For Stem Cell Researchers at UCSF, Already at the Forefront, Yamanaka's Work Galvanizes Field

Editor's Note: This story was updated Sept. 30, 2011, after first version appeared on Nov. 21, 2010.

Maybe you can’t teach an old dog new tricks, but thanks to stem cell research advances, we may one day be able to rejuvenate our tired old hearts, minds and tissues of all types by reprogramming the cells within them.

Many scientists never thought it was possible to do what Shinya Yamanaka, MD, PhD, has done. Yamanaka transformed and immortalized cells by manipulating just four genes, first in mouse skin cells in 2006, and then in human skin cells in 2007. In doing so, he also transformed the field of stem cell research.

Development of multicellular organisms had always been thought to move in the direction of greater cell specialization and a more constrained fate – the egg becomes an adult animal with hundreds of cell types, each with its own job to do.

But Yamanaka showed that one could turn back the clock and start over, converting adult cells into cells that act like cells from the early embryo. The stem cells created this way are known as induced pluripotent stem cells, or just iPS cells. Pluripotent means that the stem cells can give rise to many different types of daughter cells.

By showing how it may be possible to make any type of cell among the 200 or so kinds that make up a human being, Yamanaka – who divides his time between Kyoto University and UCSF’s Mission Bay campus, where he a member of the UCSF-affiliated J. David Gladstone Institutes as well as a UCSF professor of anatomy – has given hope to scientists, doctors and patients with all types of life-threatening, chronic diseases.

At UCSF and around the world, the amount of research effort that goes into studying iPS cells made by manipulating skin cells and other adult cell types now at least rivals the energy devoted to studies of stem cells from embryos.

Unlike embryonic stem cells, iPS cells can be derived from cells taken from adults who already have a disease. IPS cells already are being used in the lab to learn more about the development of human ailments, using human cells instead of relying solely on animal cells and animal disease models.

It’s well known that our genetic makeup can make us vulnerable to disease and even cause certain diseases. Using iPS techniques, researchers can observe how the genetic hands dealt to afflicted individuals play out in cells growing in the lab, shedding light on biochemical mechanisms of disease and new drug targets.

In the development of cell therapies to regenerate tissue, the capability of using iPS cells derived from the patient’s own tissue for transplantation should eliminate the risk of transplant rejection.

“The development of iPS cells is an advance that has really captured the imagination of the entire field and galvanized laboratories around the world,” said Arnold Kriegstein, MD, PhD, director of the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at UCSF.

iPS Cells Used to Shed Light on Development of Human Ailments

At UCSF and around the world, the amount of research effort that goes into studying iPS cells made by manipulating skin cells and other adult cell types now at least rivals the energy devoted to studies of stem cells from embryos.

Unlike embryonic stem cells, iPS cells can be derived from cells taken from adults who already have a disease. IPS cells already are being used in the lab to learn more about the development of human ailments, using human cells instead of relying solely on animal cells and animal disease models.

It’s well known that our genetic makeup can make us vulnerable to disease and even cause certain diseases. Using iPS techniques, researchers can observe how the genetic hands dealt to afflicted individuals play out in cells growing in the lab. By doing so, they hope to shed light on biochemical mechanisms of disease and to identify new drug targets.

In the development of cell therapies to regenerate tissue, the capability of using iPS cells derived from the patient’s own tissue for transplantation should eliminate the risk of transplant rejection.

Yamanaka was not the first to restart the developmental program with DNA from adult cells. Before Yamanaka pioneered iPS techniques, researchers had learned how to reprogram an adult cell by extracting the cell’s DNA-containing nucleus and injecting it into an egg cell from which the nucleus had been removed.

This is a more difficult and less successful reprogramming technique used to create Dolly the sheep and other cloned animals. Nobody has yet created a human stem cell line by this method. The technique requires many donated eggs, and as with iPS-cell research, attempts at cloning a human are widely regarded as unethical as well as unlikely to succeed. This so-called reproductive cloning is outlawed in many states, including California.

iPS Cells Compared to Embryonic Stem Cells

IPS cells do not engender the same ethical concerns posed by the use of even the earliest-stage, egg-like embryos, and studies using iPS cells are unlikely to be compromised as a result of legal or legislative actions arising from those ethical concerns.

