Figure 2

WJCs characterization. A. Light microscopic micrographs of WJCs in monolayer culture. In monolayer culture, the cells assumed a polymorphic, fibrobroblast-like morphology, which was maintained throughout the passaging processes. 10 days (left panel), 15 days of culture (central panel) and cells at passage 1, prior to reach confluence (right panel). Original magnification ×100. B. Analyses of surface antigen marker by flow cytometry. Cell suspensions were stained with specific mouse anti-human monoclonal antibodies (Mabs) as indicated in filled histograms. The empty histogram is the respective IgG isotype control. C. Ability to differentiate into several lineages. Osteogenic differentiation (left panel) was indicated by the increase in alkaline phosphatase (magnification 200×). Adipogenic differentiation (central panel) is visually marked by accumulation of neutral lipid vacuoles in cultures (red oil staining) (magnification 200×). Chondrogenic differentiation (right panel) is visually marked by toluidine blue staining (magnification 100×). D. Flow cytometry of cells showing DNA stained with propidium iodide. G1 and G2/M indicate 2n and 4n cellular DNA content, respectively. S indicates cells undergoing DNA synthesis, intermediate in DNA content between 2n and 4n. In the phase contrast image, arrow indicates one mitotic event. E. Telomere length assay on WJCs at different culture passages. The maintenance of long telomeres is a key feature of stem cells, ensuring the capability to undergo several cell cycles of replication. Molecular weight marker (M), control DNA with short telomeres (S) and control DNA with long telomeres (L). p4, p8 and p12 DNA extracts from WJCs at different 4^th 8^th and 12^th passages showing long telomeric ends. F, G. Growth Features of WJCs during expansion period. Proliferation (F) and doubling time (G) at different passages of WJCs in vitro culture calculated by Trypan blue exclusion test.