Peripheral venous blood mononucleated cells have potential to commit to progenitor endothelial cells when exposed to vascular endothelial growth factor

Abstract Introduction The endothelium plays a pivotal role in the regulation of homeostasis through expression of the antithrombotic and prothrombotic factor. Transdifferentiation requires lesser cell subcultures and can reduce the chance of alterations. We describe in the current article the transdifferentiation of blood mononucleated cells to committed progenitor endothelial cells. Materials and methods Experiments were set up for mononucleated cells culture, transdifferentiation of mononucleated cells to committed progenitor endothelial cells, characterization of mononucleated cells to committed progenitor endothelial cells to terminal transdifferentiated endothelial cells. Results It was confirmed that 10 weeks of maintenance of mononucleated cells in culture with supplementation of vascular endothelial growth factor in endothelial conditioning growth medium brings about mononucleated cells to terminal transdifferentiation of endothelial cells, which was confirmed finally by cell characterization (immunocytochemistry) using the specific marker vWF+. Conclusion Vascular endothelial growth factor has the potential signalling cascade that can transdifferentiate mononucleated cells to endothelial cells in culture.


Introduction
The endothelium plays a pivotal role in the regulation of homeostasis through expression of the antithrombotic and prothrombotic factor.Transdifferentiation requires lesser cell subcultures and can reduce the chance of alterations.We describe in the current article the transdifferentiation of blood mononucleated cells to committed progenitor endothelial cells.

Materials and methods
Experiments were set up for mononucleated cells culture, transdifferentiation of mononucleated cells to committed progenitor endothelial cells, characterization of mononucleated cells to committed progenitor endothelial cells to terminal transdifferentiated endothelial cells.

Results
It was confirmed that 10 weeks of maintenance of mononucleated cells in culture with supplementation of vascular endothelial growth factor in endothelial conditioning growth medium brings about mononucleated cells to terminal transdifferentiation of endothelial cells, which was confirmed finally by cell characterization (immunocytochemistry) using the specific marker vWF + .

Introduction
Vascular endothelial cells develop as a nonthrombogenic monolayer, lining the entire interior surface of the blood vessels 1 .At embryogenesis, new blood vessel formation occurs, which leads to branching of capillaries and microvasculature often termed as angiogenesis.It is very well understood that mononucleated cells (MNCs) can be proliferated and differentiated into different cell lineages 2 .Differentiation is the process leading to the expression of phenotypic property characteristics of the functionally mature cell in vitro.There are pathways to differentiation in adult organism.In constantly renewing tissues like blood, a small population of totipotent or pluripotent undifferentiated stem cells are capable of self-renewal that gives rise to various progenitor cells that will progress towards differentiation, losing their capacity to divide as they reach the terminal stages.As differentiation progresses, cell division is reduced and eventually ceases.In most cell systems, cell proliferation is incompatible with the expression of differentiated properties.Progression from stem cell to a particular pathway of differentiation traditionally implied a rapid increase in commitment.Commitment is regarded as a point between the stem cell and a particular progenitor stage where stem cell or its progeny can no longer transfer to a separate lineage.Transdifferentiation is a process by which a particular cell converts to another cell type.In cardiovascular patients with atherosclerosis and heart attacks, it has already been reported that progenitor endothelial cells are found in the circulating blood.But healthy individuals have extremely little or are devoid of progenitor endothelial cells 3 .The aim of this study was to understand the differentiation of MNCs to committed progenitor endothelial cells to terminal transdifferentiated endothelial cells.The model of our investigation is differentiation of MNCs to committed progenitor and terminal transdifferentiation of endothelial cells.

Isolation of peripheral venous blood mononucleated cells Institutional ethical committee (Committee for Human and Animal
Research Ethics) issue informed consent was taken from the donor.Venous peripheral blood was collected from the healthy donor and processed for MNC isolation with density-dependent centrifugation process using Histopaque as a gradient.Mononuclear cells were obtained from diluted blood using Histopaque (Sigma Chemical Co., USA) following manufacturer's protocol.Isolated cells were refreshed and centrifuged twice at 250 g for 10 min, the cell pellet was further resuspended in the cell culture medium DMEM and re-centrifuged at 250 g for 10 min.The supernatant was discarded and the cells were seeded with 20% Dulbecco's Modified Eagle Medium (low glucose).The plate was incubated at 37°C in 5%CO 2 .Culture medium was changed after 24 h 4 , unattached cells and debris were removed by refreshing cells with phosphate buffer saline (PBS 1×) and fresh complete medium was fed to the cells 5 .

