Pluripotent human embryonic
stem cells (hESCs) have an unlimited capacity for self-renewal and the ability to differentiate in culture and in vivo into tissues derived from all 3 embryonic germ layers. To date, most hESC lines have been characterized by their expression of cell surface antigens [1
]. These studies have identified a battery of glycolipids and glycoproteins that are found on a high percentage of undifferentiated hESCs, including the stage-specific antigens, SSEA-3 and SSEA-4, and the keratin sulfate-related antigens, Tra-1–60 and Tra-1–81, among others [2
]. These antigens are commonly used to assess the pluripotency of hESCs, for within days upon the induction of differentiation their expression dramatically decreases [3
]. It also has been appreciated that low levels of spontaneous differentiation occur within hESC cultures grown under proliferation conditions, and that cells within proliferating colonies can express early markers of specific embryonic germ layers [4
]. As such, the presence of these cells may bias the study of mechanisms of pluripotency in proliferating hESC colonies. Nuclear transcription factors such as Oct4 and Nanog have been implicated in pathways regulating pluripotency [5
]; however, expression of these proteins is more difficult to assess in live cells. Virally transduced reporters have been shown to be specific and efficient for this purpose [7
]; however, these have the potential to alter cell behavior, especially when randomly integrated into the cell genome.
Molecular beacons are single-stranded oligonucleotides that have been employed to assay gene expression in vitro, as in real-time polymerase chain reaction (PCR), and in single cells using microscopy [8
]. These consist of short sequences capable of forming stem-loop structures bearing a fluorescent reporter group at one end and a fluorescent quencher at the opposite end [8
]. In the absence of a target sequence, the oligonucleotide self-anneals, forming a stem that brings the reporter and quencher in close proximity, thereby quenching fluorescence. In the presence of a target sequence, the oligonucleotide anneals to the target, separating the reporter and quencher, thereby allowing fluorescence. To test the potential of this technology for identifying and isolating live pluripotent hESCs in a high-throughput manner, we developed a fluorescence-activated cell sorting (FACS)-based, dual fluorescence resonance energy transfer (FRET) molecular beacon system that utilizes pairs of molecular beacons containing donor and acceptor fluorescent groups. FRET results when the 2 fluorescent groups are brought into proximity by both beacons annealing to a target sequence, thus increasing specificity by requiring recognition by both oligonucleotides. The probes are synthesized using O-methylated nucleotides, which are not recognized by ribonucleases and avoid activating the RNA interference system [9
]. FACS allows for excitation of the donor group, detection of emission from the acceptor group, and high-throughput sorting of cells expressing the target nuclear protein based on FRET. Using this approach, we developed a high-throughput method for isolating live hESCs based on expression of intracellular proteins, without altering the functional or genomic characteristics of the cells.