ABSTRACT
Skin stem cells are very important in cosmetics, pharmacological and regenerative medicine and burn cases. Foreskin samples surgically removed after circumcision from boys below 7 years were collected and primary epidermal cells were prepared by enzymatic and mechanical tituration method. Selecting CD133 (prominin-1) multipotent stem cell marker, enriched stem cells were analyzed by MACS using CD133 antibodies conjugated with magnetic beads. CD133 positive and negative cells with specific skin stem cells markers like - CD34 (Universal stem cells marker), CD29 (integrin beta-1) and CD49f (integrin alpha-6) immunophenotypes were screened and sorted in flowcytometer. Further the expression of four embryonic genes or transcription factors of pluripotent stem cells were analyzed for pluripotent character of sorted cells. It was found that skin stem cell markers associated with CD133 cells, differentially expressed CD34, CD29 and CD49f immunophenotyes on both positive and negative CD133 cells in FACS analysis. The embryonic stem cell markers (induced pluripotent stem cell markers) like Oct4, SOX2, Notch-2 and K19 genes were expressed in CD133 positive epidermal cells. It is therefore evident that foreskin derived epidermal stem cells showed pluripotent or multipotent nature. This finding opens up avenues for new uses of these stem cells for direct cell seeding in wound healing, surgical suturing and drug screening.
Subject(s)
Antigens, CD/metabolism , Biomarkers/metabolism , Cell Lineage/genetics , Cell Separation , Cell Survival/genetics , Child , Epidermis/cytology , Flow Cytometry , Gene Expression Profiling , Gene Expression Regulation , Glycoproteins/metabolism , Humans , Immunophenotyping , Male , Peptides/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Propidium/metabolism , Skin/cytology , Staining and LabelingABSTRACT
Evaluation of apoptosis by flow cytometry is generally accomplished by methods that use annexin V-FITC as vital dye, which access phosphatidylserine exposed on the external membrane at the beginning of this process. In addition, the concomitant use of propidium iodide makes possible to verify the characteristic nuclear alterations in the late stages of apoptosis, as a consequence of the increase in membrane permeability. On the other hand, the use of calcein-AM in association with ethidium homodimer (EthD-1) allows the evaluation of cell apoptosis through detection of esterase activity and cellular membrane physical and chemical alterations. The aim of this study was to compare the sensibility of calcein-AM and EthD-1 with annexin V-FITC and propidium iodide for early apoptosis evaluation in peripheral blood mononuclear cell culture, obtained from HIV-infected patients. Apoptosis and cellular viability were detected and quantified by flow cytometry after 24 and 48 hours incubation times. Our results showed that calcein-AM/EthD-1 was more sensitive for apoptotic cell quantification in both incubation times than annexin V-FITC/propidium iodide (mean of 46.95 percent ± 3.56, p < 0.0001, for 24 hours and mean of 37.67 percent ± 2.47, p < 0.0014 for 48 hours), besides allowing to clearly define viable, apoptotic and dead cell populations.