Purification of cell mixtures using molecular beacons targeting cell specific RNA
This disclosure is in the area of research and therapeutics. In certain embodiments, it provides methods to assist in the purification of cell mixtures, e.g., cardiomyocytes, using molecular beacons targeting cell-type specific RNA, e.g. mRNA.
1. A method for purifying cardiomyocytes comprising,
a) introducing a molecular beacon comprising a loop sequence having SEQ ID NO: 4 into a mixture of cardiomyocytes and pluripotent stem cells under conditions such that the molecular beacon hybridizes to mRNA in the cardiomyocytes providing molecular beacon bound fluorescent cardiomyocytes; and
b) purifying the molecular beacon bound fluorescent cardiomyocytes by fluorescence activated cell sorting providing a purified composition of cardiomyoctyes.
2. The method of claim 1 , wherein the mixture of cardiomyocytes and pluripotent stem cells have been differentiated by a method comprising:
a) culturing undifferentiated pluripotent stem cells under conditions such that a monolayer of the undifferentiated stem cells is formed,
b) applying bone morphogenetic protein 4 (BMP4), Activin A, and fibroblast growth factor 2 (FGF2) to the undifferentiated stem cells under conditions such that cardiac linage cells are formed, and
c) treating the cardiac linage cells with isoproterenol to generate spontaneous beating cardiomyocytes.
3. The method of claim 1 , wherein pluripotent stem cells are mammalian pluripotent stem cells.