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Research Training at Gladstone—Learning to Follow the Science
Unexpected scientific results lead to Shinya Yamanaka's stem cell breakthrough

Shinya YamanakaThe year was 1993, and no one was returning Shinya Yamanaka's phone calls. The physician and recent PhD graduate of Osaka City University had given up a promising career as an orthopedic surgeon to pursue his dream as a research scientist. But now he wasn't getting any traction for his application to become a postdoctoral research fellow.

Until, that is, Dr. Yamanaka's paperwork came across the desk of Tom Innerarity, PhD, who at that time was a senior investigator focused on cardiovascular research at the Gladstone Institutes in San Francisco.

"I applied to many, many research institutes in the United States, but I received no responses," recalled Dr. Yamanaka. "The one exception was Tom, who chose me to come to Gladstone and be a part of his research laboratory."

Smart Selection
It was a wise choice. The work Dr. Yamanaka undertook as a Gladstone postdoc—and the Gladstone notion that researchers must follow science wherever it leads—would form the basis for his subsequent discovery of how to reprogram adult skin cells into embryonic-like stem cells. Dr. Yamanaka called these cells induced pluripotent stem cells, or iPS cells, and they have since ushered in a new era of regenerative medicine worldwide. Now back at Gladstone as a senior investigator, this former Gladstone postdoc has won the 2012 Nobel Prize in Physiology or Medicine.

But when Dr. Yamanaka first arrived at Gladstone 19 years ago, stem cells were far from his mind. He came to Gladstone to train in biomedical research. Founded in 1979, the independent and nonprofit Gladstone was at that time focused primarily on research into cardiovascular and viral diseases (later in the 90s, it added a third focus—on neurological diseases). In the Gladstone research and training labs, Dr. Yamanaka's focus was on finding new ways to lower so-called "bad cholesterol," a major risk factor for heart disease. It was widely known among scientists that a protein called apoB was the principle component of bad cholesterol, which is also known as low-density lipoprotein.

Fighting Cholesterol
Gladstone scientists had previously discovered that two forms of the apoB protein existed in the body. The longer form, normally found in the liver, is involved with the build-up of bad cholesterol. Another form, found in the intestine, is shorter and relatively harmless. Dr. Yamanaka sought to understand how both forms were constructed. If he could understand that, he reasoned, he could then find ways to change the longer form so that it would resemble the shorter and harmless form.

In early experiments, Dr. Yamanaka identified an enzyme called APOBEC-1, which shortens the apoB protein in the intestines, making it less harmful. In the liver, APOBEC-1 is normally inactive. Working under Dr. Innerarity's guidance, Dr. Yamanaka and the other scientists in the lab thought that by activating the APOBEC-1 enzyme in the liver, they could mimic the intestinal process to create a shortened apoB protein, which would reduce bad cholesterol.

The entire lab began planning and carrying out the experiments needed to test this hypothesis. Nearly every item needed for the experiments—things that today can be bought off-the-shelf, such as reagents—had to be made in house.

"Designing and carrying out our experiments 15 years ago was quite labor intensive," recalled Reeny Balestra, a senior research associate at Gladstone who worked closely with Dr. Yamanaka during his postdoc years. "We thought we were on the right track. I remember our small team lining up along the entire lab bench like an assembly line, each person taking on a different step in the experiment. And Dr. Yamanaka was right in the thick of it with us."

Unexpected Results
The team's experiments on mice did show a reduction in bad cholesterol. But the experiments also had an unintended side effect—liver cancer.

"The fact that the mice developed cancer was a big blow, and all of us were disappointed," said Ms. Balestra. "Dr. Yamanaka had worked the hardest of all of us on this project, but instead of getting discouraged, he got curious. He wanted to understand what went wrong."

At Gladstone, scientists are trained and funded to follow the science wherever it leads—even if that means switching a key focus, primary methods or disease expertise. So for Dr. Yamanaka, this latest turn of events didn't constitute a failed experiment. It was the beginning of a new project. He wondered if switching on APOBEC-1 in the liver was responsible for tumor growth. Further research refined his theory: switching on APOBEC-1 in the liver altered NAT1, a protein that, when modified, causes tumors to develop. Dr. Yamanaka had found the cause of the tumor growth—a dysfunctional NAT1. Now he wanted to find the solution.

Following the Science
"The Gladstone philosophy has always been to allow scientists like Shinya Yamanaka the freedom to follow wherever their curiosity—and the science—leads," said Robert Mahley, MD, PhD, Gladstone's founding scientist, who is currently a senior investigator and president emeritus. "As a postdoc, Dr. Yamanaka embraced that philosophy, which I think has played a big part in making him the scientist and the person he is today."

Dr. Yamanaka wanted to study mice that lacked NAT1, to see whether they would still develop cancer. But to do so, he needed to develop genetically modified mice—a process that required embryonic stem cells. Embryonic stem cells are called pluripotent, meaning that they have the ability to develop into any type of cell, such as skin cells, muscle cells and blood cells.

To get the cells he needed, Dr. Yamanaka called upon his friend Robert Farese, MD, who at that time was a Gladstone Assistant Investigator. Dr. Farese, in turn, introduced Dr. Yamanaka to research associate Heather Myers. Ms. Myers was an expert at developing stem cells. And when Dr. Yamanaka came to her asking for help in developing mice without NAT1, he wanted to learn every aspect of the process.

Unconventional Skills
"At the time, the Farese lab was the only lab at Gladstone with the tools to develop embryonic stem cell cultures, and a lot of scientists would come asking us for help," said Ms. Myers. "But Dr. Yamanaka was one of the few who wanted to take part in every step. He kept saying he wanted to learn it all himself so he wouldn't have to keep asking me for help all the time. But I honestly think he just enjoyed discovering something new."

Even so, Dr. Yamanaka's second round of experiments also yielded disappointing results.

"The embryonic stem cells lacking NAT1 that Heather and I had developed never matured," said Dr. Yamanaka. "They just multiplied over and over again. By accident, I had discovered that NAT1 was key to helping stem cells transform into individual cell types."

But with that, he was hooked. Dr. Yamanaka still credits Ms. Myers with teaching him everything he knows about stem cells, and for inspiring him to not think of stem cells as just a tool, but as the central focus of his research.

"He gives me too much credit," laughed Ms. Myers. "I just nudged him in the right direction."

Applied Knowledge
His new direction eventually took him back to Japan where, armed with the expertise learned at Gladstone, he undertook what seemed to most, at the time, to be an impossible undertaking: to find out which genetic factors from among millions of possibilities instruct embryonic stem cells to become other types of cells. This laborious task involved sorting through many different factors that influence cell development. Dr. Yamanaka selected the most likely ones and began examining them in various combinations. With what was widely seen as stunning speed, Dr. Yamanaka first announced the four genetic factors behind his iPS breakthrough in mice in 2006. The following year, he reported using the same four factors to do the same with human cells. That same year, 2007, Dr. Yamanaka returned to Gladstone, this time as a senior investigator and a professor of anatomy at the University of California, San Francisco, with which Gladstone is affiliated.

Today he splits his time between San Francisco and Japan, where he's the director of the Center for iPS Cell Research and Application at Kyoto University. After years of hard work and international recognition, his principal desire—to find workable solutions to pressing medical problems—has never wavered.

"What I admire most about Dr. Yamanaka is that he is simply trying to solve a problem," said Dr. Farese. "The awards and recognition he's received have paled in comparison to what he has always been most interested in doing—saving lives."