Researchers from Kyushu University in Japan and Harvard Medical School in the US have identified genes that can “reprogram” the fibroblasts – the most common type of skin and connective tissue cell – into cells with properties similar to those of limb progenitor cells. The new findings published in the scientific journal “Developmental Cell” offer valuable knowledge about the development of human (and not only) limbs and pave the way for their regeneration.

60 million amputees worldwide

Worldwide, almost 60 million people are amputated due to various medical reasons such as tumors, infections and genetic defects, but also due to road or work accidents as well as natural disasters such as earthquakes. Many of these people live with prosthetic limbs however, researchers around the world are studying the process limb development with a view to giving them their lost limbs through her regenerative medicine.

Finding an easy way to create progenitor cells

“During the process of limb development their progenitor cells differentiate into most of the limb tissues such as bone, muscle, cartilage and tendon tissues. It is therefore important to find an easy way to create these cells,” said Dr Yuji Atsutaleading the study that he began while working at Harvard Medical School and continues now as a lecturer at Kyushu University’s School of Science.

Existing reprogramming methods

Today a common method for scientists to create limb precursor cells in the laboratory involves the use of embryos which, in the case of human embryos, raises ethical issues. Alternatively, these progenitor cells can be created using induced pluripotent stem cells (iPS) – these are adult cells that are reprogrammed to return to the pluripotent stage of embryonic cells and then transform into a multitude of different cell types in the body .

The advantages of the new method

The new method developed by Dr. Atsutas and his colleagues, which directly reprograms them fibroblasts in limb progenitor cells, simplifies the reprogramming process and significantly reduces its cost. At the same time, it overcomes the “scope” of the possible transformation of cells into cancer, which is often associated with iPS cells.

In the initial phase of the study the researchers looked for genes expressed during early limb development in mouse and chicken embryos. They identified 18 genes that were more highly expressed in the developing limb tissues of the embryos compared to the other tissues.

Modified fibroblasts

They then cultured fibroblasts from mouse embryos and introduced the 18 genes into them using viruses as “transport vehicles”. They saw that the modified fibroblasts acquired the properties of limb progenitor cells and showed similar gene expression to the specific cells.

The three “key” genes

In another series of experiments the research team determined that three of the 18 genes were sufficient for fibroblast reprogramming: Prdm16, Zbtb16 and Lin28a.

In vitro and in vivo experiments

In fact, the researchers confirmed that the reprogrammed limb progenitor cells had similar gene expression to the natural limb progenitor cells and similar properties. “These reprogrammed progenitor cells are not just molecular mimics. We confirmed their ability to transform into specialized limb tissues both in the laboratory – in vitro – and in living organisms – in vivo” noted Dr. Atsuta and added that “the in vivo experiments were a special challenge for us as we had to transplant the reprogrammed mouse cells in the developing limbs of chicken embryos’.

In Search of Safer “Vehicles of Transport”

In these experiments, scientists used lentiviruses as vehicles to transfer genes directly into the genome of cells, which increases the risk of cells becoming cancerous. They are now looking for other, safer gene “carriers” such as adenoviruses or plasmids that carry the genes into cells without inserting the genes into their genomes.

Maybe one day snakes will get… legs

Professor Atchuta’s team is currently trying to apply the new method to human cells with the aim of developing regenerative therapies but also to … snakes whose ancestors had limbs that were lost during the evolution process. “The reprogrammed limb progenitor cells we created also led to the creation of organelles associated with limb development. Thus it seems possible that we may one day be able to create limb tissues in species that no longer possess limbs. Studying snakes and trying to regenerate limbs in this species can reveal new pathways and lead to valuable knowledge about evolutionary biology.”