185 Overexpression or CRISPr/Cas9-mediated inhibition of prostaglandin E2 receptors EP2 and EP4 in equine adipose mesenchymal stem cells: implications for cell migration and cellular therapies

2019 ◽  
Vol 31 (1) ◽  
pp. 217
Author(s):  
A. C. F. Mançanares ◽  
J. O. Manríquez ◽  
J. Cabezas ◽  
F. Telleria ◽  
L. Rodriguez ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Prostaglandin E2 ◽  
Lentiviral Vector ◽  
Inflammatory Diseases ◽  
Calf Serum ◽  
Negative Control ◽  
Cell Surface Receptor ◽  
Surface Receptor

Prostaglandin E2 (PGE2) acts through 4 cellular receptors: EP1, EP2, EP3, and EP4; only EP2 and EP4 are relevant for immunomodulation and migration in immune cells. Besides those, several cells express these receptors on their surface, including mesenchymal stem cells. Pharmacological inhibition of the EP2 receptor prevents migration of immune system cells to inflamed sites, where the concentration of PGE2 is high. Based on this, we hypothesised that overexpression of EP2 or EP4 receptors in equine mesenchymal stem cells (eMSC) will improve their migration to inflammatory sites and subsequent homing capability. Conversely, their suppression will lead to low or no migration, favouring the paracrine properties of MSC in the processes of tissue regeneration and reduction of inflammation. To test this, we manipulated the PGE2-EP2-EP4 axis and evaluated the effect of such modifications on transgenic cells in vitro. Equine MSC from adipose tissue were obtained from 5 animals. The coding sequences of both receptors were synthesised (GenScript, Hong Kong) based on the published horse genome (National Center for Biotechnology Information; https://www.ncbi.nlm.nih.gov/) and cloned into pcDNA3.1 overexpression vectors (Addgene). The resulting constructs were lipofected into naïve adipose eMSC. For knockouts, we PCR-amplified and sequenced horse EP2 and EP4 receptors, and gRNAs were created based on the obtained sequences and ligated into LentiCRISPRV2 plasmid (Addgene, Cambridge, MA, USA). The lentiviral vector plus helping packaging plasmids were co-transfected into HEK293FT cells and the produced viral particles were harvested and transduced into adipose eMSC. After 48h of transfection (for overexpression) or transduction (for knockout, KO), cells were probed for the presence/absence of EP2 and EP4 receptors by immunohistochemistry and/or quantitative (q)PCR. Mitomycin-C-treated cells of both phenotypes and naïve, were subjected to migration in scratch assay, towards 3mM PGE2. Fetal calf serum (10 or 0%) was used as positive or negative control, respectively, in migration experiments. Receptors EP2 and EP4 were clearly overexpressed after transfection as determined by immunocytochemistry or qPCR assays (phenotype MSC-EP2+/EP4+), whereas in the cells that underwent KO, little or no expression of EP2 and EP4 was detected (phenotype MSC-EP2ko/EP4ko) compared with unmanipulated cells (naïve MSC-Ctr). In the migration experiments towards 3mM of PGE2, MSC-EP2+/EP4+ cells at 24h filled the scratch faster (P<0.05) than MSC-EP2ko/EP4ko or MSC-Ctr. These results showed that manipulation of PGE2-EP2/EP4 axis receptors led to changes in cell surface receptor availability and increased the migration pattern in overexpressed cells compared with KO and unmanipulated cells. These factors may affect the design of cellular therapeutic tools for inflammatory diseases in the equine species. This research was supported by FONDECYT 3170390 to ACFM, Ministry of Education, Chile.

scholarly journals Successful Treatment of Plaque Psoriasis with Allogeneic Gingival Mesenchymal Stem Cells: A Case Study

2020 ◽  
Vol 2020 ◽  
pp. 1-4 ◽  
Author(s):  
Sebo Gene Wang ◽  
Nicholas C. Hsu ◽  
Sebo Michelle Wang ◽  
Fu Nan Wang
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Plaque Psoriasis ◽  
Inflammatory Diseases ◽  
Treatment Options ◽  
Gingival Tissue ◽  
Complete Regression ◽  
Allogeneic Mscs ◽  
Human Gingiva

Plaque psoriasis is the most common type of psoriasis that manifests as red scaly patches with white scales affecting body areas including scalp, elbows, knees, trunk, and buttocks. Although many treatment options are available including novel biologics, no cure is available. Mesenchymal stem cells (MSCs) have been safely used to treat a variety of human diseases. Allogeneic MSCs possess unique characteristics including hypoimmunogenicity, immunomodulatory, and anti-inflammatory properties, and they are currently being explored for potential therapeutic use for many systemic inflammatory diseases. The human gingival tissue is an easily accessible and obtainable source for the isolation of MSCs. MSCs from adult human gingiva are of fetal-like phenotype, multipotent, and easy to isolate and expand in vitro. Herein, we report a case of a 19-year-old man with a 5-year history of severe plaque psoriasis refractory to multiple topical and systemic therapies who was treated with allogeneic human gingival MSCs. Complete regression was achieved after 5 infusions with no adverse reaction occurred. The patient has been followed for three years and has remained disease free.

