scholarly journals Edition of Prostaglandin E2 Receptors EP2 and EP4 by CRISPR/Cas9 Technology in Equine Adipose Mesenchymal Stem Cells

Animals ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 1078
Author(s):  
Ana Carolina Furlanetto Mançanares ◽  
Joel Cabezas ◽  
José Manríquez ◽  
Vanessa Cristina de Oliveira ◽  
Yat Sen Wong Alvaro ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Prostaglandin E2 ◽  
Sanger Sequencing ◽  
Surface Markers ◽  
Wild Type ◽  
Migration Ability ◽  
Knock Out ◽  
Directed Mutation

In mesenchymal stem cells (MSCs), it has been reported that prostaglandin E2 (PGE2) stimulation of EP2 and EP4 receptors triggers processes such as migration, self-renewal, survival, and proliferation, and their activation is involved in homing. The aim of this work was to establish a genetically modified adipose (aMSC) model in which receptor genes EP2 and EP4 were edited separately using the CRISPR/Cas9 system. After edition, the genes were evaluated as to if the expression of MSC surface markers was affected, as well as the migration capacity in vitro of the generated cells. Adipose MSCs were obtained from Chilean breed horses and cultured in DMEM High Glucose with 10% fetal bovine serum (FBS). sgRNA were cloned into a linearized LentiCRISPRv2GFP vector and transfected into HEK293FT cells for producing viral particles that were used to transduce aMSCs. GFP-expressing cells were separated by sorting to obtain individual clones. Genomic DNA was amplified, and the site-directed mutation frequency was assessed by T7E1, followed by Sanger sequencing. We selected 11 clones of EP2 and 10 clones of EP4, and by Sanger sequencing we confirmed 1 clone knock-out to aMSC/EP2 and one heterozygous mutant clone of aMSC/EP4. Both edited cells had decreased expression of EP2 and EP4 receptors when compared to the wild type, and the edition of EP2 and EP4 did not affect the expression of MSC surface markers, showing the same pattern in filling the scratch. We can conclude that the edition of these receptors in aMSCs does not affect their surface marker phenotype and migration ability when compared to wild-type cells.

scholarly journals Mesenchymal stem cells express serine protease inhibitor to evade the host immune response

Blood ◽  
2011 ◽  
Vol 117 (4) ◽  
pp. 1176-1183 ◽  
Author(s):  
Najib El Haddad ◽  
Dean Heathcote ◽  
Robert Moore ◽  
Sunmi Yang ◽  
Jamil Azzi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
T Cell ◽  
Protease Inhibitor ◽  
Serine Protease ◽  
Serine Protease Inhibitor ◽  
Cytotoxic T Cell ◽  
Wild Type

Abstract Clinical trials using mesenchymal stem cells (MSCs) have been initiated worldwide. An improved understanding of the mechanisms by which allogeneic MSCs evade host immune responses is paramount to regulating their survival after administration. This study has focused on the novel role of serine protease inhibitor (SPI) in the escape of MSCs from host immunosurveillance through the inhibition of granzyme B (GrB). Our data indicate bone marrow–derived murine MSCs express SPI6 constitutively. MSCs from mice deficient for SPI6 (SPI6−/−) exhibited a 4-fold higher death rate by primed allogeneic cytotoxic T cells than did wild-type MSCs. A GrB inhibitor rescued SPI6−/− MSCs from cytotoxic T-cell killing. Transduction of wild-type MSCs with MigR1-SPI6 also protected MSCs from cytotoxic T cell–mediated death in vitro. In addition, SPI6−/− MSCs displayed a shorter lifespan than wild-type MSCs when injected into an allogeneic host. We conclude that SPI6 protects MSCs from GrB-mediated killing and plays a pivotal role in their survival in vivo. Our data could serve as a basis for future SPI-based strategies to regulate the survival and function of MSCs after administration and to enhance the efficacy of MSC-based therapy for diseases.

Intercellular Crosstalk of Mesenchymal Stem Cells with Prostate Cancer Cells via Microvesicles Loaded with Magnetic Nanocubes for Targeted Magnetic Hyperthermia

2019 ◽  
Vol 15 (12) ◽  
pp. 2291-2304
Author(s):  
Liqun Huang ◽  
Mengwei Chen ◽  
Chang Xu ◽  
Qishuai Feng ◽  
Jiaojiao Wu ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cancer Cells ◽  
Targeted Delivery ◽  
Magnetic Hyperthermia ◽  
Migration Ability ◽  
Hyperthermia Therapy

