Postdoc University of California, Los Angeles Los Angeles, California, United States
Introduction: Direct cell reprogramming has emerged as a revolutionary approach in biology with broad applications in cell therapies for tissue regeneration and disease treatment. However, the isolation and purification of reprogrammed cells, especially post-mitotic cells such as neurons and cardiomyocytes, from mixed cell populations remains a challenge. In addition, many cell types to be isolated do not have unique cell surface markers for traditional protein-based cell sorting. Materials and Methods: To address this challenge, we engineer a nucleic acid-based gold nanorod (NAGN) biosensor that can detect the mRNA expression of intracellular phenotypic markers and become fluorescent for cell sorting, and demonstrate the application of isolating induced neuronal (iN) cells from dermal fibroblast populations during the early stages of cell reprogramming. Here we designed complementary nucleic acid probes for mRNAs of neuronal markers such as Ascl1 and Scn2. NAGNs incorporated with mRNA probes were introduced into cells by 8 hours of incubation at day 1 after the reprogramming factors Brn2, Ascl1 and Myt1l were expressed in fibroblasts. Results and Discussion: The amounts of NAGNs were optimized to a level without any side effects on cell viability and functions. At day 2, fluorescence-activated cell sorting (FACS) was utilized to sort for Ascl1+ cells, which were further cultured to yield mature, functional neurons. 1his approach resulted in the enrichment of iN cells from 3% to 72%. In addition, Scn2-NAGN was used to sort the Ascl1+ cells on the second day of iN reprogramming, resulting in an iN cell enrichment of greater than 85%, demonstrating the feasibility of mRNA-based cell sorting of multiple markers. Furthermore, we showed that NAGN could be used in conjunction with protein-based cell sorting. Finally, an in vitro co-culture of iN cells and muscle tissue constructs demonstrated the functionality of NAGN-sorted iN cells in response to electrical stimulations. Conclusions: Taken together, these results suggest that NAGN-based cell sorting holds a great potential for cell isolation and purification that are critical for cell therapies and disease modeling. Acknowledgements: The authors are supported in part by a grant from the National Institute of Health (HL121450 and GM143485).
