American researchers at the Stanford University School of Medicine have discovered a protein that plays a crucial role in reprogramming mature cells into induced pluripotent stem cells (iPSCs). The results were published on July 16, 2018 in Nature Cell Biology.
Earlier studies have shown that the NKX3-1 protein is involved in prostate development and tumor suppression. It can replace one of the four proteins that was first identified in 2007 by the stem cell researcher Dr. Shinya Yamanaka, and can successfully use in the reprogramming of mature cells in iPSCs.
This discovery sheds light on cellular transformation and can be a new method of creating iPSCs. The researchers used a unique laboratory model for cell reprogramming, that synchronizes the earliest stages of the process.
“This is a crucial regulator that would not have been discovered any other way,” – said Helen Blau, Ph.D., professor of microbiology and immunology. “It appears within two hours of the initiation of reprogramming, and then it’s gone. But it’s absolutely critical. If we eliminate it, reprogramming doesn’t happen.”
Yamanaka received the Nobel Prize in 2012 for the discovery of a method for reprogramming mature cells types such as skin cells by the addition of just four proteins called Yamanaka factors. Since then, many researchers worldwide have used this method of obtaining iPSC for research or potential clinical use.
The Yamanaka factors (Oct4, Sox2, cMyc, and Klf4) were identified because they are actively expressed in embryonic stem cells of mice and humans. Mature cells under their influence reverse they development and return to the state of stem cells, starting to express Oct4 independently.
However, this process causes fears of scientists, since cMyc and Oct4 are oncogenes that can cause cancers in normal cells when overexpression. Researchers believe that understanding the reprogramming mechanism will create new ways to produce stem cells that are safer for clinical use.
Unfortunately, much of what goes on during the first hours of reprograming has remained a mystery, part of the reason for this lies in the fact that only 1 out of 1000 cells treated with Yamanaka factors successfully undergoes the transition. In addition, for each cell this process is individual and differs in time.
“It’s been difficult to get a handle on early regulators of reprogramming to pluripotency”, – Blau said. “The process is highly heterogeneous and asynchronous, so the earliest events have been hard to study.”
To solve this problem, lead author of the study Dr. Thach Mai used a successful model of cell fusion, developed by Blau in the 1980s. This method demonstrated that adult specialized human cells, such as hepatocytes and skin cells, after combining with mouse muscle fibers could express muscle-specific genes specific for muscles. This was the first evidence that mature cells could be directed to different pathways of development under certain conditions.
In a new study, Mai fused human skin cells (fibroblasts) to mouse embryonic stem cells. After fusion, stem cell factors quickly and efficiently reprogrammed the fibroblast nucleus along a predictable, research-amenable timeline. The fused cells are called heterocaryons. They allowed Mai and his colleagues to closely track patterns of gene expression and DNA modification during the first 24 hours of reprogramming.
The use of the heterocarion model allowed a group of scientists to discover that NKX3-1 is expressed within about two hours after the start of reprogramming, but then quickly dissipates.
When the expression of this protein is blocked, the Yamanaka factors are no longer able to reprogram human fibroblasts, which indicates the important role of NKX3-1 in the transformation of mature cells into stem cells. The researchers also found that the artificial addition of NKX3-1 can replace Oct4 in reprogramming cells without losing effectiveness.
In addition, scientists also showed that NKX3-1 expression was necessary to activate the production of its own Oct4 protein in cells and to promote other genetic changes that facilitate reprogramming.
Currently, Blau and her colleagues intend to continue studying the earliest stages of reprogramming cells into pluripotent cells using a new approach.
“Reprogramming completely changes a cell’s fate. We want to understand the mechanistic and signaling pathways that mediate such a remarkable change”, – Blau said.