8th April 2009

Adding factors

Stem cells from more efficient reprogramming

stemcells

Contribution of integration-free iPSCs to somatic tissue and germ line in chimeras. (a,b) Chimeric embryos at 10.5 d.p.c. (a) and postnatal day 5 pups (b) derived from integration-free iPSCs with constitutive eGFP expression. Non-chimeric embryo (a, right) and pup (b, top) are shown as nonfluorescent controls. Fluorescence images are shown at the bottom.

Only four weeks ago, two reports outlined better approaches to produce embryonic stem cells by reprogramming adult cells. Today, work from labs in the UK and Japan take this one step further.

The new work adds one more factor to the mix to double the efficiency of producing the new stem cells. Moreover, the team added a selection step that enables efficient removal of the reprogramming factors from the stem cells after they have completed their task. Removing these factors ensures the genetic make up of these cells is an exact copy of the donor.

Embryonic stem cells are most often derived from early embryos, because early in development the cells of all animals retain the ability to form any tissue. As cells of an embryo develop, their ability to form any tissue becomes less potent.

It can be difficult to establish large numbers of embryonic stem cells and for human cell lines the availability of embryos has been limiting. For many years, researchers have tried to reverse loss of potency of other cell types, to induce them back to their embryonic state so that they make all or many cell types. Such cells are called induced pluripotent stem cells, or iPSCs.

"Through the two steps we have taken, we have improved the efficiency of iPSC production," says Allan Bradley, Director of the Wellcome Trust Sanger Institute. "We have matched the highest levels seen with other, less robust systems."

"It is a remarkable achievement."

" Through the two steps we have taken, we have improved the efficiency of iPSC production. We have matched the highest levels seen with other, less robust systems. It is a remarkable achievement "

Prof Allan Bradley

In the previous research, scientists added genes for four genetic factors to reverse the loss of potency of human and mouse cells. These factors reprogram the genetic machinery of the cell and, over several weeks, take the cell back to a more embryonic-like state. Once reprogrammed, the genes can be removed, leaving the original genome of the cell undisturbed.

The new research answers two questions: can we improve the efficiency of recovering cells with the original-state genome (typically, it is around one in 1000) and can we improve on the efficiency of the four factors?

In the latest work, published in Nature Methods, the research team used a trick of adding a gene that will kill the cell when a chemical is added to the growth solution. Only if the poison gene is removed along with the factors and the original cell genome restored, will cells survive the selection. If the poison gene is still there, they will die. This step improves the recovery of correctly modified iPSCs.

The second improvement was to add a fifth genetic factor to the cells, which improved the production of iPSCs twofold.

"It might be that other factors can add yet more to the efficiency," explains Kosuke Yusa, Sanger Institute investigator and lead author on the study, "and we are looking into that. Combined with our robust selection step, we can much more rapidly develop and explore iPSCs."

The genetic factors are carried into the genomes of cells in a special DNA vehicle called a piggyBac that can itself be precisely excised when the reprogramming is complete.

Notes to Editors

Publication details

  • Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon.

    Yusa K, Rad R, Takeda J and Bradley A

    Nature methods 2009;6;5;363-9

Funding

This work was supported by the Wellcome Trust.

Participating Centres

  • Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK
  • Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan



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