Researchers fix mutation in reprogrammed stem cell, create functional liver

A group led by the Sanger Institute and the University of Cambridge, working with the Sangamo Biosciences, has shown that it's possible to fix mutations in reprogrammed cells. This work, which was published in Nature, takes two previous advances and combines them into one proof-of-concept.

Since 2007 stem cell scientists have been able to reprogram adult cells such as skin back into an embryonic-like state. These so-called iPS cells can then mature into any cell type in the body, much like embryonic stem cells.

Other groups have shown that it's possible to take adult stem cells such as those from the bone marrow, correct mutations, and create mutation-free cells that, at least in animal models, can fix diseases. That's the idea behind CIRM's two HIV/AIDS disease team awards (described here and here) and a sickle cell disease team. In fact, one of the HIV/AIDS disease teams is also working with technology developed by Sangamo to fix the mutations.

In the work reported in Nature, the team created iPS cells from a person with a genetic liver disease, fixed the mutation, then matured the iPS cells into functional liver cells. A Reuters story quotes Allan Bradley, director of the Sanger Institute:

"These are early steps, but if this technology can be taken into treatment, it will offer great possible benefits for patients," he added.

Reuters quoted David Lomas, who was part of the team from Cambridge, saying that the liver cells survived when transplanted into mice.

The researchers said it could be another five to 10 years before full clinical trials of the technique could be run using patients with liver disease. But if they succeed, liver transplants -- costly and complicated procedures where patients need a lifetime of drugs to ensure the new organ is not rejected -- could become a thing of the past.

"If we can use a patient's own skins cells to produce liver cells that we can put back into the patient, we may prevent the future need for transplantation," said Lomas.

A.A.

New cells lines made using “cloning” technique valuable for research

Yesterday a New York Stem Cell Foundation team reported for the first time that they had created two new embryonic stem cell lines through a technique known as somatic cell nuclear transfer (SCNT), which is sometimes called therapeutic cloning. They reported their findings in the journal Nature.

SCNT is a third avenue for creating cell lines able to form all tissues in the adult body – called being “pluripotent.” Interestingly, SCNT borrows from the two other techniques used to date.

Pluripotent cell lines were first created by extracting them from 5-6 day old human embryos left over after in vitro fertilization – hence their name human embryonic stem cell lines.

Pluripotent cell lines were later created by reverting skin cells to a pluripotent state through a process called “reprogramming” – commonly referred to as induced pluripotent cells.

SCNT is a reprogramming method that involves the creation of an embryo as a first step.  In this case, scientists took DNA from a human skin cell and placed it in a human egg, which they then stimulated to form a 5-6 day old embryo. In this environment, the DNA was reprogrammed to an earlier state and the resulting cells were extracted to create human embryonic stem cell lines.

The fact that SCNT-derived stem cell lines have so much in common with other forms of pluripotent stem cells has some opponents of the research asking why bother? Here’s why. CIRM held a conference in June 2010 to discuss the value of pursuing SCNT and posted a report on the findings in November, 2010. 

That report suggests three areas where embryonic stem cell lines generated through SCNT would clearly be valuable in three ways:

• Understanding how you reprogram any cell to become pluripotent could help us optimize the creation of iPS cells, which are so far inefficient to create in addition to being incompletely reprogrammed.
• Understanding and treating the rare diseases that are passed on from those few genes that reside outside the nucleus in the cellular organ called the mitochondria.
• Studying the very early stages of human development, which are poorly understood now, and which is when some human diseases are thought to originate.

The fact that the New York team got the technique to work in humans is a significant advance that has value for all three of those potential areas of research. However, the two cell lines reported yesterday aren’t exactly ready for therapies. Rather than having two copies of each gene, as all of us do, these cells have three copies of every gene and are therefore biologically abnormal. The Wall Street Journal described the problem like this:

While such cloning experiments have been successful in various mammals, the "de-nucleated" egg approach hasn't worked so far in humans. Now, Dr. Egli and his colleagues have—partially—achieved it via a simple move: They didn't remove the egg's own nucleus.

