STANFORD, Calif.—Using a technique based on standard molecular biology practices, scientists at Stanford University have discovered a safer and easier way to make induced pluripotency stem cells (iPS), a finding that could produce a starting point for research on many human diseases.
In a study published online in February in Nature Methods, the researchers describe how they used "minicircles"—rings of DNA about one-half the size of those usually used to reprogram cells—to induce pluripotency in stem cells from human fat. This is in contrast to other commonly used techniques, which use viruses to introduce genes into cells or permanently alter a cell's genome, says Dr. Joseph Wu, a cardiologist and assistant professor of medicine and radiology at the Stanford University School of Medicine, and a senior author of the study. Even better, because of the ease of the researchers' technique and its relative safety, scientists hope to quickly gain the necessary approvals from the U.S. Food and Drug Administration (FDA) so its clinical trial potential can be realized, Wu says.
"The main advance of our paper is that we show a non-integrating, non-viral way of making iPS cells, which the FDA may be more amenable to approving," Wu says.
Wu notes that the paper is actually the multidisciplinary result of three different Stanford research teams coming together. While Wu was searching for ways to create patient-specific cell lines to study some of the common heart problems he was seeing in the clinic, two other researchers formed the other pieces of this puzzle. Dr. Mark Kay, a genetics professor, invented the minicircles several years ago in a quest to develop suitable gene therapy techniques. Finally, study co-author Dr. Michael Longaker, a Stanford surgery professor, was discovering the unusual prevalence and developmental flexibility of stem cells from human fat.
Previous work in Wu and Longaker's lab showed that the stem cells from human fat are numerous, easy to isolate and amenable to the iPS transformation, probably because of the naturally higher levels of expression of some reprogramming genes. This made reprogramming the minicircles more efficient, Wu says. In fact, the researchers found that about 10.8 percent of the stem cells took up the minicircles and expressed the green fluorescent protein (GFP), versus about 2.7 percent of cells treated with a more traditional DNA plasmid.
"About three years ago, Mark gave a talk and I asked him if we could use minicircles for cardiac gene therapy," says Wu. "And then it clicked for me, that we should also be able to use them for non-viral reprogramming of adult cells."
In addition, the minicircle reprogramming vector works so well because it is made of only the four genes needed to reprogram the cells (plus a gene for a green fluorescent protein to track minicircle-containing cells). Unlike the larger, more commonly used DNA circles called plasmids, the minicircles contain no bacterial DNA, meaning that the cells containing the minicircles are less likely than plasmids to be perceived as foreign by the body. The expression of minicircle genes is also more robust, and the smaller size of the minicircles allows them to enter the cells more easily than the larger plasmids. Finally, because they don't replicate they are naturally lost as the cells divide, rather than hanging around to potentially compromise any subsequent therapeutic applications.
When the researchers isolated the GFP-expressing cells and grew them in a laboratory dish, they found that the minicircles were gradually lost over a period of four weeks. To be sure the cells got a good dose of the genes, they reapplied the minicircles at days four and six. After 14 to 16 days, they began to observe clusters of cells resembling embryonic stem cell colonies, some of which no longer expressed GFP.
They isolated these GFP-free clusters and found that they exhibited all of the hallmarks of induced pluripotent cells: they expressed embryonic stem cell genes, they had similar patterns of DNA methylation, they could become multiple types of cells and they could form tumors called teratomas when injected under the skin of laboratory mice. They also confirmed that the minicircles had truly been lost and had not integrated into the stem cells' DNA.
Altogether, the researchers were able to make 22 new iPS cell lines from adult human adipose stem cells and adult human fibroblasts. Although the overall reprogramming efficiency of the minicircle method is lower than that of methods using viral vectors to introduce the genes (about 0.005 percent vs. about 0.01 to 0.05 percent, respectively), it still surpasses that of using conventional bacterial-based plasmids. Furthermore, stem cells from fat, and, for that matter, fat itself, are so prevalent that a slight reduction in efficiency should be easily overcome, Wu says.
The researchers are now working on using these cells to better understand human heart disease, but Wu says this new way of producing iPS cells could have applications for research on a wide range of human diseases. He stresses, however, that the study is based on basic molecular biology, and further study and regulatory approvals will be needed before clinical researchers can begin to use this new method.
"From a cardiology standpoint, you can make iPS cells and differentiate cardiac cells and use them as a drug screening platform to evaluate the safety of different drugs, for example," he says. "But in terms of injecting the iPS cells unto humans and seeing any clinical cure, that is a few years away."
The study, "A Non-Viral Minicircle Vector for Deriving Human iPs Cells," was funded by the Mallinckrodt Foundation, the National Institutes of Health, the Burroughs Wellcome Foundation, the American Heart Association, the California Institute of Regenerative Medicine, the Oak Foundation and the Hagey Laboratory for Pediatric Regenerative Medicine.
