Background and Objectives: Mesenchymal stem cells (MSCs) are multipotent stem cells that can be isolated from umbilical cords and are therapeutically used because of their ability to differentiate into various types of cells, in addition to their immunosuppressive and anti-inflammatory properties. Fetal bovine serum (FBS), considered as the standard additive when isolating and culturing MSCs, has a major limitation related to its animal origin. Here, we employed a simple and economically efficient protocol to isolate MSCs from human umbilical cord tissues without using digestive enzymes and replacing FBS with umbilical cord blood serum (CBS). Methods and Results: MSCs were isolated by culturing umbilical cord pieces in CBS or FBS supplemented media. Expansion and proliferation kinetics of cells isolated by explant method in the presence of either FBS or CBS were measured, with morphology and multi-differentiation potential of expanded cells characterized by flow cytometry, RT-PCR, and immunofluorescence. MSCs maintained morphology, immunophenotyping, multi-differentiation potential, and self-renewal ability, with better proliferation rates for cells cultured in CBS compared to FBS supplement media. Conclusions: We here present a simple, reliable and efficient method to isolate MSCs from umbilical cord tissues, where cells maintained proliferation, differentiation potential and immunophenotyping properties and could be efficiently expanded for clinical applications.

PurposeArticular cartilage has a poor capacity for self-repair, and thus still presents a major challenge in orthopedics. Mesenchymal stem cells (MSCs) are multipotent stem cells with the potential to differentiate into chondrocytes in the presence of transforming growth factor beta (TGF-β). Platelet lysate (PL) contains a relatively large number of growth factors, including TGF-β, and has been shown to ameliorate cartilage repair. Here, we investigated the ability of PL to direct chondrogenic differentiation of MSCs along with other standard differentiation components in a pellet culture system.MethodsWe isolated and expanded MSCs from human umbilical cords using a PL-supplemented medium and characterized the cells based on immunophenotype and potential for differentiation to adipocytes and osteocytes. We further cultured MSCs as pellets in a chondrogenic-differentiation medium supplemented with PL. After 21 days, the pellets were processed for histological analysis and stained with alician blue and acridine orange. The expression of SOX9 was investigated using RT-PCR.ResultsMSCs maintained their stemness characteristics in the PL-supplemented medium. However, the distribution of cells in the pellets cultured in the PL-supplemented chondrogenic differentiation medium had a greater similarity to cartilage tissue-derived chondrocytes than to the negative control. The intense alician blue staining indicated an increased production of mucopolysaccharides in the differentiated pellets, which also showed elevated expression of SOX9.ConclusionsOur data suggest that MSCs could be differentiated to chondrocytes in the presence of PL and absence of exogenous TGF-β. Further research needs to be conducted to understand the exact role and potential of PL in chondrogenic differentiation and chondrocyte regeneration.

Background: Mesenchymal stem cells (MSCs) are multipotent cells which can be isolated from many sources including umbilical cord. Isolation protocols are depended on either explant or enzymatic methods. Although fetal bovine serum (FBS) is used as a supplement in isolation and expansion of MSCs, human blood derivatives such as cord blood serum (CBS) and platelet lysate (PL) are attractive substitutes of FBS which overcome impediments of using FBS in clinical setups. Methods: Here we compared the effect of using CBS, PL and FBS supplemented media in isolation of umbilical cord tissue derived MSCs by using explant method. To do that we cultured umbilical cord tissue explant in either CBS or PL or FBS supplemented media. Isolated cells were quantified, their morphology was assessed and Cells in passage 3 were characterized based on their immunophenotyping and their potential for differentiation into adipocytes and osteocytes. Moreover, proliferation of cells was assessed by crystal violet staining. Results: All the three media succeeded to isolate MSCs and maintain their stemness characteristics. However, the highest number of isolated cells were obtained using CBS, ~10-fold more than FBS, while the number of isolated cells obtained using PL was ~2-fold more than FBS. Moreover, crystal violet showed that both PL and CBS promote proliferation of MSCs more than FBS. Conclusions: Our data suggest that, although all supplements maintain stemness characteristics of MSCs when used to isolate those cells by explant method, using human blood derived supplements is more effective than FBS. In the same context CBS is more effective than PL.