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Abstract Mesenchymal stem cells (MSCs) are heterogenous population of stem/ progenitor cells with the capacity to differentiate into mesodermal and non-mesodermal cell lineages. MSCs can self renew, have multi-potent differentiation potential and represent an attractive cell source for regeneration of damaged tissues in tissue engineering. They can be isolated from different tissue sources such as bone marrow, adipose tissue, umbilical cord, synovium, periosteum and placenta. Using specific media, MSC can be induced to differentiate in vitro into different cell lineages such as adipogenic, osteogenic, chondrogenic, and myogenic lineages. For many years, bone marrow has been considered the ―gold standard‖ for the derivation of MSCs for human stem cell engineering. However, extraction comes with ethical constraint and problems associated with painful harvesting and donor site morbidity. Umbilicalcords appear to show potential as a source of MSCs for a number of reasons; easy isolation, they are considered medical waste, and therefore their use in research has little ethical concern, they proliferate rapidly in culture and are thought to be immune privileged. The aim of this study is to investigate isolation and in vitro differentiation of umbilical cord mesenchymal stem cells into different cell lineages such as osteocytes, adipocytes and chondrocytes. The present study included 35 UC samples, 10 of them excluded due to use of FBS and contamination. All samples were subjected to the followings: isolation of UC-MSCs was done using explants method. The cells then were identified by their morphology (fibroblastic like cells) and flowcytometric analysis of cell surface markers (CD34-, CD44+ and CD73+). Moreover, osteogenic, adipogenic and chondrogenic differentiation were induced using osteogenic, adipogenic and chondrogenic differentiation supplement respectively and were identified by morphology and ICC. The results revealed that: As regards UC-MSCs morphological changes: In the first five days, the adherent cells grew in a round shape with few morphological changes. After 7 days of primary culture, UC-MSCs adhered to the plastic surface of the culture flasks and presented as a small population of single cells with spindle-like shape. On day 10 after initial culture, the cells exhibited long spindleshapedfibroblastic cells; they had begun to form colonies and were confluent. By day 14, UC-MSCs were duplicating rapidly and the cell morphology was mainly spindle-shaped with triangular appearance. As regards flowcytometric detection of UC-MSCs: Isolated MSCs showed positive CD44 (R= 55.0 – 90.0%) with mean value (71.2±11.1), CD73 (R=60.0–85.0%) with mean value (73.8± 8.7) and negative CD34 (R=0.3 –3.5%) with mean value (2.3 ± 0.9). As regards post-induction morphological changes: Cells induced with the osteogenic differentiation medium acquired stellate- shape with cellular processes seen. Cells induced with the adipogenic differentiation medium developed lipid droplets that accumulated in the cells. Cells induced with the chondrogenic differentiation medium changed from differentiated round and polygonal cells at day 7 to reach elongated fibroblastic-like phenotype at day 14. As regards post-induction ICC analysis: Immunocytochemical analysis of differentiated osteocytes, adipocytes and chondrocytes shows that cells were stained red and the nuclei were counterstained blue. |