111 EQUINE AMNIOTIC EPITHELIAL OR BONE MARROW MESENCHYMAL STEM CELLS DIFFERENTLY SUPPORT IN VITRO EMBRYO DEVELOPMENT IN A BOVINE IN VITRO CULTURE MODEL

2009 ◽  
Vol 21 (1) ◽  
pp. 156 ◽  
Author(s):  
F. Cremonesi ◽  
V. Maggio ◽  
A. Lange Consiglio
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
In Vitro Culture ◽  
Calf Serum ◽  
Biologically Active ◽  
Bovine Embryo ◽  
Epithelial Stem Cells ◽  
Marrow Mesenchymal Stem Cells

There are indications that the culture system and the medium composition can affect embryo quality. In fact, various studies have been shown that the in vitro culture environment is one of the key determinants of the blastocyst output. In light of this, recently, some studies used co-culture with mouse embryonic fibroblasts in the effort to improve the development of bovine and ovine in vitro-derived embryos. Despite the progress in equine IVM and ICSI technologies and the different culture conditions reported for preimplantation development of ICSI fertilized horse oocytes, the yield of blastocysts remained low. In the present study we investigated the benefits of co-culturing bovine embryos with equine bone marrow mesenchymal stem cells (BM-MSC) or equine amniotic epithelial stem cells (AE-SC) on blastocyst development. This study employed the bovine embryo as a model and represents the initial step towards standardization of a protocol for the culture of equine embryos in our laboratory. BM specimens were obtained aseptically from sternal aspirates of horses under local anaesthesia and layered over Hystopaque™ 1.080, then centrifuged for 20 min at 400g and 4°C. Cell pellets were resuspended in 10 mL Dulbecco Modified Earle’s Medium supplemented with 10% fetal calf serum, 1% non-essential amino acids, penicillin (100 U mL–1) and streptomycin (100 μg mL–1) and seeded in 24-well plates. Amniotic membranes were obtained from fresh placentas and, to release the AE cells, amniotic fractions were incubated at 37°C with 0.05% trypsin for 45 min. Separated AE cells were plated on 25 cm2 flask in standard culture media containing 10 ng mL–1 epidermal growth factor. Seven hundred fifty cumulus–oocyte complexes with a homogeneous cytoplasm and two or more layers of cumulus cells were used. After IVM and IVF cumulus-free presumptive zygotes were randomly transferred into one of three co-culture systems in which they were cultured for up to Day 7: 1) co-culture with granulosa cells (control); 2) co-culture with BM-MSC; 3) co-culture with AE-SC. The culture medium was TCM 199 + 10% fetal bovine serum, pyruvate and gentamicin at 38.5°C in 5% CO2. Statistical analyses was performed by chi square test. Blastocysts developmental rates were similar among control, AE-SC and BM-MSC (35%, 41% and 30%, respectively), but the co-culture with AE-SC gave a significantly greater percentage of blastocysts compared to BM-MSC (P < 0.05). In conclusion, despite the absence of a significant increment in blastocysts attainment using stem cells as feeders for embryo culture, the AE-SC monolayer create a more suitable microenvironment necessary for inducing local cell activation and proliferation of the growing embryos in comparison with BM-MSC. It can be suggested that these cells secrete biologically active substances including signaling molecules and growth factors of epithelial nature different from those of the BM cells of mesenchymal origin. Regione Lombardia is acknowledged for the “Dote Ricercatori” fellowship to V.M.

