الفهرس 
Chapter I: Stem CellOnly 14 pages are availabe for public view
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Abstract
Vascular endothelial cells line the entire circulatory system, from the heart to the smallest capillaries. These cells have unique functions in vascular biology. These functions include fluid filtration, blood vessel tone, hemostasis, neutrophil recruitment, and hormone trafficking. ECs are of great interest because of their potential in cell therapy for vascular diseases and ischemic tissue, tissue engineering for vascular grafts and vascularized tissue beds, and modeling for pharmaceutical transport across endothelial barriers. EC derived from stem cells could potentially lead to a variety of clinically relevant applications. These cells could be used in therapeutic strategies for the repair and revascularization of ischemic tissue in patients exhibiting vascular defects.Mesenchymal stem cells (MSCs), known as multipotent mesenchymal stromal cells, are self-renewing cells which can be found in almost all postnatal organs and tissues. Bone marrow is considered as the gold standard for MSC however, BM has several limitations. So that MSCs obtained from the Wharton’s Jelly (WJ) of umbilical cords (UC) have gained much attention since they can be easily isolated, without any ethical concerns, from a tissue which is discarded after birth. This study aimed to investigate the in vitro differentiation of mesenchymal stem cells into cells of the endothelial lineage as future revascularization therapy of ischemic tissue.This study was conducted at Clinical Pathology department in collaboration with Obstetrics & Gynecology Departments at Faculty of Medicine, Menuofia University during the period from January 2015 to February 2016. Twenty bone marrow samples were used for isolation of MSCs. MSCs were isolated from 75% (15/20) samples and 5 samples failed due to contamination.BM samples were subjected to mononuclear cell separation by lymphocytes separation medium then MNC suspension were seeded at a concentration of (1x106 cells /ml) in plastic tissue culture flask 25cm2, incubated for 7 days in 5 ml of fresh complete medium . In addition to ten umbilical cord samples were used for collection of WJ in order to isolation MSCs and to obtain cord blood serum to replace FBS. The success rate in isolating and culturing of MSCs were 60% (6/10). Failure due to contamination and use of FBS.Umbilical cord tissue cultured by explant method in which the cord was divided into small segments then opened longitudinally, vessels were dissected and removed then the cord tissues were washed with saline. WJ was cut into small pieces of about 1.5-2.5mm then cultured in plastic tissue culture flask 25cm2, incubated for 7 days in 5 ml of fresh complete medium. At day 7, the tissue removed by changing the medium. Adherent cells were kept in culture and were fed with fresh complete nutrient medium about 1 week later. MSCs at the base of the flasks were harvested by trypsinization and identified by morphology and flowcytometric analysis of CD44, CD34 and CD31.flowcytometric analysis revealed that the isolated MSCs showed positive expression of CD 44 for BM (81.2±12.4) and for WJ (77.1±12.7), and negative expression of CD34 for BM (1.97±1.5 ) and for WJ (1.7± 1).During cell proliferation, the MSCs were cultured up to passage 5 .The WJ-MSCs displayed the higher cumulative cell population and shorter doubling time than BM-MSCs. Confluent MSC cells were induced to differentiate into endothelial cells by culture in the presence of complete medium supplemented with 50 ng/ml VEGF for 7 days. Medium was changed every 3 days. MSCs induced with VEGF successfully differentiated into endothelial cells. ECs proved by morphology, flowcytometric analysis of CD31and CD34, immunocytochemical analysis of VE- cadherin and hematoxylin and eosin staining. EC showed spindle shape with no much morphology difference form MSCs. Positive expression of CD31 for BM (74.2±12) and for WJ ( 87.9±4.5) and positive expression of CD34 for BM (83.4 ±10.4) and for WJ (83.7±9.3). By immunocytochemical analysis the harvested endothelial cells at day 8 were positive for vascular endothelial cadherin (CD144). The harvested endothelial cells were identified by routine stain, using hematoxylin and eosin stain. The endothelial cells appear typically spindle shape with pink cytoplasm and blue nucleus.