Virus-free Pluripotency for Human Cells

The team of researchers from the UK and Canada say their discovery could ultimately spell an end to the need for human embryos as a source of stem cells. Their findings are published today in two papers in Nature online.

The two teams involved, led by Dr Keisuke Kaji from the Medical Research Council (MRC) Centre for Regenerative Medicine, at the University of Edinburgh, and Dr Andras Nagy from the University of Toronto, are the first to get human skin cells to act like embryonic stem cells without needing to use viruses in the process.

Reprogramming cells using viruses modifies their DNA in such a way that they cannot be given to patients without greatly increasing their risk of cancer.

This new method avoids using these potentially harmful viruses. It also allows for the four genes inserted to affect cell reprogramming to be removed once this is complete. This should help avoid any abnormalities in how the cells develop.

The researchers were initially working separately on reprogramming. It was only after a chance encounter between Dr Kaji and Dr Nagy that they discovered they had each solved a separate half of the challenge – and their combined method was both safer and more efficient.

The group developed their techniques in both mouse and human skin cells. Tests on the reprogrammed cell lines show they behave exactly like embryonic stem cells.

MRC research fellow Dr Kaji said: “I was very excited when I found stem cell-like cells in my culture dishes. Nobody, including me, thought it was really possible. This new method will advance the field of regenerative medicine, and should help understand diseases and test new drugs.”
 

“It is a step towards the practical use of reprogrammed cells in medicine, perhaps even eliminating the need for human embryos as a source of stem cells,” he said.
The induced pluripotent stem (iPS) cells are created by causing specialised cells to act like embryonic stem cells – and therefore have the ability to become any type of cell in the body.

Human cell reprogramming was first achieved in 2007, but the use of multiple viruses to deliver the four crucial genes - which can reverse the adult cell’s fate - into the cells’ DNA also made them liable to switch on cancer-causing genes. This has previously made them unsafe for transplantation into patients. Prior to this new study, attempts at non-viral reprogramming had only succeeded with mice cells, and were generally inefficient.

Before their chance encounter, Dr Kaji had found a non-viral method to deliver the four genes in a single fragment, and a way to remove them after reprogramming. Being able to remove the genes he inserted into the cells once reprogramming was complete overcame a significant safety hurdle as their presence could otherwise lead to subsequent growth abnormalities.

However, Dr Kaji could not find a way of removing all trace of reprogramming from the genome, and this remaining ‘footprint’ could disrupt other genes at the site of insertion.

Dr Nagy’s group, meanwhile, had developed a reprogramming system that allowed the removal of inserted genes without a trace - but because their method delivered the four genes into different parts of the genome they had not yet managed to remove all of them.

The two research teams’ breakthrough came when they combined their methods - Dr. Kaji’s system, which used just one fragment of DNA including all four genes - c-Myc, Klf4, Oct4 and Sox, and Professor Nagy’s ‘footprint-less’ removal system.

The findings show how the group have allowed delivery of genes into the cell by safer, more conventional methods, avoided the use of viruses to reprogramme the cells, and created the iPS cells without significantly modifying their genetic structure.

Dr Kaji said: “Using our method, it will be possible to remove not only the inserted genes, but all traces of our genetic modification from the human iPS cells, although we still need to improve the efficiency.”
Dr Nagy, Senior Investigator at the Samuel Lunenfeld Research Institute of Mount Sinai Hospital and Canada Research Chair in Stem Cells and Regeneration, said: “We hope that these stem cells will form the basis for treatment for many diseases and conditions that are currently considered incurable. We have found a highly efficient and safe way to create new cells for the human body which avoids the challenge of immune rejection.”
                                                                      
Professor Sir Ian Wilmut, Director of the MRC Centre for Regenerative Medicine, said: “It will still take time before these iPS cells can be given to patients. Crucially, we need to have a method to generate the desired cell types from these stem cells. But I believe the team has made great progress and combining this work with that of other scientists working on stem cell differentiation, there is hope that the promise of regenerative medicine could soon be met.”

Original Articles
K. Kaji et al. Virus-free induction of pluripotency and subsequent excision of reprogramming factors, 2009, Nature doi:10.1038/nature07864;

K. Woltjen et al. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells, 2009, Nature doi:10.1038/nature07863;