The targeted delivery of nanomedicines into solid tumors remains challenging in cancer treatment. Stem cells with tumortropic migration ability are promising as biocarriers to transport nanomedicines. The transportation of nanomedicines into cancer cells is the key step for tumor targeted delivery via stem cells. In this study, we designed a magnetic nanocube (scMNP) loaded in mesenchymal stem cells for magnetic hyperthermia of prostate cancer, and the delivery and transportation pathways into the cancer cells were fully investigated. The MSCs acted as the carrier of the loaded scMNPs along with the upregulation of CXCR4 for the migration to cancer cells. The therapeutic effect was mainly due to scMNPs via magnetic hyperthermia. Stem cell-derived microvesicles containing scMNPs played an essential role in the crosstalk between stem cells and cancer cells for targeted delivery. Both in vitro and in vivo studies demonstrated that the system showed satisfactory therapeutic efficiency under magnetic hyperthermia therapy. Our investigation presents a comprehensive study of magnetic nanoparticles in combination with MSCs and their extracellular microvesicles and is promising as an effective strategy for magnetic hyperthermia therapy of prostate cancer.

scholarly journals Stem cell antigen/Ly6a protects against bladder fibrosis in mice

2019 ◽  
Vol 317 (6) ◽  
pp. F1503-F1512 ◽  
Author(s):  
Nicholas M. Tassone ◽  
Belinda Li ◽  
Mehul S. Patel ◽  
Megan Y. Devine ◽  
Paula R. Firmiss ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Outlet Obstruction ◽  
Specific Protein ◽  
Male Mice ◽  
Wild Type ◽  
Cell Antigen ◽  
Steady State Levels

We have defined a population of stem cell antigen (Sca)-1+/CD34+/lin− mesenchymal stem cells in the mouse urinary bladder. These cells are reduced after partial bladder outlet obstruction (PO). To test the role of Sca-1 expressed by these cells, we analyzed bladders from Sca-1 knockout (KO) mice in both uninjured male mice and male mice subjected to PO. We found that loss of Sca-1 alone had little effect on bladder development or function but reduced the total number of mesenchymal stem cells by 30%. After PO, bladders from Sca-1-null KO male mice were larger, with more collagen and less muscle, than obstructed wild-type mice. Steady-state levels of caldesmon were significantly reduced and levels of fibroblast-specific protein 1 were significantly increased in Sca-1 KO mice compared with wild-type mice after PO. In investigating the effects of PO on cell proliferation, we found that loss of Sca-1 changed the timing of cell division in CD34+/lin−, collagen-producing, and smooth muscle cells. PO in combination with loss of Sca-1 drastically reduced the ability of CD34+/lin− cells to form colonies in vitro. Our findings therefore support the hypothesis that Sca-1 protects the bladder from fibrotic remodeling after obstruction, in part by influencing the proliferation of cells responding to the injury.

scholarly journals Adipose-derived mesenchymal stem cells modulate CD14++CD16+ expression on monocytes from sepsis patients in vitro via prostaglandin E2

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Guanguan Qiu ◽  
Guoping Zheng ◽  
Menghua Ge ◽  
Lanfang Huang ◽  
Haijiang Tong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Prostaglandin E2 ◽  
Cd16 Expression

Immunogenicity of human umbilical cord mesenchymal stem cells during ex vivo expansion

2016 ◽  
Vol 50 (2) ◽  
Author(s):  
Mingli Ji ◽  
LiPing Guan
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Major Histocompatibility Complex ◽  
Ex Vivo ◽  
Human Umbilical Cord ◽  
Surface Markers ◽  
Histocompatibility Complex ◽  
Major Histocompatibility Complex Expression ◽  
Complex Expression

The aim of this study was to investigate the immunogenicity of human umbilical cord mesenchymal stem cells (hUCMSCs) cultured <italic>in vitro</italic> by detecting changes in major histocompatibility complex expression. Major histocompatibility complex expression was detected by RT–PCR and flow cytometry. Morphologically, hUCMSCs exhibited no visible changes before the 5 st passage of the <italic>in vitro</italic> culture. In addition, At the molecular level, the cells continued expressing the specific positive surface markers, CD105, CD73 and CD90, and did not express the negative surface markers, CD14, CD34 or CD45, during culture <italic>ex vivo</italic>. Furthermore, the hUCMSCs exhibited low immunogenicity, which was maintained when cultured for five passages. Taken as a whole, these results suggested that hUCMSCs cultured in vitro may be safely for transplanted because of low immunogenicity before the fifth passage.

scholarly journals An Improved Harvest andin VitroExpansion Protocol for Murine Bone Marrow-Derived Mesenchymal Stem Cells

2010 ◽  
Vol 2010 ◽  
pp. 1-10 ◽  
Author(s):  
Song Xu ◽  
Ann De Becker ◽  
Ben Van Camp ◽  
Karin Vanderkerken ◽  
Ivan Van Riet
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Surface Markers ◽  
Differentiation Potential ◽  
Murine Bone Marrow ◽  
Expansion Time ◽  
Expansion Protocol ◽  
In Vitro Expansion

