Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass (ICM) of preimplantation embryos and are capable of unlimited, undifferentiated proliferation in vitro under appropriate cell culture conditions. Undifferentiated embryonic stem cells are characterized by high expression levels of stage-specific embryonic antigens (SSEA-1, 3 and 4), tumor rejection antigens (TRA-1-60 and TRA-1-81), OCT-4 and alkaline phosphatase. These undifferentiated markers are downregulated upon spontaneous or induced differentiation.
Alkaline phosphatase (ALP) is a hydrolase enzyme responsible for dephosphorylating molecules such as nucleotides, proteins, and alkaloids under alkaline conditions. The enzyme is present within all tissues of the body but is elevated in cells of the liver, kidney, bone, placenta, embryo and under specific disease states. Under alkaline conditions (pH>10), ALP can catalyze the hydrolysis of p-nitrophenylphosphate (p-NPP) into phosphate and p-nitrophenol, a yellow colored by-product of the catalytic reaction. The amount of p-nitrophenol produced is proportional to the amount of alkaline phosphatase present within the reaction. The amount of ALP can thus be reliably quantified by reading the amount of p-nitrophenol amassed after the catalytic reaction at 405 nm on a spectrophotometer.