Not removing the egg’s nucleus resulted in the triple copy of chromosomes (one from the egg and two from the donor’s nucleus) that left the cells as “research only” cell lines. Many news stories about the work have referred to the new lines as coming from “cloned embryos”. However, because the cell lines contain more chromosomes than the donor cell they are not truly clones.

Understanding what factors in the nucleus aided in getting SCNT to work could provide clues about factors that might aid in making iPS cells more efficiently, and also provide clues as to how to create SCNT-derived lines with normal numbers of chromosomes.

The Wall Street Journal story quotes George Daly, a stem-cell researcher at Children's Hospital Boston , who summarizes the findings as “a landmark even if it isn’t a complete victory.”

In coming months we should watch for advances that turn this landmark into a victory for people hoping to use the SCNT-derived stem cells to study the earliest stages of development, understand and treat mitochondrial diseases, and learn how to create better iPS cells.

DG

Stem cell banking and the making of a patient "advocist"

In 2005 Chris Hempel gave birth to twin daughters Cassi and Addi. In 2007, she and her husband learned that their girls had a rare, fatal disease called Neiman Pick Type C.

Four years later, Hempel describes herself as a "advocist" for rare diseases. She's an advocate for scientific research but also an activist seeking to bridge the gap between patients and researchers. One of her primary messages is this: If patients donate tissue (skin, in her case) that contribute to science, then they should get to know the results and participate in the research.

That sounds easy, but has proven difficult. She spoke to CIRM's Standards Working Group in April to discuss her experiences and encourage CIRM to take a leadership role in creating policies that engage and inform the tissue donors.

During her talk, she said, "If the entire goal is really to use a patient's own cells to cure them, well, you really can't cure a patient if they are just a number."

CIRM funds several awards that have the intention of creating reprogrammed stem cell lines from skin samples to better understand genetic diseases, much like the research Hempel participated in. These scientists have creating disease-in-a-dish models of schizophrenia, Parkinson's disease and autism using this approach.

One of the issues the working group discussed is the types of standards that should be in place to protect the rights of the people who donate tissues for these and other CIRM studies. In addition there was discussion about ways of providing information to potential donors, research participants and the public.

The agenda from that meeting contains additional information about tissue donation and iPS banking: available here.

Here is Hempel's talk:



A.A.

Stem cell hope, hype, and hypocrisy according to Arthur Caplan

Ethicist Arthur Caplan had an excellent piece about stem cell hype last week on Science Progress, a publication of the Center for American Progress. Caplan is Director of the Center for Bioethics and the Sidney D. Caplan Professor of Bioethics at the University of Pennsylvania.

He starts by saying that yes, some have over-hyped the promise of stem cell research, saying:

Anyone who has followed my advocacy for embryonic stem cell research would know I have long been critical of claims that funding today means people tomorrow will leap from their wheelchairs and walk.

However, he goes on to say that some of the most over-hyped claims in stem cell research come from those who oppose work with human embryonic stem cells. His list includes:

• Hyped claim #5: The Bush “compromise”
• Hyped claim #4: Adult stem cells can do it all
• Hyped claim #3: If embryonic stem cell research is so promising, then why isn’t private research behind it?
• Hyped claim #2: IPS cells are the magical solution to the embryonic stem cell quandary
• Hyped claim #1: Frozen embryos should be put up for adoption rather than used as sources of stem cell lines. Of this hype, Caplan adds:

While I am on this particular bit of hype, I should add that those who do not favor the use of unwanted and certain-to-be-destroyed frozen embryos languishing in clinics worldwide never ever say what they propose be done with them. Conservatives say destruction is unthinkable, however, since it is inevitable then what are they talking about? ( I suppose this constitutes hypocrisy and not hype.)

His arguments regarding items on the list are worth a read. He says:

There is plenty more hype to be had from what has passed as debate over the past decade or so since human embryonic stem cells were first isolated. I don’t mean to suggest that most of the hype has come from critics rather than proponents. I do mean to suggest, however, that those who live in very fragile houses often constructed of hype ought not be quick to cast stones.

CIRM funds work with adult stem cells and iPS cells in addition to embryonic stem cells because until people in wheel chairs can get up and walk it's too soon to start ruling out therapeutic options.