Compared to bone marrow (BM) derived mesenchymal stem cells (MSCs) from human origin or from other species, the in vitro expansion and purification of murine MSCs (mMSCs) is much more difficult because of the low MSC yield and the unwanted growth of non-MSCs in the in vitro expansion cultures. We describe a modified protocol to isolate and expand murine BM derived MSCs based on the combination of mechanical crushing and collagenase digestion at the moment of harvest, followed by an immunodepletion step using microbeads coated with CD11b, CD45 and CD34 antibodies. The number of isolated mMSCs as estimated by colony forming unit-fibroblast (CFU-F) assay showed that this modified isolation method could yield 70.0% more primary colonies. After immunodepletion, a homogenous mMSC population could already be obtained after two passages. Immunodepleted mMSCs (ID-mMSCs) are uniformly positive for stem cell antigen-1 (Sca-1), CD90, CD105 and CD73 cell surface markers, but negative for the hematopoietic surface markers CD14, CD34 and CD45. Moreover the immunodepleted cell population exhibits more differentiation potential into adipogenic, osteogenic and chondrogenic lineages. Our data illustrate the development of an efficient and reliable expansion protocol increasing the yield and purity of mMSCs and reducing the overall expansion time.

scholarly journals Platelet-Derived Microparticles Increase Expression of hTERT in Umbilical Cord Mesenchymal Stem Cells

2018 ◽  
Vol 5 (4) ◽  
pp. 31 ◽  
Author(s):  
Maryam Samareh Salavati Pour ◽  
Fatemeh Hoseinpour Kasgari ◽  
Alireza Farsinejad ◽  
Ahmad Fatemi ◽  
Roohollah Mirzaee Khalilabadi
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Life Span ◽  
Real Time ◽  
Umbilical Cord ◽  
Control Group ◽  
Surface Markers

Introduction: Mesenchymal stem cells (MSCs) are widely studied due to their self- renewal potential and capacity to differentiate into multiple tissues. However, they have a limited life span of several divisions in vitro, which alters various cellular characteristics and reduces their application. Aim: We evaluated the effect of platelet-derived microparticles on gene expression of hTERT, one of the main factors involved in aging and cell longevity. Materials and methods: Umbilical cord MSCs were used for this study. Cells were characterized by evaluating morphology via inverted microscope and identifying associated surface markers using flow cytometry. Platelet-derived microparticles were prepared by centrifuging platelet bags at varying speeds, and their concen- trations were determined by Bradford assay. At 30% confluency, MSCs were treated with 50 μg/mL of microparticles for five days. Then, RNA was extracted and cDNA was synthesized. Quantitative expression of hTERT was assessed using real-time polymerase chain reaction (PCR). Results: Fibroblast-like cells were isolated from umbilical cord tissue and MSCs were identified by the presence of mesenchymal surface markers via flow cytometry. Real- time PCR showed that gene expression of hTERT increased by more than three times when treated with platelet-derived microparticles, in comparison to expression of the control group. Conclusion: We concluded that platelet-derived microparticles may be a potentially safe and effective method to increase hTERT gene expression in MSCs, ultimately prolonging their life span in vitro. 

scholarly journals Manufacturing and Banking Canine Adipose-Derived Mesenchymal Stem Cells for Veterinary Clinical Application

2020 ◽  
Author(s):  
Huina Luo ◽  
Dongsheng Li ◽  
Zhisheng Chen ◽  
Bingyun Wang ◽  
Shengfeng Chen
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Clinical Applications ◽  
Surface Markers ◽  
Clinical Use ◽  
Therapeutic Application ◽  
Differentiation Potential ◽  
Safety And Efficacy

Abstract BACKGROUND: Mesenchymal stem cells (MSCs) have generated a great amount of interest in recent years as a novel therapeutic application for improving the quality of pet life and helping them free from painful conditions and diseases. It has now become critical to address the challenges related to the safety and efficacy of MSCs expanded in vitro. In this study, we establish a standardized process for manufacture of canine adipose-derived MSCs (AD-MSCs), including tissue sourcing, cell isolation and culture, cryopreservation, thawing and expansion, quality control and testing, and evaluate the safety and efficacy of those cells for clinical applications. RESULTS: After expansion, the viability of AD-MSCs manufactured under our standardized process was above 90 %. Expression of surface markers and differentiation potential was consistent with ISCT standards. Sterility, mycoplasma, and endotoxin tests were consistently negative. No adverse events were noted in two cases treated with intravenously AD-MSCs. CONCLUSION: Herein we demonstrated the establishment of a feasible bioprocess for manufacturing and banking canine AD-MSCs for veterinary clinical use.

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