scholarly journals Molecular Profiling and Gene Banking of Rabbit EPCs Derived from Two Biological Sources

Genes ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 366
Author(s):  
Jaromír Vašíček ◽  
Andrej Baláži ◽  
Miroslav Bauer ◽  
Andrea Svoradová ◽  
Mária Tirpáková ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Morphological Changes ◽  
Umbilical Vein ◽  
Differentiation Potential ◽  
Neuronal Markers ◽  
High Viability ◽  
Droplet Pcr ◽  
Passage Number ◽  
Umbilical Vein Endothelial Cells

Endothelial progenitor cells (EPCs) have been broadly studied for several years due to their outstanding regenerative potential. Moreover, these cells might be a valuable source of genetic information for the preservation of endangered animal species. However, a controversy regarding their characterization still exists. The aim of this study was to isolate and compare the rabbit peripheral blood- and bone marrow-derived EPCs with human umbilical vein endothelial cells (HUVECs) in terms of their phenotype and morphology that could be affected by the passage number or cryopreservation as well as to assess their possible neuro-differentiation potential. Briefly, cells were isolated and cultured under standard endothelial conditions until passage 3. The morphological changes during the culture were monitored and each passage was analyzed for the typical phenotype using flow cytometry, quantitative real–time polymerase chain reaction (qPCR) and novel digital droplet PCR (ddPCR), and compared to HUVECs. The neurogenic differentiation was induced using a commercial kit. Rabbit cells were also cryopreserved for at least 3 months and then analyzed after thawing. According to the obtained results, both rabbit EPCs exhibit a spindle-shaped morphology and high proliferation rate. The both cell lines possess same stable phenotype: CD14-CD29+CD31-CD34-CD44+CD45-CD49f+CD73+CD90+CD105+CD133-CD146-CD166+VE-cadherin+VEGFR-2+SSEA-4+MSCA-1-vWF+eNOS+AcLDL+ALDH+vimentin+desmin+α-SMA+, slightly different from HUVECs. Moreover, both induced rabbit EPCs exhibit neuron-like morphological changes and expression of neuronal markers ENO2 and MAP2. In addition, cryopreserved rabbit cells maintained high viability (>85%) and endothelial phenotype after thawing. In conclusion, our findings suggest that cells expanded from the rabbit peripheral blood and bone marrow are of the endothelial origin with a stable marker expression and interesting proliferation and differentiation capacity.

Distinct signals via Rho GTPases and Src drive shape changes by thrombin and sphingosine-1-phosphate in endothelial cells

2002 ◽  
Vol 115 (12) ◽  
pp. 2475-2484 ◽  
Author(s):  
Valérie Vouret-Craviari ◽  
Christine Bourcier ◽  
Etienne Boulter ◽  
Ellen Van Obberghen-Schilling
Keyword(s):  
Endothelial Cells ◽  
Rho Gtpases ◽  
Actin Polymerization ◽  
Morphological Changes ◽  
Umbilical Vein ◽  
Cell Spreading ◽  
Actin Binding ◽  
Sphingosine 1 Phosphate ◽  
And Migration ◽  
Umbilical Vein Endothelial Cells

Soluble mediators such as thrombin and sphingosine-1-phosphate regulate morphological changes in endothelial cells that affect vascular permeability and new blood vessel formation. Although these ligands activate a similar set of heterotrimeric G proteins, thrombin causes cell contraction and rounding whereas sphingosine-1-phosphate induces cell spreading and migration. A functional requirement for Rho family GTPases in the cytoskeletal responses to both ligands has been established, yet the dynamics of their regulation and additional signaling mechanisms that lead to such opposite effects remain poorly understood. Using a pull-down assay to monitor the activity of Rho GTPases in human umbilical vein endothelial cells, we find significant temporal and quantitative differences in RhoA and Rac1 activation. High levels of active RhoA rapidly accumulate in cells in response to thrombin whereas Rac1 is inhibited. In contrast, sphingosine-1-phosphate addition leads to comparatively weak and delayed activation of RhoA and it activates Rac1. In addition, we show here that sphingosine-1-phosphate treatment activates a Src family kinase and triggers recruitment of the F-actin-binding protein cortactin to sites of actin polymerization at the rim of membrane ruffles. Both Src and Rac pathways are essential for lamellipodia targeting of cortactin. Further, Src plays a determinant role in sphingosine-1-phosphate-induced cell spreading and migration. Taken together these data demonstrate that the thrombin-induced contractile and immobile phenotype in endothelial cells reflects both robust RhoA activation and Rac inhibition, whereas Src- and Rac-dependent events couple sphingosine-1-phosphate receptors to the actin polymerizing machinery that drives the extension of lamellipodia and cell migration.