- A.A.

Guest blogger Alan Trounson - April's stem cell highlights

Alan Trounson is President of CIRM

Since I arrived at CIRM late in 2007 I have maintained a tradition of presenting some of the top science journal papers from the previous month or two at each of our Board meetings. Beginning last month, I decided this would be easier to digest in a written document than in PowerPoint slides amid a harried board meeting. You can see an archive of these periodic stem cell reports on our website.

This month I want to start a second part of the new tradition, a brief blog note to let you know why I, as someone who toiled in stem cell labs for many years, chose these items as some of the most important papers in the field in the past month or so.

The first paper is a true breakthrough, something no one had accomplished before. A Japanese team was able to create an “organized” tissue in a dish, not just drive stem cells to become a specific adult cell, but rather two types of cells in two distinct layers. In this case they created an optic cup that resembled a post-natal retina. With one of the holy grails of stem cell research being the ability to replace complex organs, this was a brilliant paper to see.

You will see that in last month’s stem cell report I discussed “this year’s problem” with iPS, or reprogrammed cells, which is their much higher rate of genetic anomalies compared to embryonic stem cells (as we blogged about here). Well, this month I am discussing “last year’s problems” with iPS cells. For the past couple years there has been much hand wringing about the possibility that the transcription factors used to reprogram cells, if left in the cells, could be turned on at the wrong time and lead to cancer, and that the reprogramming processes were all hugely inefficient. Now, only five years after the first iPS cells were created in mice, a number of papers came out this month showing major strides to reprogramming with only transient integration of the reprogramming factors and exponential improvement in efficiency in creating iPS cells. I have to hope that “this year’s” iPS problem will be even more quickly solved or at least its relevance determined.

Last, I chose a paper that does two things: it explains a clinical result that had many purists in the fields shaking their heads in doubt and points the way to another major goal of the field, a way to stimulate endogenous stem cells to make repairs when needed. The study found a protein that can induce endogenous stem cells in heart attack patients and may explain why certain bone marrow stem cells, ones that have no ability to form heart tissue, nonetheless seem to offer some small but genuine improvement for many patients.

Alan Trounson

CIRM a leader in iPS cell publications

Yesterday, stem cell blogger and newly tenured CIRM grantee at UC Davis Paul Knoepfler had an interesting blog entry on iPS cell publications.

After mining the literature for publications with the phrases iPS cells, induced pluripotent stem cells, induced pluripotent or induced pluripotency in the title, he found a consistent increase in publications each year after the first creation of mouse iPS cells in 2006 by Shinya Yamanaka. That is, a consistent increase until this year, where the first third of the year contained fewer than expected publications. Knoepfler doesn't speculate on what this decrease means—and by the end of the year the discrepancy might disappear.

He did find more diversity in the researchers publishing in the iPS field and in the journals where those papers were published. That makes sense for a field that is becoming ever more mainstream. Knoepfler writes:

I think this is a good thing as the iPS cell field grows. The range of journals publishing iPS cell papers has greatly broadened, which is also a positive for the field as it matures.

Knoepfler doesn't speculate on what his findings mean for the field of iPS cells, either as potential therapies or as disease in a dish models. The cells have been the source of much consternation recently as they are shown to differ in significant but clinically unknown ways from embryonic stem cells (as we blogged about here). At the same time, they are also proving their worth in mimicking genetic disease (blogged about here, here, and here).

One discovery that stands out is CIRM's rank as second most prominent funder of iPS papers, following only the NIH. CIRM funds 4.8% of papers that Knoepfler found in his search. Coming in third was the National Natural Science Foundation of China.

CIRM's searchable grants database shows 66 awards to grantees working with iPS cells, worth a total of $146,882,748 or 12% of CIRM funding. You can see those awards here. By contrast, CIRM provides $384,709,412 toward awards working with embryonic stem cells, or 32% of our funding, and $194,221,598 or 16% toward grants working with adult stem cells.

Some CIRM grants fund work using more than one type of stem cell, including several awards to grantees trying to understand differences between iPS and embryonic stem cells.

- A.A.