Mononucleated cell culture
Plated MNCs were fed with complete medium (DMEM) after 24 h till the culture reached confluence, and the first subculture was done after 8 days.Cell count was taken on subculture using a cell counter 5 .

Differentiation of MNCs to committed progenitor endothelial cells
MNCs were seeded singly after trypsinization, and the cells were fed again with 20% endothelial growth condition medium (ECGM) supplemented with endothelial supplement mix, 1 ng/ml vascular endothelial growth factor (VEGF), 2 mM L-glutamine, penicillin 5 units/ml and streptomycin 5 mg/ ml.Cultured cells were maintained for 10 weeks in the ECGM complete medium.

Immunocytochemistry for phenotypic expression of committed progenitor and terminal transdifferentiated endothelial cells
For immunocytochemistry, the same protocol was adapted as above 6 .MNCs were maintained for 10 weeks in complete ECGM supplemented with 1 ng/ml VEGF.Cells were characterized for their phenotypic expression of committed progenitor and terminal transdifferentiated endothelial cells using endothelial cellspecific marker such as von-Willebrand factor (vWF (-/+) ).

Results
Isolation and MNC culture and subculture Isolated MNCs were cultured in a complete medium DMEM.The cells were attached singly round and adhered to the plate surface and further become flattened and started expanding in the culture.The first subculture was done on the ninth day as MNCs reached confluence.The cell count was 1.2 × 10 5 cells/ml (Figure 1).

MNCs to committed progenitor and terminal transdifferentiation of endothelial cells
About 5 × 10 4 cells/ml MNCs were plated for the terminal transdifferentiation experiment.MNCs were maintained for 10 weeks in the complete ECGM medium supplemented with 1 ng/ml VEGF.During the experiment, the majority of MNCs were shed off from the plate surface till they reached the seventh week and the proliferation of MNCs was extremely reduced (halted) and showed round colony-forming cells (CFCs) in a scattered cluster.On the tenth week, approximately 100 cells were present on the plate surface, with changed cellular morphology that resembles single round to pebble shape (Figure 2).

Cell characterization experiments (phenotypic expression)
MNCs that were grown in DMEM without any supplements showed negative expression of vWF (-) -that is, absence of vWF (-) (Figure 3) on the tenth week.
MNCs that were maintained for 10 weeks (CFC) in ECGM along with supplementation of 1 ng/ml VEGF (Sigma) (Figure 4) showed positive phenotypic expression of vWF + and further progress towards lineagespecific committed progenitor and terminal transdifferentiation to endothelial cells (vWF + ).important role during vasodilatation 1 .The main goal of this research was to improve the efficiency of endothelial progenitor cell recovery or harvest from human peripheral blood at in vitro conditions.Cells platted on day 1 showed complete round cells in many clusters, further resembling expansion.The MNCs were subcultured and re-plated for experiments of differentiation.We tried to understand the mechanism of blood MNCs to phenotypic expression of vWF and progress towards committed progenitor and terminal transdifferentiated endothelial cells.The experiments revealed to us that MNCs when cultured in complete DMEM till they reached confluence do not express the endothelial-specific marker vWF (-) , but when MNCs were maintained for 10 weeks (CFC) in ECGM exposed specifically to VEGF and endothelial growth supplement mix, there was terminal transdifferentiation of MNCs with positive phenotypic character expression, that is vWF (+) .

Conclusion
VEGF has a potential signalling cascade mechanism towards MNCs to form committed progenitor endothelial cells.This even emphasizes that VEGF signalling can be induced to isolated and cultured MNCs, which can be further converted to committed progenitor followed by terminal transdifferentiation endothelial cells.

Discussion
Vascular endothelial cells are recruited from the bone marrowreleased hematopoietic stem cells, which further get differentiated into progenitor endothelial cells upon receiving specific signalling cascade in the circulatory system.Endothelial cells line the entire blood vascular and circulatory system and play an

Figure 4 :
Figure 4: MNCs to terminal transdifferentiation of endothelial shows phenotypic expression of vWF (+) staining in cytosol area.