Laser delivery of microRNAs into cells
Cell therapy represents a new revolutionary approach for the regeneration of tissues. Induction of a pluripotent state in adult somatic cells resulted in exciting work in clinical therapy and disease modelling. Recently, the concept of direct transition from one determined cell type into another (transdifferentiation) has emerged. It was established that overexpression of specific transcription factors and microRNAs (miRs) can be used for transdifferentiation, bypassing the stage of pluripotency. The EU-funded MIR-OPTOFECTARRAY (Delivering non-viral silencing RNA (microRNA) using automated femtosecond lasers into stem cells for cardiac reprogramming and characterization using non-destructive optical techniques) project aimed to develop techniques for laser-guided delivery of miRs into cells to reprogram them into cardiomyocytes. Researchers used femtosecond laser optotransfection to create transient nanopores in the membrane for the efficient entry of microRNAs into the cells. They optimised protocols for the pre-conditioning of embryonic fibroblasts to make them more susceptible towards epigenetic re-programming. This protocol has been applied successfully to mouse embryonic fibroblasts and human foetal cardiac fibroblasts. Delivery of the transcription factors GATA4, Mef2c and Tbx5 using laser optoporation increased the number of cells expressing cardiomyocyte specific markers. During the project, researchers obtained qualitative images of cells via fluorescent lifetime imaging microscopy (FLIM). This enabled them to evaluate cellular metabolic activity and detect exogenous fluorescence. In the end, the laser optoporation pathway was modified to coincide with different imaging modalities. This allowed the simultaneous imaging of cells for the identification of cells expressing fluorescent markers. MIR-OPTOFECTARRAY demonstrated the potential of optoporation for induction of cell reprogramming towards cardiomyogenesis. This novel system could be expanded for different cell types and investigated further for efficient application of miRs to achieve transdifferentiation in cells.
Keywords
MicroRNA, transdifferentiation, MIR-OPTOFECTARRAY, cardiomyocytes, optoporation
