Molecular beacons (MBs) are dual-labeled oligonucleotides that fluoresce only in the presence of complementary mRNA. The use of MBs to target specific mRNAs allows sorting of specific cells from a mixed cell population. Specifically, we designed and validated MBs targeting cardiac troponin T (cTNT or TNNI3) or myosin heavy chain genes (a/ßMHC or MYH6/7) mRNAs respectively that are highly expressed only in CMs. MBs were then delivered into differentiating mouse or human PSCs using nucleofection followed by FACS sorting of CMs based on MB signal. Control studies confirmed that CM specific MBs had low background signal in all non-CM cells in the differentiating culture of PSCs, confirming detection specificity. We found that 97% of mouse or human cells isolated using FACS based on MB signal demonstrated CM-like characteristics in assays including electrophysiology, flow cytometry, and immunocytochemistry. This approach, using a FACS sorter to purify CMs based on MB signal from specific mRNA expression, is very versatile and can be adopted for the isolation of other cell types from differentiating PSCs.
Nucleofection tools: Nucleofector™ 2b Device (Lonza) and Cell Line Nucleofector Kit V (Lonza, cat. no. VACA-1003)
Molecular beacons (MBs) are dual-labeled oligonucleotides that fluoresce only in the presence of complementary mRNA. The use of MBs to target specific mRNAs allows sorting of specific cells from a mixed cell population. In contrast to existing approaches that are limited by available surface markers or selectable metabolic characteristics, the MB-based method enables the isolation of a wide variety of cells. For example, the ability to purify specific cell types derived from pluripotent stem cells (PSCs) is important for basic research and therapeutics. In addition to providing a general protocol for MB design, validation and nucleofection into cells, we describe how to isolate a specific cell population from differentiating PSCs. By using this protocol, we have successfully isolated cardiomyocytes differentiated from mouse or human PSCs (hPSCs) with ~ 97% purity, as confirmed by electrophysiology and immunocytochemistry. After designing MBs, their ordering and validation requires 2 weeks, and the isolation process requires 3 h.