A better way to make iPSCs?

Embryonic stem cells can develop into most cell types and have much therapeutic potential. However, its method of extraction from human embryos remains highly controversial.

That is what made Shinya Yamanaka’s invention of induced pluripotent stem cells (iPSC) a Nobel-winning achievement.

iPSCs are body (somatic) cells which have been reprogrammed to function like embryonic stem cells, thereby sidestepping the controversial use of killing the embryos while harvesting the stem cells. This is done by introducing four regulatory factors (pieces of DNA) into the cells.

“Each of the ‘Yamanaka’ factors, when expressed in cells will activate or repress target genes. Expressed together they can, at low- frequency, convert cells to a relatively stable state of gene-expression similar to that seen in embryonic stem cells,” Dr. K. VijayRaghavan pointed out in an email to this correspondent. Dr. VijayRaghavan is the Secretary, Department of Biotechnology, and former Director of Bangalore-based NCBS. He was not involved in the work.

However, the efficiency of iPSC production is traditionally quite low. Only about 0.01 per cent of the cells successfully become iPSC.

Duanqing Pei and team at the Guangzhou Institutes of Biomedicine and Health, China, may have found a way to improve this performance. The results were published on May 27 in Nature Cell Biology. They suspected that the four factors may counteract each other when introduced together, making iPSC production less efficient.

So the team used the same four Yamanaka factors — Oct4, Klf4, c-Myc and Sox2 — but experimented with the sequence, timing and combination of their introduction.

They found that an OK+M+S (Oct4+Kllf4 followed by c-Myc and then Sox2) combination achieved the highest reprogramming efficiency among all the other combinations they tried. The sequential introduction showed five times more efficiency than the simultaneous introduction protocol. This better performance was recorded both in mouse and human differentiated cells.

“We can generate iPSCs with less money and time and higher efficiency,” said Dr. Pei in an email to this correspondent.

More importantly, such experiments shed light on the individual roles of the Yamanaka factors in reprogramming — something that according to Dr. Pei is still not very clear at molecular or mechanistic level.

What is known is that the Yamanaka factors turn on/off target genes. In this way, they reprogramme the DNA of the somatic cell such that it attains pluripotency — the ability to develop into all cell types that make up the body, just like embryonic stem cells. The research shows that it is not enough that these factors just function, but it is also crucial when and how much they do.

“The correct balance of on/off genes that switch a cell to an iPS fate is achieved only by chance in very few cells. The sequential induction of the Yamanaka factors seems to increase this chance,” elaborated Dr. VijayRaghavan.

Further, the team confirmed a 2010 discovery that a process called mesenchymal-to-epithelial-transition (MET) is involved in the transformation of a mouse fibroblast cell into an iPSC. The current work showed that the cells were actually undergoing a reverse process —early EMT followed by MET — before finally attaining pluripotency.

The Yamanaka factors were shown to play roles in the EMT-MET processes. “Now, we know that each of the factors appear to perform a specific task to convert a fibroblast to iPSC, a division of labour,” said Dr. Pei.

Understanding how induced pluripotent cells are made will allow us to program human cells in a particular direction. As Dr. VijayRaghavan emphasised, “This is of fundamental importance in understanding how humans develop and in our understanding and treatment of disease and in regeneration.”