MicroRNA Switches Improve Cell Purification

Stem cell-based therapies usually require the differentiation of stem cells into various cell types that are used for regenerative therapies. Such a strategy requires that the differentiated cells be purified from the rest of the cells. Typically, cell surface proteins are used as the means to distinguish cell types. Unfortunately, many undesired cell types may also share the same cell surface receptors, which will badly compromise the efficiency of cell purification.

Hirohide Saito from the Center for iPS Cell Research and Application (CiRA) at Kyoto University has designed a new way to isolate differentiated cells using microRNAs. This technique appears to be better than using cell surface proteins and it may revolutionize stem cell science.

Readers of this blog will recognize the term induced pluripotent stem cells or iPSCs, but for newer readers, I will provide a brief explanation of these cells. Induced pluripotent stem cells are made from mature, adult cells by means of genetic engineering and cell culture techniques. When the expression of four different genes (Oct4, Klf4, Sox2 and c-Myc) is forced in adult cells, a fraction of the cells de-differentiate and become like young, embryonic cells. When these cells are cultured ion special culture systems, they will aggregate and grow into an iPSC cell line. These cells have many, though not all, the features of embryonic stem cells, and they can, theoretically, differentiate into any adult cell type.

iPSCs are so popular in medical research because they are derived from a patient’s own body and they can be differentiate into any cell type. However, the protocols that are normally used to differentiate iPSCs lead to a mixed population of cells that are very heterogeneous, and the desired cell type has to be isolated from this mixture. Normally, antibodies that bind to surface receptors unique to the desired cell type are used for this purpose but in many cases such purification strategies are inefficient and the cell yield is rather poor. Also, these cell purification techniques have a tendency to damage cells.

New RNA-based procedures designed at CiRA may avoid these problems. Hirohide Saito and his colleagues designed tiny RNA molecules (microRNAs or miRNAs) that are designed to detect and sort live cells not by surface receptors, but by miRNAs. MicoRNAs are better markers of cell types and can improve purity levels. These “miRNA switches” as they are called, consist of synthetic mRNA sequences that include a recognition sequence for miRNA and an open reading frame (ORF) that codes a desired gene, such as 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, which helps distinguish one cell type from others that do not contain the miRNA and express the protein.

Senior Lecturer Yoshinori Yoshida, a heart muscle specialist who works with Professor Saito, immediately saw the potential of this technology. Dr. Yoshida has been studying how iPS cells can be used to combat cardiac diseases, but he has been stifled by unsatisfactory cell purification protocols. Heart muscle cells (cardiomyocytes) are especially difficult to purify because they do not possess unique cell surface proteins. So Professor Saito and Dr. Yoshida put their heads together to test the effectiveness of miRNA switches for isolating differentiated heart muscle cells from iPSCs.

First, they went to established heart muscle cell lines (which, by the way, are a colossal pain in the neck to deal with). They used these cells to define the miRNAs that are unique to cardiomyocytes. Then they designed several miRNA switches that contained sequences complementary to these miRNAs. After constructing the miRNA switches, they used them to isolated differentiated heart muscle cells from iPSCs.

The results were remarkable. Dr. Yoshida saw far better purification than he ever seen with standard methods. Furthermore, because this technology is RNA-based, it does not integrate into the genome and cause mutations. This could potentially make the cells eligible for clinical application.

Yoshida sees this tool as remarkably 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,” he said. To prove this point, he and Saito used their miRNA switches to purify liver cells and pancreatic cells from iPSCs. This is significant, because neither of these cell types possess unique cell surface markers, but miRNA switches wre able to effectively purify them.

Intriguingly, the performance of different miRNA switches varied with the stages of cell development. This suggests that strategic selection of miRNAs could separate heart muscle cells that are at different developmental stages, which could also lead to even more homogeneous cell pools and potentially better cell therapy outcomes.

Saito believes that with further 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,” he said.