Researchers have developed a new method for generating induced pluripotent stem cells (iPSCs) from adult mouse and human cells that doesn’t require the use of transcription factors. The approach used by University of Pennsylvania School of Medicine scientists instead uses miRNAs to trigger the transformation of fibroblasts into iPSCs.
The team suggests that while traditional transcription factor-based methods can generate perhaps a small handful of iPSCs from every 100,000 cells reprogrammed, their miRNA-mediated method results in about 10,000 iPSCs from every 100,000 starting cells. Their work is published in Cell Stem Cell in a paper titled “Highly Efficient miRNA-Mediated Reprogramming of Mouse and Human Somatic Cells to Pluripotency.”
There are currently several limitations to iPSC generation, including the low (0.2%–1.0%) efficiency of the process and the requirement to force the expression of at least one pluripotent stem cell transcription factor, according to the Penn research team, led by Edward Morrisey, Ph.D., director of the Penn Institute for Regenerative medicine, and professor in the departments of medicine and cell and developmental biology.
The current standard strategy for iPSC generation thus relies upon ectopic expression of Oct4, Sox2, Klf4, and Myc (known as OSKM). Although there are several alternatives to some of these factors, including the use of other transcription factors, signaling factors, and pharmacological molecules, at least one pluripotent stem cell transcription factor, usually Oct4, is required for efficient iPSC reprogramming, they point out. “These limitations hamper the use of iPSC technology in high-throughput formats such as generation of human iPSC clones from large patient populations.”
Conversely, several miRNAs have recently been shown to enhance iPSC reprogramming when expressed along with combinations of the OSKM factors. These miRNAs belong to families that are expressed preferentially in embryonic stem cells (ESCs) and are thought to help maintain the ESC phenotype. Of the miRNAs expressed at high levels in ESCs and iPSCs, the miR302/367 cluster has been shown to be a direct target of Oct4 and Sox2.
Working with mouse cells initially, the Penn team showed that expression of the miR302/367 miRNAs can directly reprogram both mouse and human somatic cells to a pluripotent stem cell state without the requirement for any transcription factors. The iPSCs were even capable of being redifferentiated into germline cells.
The team then moved on to see whether the miRNA method would work with human cells. They found that in comparison with the OSKM transcription factor method, the miRNA expression technique was about two orders of magnitude more efficient at generating iPSCs from human fibroblasts. “Based on the cell counts, approximately 10% of human fibroblasts used for viral transduction produce iPSC clones,” they state. “These data indicate that miR302/367 can reprogram human as well as mouse fibroblasts to an iPSC state with greatly increased efficiency.”
Interestingly, for the technique to work with mouse cells, the researchers needed to combine miRNA expression with the Hdac inhibitor valproic acid (VPA). In human fibroblasts, however, the generation of iPSCs could proceed without VPA.
The iPSCs generated using the miRNA method can generate most of the tissues in the developing mouse, including germ cells, the researchers state. They are now working with collaborators to redifferentiate the iPSCs into cell types including cardiomyocytes, hematopoietic cells, and hepatocytes.
“Its a game changer,” Dr. Morrisey concludes. “This is the first time we’ve been able to make induced pluripotent stem cells without the four transcription factors and increase the efficiency by 100-fold. The upshot is that we hope to be able to produce synthetic miRNAs to transform adult cells into iPSCs, which could eventually then be redifferentiated into other cell types.”
The team hopes that it will be possible in the future to use synthetically generated mimics or precursor miRNAs, which can be easily introduced into cells at high levels and allow for a nongenetic method for generating iPSCs, Dr. Morrisey states.