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.

scholarly journals Effects of MRI on Stemness Properties of Wharton’s Jelly Derived Mesenchymal Stem Cells

2021 ◽  
Author(s):  
Mahnaz Tashakori ◽  
Fatemeh Asadi ◽  
Faezeh-Sadat Khorram ◽  
Azita Manshoori ◽  
Ali Hosseini-Chegeni ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Doubling Time ◽  
Expression Patterns ◽  
Wharton’S Jelly ◽  
Population Doubling ◽  
Control Group ◽  
Differentiation Potential ◽  
Wharton's Jelly ◽  
Promising Source

Abstract BackgroundMesenchymal stem cells (MSCs), derived from various tissues, have served as a promising source of cells in clinic and regenerative medicine. Umbilical cord-Wharton’s jelly (WJ-MSCs)-derived MSCs exhibit advantages over those from adult tissues, such as no ethical concerns, shorter population doubling time, broad differentiation potential, readily available non-invasive source, prolonged maintenance of stemness properties. Material and methodsThe aim of this study was to evaluate the effect of MRI (1.5 T, 10 min) on stemness gene expression patterns (OCT-4, SOX-2, NANOG) of WJ-MSCs. In addition, we assessed cell viability, growth kinetics and apoptosis of WJ-MSCs after MRI treatment. ResultsThe quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) data showed that transcript levels of SOX-2, NANOG in MRI-treated WJ-MSCs were increased 32- and 213-fold, respectively. MTT assay was performed at 24, 48, and 72 hours post-treatment and the viability was not significantly difference between two groups. The doubling time of MRI group was markedly higher than control group. In addition, the colony formation ability of WJ-MSCs after MRI treatment significantly increased. Furthermore, no change in apoptosis was seen before or after MRI treatment. ConclusionsOur results suggest the use of MRI can improve quality of MSCs and may enhance the efficacy of mesenchymal stem cell-based therapies.

scholarly journals Characterization and plasticity of wharton's jelly mesenchymal stem cells of goat

2021 ◽  
Vol 37 ◽  
pp. e37002
Author(s):  
Gustavo Cardoso da Silva Neves ◽  
Napoleão Martins Argôlo Neto ◽  
Maíra Soares Ferraz ◽  
Clautina Ribeiro de Moraes Da Costa ◽  
Andressa Rêgo Da Rocha ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Umbilical Cord ◽  
Growth Curve ◽  
Invasive Procedure ◽  
Wharton’S Jelly ◽  
Wharton's Jelly ◽  
Undifferentiated State

Mesenchymal stem cells (MSCs), obtained from several anatomical sites, have already been described, characterized and used in therapeutic models for tissue repair. The umbilical cord mesenchymal stem cells, represented by cells from arteries and veins walls, as well as Wharton's jelly are easy to be obtained, highly available, require no invasive procedure, do not present risk to donors and do not present ethical limitation. The aim of this research was to analyze the plasticity of Wharton's jelly mesenchymal stem cells (WJ-MSCs) of goat, evaluating their behavior in vitro and characterizing them immunophenotypically. Thus, tests were performed on colony forming units, viability and cell growth curve, flow cytometry analysis and plasticity potential. Goat umbilical cord matrix cells exhibited fibroblastoid morphology with colony formation and self-renewal ability, always maintaining their undifferentiated state up to the eighth passage (P8). The growth curve kinetics exhibited the LAG, LOG, and DECAY phases, without displaying a PLATEAU phase. The plasticity assay demonstrated positive differentiation for osteogenic, adipogenic and chondrogenic lines, characterized by the synthesis of intracytoplasmic granules or extracellular matrix with the presence of calcium, lipids and proteoglycans. Flow cytometry demonstrated the expression of CD90 and CD105; absence of CD14 expression.  It is concluded that the cell population isolated from the Wharton's  jelly of goat constitutes a representative sample of mesenchymal stem cells, with great possibilities in the field of regenerative and reproductive medicine.

scholarly journals Histomorphology of Pancreatic Islet in Physiological Aging Female Rats Post Intravenous Human Wharton’s Jelly Mesenchymal Stem Cell Injection

2019 ◽  
Vol 3 (1) ◽  
Author(s):  
Wining Astini
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Treatment Group ◽  
Pancreatic Islet ◽  
Aging Treatment ◽  
Wharton’S Jelly ◽  
Female Rats ◽  
Control Group ◽  
Female Rat ◽  
Wharton's Jelly

The increasing population of aged people will have the important role in the life, but the function of their bodies will decrease because of aging. Aging will increase the risk of degenerative disease, one of example is diabetes. The disease is related to the aging in the pancreatic organ which progressively declines by age. The aimed of the experiment was to determine the effect of human wharton’s jelly mesenchymal stem cells by injecting intravenously in aging female rats. This study used 3 young female rats (3 months) and 6 aging female rats (24 months). The experiment consisted of three groups. The young control group (A), the aging control group (B) that received NaCl (0.9%) 0,4 mL, the aging treatment group (C) received 1 x 106 cells/kg of human wharton’s jelly mesenchymal stem cells 0,4 mL. The aging control and the aging treatment group were injected 4 times with the interval in 3 months. The end of the experiment (12 months), the rats were anesthetized and sacrificed. The pancreatic tissues were collected to examine the pancreatic islets by histology studies. Changes of the pancreatic islet in control and treated groups were examined using hematoxylin and eosin staining. These findings conclude that injecting human wharton’s jelly mesenchymal stem cell increase the diameter and total pancreatic islet in the treatment group. In other side, the cell population of pancreatic islet also have significant differences (P<0.05) in treated physiological aging female rat groups than control aging female rat group.

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.

Identify the role of Human Wharton's Jelly Mesenchymal Stem Cells in repairing injured uterine of rat

2021 ◽  
Vol 47 (1) ◽  
pp. 320-328
Author(s):  
Hezhu Wang ◽  
Xiaoqing Yang ◽  
Xiaojing Chen ◽  
Huihui Xie ◽  
Junxia Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Wharton’S Jelly ◽  
Wharton's Jelly

scholarly journals Exosomes derived from human umbilical cord Wharton's jelly mesenchymal stem cells ameliorate experimental lymphedema

2021 ◽  
Vol 11 (7) ◽  
Author(s):  
Zhao Ting ◽  
Yan Zhi‐xin ◽  
Tan You‐wen ◽  
Yang Fu‐ji ◽  
Sun Hui ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Umbilical Cord ◽  
Wharton’S Jelly ◽  
Human Umbilical Cord ◽  
Wharton's Jelly
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