Abstract 023: Development Of Molecular Beacons Targeting Atrial Natriuretic Factor Mrna For Live Cell Sorting

Abstract

Introduction: Human embryonic stem cell (hESC) differentiation protocols generate both working and nodal cardiomyocytes, however this mixed cell population is not ideal for clinical applications. No surface markers have been used for the separation of live nodal or working cells to date. Molecular beacons are hairpin oligonucleotide probes with a reporter fluorophore at one end and a quencher at the other; they are designed to fluoresce when hybridized to a complementary target and can be used to detect mRNA in live cells. Atrial natriuretic factor (ANF) is expressed significantly more in working cardiomyocytes than nodal cardiomyocytes. We hypothesize that molecular beacons targeting ANF mRNA may be used to separate working cells from differentiated hESCs. Methods and Results: We designed molecular beacons to target regions of ANF mRNA predicted to be accessible using RNA secondary structure prediction programs. We then developed a positive control cell line using chinese hamster ovary (CHO) cells to select functional beacons. CHO cells were transfected with a plasmid containing the ANF gene and puromycin resistance. Transfected cells were sorted into separate wells and clonal populations were exposed to puromycin for 2 weeks. Surviving colonies were assayed for ANF expression and used as a positive control for beacon characterization. We used this system to screen 10 molecular beacons targeting ANF and found 3 beacons that increased in fluorescence in more than 60% of ANF-CHO cells but less than 5% of untransfected cells. To examine whether molecular beacons are suitable for differentiated cultures, we carried out cardiomyocyte differentiation from hESCs using growth factors. ANF expression was up-regulated in differentiated cultures compared with undifferentiated cells. Transfection of differentiated hESCs using the Neon system was optimized using fluorophore labeled oligonucleotides, a positive control for beacon delivery. Studies are currently underway to determine ANF beacon sensitivity and specificity in hESCs. Conclusions: We have developed several molecular beacons with potential for the use of separating live cells based on the expression of ANF mRNA which could represent a powerful technique for stem cell sorting.