Stem cells from very early embryos are immortal and can spin off daughter cells with the potential to become any type of cell. They have been the early focus of stem cell research aimed at new treatments. In fact, Bay Area company Geron Corp. enrolled the first patient into a US stem cell clinical trial a year ago. The researchers are using nerve-insulating cells derived from embryonic stem cells to treat recent spinal cord injury. The US Food and Drug Administration has given the go-ahead to launch just a handful of clinical trials so far.

Embryonic stem cells still are the gold standard to which iPS cells are compared. In most respects, it appears that – depending on lab methods and on which type of adult cells one starts out with to make iPS cells – the potential fates of daughter cells spun off from those iPS cells may be biased toward cell types more similar to the original type of adult cell from which the iPS cells were created.

But following Yamanaka’s example, researchers the world over are quickly learning more and more about which molecular routing switches guide cells down particular developmental paths. They are using this new knowledge to more successfully guide the developmental fate of stem cell progeny.

To induce an adult cell to become a stem cell, many genes that are active must be silenced, and many genes that are silent must become active. Yamanaka originally found that this reprogramming could be set in motion by using a virus to insert just four genes into the cell and forcing these genes to become active, or expressed. Since then, he has developed another genetic reprogramming technique that employs circular bits of bacterial DNA to insert genes into the cellular genome, without otherwise perturbing the cell’s DNA in the way that inserting viral DNA can.

“It’s likely that every stem cell laboratory in the world right now is working on some aspect of the technology that Dr. Yamanaka initially described,” said Deepak Srivastava, MD, director of the Gladstone Institute of Cardiovascular Disease and UCSF professor of pediatrics and of biochemistry and biophysics.

Yamanaka’s Work Spurs Campus Colleagues

Yamanaka is not done advancing stem cell research. It’s only been a few years since his revolutionary experiments, and he and his Gladstone and UCSF colleagues are closer to the beginning than to the end of their research journey. Scientists have continued to make remarkable progress.

In 2010, for instance, Srivastava and his lab group, led by former postdoctoral fellow Masaki Ieda, MD, PhD,reported success in transforming structural cells known as fibroblasts into cardiomyocytes – the beating muscle cells of the heart. This represents significant progress in efforts aimed at developing regenerative therapies for heart disease.

“Our work reprogramming fibroblasts directly to cardiomyocytes was inspired by the approach Shinya took in reprogramming fibroblasts to iPS cells,” Srivastava said. In the same way that Yamanaka originally homed in on just four genes that are key to converting an adult stem cell to an iPS cell, Srivastava’s lab team homed in on the genes that are essential for cardiomyocytes.

“We took a group of master regulatory genes enriched in developing cardiomyocytes compared to fibroblasts, and overexpressed all of them in fibroblasts containing a reporter system that would indicate the conversion to a myocyte,” Srivastava said. “It worked, and we then narrowed the minimal cocktail to just three factors, which did the trick very efficiently, converting almost 20 percent of cells to muscle cells. We also found that the cells directly became mature cardiomyocytes without ever passing through a stem cell stage.”

Fundamental Questions of Cell Identity, Including Cancer Cell Identity

Before Yamanaka pioneered iPS techniques, researchers had learned how to reprogram an adult cell by extracting the cell’s DNA-containing nucleus and injecting it into an egg cell from which the nucleus had been removed – a more difficult and less successful reprogramming technique used to create Dolly the sheep and other cloned animals. But nobody has done this with human cells.

Yamanaka’s work has been important to UCSF’s Robert Blelloch, MD, PhD, who is interested in the fundamental question of how cells become specialized, or differentiated, and conversely, how they can lose their specialization and become like stem cells, a process called de-differentiation.

“Stem cells exist both in the developing embryo and in many organs of the adult,” according to Blelloch. Adult stem cells can spin off a range of cell types, but unlike embryonic stem cells, they cannot give rise to every cell type.