The right tool for the job: is it iPS, ES or adult? Answer: It depends

Two stem cell stories in the news today bring to mind yesterday's interview on NPR's Fresh Air, in which veteran journalist Matthew Wald of the New York Times said of the decision to store spent nuclear waste in Yucca Mountain, NV:

Yucca was chosen by the finest geologists in the United States Senate, which is to say they may not have made the best technical choice.

A similar statement could be made about stem cell research policies, which are to some degree being made by the best stem cell scientists in politics.

People who oppose embryonic stem cell research point to reprogrammed iPS cells and adult (or more accurately tissue-specific) stem cells as perfect replacements. These arguments are winning some political advocates around the world including France where they are debating a ban on human embryonic stem cell research and in Ireland (which we've blogged about recently), but aren't borne out by science.

Just to be clear, we here at CIRM are big fans of reprogrammed and tissue-specific stem cells, which is why we fund so much of that work (you can see all of our adult stem cell grants here and our reprogrammed iPS cell grants here). But we're also big fans of the right tool for the right job, and just because we love our hammer and screwdriver doesn't mean we don't still need a few wrenches to get the job done.

Today's news brings a story from Nature about a paper published in Cell Stem Cell in which scientists in France used embryonic stem cells to learn how a mutation leads to the muscle wasting disease myotonic dystrophy. The discovery could help scientists understand and treat the disease. They quote Marc Peschanski, director of the Institute for Stem Cell Therapy and an author of the latest paper.

Peschanski runs a large iPS-cell research programme in addition to his hES-cell work. "We make iPS cells to model particular diseases when we don't have access to the relevant hES cells — which remain our gold standard," he says.

Politicians who oppose hES-cell research often — wrongly — insist that iPS cells can always substitute for hES cells, says Peschanski. He is frustrated that the lower house of the French parliament invoked this argument when proposing a ban on hES-cell research in France. Peschanski has since been working with other French scientists to persuade the Senate to overturn the proposal next week.

A related story from Reuters cites several recent papers showing significant differences between iPS and embryonic stem cells. They write:

Stem cell scientists are not giving up on iPS cells, but instead of a replacement for embryonic stem cells, they see them filling a unique research role.

We've written quite a bit about the role of iPS cells (The confusing (and ongoing) story of iPS vs. embryonic stem cells) and their clear value in generating disease in a dish models for understanding diseases and testing drugs. The Reuters story goes on to quote George Daley of the Harvard Stem Cell Institute and Harvard Medical School:

"It has not ever been a scientifically driven argument that iPS cells are a worthy and complete substitute for embryonic stem cells," Daley said. "Those arguments were always made based on political and religious opposition to embryonic stem cells."

CIRM grantee Jeanne Loring of The Scripps Research Institute in La Jolla has said that what's not known is what these differences between the cell types mean (here's our blog entry on that work). Are they deal breakers in terms of using the cells therapeutically, or are they just temporary set backs while scientists work to develop better iPS cells? For now that's not known.

Which is all to say that in order to get the job done of understanding and treating diseases, scientists need all the tools at their disposal. Sometimes tissue-specific stem cells are going to be ideal. Blood-forming stem cells in bone marrow have certainly proven their worth in treating a number of blood diseases. And iPS cells are becoming valuable tools for studying diseases in a dish. But I wouldn't want to build a house with just a hammer, and I'd hate to see stem cell scientists trying to generate new cures without a full toolbox of cells to work with.

- A.A.

More questions raised about iPS cells safety

Much has been written over the past few days about a spate of new papers by CIRM grantees showing significant differences between reprogrammed iPS cells and embryonic stem cells (see the San Diego Union Tribune,  Discover, Technology Review) and CIRM grantee Paul Knoepfler at UC Davis had an insightful blog entry on the topic.

What's causing the stir is the fact that when scientists first reprogrammed skin cells into embryonic-like iPS cells in 2006, those iPS cells seemed like the ultimate solution -- all the power of embryonic stem cells without the embryos. Everybody wins!