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

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 Lentiviral vector mediated modification of mesenchymal stem cells & enhanced survival in an in vitro model of ischaemia

2011 ◽  
Vol 2 (2) ◽  
pp. 12 ◽  
Author(s):  
Lisa McGinley ◽  
Jill McMahon ◽  
Padraig Strappe ◽  
Frank Barry ◽  
Mary Murphy ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
In Vitro Model ◽  
Lentiviral Vector ◽  
Vitro Model

EFFECTS OF SHORT-TERM CYCLIC HYDROSTATIC PRESSURE ON INITIATING AND ENHANCING THE EXPRESSION OF CHONDROGENIC GENES IN HUMAN ADIPOSE-DERIVED MESENCHYMAL STEM CELLS

2014 ◽  
Vol 14 (04) ◽  
pp. 1450054 ◽  
Author(s):  
FARZANEH SAFSHEKAN ◽  
MOHAMMAD TAFAZZOLI SHADPOUR ◽  
MOHAMMAD ALI SHOKRGOZAR ◽  
NOOSHIN HAGHIGHIPOUR ◽  
SEYED HAMED ALAVI
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Growth Factor ◽  
Hydrostatic Pressure ◽  
Chondrogenic Differentiation ◽  
Negative Control ◽  
Cartilage Tissue ◽  
Control Group

Cartilage tissue engineering is a promising treatment for damaged or diseased cartilage that requires thorough understanding of influential parameters involved in chondrogenic differentiation. This study examined how 4-h application of cyclic hydrostatic pressure (CHP) of 5 MPa at 0.5 Hz could modulate chondroinduction of human adipose-derived mesenchymal stem cells (hAMSCs) in vitro. Four groups were examined including a negative control group, a chemical group treated by growth factor for 10 days, a mechanical group exposed to 4-h loading on the 10th day of pellet culture without any chondrogenic stimulator, and finally a chemical-mechanical group subjected to both growth factor and loading. Application of cyclic hydrostatic pressure increased the expression of chondrogenic genes, including sox9 and aggrecan to higher levels than those of the chemical group. This study indicates that cyclic hydrostatic pressure initiates and enhances the chondrogenic differentiation of mesenchymal stem cells with or without growth factors in vitro and confirms the important role of hydrostatic pressure during chondrogenesis in vivo.

Study on Telomerase Activity of Human Bone Marrow Mesenchymal Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4255-4255
Author(s):  
Jingyuan Li ◽  
Xiaoyu Lai ◽  
Huang He
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Telomerase Activity ◽  
Negative Control ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Significant Difference

Abstract Human mesenchymal stem cells(hMSCs) have multiple differentiate potential, and it can differentiate into adipocytes, osteogenic cells, chondrocyte and neural cells et al. It has been reported that telomerase activity in hMSCs is negative, but it is still controversial and telomerase activity in hMSCs-derived adipocytes has not been reported. We investigate the telomerase activity in hMSCs before and after their committed differentiation into adipocytes in vitro and cryopreservation. hMSCs were isolated from normal human bone marrow fellowed by cell culture in DMEM with low glucose containing 10% FBS. The FACS was performed to examine the expression of cell surface molecules and analyze cell cycle of primary hMSCs.Then some of hMSCs were induced to differentiate into adipocytes in vitro by being treated with adipocytic medium fellowed by being stained with oil red O, and the others were cryopreserved in liguid nitrogon for three months. TRAP assay(telomerase repeat amplification protocol assay)was employed to detect telomerase activity in those hMSCs. T293 cells and α-Interferon were analyzed with each test as an additional positive control and negative control respectively. Telomerase activity was expressed as a percentage of the relative telomerase activity (RTA) of the hMSCs relative to the RTA of T293 cells. The results indicated the cells were positive for SH2, SH3, CD90 and negative for CD34, CD45, AC133. It was showed that the majority of primary hMSCs(85%) was at cell cycle of G0/G1 phase and the minority of hMSCs was at S, G2 or M phase. 80% hMSCs was orange adipocytes after they were treated with adipocytic medium for 3–4weeks. Telomerase activity was negative in hMSCs both at the beginning of culture and at the later stages during cell expansion,telomerase activity in hMSCs-passage 1–3(n=10) and hMSCs-passage 4–7(n=9) made no significant difference(1.46±0.83% vs 1.46±0.67%, p=0.99). Cryopreservation did not affect the telomerase activity in hMSCs. Telomerase activity in fresh hMSCs(n=13) and frozen hMSCs(n=6) made no significant difference(1.41±0.44% vs 1.51±1.07%, p=0.64). Telomerase activity in hMSCs-derived adipocytes(n=3) was significantly higher than in hMSCs(n=19)( 11.8±2.52% vs 1.46. ±0.67%, p&lt;0.00001). It is concluded that hMSCs are telomerase-negative, and the stage of culture or cryopreservation does not affect their telomerase activity. After being induced to differentiated into adipocytes, hMSCs telomerase activity is upregulated.

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