“Differentiation of stem cells is tightly regulated, so that as they become increasingly specialized, they lose the potential to revert or transform into other cell types,” Blelloch said. “This regulation is very important both to maintain organ function and to avoid the possibility of uncontrolled cell growth, the basis of cancer. Very little is known about the mechanisms that control and lock in cell differentiation.”

Before Yamanaka developed iPS techniques, there was no way to study the determinants of cellular differentiation in humans,Blelloch said. “Why does an adult cell of any type – a skin cell, let’s say – remain a skin cell? If skin cells just randomly de-differentiated, becoming unspecialized, then you would end up with cancers. De-differentiation is probably an important underlying mechanism behind cancers. We are learning a lot about cancer by reprogramming because in cancer itself, cells are reprogrammed – although perhaps not all the way toward becoming an embryonic stem cell.

“The more we know about how a cell maintains its properties, the more likely it is that we will be able to manipulate cells and change one type of cell into another. That would be a dream realized for regenerative medicine because then we can replace one type of damaged tissue with another type of tissue that isn’t damaged.”

Once Yamanaka demonstrated that it was possible to manipulate the differentiation and de-differentiation of cells, it made sense to begin looking for other ways to do it, Blelloch said.

Blelloch and other scientists are pioneering the manipulation of cell characteristics by turning genes on and off with small molecules, including the same kinds of microRNA molecules that play a natural role in helping to guide the differentiation of cells.

“The same family of microRNAs that regulates embryonic stem cells can also promote the de-differentiation of adult cells to embryonic stem cells,” Blelloch said. “In addition, the same family of microRNAs is active in cancer, and my lab group and others have data to show that these microRNAs are functionally important in tumor cells.”

In 2009 Blelloch and graduate student Robert Judson last year showed that microRNAs that normally are present in embryonic stem cells can be used with three of the four factors first identified by Yamanaka to increase the efficiency of the reprogramming of adult cells into iPS cells. The UCSF scientists left out the fourth factor, called c-Myc, which has been blamed for unwanted variability between iPS cell colonies and even for the development of tumors in mice transplanted with iPS cells.

Yamanaka’s earlier experiments demonstrating how adult cells can be successfully deprogrammed and reprogrammed to become another cell type using just a few proteins has recently inspired others to develop techniques for accomplishing this that don’t require putting cells through the induced pluripotent stem cell stage. The expectation is that avoiding pluripotency will prevent the genesis of rogue cells that could form tumors.

A Stanford University team announced a shortcut for converting mouse skin cells directly into neurons in January, 2010, and since then several scientific teams, including one led by Sheng Ding, PhD, of the Gladstone Institutes, have had success working with similar techniques and human skin cells.

Ding also has used another strategy to convert adult skin cells into either cardiac stem cells or neural stem cells without putting cells through the pluripotent stage. Similar techniques developed by Ding make it possible to generate large numbers of cells for research, and such techniques may one day be used to provide cells for clinical trials.

Using iPS Cells to Develop Models for Studying Disease

Type 2 diabetes, which now is epidemic, thanks to a rising tide of obesity, also is very much a genetic disease. That fact is easy to appreciate when one considers that, although obesity is a strong risk factor for the disease and most people with type 2 diabetes are obese, only a minority of the obese become diabetic. The risk varies greatly among individuals, depending on variations in their genetic makeup. However, type 2 diabetes is genetically complex. Many combinations of genetic variants are likely to contribute to the risk of developing the disease.

Matthias Hebrok, PhD, director of the Diabetes Center at UCSF, very much appreciates the value of embryonic stem cells, as he and his colleagues are well on their way to successfully programming them to produce insulin-secreting cells for transplantation in type 1 diabetes, a disease in which pancreatic islet cells are destroyed by the immune system.

But to better understand how type 2 diabetes arises, the Diabetes Center is committed to working more with iPS cells, he said.

