals Effect of Silicon, Titanium, and Zirconium Ion Implantation on NiTi Biocompatibility

2012 ◽  
Vol 2012 ◽  
pp. 1-16 ◽  
Author(s):  
L. L. Meisner ◽  
A. I. Lotkov ◽  
V. A. Matveeva ◽  
L. V. Artemieva ◽  
S. N. Meisner ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Ion Implantation ◽  
Cell Counting ◽  
High Dose ◽  
Surface Layers ◽  
Test Flow ◽  
Ion Implanted

The objective of the work was to study the effect of high-dose ion implantation (HDII) of NiTi surface layers with Si Ti, or Zr, on the NiTi biocompatibility. The biocompatibility was judged from the intensity and peculiarities of proliferation of mesenchymal stem cells (MSCs) on the NiTi specimen surfaces treated by special mechanical, electrochemical, and HDII methods and differing in chemical composition, morphology, and roughness. It is shown that the ion-implanted NiTi specimens are nontoxic to rat MSCs. When cultivated with the test materials or on their surfaces, the MSCs retain the viability, adhesion, morphology, and capability for proliferationin vitro, as evidenced by cell counting in a Goryaev chamber, MTT test, flow cytometry, and light and fluorescence microscopy. The unimplanted NiTi specimens fail to stimulate MSC proliferation, and this allows the assumption of bioinertness of their surface layers. Conversely, the ion-implanted NiTi specimens reveal properties favorable for MSC proliferation on their surface.

Expression and intracellular localization of Nanos2-homologue protein in primordial germ cells and spermatogonial stem cells

Zygote ◽  
2019 ◽  
Vol 27 (02) ◽  
pp. 82-88 ◽  
Author(s):  
Vivek Pandey ◽  
Anima Tripathi ◽  
Pawan K. Dubey
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Germ Cells ◽  
Expression Patterns ◽  
Intracellular Localization ◽  
Primordial Germ Cells ◽  
Type A ◽  
Spermatogonial Stem Cells ◽  
Single Type ◽  
Rt Pcr

SummaryThe decision by germ cells to differentiate and undergo either oogenesis or spermatogenesis takes place during embryonic development and Nanos plays an important role in this process. The present study was designed to investigate the expression patterns in rat of Nanos2-homologue protein in primordial germ cells (PGCs) over different embryonic developmental days as well as in spermatogonial stem cells (SSCs). Embryos from three different embryonic days (E8.5, E10.5, E11.5) and SSCs were isolated and used to detect Nanos2-homologue protein using immunocytochemistry, western blotting, reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. Interestingly, Nanos2 expression was detected in PGCs at day E11.5 onwards and up to colonization of PGCs in the genital ridge of fetal gonads. No Nanos2 expression was found in PGCs during early embryonic days (E8.5 and 10.5). Furthermore, immunohistochemical and immunofluorescence data revealed that Nanos2 expression was restricted within a subpopulation of undifferentiated spermatogonia (As, single type A SSCs and Apr, paired type A SSCs). The same results were confirmed by our western blot and RT-PCR data, as Nanos2 protein and transcripts were detected only in PGCs from day E11.5 and in undifferentiated spermatogonia (As and Apr). Furthermore, Nanos2-positive cells were also immunodetected and sorted using flow cytometry from the THY1-positive SSCs population, and this strengthened the idea that these cells are stem cells. Our findings suggested that stage-specific expression of Nanos2 occurred on different embryonic developmental days, while during the postnatal period Nanos2 expression is restricted to As and Apr SSCs.

scholarly journals Isolation and Characterization of Human Synovial Fluid-Derived Mesenchymal Stromal Cells from Popliteal Cyst

2020 ◽  
Vol 2020 ◽  
pp. 1-15
Author(s):  
Fang Li ◽  
Jianglin Chen ◽  
Mengjia Gong ◽  
Yang Bi ◽  
Chengchen Hu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Synovial Fluid ◽  
Stromal Cells ◽  
Pediatric Patients ◽  
Cyst Fluid ◽  
Popliteal Cyst ◽  
Cell Marker ◽  
Related Proteins

Mesenchymal stem cells (MSCs) are multipotent progenitor cells in adult tissues. The aim of this study is to isolate and identify synovial fluid-derived mesenchymal stromal cells (SF-MSCs) from the popliteal cyst fluid of pediatric patients. SF-MSCs were collected from the popliteal cyst fluid of pediatric patients during cystectomy surgery. After cyst fluid extraction and adherent culturing, in vitro morphology, growth curve, and cell cycle were observed. The expression of stem cell surface markers was analyzed by flow cytometry, and expression of cell marker protein was detected by immunofluorescence. SF-MSCs were cultured in osteogenic, adipogenic, and chondrogenic differentiation medium. The differentiation potential of SF-MSCs was analyzed by alkaline phosphatase (Alizarin Red), Oil Red O, and Alcian blue. Antibody detection of human angiogenesis-related proteins was performed compared with bone marrow mesenchymal stem cells (BM-MSCs). The results show that SF-MSCs from the popliteal cyst fluid of pediatric patients showed a shuttle appearance and logarithmic growth. Flow cytometry analysis revealed that SF-MSCs were negative for hematopoietic lineage markers (CD34, CD45) and positive for MSC markers (CD44, CD73, CD90, and CD105). Interstitial cell marker (vimentin) and myofibroblast-like cell marker alpha-smooth muscle actin (α-SMA) were positive. These cells could differentiate into osteogenic, adipogenic, and chondrogenic lineages, respectively. Several types of human angiogenesis-related proteins were detected in the cell secretory fluid. These results show that we successfully obtained SF-MSCs from the popliteal cyst fluid of pediatric patients, which have the potential to be a valuable source of MSCs.

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