Using Plasmids to make induced pluripotent stem cells

Induced Pluripotent Stem Cells (iPSCs) are made from normal body cells. Normal body cells are engineered with specific genes that force the cells into a “more primal” or less differentiated state. These cells behave very much like embryonic stem cells (ESCs). Although there are some differences between iPSCs and ESCs, they are similar enough to each other to suggest that iPSCs can replace ESCs as the great hope for regenerative medicine.

A major concern of IPSCs is the manner in which they are made. Body cells are infected with recombinant retroviruses and these retroviruses introduce the genes necessary to transform the body cells into iPSCs. However, retroviruses tend to insert genes into the human genome, largely at random, and this can produce mutations that can kill cells or even convert them into cancer cells. To deal with this problem, scientists have tried to produce iPSCs with other strategies.

First, scientists have used viruses that do not insert genes into the genome. Adenoviruses, for example, do not insert genes into the genome of the cells they infect, and once the infection is complete they, in this case, do not persist. Researchers have used engineered adenoviruses to convert body cells into iPSCs (Matthais Stadfeld et al., Science 322 (2008): 945-9). Other researchers have even found ways to convert body cells into IPSCs without even using viruses (Keisuke Okita et al., Science 322 (2008): 949-53; Knut Woltjen et al., Nature 458 (2009): 766-70). Also, scientists have found a way to use retroviruses that self-destruct after they have inserted into the genome (Keisuke Kaji et al., Nature 458 (2009): 771-5).

A new report now shows that it is possible to make iPSCs by using genes that are loaded into small circles of DNA. Michael Longaker, professor of surgery at Stanford University, used “minicircles” of DNA that did not contain any bacterial DNA to reprogram human fat cells into iPSCs.  The senior author of this research, Joseph Wu, said, “Imagine doing a fat or skin biopsy from a member of a family with heart problems, reprogramming the cells to pluripotency and then making cardiac cells to study in a laboratory dish.”  He continued, “This would be much easier and less invasive than taking cell samples from a patient’s heart.”  This article was published online Feb. 7 in Nature Methods.

This protocol works quite well because the minicircle vector with the reprogramming genes on it quite small; it only contains the four genes needed to reprogram the cells (plus a gene for a green fluorescent protein to track minicircle-containing cells).  The expression of the reprogramming genes on the minicircles is quite robust, and the smaller size of the minicircles allows them to enter the cells more easily than the larger segments of DNA.  Also, these minicircles do not replicate and are naturally lost as the cells divide.  Therefore they do not remain in the cell and cannot cause any later problems.

These researchers chose to use fat cells because they are numerous, easy to isolate and amenable to the iPS transformation.  In this work, they found that about 10.8 percent of the stem cells took up the minicircles and expressed the green fluorescent protein, or GFP, versus about 2.7 percent of cells transformed by more traditional means.

It gets better though.  Isolation of the GFP-expressing cells showed that the minicircles were gradually lost over a period of four weeks.  After a second dose of the minicircles 4 to 6 days  and after 14 to 16 days, they observed clusters of cells resembling embryonic stem cell colonies – some of which no longer expressed GFP.

This new procedure is a remarkable advance towards safety for iPSCs and places them a step close to being used in therapeutic trials.