Tiny circles of DNA are the key to a new and easier way to transform stem cells from human fat into induced pluripotent stem cells for use in regenerative medicine, say scientists at the Stanford University School of Medicine. Unlike other commonly used techniques, the method does not use viruses to introduce genes into the cells or permanently alter a cell's genome. Details were published online February 7 in Nature Methods.
Altogether, the researchers were able to make 22 new iPSC 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% vs. about 0.01–0.05%, respectively, it still surpasses that of using conventional bacterial-based plasmids. The researchers believe that this reduction in efficiency should be easily overcome since stem cells from fat and fat itself are so prevalent.
“This technique is not only safer, it's relatively simple,” points out Stanford surgery professor Michael Longaker, M.D., and co-author of the paper. “It will be a relatively straightforward process for labs around the world to begin using this technique. We are moving toward clinically applicable regenerative medicine.”
The Stanford researchers used the 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. The minicircle reprogramming vector works so well because it comprises only the four genes needed to reprogram the cells plus a gene for a green fluorescent protein (GFP) to track minicircle-containing cells, the team explains.
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, the scientists add. Finally, because they don't replicate they are naturally lost as the cells divide, rather than hanging around to potentially muck up any subsequent therapeutic applications.
The investigators found that about 10.8% of the stem cells took up the minicircles and expressed the GFP versus about 2.7% of cells treated with a more traditional DNA plasmid. When they isolated the GFP-expressing cells and grew them in a laboratory dish, they observed 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 the hallmarks of induced pluripotent cells: They expressed embryonic stem cell genes, had similar patterns of DNA methylation, could become multiple types of cells, and 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.