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 kit comprising a molecular beacon comprising a loop sequence having SEQ ID NO: 2 and a fluorophore.
2. The kit of claim 1 further comprising instructions for purifying cardiomyocytes.
3. The kit of claim 1 further comprising bone morphogenetic protein 4.
4. The kit of claim 1 further comprising activin A.
5. The kit of claim 1 further comprising basic fibroblast growth factor.
6. The kit of claim 1 further comprising isoproterenol.
7. The kit of claim 1 , wherein the molecular beacon is enclosed in a container.
8. The kit of claim 7 , wherein the container is a vial or ampoule.
9. A method for purifying cardiomyocytes comprising,
a) introducing a molecular beacon comprising a loop sequence having SEQ ID NO: 2 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 to provide a purified composition of cardiomyocytes.
10. The method of claim 9 , wherein the mixture of cardiomyocytes and pluripotent stem cells have been differentiated by a method comprising:
a) culturing undifferentiated 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 lineage cells are formed, and
c) treating the cardiac lineage cells with isoproterenol to generate spontaneous beating cardiomyocytes.
11. The method of claim 9 , wherein the pluripotent stem cells are mammalian pluripotent stem cells.