Dual-labeled antisense oligonucleotide probes for fluorescent activated cell sorting (FACS) of pluripotent stem cells.
- Allows for the purification of a homogeneous cell population compared to currently available techniques which can only produce heterogeneous mixtures.
- Binds intracellular RNA using standard delivery techniques eliminating the need for known surface markers to be present within the cell population.
- Specific probes can be generated for different cell types.
The global market for flow cytometry products and reagents was estimated at $2.6 million in 2010. The reagents market comprised the second largest share at $0.8 billion, a number that is expected to reach $1.5 billion by 2015. Flow cytometry sorting techniques have previously used a mitochondrial dye for the purification of mature cardiomyocytes, but these techniques suffer from a lack of specificity and sensitivity. Additional approaches have relied on cell specific proteins and antibodies to enrich a homogeneous population. In this instance purification is often hindered by a lack of known markers.
The inventors have described cardiomyocyte-specific molecular probes, termed molecular beacons, consisting of an oligonucleotide with a fluorescent emitter at one end and a fluorescent quencher at the other. These dual-labeled molecular beacons are designed to fold back on themselves in the absence of a complementary cellular nucleotide sequence. In the presence of a complementary sequence, the beacon will hybridize with the targeted mRNA sequence and produce a fluorescent signal. The differentiated cells can then be sorted via FACS with high efficiency and specificity using flow cytometry. Because these molecular beacons bind intracellular RNA through traditional delivery systems, the need to first identify a surface protein specifically expressed on the target cell is eliminated. The technology therefore provides a method of purifying cell populations that lack known surface markers.
A homogeneous population of cardiomyocytes has been purified from differentiating mouse and human pluripotent stem cells.
Publications: Ban K et al. Circulation. 126:A18585. (2012).