High Efficiency Live Cell Purification Uses MicroRNA Switches

Pluripotent stem cells can be differentiated into any cell type, so they are in demand in medical research.

But conventional differentiation protocols result in a heterogeneous population from which the desired type need to be purified.

Typically, antibodies that react to surface receptors specific to the desired cell are used for this purpose. In many cases, unfortunately, purification levels remain low and cells can be damaged. A new RNA technology from Kyoto University’s Center for iPS Cell Research and Application may get around these issues.

Professor Hirohide Saito, a bioengineer who makes tools for cell researchers, developed the microRNA (miRNA) switch. It is designed to detect and sort live cells not by surface receptors, but by miRNAs.

miRNA is a Better Marker of Cell Types

The miRNA switches are made of synthetic mRNA sequence, with a recognition sequence for miRNA and an open reading frame that codes a desired gene, like a regulatory protein that emits fluorescence or promotes cell death.

If the miRNA recognition sequence binds to miRNA expressed in the desired cells, the expression of the regulatory protein is suppressed, marking the cell type as distinct from others that do not contain the miRNA and express the protein.

Senior Lecturer Yoshinori Yoshida, Kyoto University cardiomyocyte specialist, immediately saw the possibilities inherent in this technology. He has been studying how iPS cells can be used to combat cardiac diseases, but has been blocked by unsatisfactory purity levels.

Cardiomyocytes are particularly tricky to purify- they do not express unique surface receptors. The two scientists collaborated to investigate the effectiveness of miRNA switches for these cells.

Yoshida sees this tool as amazingly simple and something that can be used by stem cell researchers studying any organ:

“It is just synthesizing RNA and transfecting them. It is not difficult.”

Hepatocytes and Pancreatic Cells

To drive home the point, he and Saito applied their miRNA switches to the purification of hepatocytes and pancreatic cells, neither of which has unique cell surface markers, finding miRNA switches effective there too.

Saito thinks that with more development, miRNA switches will be applicable to all cell types at all cell stages:

“We want to make an active miRNA dictionary for each cell type, so that if we want to isolate this kind of cell type, we know how to use this kind of switch.”

Yoshinori Yoshida et al.
Efficient Detection and Purification of Cell Populations Using Synthetic MicroRNA Switches.
Cell Stem Cell, May 2015 DOI: 10.1016/j.stem.2015.04.005

Photo: Vasanta Subramanian, Wellcome Images Creative Commons by-nc-nd 4.0