Creating human induced pluripotent stem (iPS) cells has until now required the right mix of several key developmental genes. Now, investigators from Harvard Medical School and McLean Hospital have used the proteins made by the genes to create human iPS cells.
The study provides proof that the concept is feasible. And the first attempt, published in the June edition of Cell Stem Cell, shows that it works. Kwang-Soo Kim, PhD, director of the molecular neurobiology laboratory at Harvard's McLean Hospital and one of the study investigators, said that the technique, if replicated in adult human cells, could eliminate problems associated with delivering genes into the cell and serve as a safer source of patient-specific stem cells.
“It was difficult to find the right conditions to generate human iPS cells by direct protein delivery because we used the whole cell extracts,” Dr. Kim said. However, the iPS cells that the investigators finally characterized had all the markers of embryonic stem cells and the cells were free of transgenes.
“Our work shows that any type of human somatic cell can be reprogrammed to an iPS-like cell,” he said.
“iPS cells have unprecedented potential for disease mechanism studies for diverse human disorders, including neurodegenerative diseases,” Dr. Kim explained. “If we can find some interesting disease-related phenotype, it will provide us with a new way to understand the disease being studied and a new platform for drug screening. I believe that our work has overcome major obstacles and could make customized cell therapy by iPS cell therapy a reality sooner.”
In 2006, Shinya Yamanaka, MD, PhD, a professor at the Institute for Integrated Cell-Material Sciences at Kyoto University in Japan, reprogrammed somatic cells to iPS cells by using viral vectors to express the transcription factors Oct4, Sox2,Klf4, and c-Myc into fibroblasts, or skin cells. Since then, various types of mouse and human somatic cells were reprogrammed to their pluripotent state, said Dr. Kim.
But while there is great potential for the technique, the multiple viral vectors used to express the factors could introduce new problems, including “unpredictable genetic dysfunction” which can cause tumor formation.
Dr. Kim and his colleagues wanted to determine whether they could generate human iPS cells without using viral vectors to avoid oncogenic effects or immune reactions. In focusing on reprogramming proteins, they needed a way to shuttle the proteins inside the cell. The work on the AIDS virus held the key. Two decades ago, scientists discovered a peptide called cell-penetrating peptide in HIV TAT proteins. This peptide allowed the HIV protein to enter the cell.
Once the proteins made their way inside the cell, they started reprogramming and the scientists could grow stable iPS cell lines in the lab. They used an array of fluorescent dyes to determine whether the reprogramming proteins were expressed where they needed to be — inside the nucleus where reprogramming occurs.
The first attempts failed. The problem was that the cells stopped reprogramming and therefore cell growth was halted. They decided to repeat the protein treatment cycle, which was 16 hours of protein treatment followed by incubation in special media for six days. The more treatment cycles, the more reprogrammed cells they had. They were able to establish five colonies.
The investigators said it took two months to establish two colonies and the yield of viable cells was much lower than that seen with the original technique of infecting viral vectors with the transcription factors: Oct4, Sox2,Klf4, and c-Myc.
Viral-based methods produce 0.01 percent of the so-called input cells compared to 0.001 percent with this new technique to reprogram proteins. But the cell lines produced expressed embryonic stem cell markers — alkaline phasphatase, nanog, tumor-rejection antigen1-60, stage-specific embryonic antigen (SSEA) 3, and SSEA 4.
For this first attempt, they used whole protein extracts. Now they are working on purifying the reprogrammed proteins to improve their efficiency, Dr. Kim said. “We need to conduct these studies to see whether this method is completely safe and efficient.”
“This is exciting progress,” said Sally Temple, PhD, scientific director of the New York Neural Stem Cell Institute in Rensselaer, NY, who was not involved with the study. “Using proteins rather than genes to make induced pluripotent cells is likely to be a safer approach. It will eliminate problems associated with inserting and removing genes, which can produce cancer. The next major hurdle is to do this on adult rather than newborn fibroblasts — this might be a challenge as adult cells grow less efficiently. Hopefully iPS cells made this way will be less prone to tumor formation, and I am sure they are working hard to evaluate this.”
John Gearhart, PhD, the James Effron University Professor and director of the Institute for Regenerative Medicine at the University of Pennsylvania, said that “scientists in the stem cell field have felt that this is where instructing cells needs to go.”
A decade ago, Dr. Gearhart identified, isolated, and developed the first stem cells from human fetal tissue. “The real challenge is whether scientists can effectively get the proteins inside the cell and be able to generate a stable population of cells,” Dr. Gearhart said. Unlike genes, proteins are not self-replicating and don't hang around long enough and it takes weeks to reprogram these proteins. “We all feel that this is the way to go,” he said. “But we need to know whether it can be done in the human body. This is a beginning and I am sure this will be the future”.•
Kim D, Kim C-H, Kim K-S, et al. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell 2009;4(6):472–6. E-pub 2009 May 28.