scholarly journals Proliferation and differentiation potential of CD133+ and CD34+ populations from the bone marrow and mobilized peripheral blood

2010 ◽  
Vol 90 (2) ◽  
pp. 127-137 ◽  
Author(s):  
Irena Koutna ◽  
Martina Peterkova ◽  
Pavel Simara ◽  
Stanislav Stejskal ◽  
Lenka Tesarova ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Differentiation Potential ◽  
Proliferation And Differentiation ◽  
Mobilized Peripheral Blood

Impaired Clonogenic Capacity Contributes to Bone Marrow Hypoplasia and Late Hematological Recovery after Zevalin in the Low-Grade NHL Patients.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4610-4610
Author(s):  
Wojciech Jurczak ◽  
Marta Szostek ◽  
Zbigniew Rudzki ◽  
Dorota Krochmalczyk ◽  
Joanna Wegrzyn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Morphological Changes ◽  
Petri Dish ◽  
Relative Increase ◽  
Significant Inhibition ◽  
Low Grade ◽  
Bone Marrow Hypoplasia

Abstract Chemoimmunotherapy with Zevalin (anty CD20,90Y) is a new therapeutic approach in the resistant or relapsed low grade malignant lymphoma patients. The most important side effect observed in the up to date studies is related to transient peripheral blood cytopenias. In an attempt to evaluate the involved pathogenetic mechanisms of the above findings, the blood and bone marrow samples from 8 low grade NHL patients were prospectively tested for the peripheral blood count, the clonogenic capacity and the histopathology abnormalities (trephine biopsies) before and every 14 days after Zevalin administration until recovery. In the majority evaluated patients the mild thrombocytopenia (30-55000/μl) was observed 4 weeks after Zevalin therapy. Bone marrow mononuclear cells were cultured in methylcellulose assay using combination of recombinant CSF (IL-3, IL-6, stem cell factor, erythropoietin; Methocult, StemCell, Canada), at a concentration of 1x10^5 cells, in 35 mm gridded Petri dish and incubated at 37°C, in a humidified atmosphere of 5% CO2. The hematopoietic colonies (CFU-GM, BFU-E and CFU-GEMM) were evaluated on 14th d. of the culture. The in vitro CFU-Meg formation in the serum-free collagen-based semisolid culture (Megacult, StemCell, Canada) were also studied, stained after 12 days with a monoclonal antibody against CD 41. In the results of our studies the significant inhibition of colony formation was found already in 7th day for CFU-GM and BFU-E and starting from 14th day after Zevalin administration for CFU-Meg. In the subsequent cultures evaluated during 4–8 weeks after Zevalin therapy the three lineages hematopoietic clonogenic capacity returned to the pre-radiotherapy values. In the repeated trephine biopsis several morphological changes were observed: oedema of stromal cells, the relative increase of erythropoietic components with inverted proportion of granulo- to erythropoiesis. In conclusion, chemoimmunotherapy with Zevalin resulted in the reversible peripheral cytopenia parallely with transient damage to bone marrow hematopoiesis and in vitro clonogenic capacity.