“Even if we knew all the factors that play a role in type 2 diabetes, I don’t think we could replicate it,” Hebrok said – not through genetic manipulations of embryonic stem cells. “Instead, we want to get a sampling of cells from people who are obese and who have type 2 diabetes, and compare them to cells from people who are obese but who do not have diabetes,” he said. 

Growing the Huge Number of Replacement Cells Needed to Treat Liver Diseases

Holger Willenbring, MD, in the Division of Transplant Surgery at UCSF, has tested whether iPS cells from Yamanaka’s lab are capable of making cells that can cure a rare and normally fatal liver disease in a mouse model. By doing so, he has shown that it should be possible to use iPS cell-based treatments to restore liver function in a range of liver diseases.

“We in the liver cell therapy field need a highly proliferative cell type that can become a true hepatocyte – the cell in the liver that provides most of its functions,” he said. “To cure liver diseases, you have to replace more than 10 percent of the 250 billion hepatocytes in the human liver. Therefore, we would not be able to get away with anything less efficient than embryonic stem cells.

“Yamanaka has now allowed us to try to obtain the needed cells from iPS cells, and to achieve our goal in a more ethically acceptable way. On top of that, these cells, because they can be derived from patients, hold out hope for autologous transplants that will not require immunosuppressive drugs, which can have significant toxic effects over time.” 

A Way to Better Understand Dementia and Possibly to Test Drugs

Gladstone scientist Robert Farese Jr., MD, is using iPS techniques to study frontal temporal dementia, the most common cause of early-onset dementia in people under age 60. The work is a collaboration between Bruce Miller, MD, clinical director of the Memory and Aging Center at UCSF, and University of Massachusetts neuroscientist Fen-Biao Gao, PhD. 

Several different genetic mutations have independently been associated with the development of frontal temporal dementia. To learn how, Farese is using Yamanaka’s original methods to convert skin cells from patients with frontal temporal dementia into two types of cells thought to act abnormally in patients with the disease – nerve cells and microglial cells, which are a type of inflammatory cell in the central nervous system. A lack of normal secretion of the protein progranulin by these cells may play a role in their dysfunction and in the origins of the disease. The normal function of the protein is unknown.

“This is where the field is right now,” Farese said. “We’re trying to sort out whether there indeed is abnormal neuronal function, and while it appears clear that there might be abnormal inflammation, this allows us to test those ideas in human cells. Down the line, we might use these cells to test therapeutic agents.”

On Track to Build on Stem Cell Research Excellence

UCSF scientists have been leaders in stem cell research ever since biochemist Gail Martin, PhD, reported the discovery of embryonic stem cells in mice in 1981. UCSF scientists have competed very successfully for state funding that is available through the California Institute for Regenerative Medicine.

Donor support, coupled with a major grant from the California Institute for Regenerative Medicine, which was set up to administer $3 billion in voter-approved funding, made it possible to construct the Ray and Dagmar Dolby Regeneration Medicine Building. This architectural landmark, wrapped around a Parnassus Heights hillside, is the headquarters for the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at UCSF, a program that extends across UCSF’s campuses. The infrastructure, talent and funding are in place to help keep UCSF at the forefront in this exciting research field.

The Gladstone Institutes is a nonprofit, independent research and educational institution consisting of the Gladstone Institute of Cardiovascular Disease, the Gladstone Institute of Virology and Immunology, and the Gladstone Institute of Neurological Disease. Independent in its governance, finances and research programs, Gladstone shares a close affiliation and proximity with UCSF through its faculty, who hold joint UCSF appointments.

The first Gladstone Institute to be established was the Institute of Cardiovascular Disease, but as the institution has grown over the years, so has the collective range of scientific expertise found among its researchers. Today, the Gladstone Institutes is home to many scientists working at the frontiers of stem cell science.

“These institutes, while they are separate administratively from the University, are more and more fully integrated with the goals of the University,” said Keith Yamamoto, PhD, vice chancellor for research at UCSF. “There are big research questions that we have to tackle, and with the Gladstone Institutes, we have the advantage of building those scientific communities out more fully. Having a larger community of scholars going after these big issues is a real advantage for us. There’s a real synergy there.”

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