When two groups of researchers announced just a few weeks ago that they had managed to induce adult skin cells to behave like embryonic stem cells, two of the major caveats of the work were that one group used the oncogene c-Myc as part of its transformation cocktail, and that no one actually had demonstrated that the induced pluripotent or iPS cells actually could cure anything. (See BioWorld Today, Nov. 21, 2007.)

Now, follow-up studies addressing both of those issues have been published. In a Nov. 30, 2007, early online publication in Nature Biotechnology, the Japanese group that published one of the original reports followed up with a report that it was able to generate iPS cells without using c-Myc as a transforming factor.

And in the Dec. 6, 2007, issue of Sciencexpress, researchers from The Whitehead Institute, the University of Alabama at Birmingham and the Massachusetts Institute of Technology showed that in genetically engineered mice, iPS cells were successful at treating sickle cell anemia.

In the Sciencexpress paper, the researchers isolated fibroblasts from a mouse model of sickle cell anemia and converted them to iPS cells using the four transcription factors first described by Yamanaka and his colleagues in 2006. They then corrected the genetic defect in the animals, converted the cells into blood-forming stem cells and transplanted the cells into mice that had been irradiated to destroy their own bone marrow cells.

Blood tests showed that the sickle cell anemia was corrected in the treated mice, with measurements of blood and kidney functions similar to those of normal mice.

Aside from the proof of principle that the Sciencexpress paper provides, between them the two papers addressed several issues that stem from one root concern: the possibility of causing cancer with gene therapy.

That possibility was clearly demonstrated in a group of baby boys who were treated with gene therapy for X-linked severe combined immunodeficiency in 2000. In stark contrast to a priori risk assessments that estimated the risk of cancer from the therapy at one in 100,000, three of 11 patients developed leukemia. (See BioWorld Today, Jan. 16, 2003, and Aug. 11, 2006.)

The foremost step to reduce the cancer risk associated with iPS cells has been the removal of the c-Myc gene from the induction mix. In their Nature Biotechnology paper, Yamanaka and his colleagues demonstrated that some fibroblasts could be converted to iPS cells without the use of c-Myc, though the transformation was slower than when c-Myc was used.

The authors noted that their finding "does not argue that Myc is dispensable for iPS cell generation. We found that [adult skin cells] expressed c-Myc from the endogenous gene at ~20% of the levels observed in mouse ES cells. This expression continues in iPS cells." The scientists said they believe that the other factors used for the transformation "may recruit endogenous Myc proteins to induce reprogramming."

Still, not adding c-Myc showed an effect where it counts: in the rate of tumor formation from the iPS cells. Yamanaka's group generated chimeric mice from the iPS cells, and found that the absence of c-Myc in the transforming mix reduced the incidence of tumors. None of the 26 animals derived from iPS cells without c-Myc developed tumors within 100 days, whereas six of 37 animals from iPS cells with c-Myc did.

Though removing c-Myc from the mix solves one large carcinogenic problem, the use of viral vectors itself can cause cancer via a process known as insertional mutagenesis, independently of whether those viral vectors are delivering an oncogene or a more innocuous one. Such insertional mutagenesis was what caused leukemia in the French X-SCID trials.

"Ultimately, [retroviral insertion] will not be the procedure that is done in humans - those factors will have to be introduced in a different way," Sciencexpress co-author Tim Townes said in a podcast accompanying the paper. "The genes will have to be introduced in such a way that they could not cause insertional mutagenesis . . . that's the next major hurdle in my view."

Even if the procedures used to generate or implant them do not cause cancer, Townes pointed out that the transplantation of stem cells is an inherently risky proposition. "There is a fine line between self-renewal of cells that can form normal bone marrow cells . . . and a cell that self-renews and might produce leukemic cells," he said.

The transplanted cells have been working normally for several months now. "We're very optimistic, but cautious," Townes said. "We think that these cells are performing as legitimate hematopoietic stem cells, but only time will tell."