Recently, Japanese researchers described a new methodology for purification of iPS/ES cell-derived tissue-specific progenitors by genetic markers. Unlike previous molecular beacon-based approaches, the authors designed so-called miRNA switches – synthetic modified mRNAs, which target tissue-specific miRNAs.
First, they screen multiple cardiomyocyte-specific miRNAs and picked 3, which were able to purify target cell population with high efficiency. miR-208a was the best performer with purity >95% on different stages of cardiomyocytes differentiation in culture.
The beauty of this study is in designing of double switches, which contain tissue-specific switch plus inductor of apoptosis. Using this system, desired cell population could be purified by suicide gene induction without sorting. This method allowed ~90% purification of cardiomyocytes. Importantly, purified cardiomyocytes survived and engrafted into the heart of immunocompromised mice without tumorigenesis.
Researchers went ahead and designed miRNA switches for endothelial cells, hepatocytes and islet-like insulin-producing cells. Yet another beauty of the study is composite purification of few cell types together by tandem miRNA switch. If you need to make a heart tissue, you may need cardiomyocytes + endothelial cells + cardiac fibroblasts all together. Imagine that you can capture this kind of microtissue from heterogeneous iPS/ES cell culture by one molecular construct without sorting. Sounds fantastic, isn’t it? The authors of this study made the first step to it and captured cardiomyocytes + endothelial cells by single tandem miRNA switch.
I’m really impressed by this methodology, especially by possibility of using apoptosis inducers and tandem switches. Unlike typical molecular beacon-based cell purification, no sorting needed here. Still, you have to do transfection, worry about efficiency and potential immunogenicity/ toxicity of synthetic mRNAs. To this end, the authors noted that they used non-immunogenic mRNAs and showed their full degradation 48 hours after transfection. If molecular marker is not DNA integrating, non-toxic and rapidly degradable – I have no concerns about such method. Molecular marker-based cell purification especially important for the cell types without specific surface marker (ex: insulin+ pancreatic islet cells).
This system depends on only two distinct properties for purification: the miRNA switch is transfected into the cells, and specific miRNAs are used to identify the target cells. The simplicity of this method enables easy purification of desired cell types by employing appropriate miRNA switches. In addition, miR-Bim-switch, which selectively induces apoptosis in non-target cells, was used to purify cardiomyocytes without cell sorting (Figure 4B). Future clinical application of hPSC-derived cardiomyocytes requires large-scale purification that is not suitable for cell sorting. miR-Bim-switch not only provides high purity but also high yield, achieving an approximately 90% recovery rate for cardiomyocytes.