Cells derived from porcine aorta tunica media show mesenchymal stromal-like cell properties in in vitro culture

2014 ◽  
Vol 306 (4) ◽  
pp. C322-C333 ◽  
Author(s):  
Andrea Zaniboni ◽  
Chiara Bernardini ◽  
Marco Alessandri ◽  
Chiara Mangano ◽  
Augusta Zannoni ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
In Vitro Culture ◽  
Umbilical Vein ◽  
Culture Conditions ◽  
Differentiation Potential ◽  
Tunica Media ◽  
Large Numbers ◽  
Umbilical Vein Endothelial Cells ◽  
Pig Animal Model

Several studies have already described the presence of specialized niches of precursor cells in vasculature wall, and it has been shown that these populations share several features with mesenchymal stromal cells (MSCs). Considering the relevance of MSCs in the cardiovascular physiopathology and regenerative medicine, and the usefulness of the pig animal model in this field, we reported a new method for MSC-like cell isolation from pig aorta. Filling the vessel with a collagenase solution for 40 min, all endothelial cells were detached and discarded and then collagenase treatment was repeated for 4 h to digest approximately one-third of the tunica media. The ability of our method to select a population of MSC-like cells from tunica media could be ascribed in part to the elimination of contaminant cells from the intimal layer and in part to the overnight culture in the high antibiotic/antimycotic condition and to the starvation step. Aortic-derived cells show an elongated, spindle shape, fibroblast-like morphology, as reported for MSCs, stain positively for CD44, CD56, CD90, and CD105; stain negatively for CD34 and CD45; and express CD73 mRNA. Moreover, these cells show the classical mesenchymal trilineage differentiation potential. Under our in vitro culture conditions, aortic-derived cells share some phenotypical features with pericytes and are able to take part in the formation of network-like structures if cocultured with human umbilical vein endothelial cells. In conclusion, our work reports a simple and highly suitable method for obtaining large numbers of precursor MSC-like cells derived from the porcine aortic wall.

scholarly journals Transdifferentiation of α-1,3-galactosyltransferase knockout pig bone marrow derived mesenchymal stem cells into pancreatic β-like cells by microenvironment modulation

2020 ◽  
Vol 33 (11) ◽  
pp. 1837-1847
Author(s):  
Imran Ullah ◽  
Ran Lee ◽  
Keon Bong Oh ◽  
Seongsoo Hwang ◽  
Youngim Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Cpg Island ◽  
Morphological Changes ◽  
Quantitative Polymerase Chain Reaction ◽  
Differentiation Potential ◽  
Pancreatic Differentiation ◽  
Epigenetic Modifiers ◽  
Pluripotent Marker

Objective: To evaluate the pancreatic differentiation potential of α-1,3-galactosyltransferase knockout (GalTKO) pig-derived bone marrow-derived mesenchymal stem cells (BM-MSCs) using epigenetic modifiers with different pancreatic induction media.Methods: The BM-MSCs have been differentiated into pancreatic β-like cells by inducing the overexpression of key transcription regulatory factors or by exposure to specific soluble inducers/small molecules. In this study, we evaluated the pancreatic differentiation of GalTKO pig-derived BM-MSCs using epigenetic modifiers, 5-azacytidine (5-Aza) and valproic acid (VPA), and two types of pancreatic induction media – advanced Dulbecco's modified Eagle's medium (ADMEM)-based and N2B27-based media. GalTKO BM-MSCs were treated with pancreatic induction media and the expression of pancreas-islets-specific markers was evaluated by real-time quantitative polymerase chain reaction, Western blotting, and immunofluorescence. Morphological changes and changes in the 5'-C-phosphate-G-3' (CpG) island methylation patterns were also evaluated.Results: The expression of the pluripotent marker (POU class 5 homeobox 1 [OCT4]) was upregulated upon exposure to 5-Aza and/or VPA. GalTKO BM-MSCs showed increased expression of neurogenic differentiation 1 in the ADMEM-based (5-Aza) media, while the expression of NK6 homeobox 1 was elevated in cells induced with the N2B27-based (5-Aza) media. Moreover, the morphological transition and formation of islets-like cellular clusters were also prominent in the cells induced with the N2B27-based media with 5-Aza. The higher insulin expression revealed the augmented trans-differentiation ability of GalTKO BM-MSCs into pancreatic β-like cells in the N2B27-based media than in the ADMEM-based media.Conclusion: 5-Aza treated GalTKO BM-MSCs showed an enhanced demethylation pattern in the second CpG island of the OCT4 promoter region compared to that in the GalTKO BM-MSCs. The exposure of GalTKO pig-derived BM-MSCs to the N2B27-based microenvironment can significantly enhance their trans-differentiation ability into pancreatic β-like cells.

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