Since their introduction, many papers have been published announcing better ways of generating the cells and comparing the cells to their embryonic counterparts. What's emerging is a somewhat complicated story in which there are some clear wins, but also some questions. We reported yesterday and a few weeks ago on some of the wins: iPS cells have been proving themselves ideal for mimicking a disease in a dish.

However, the cells do appear to be significantly different than embryonic stem cells. My colleague Zachary Scheiner in our science office had this to say about the various papers that came out this week:

There are many similarities between embryonic and reprogrammed stem cells, but a number of recent papers have highlighted differences that could affect the utility of iPS cells for therapies. In one paper, Lister et al. examined a chemical alteration to DNA, called methylation, in a variety of cell types including embryonic stem cells, iPS cells, and adult skin and fat cells. DNA methylation is a normal biological process and helps determine which genes in the DNA get made into proteins in the cell. The DNA in your skin cells, for example, has different methylation than that in your liver cells because those two types of cells need to make different proteins.

Lister et al. found that reprogramming adult skin and fat cells to iPS cells caused hundreds of locations in the genome to have unusual methylation compared to embryonic stem cells. Importantly, these differences remain after the cells are matured into other cell types. This finding suggests that these aberrant DNA modifications could affect the function of cells derived from iPS cells for therapeutic purposes, such as transplantation into patients.

In a complementary paper, Gore et al. examined iPS cells for genetic changes, or mutations, in the DNA code itself, which can have profound effects on the safety of the cells. They found that the process of creating iPS cells introduced an average of six gene mutations per cell line, many more than would be predicted from normal cell culturing. Further, they found that 40% of the mutations discovered were in genes previously found to be mutated in cancers.

Let's review that last sentence: 40% of the mutations discovered in iPS cells were in genes associated with cancer.

In the San Diego Union Tribune, Keith Darce quotes CIRM grantee Jeanne Loring of Scripps Research Institute, who has published several papers showing genetic differences between the two cell types:

“The big question is, is there anything wrong with this stuff happening? We have no idea.”

My colleague Zachary Scheiner summed it up like this:

Taken together, these two papers raise cautionary flags for researchers seeking to develop cell therapies from iPS cells. However, they also empower these researchers by revealing the types of abnormalities that exist in these cells. Armed with this knowledge, researchers should be better able to assess and assure the safety of iPS cell-derived therapies prior to clinical translation.

The great thing about giving money to smart people (that would be our grantees) is that we can now hope to see papers investigating safety issues that result from these genetic changes, or developing ways of creating iPS cells with fewer anomalies.

CIRM funding:
Nature, March 3: Ronald Evans (RB2-01530)
Nature, March 3: Athurva Gora (TG2-01154) Lawrence Goldstein (RC1-00116)

- A.A.

Disease in a dish model provides insight on aging

Normal aging takes many decades to create major changes in our cells, so it is very difficult to study. As a result, very little is known about this fundamental inevitability of life. But that may change with the help of an unfortunate child, who by the bad luck of a single point mutation developed a rare disease that results in aging at eight to 10 times the normal pace.

A Salk Institute research team lead by Juan-Carlos Izpisua Belmonte has reprogrammed skin cells from the child, who has Hutchinson-Gifford progeria, into induced pluripotent (iPS) stem cells and then forced them to mature into smooth muscle cells in a dish that displayed all the characteristics of aging cells, a model for aging in a dish.

In a Salk press release Belmonte said:

Having a human model of accelerated aging may give us new insights into how we age. It may also help prevent or treat heart disease in the general aging population.

In a paper in Nature, the Salk team noted that this progeria is caused by a single point mutation in the gene encoding lamin A, and that there is evidence that defective lamin A also accumulates in the normal aging process via sporadic gene splicing.

The beauty of this model is the researchers were able to provide evidence for the impact of the defective protein. When the reprogrammed cells were in the embryonic-like state the lamin A was silenced, but when those cells were differentiated into smooth muscle the signs of premature aging appeared.

CIRM funding: Guang-Hui Liu  (TG2-01158)
Nature, February 23, 2011

D.G.

The confusing (and ongoing) story of iPS vs. embryonic stem cells

It appears we weren't the only people to notice last week's convergence of reprogrammed iPS cell news -- first they are made better, then they are suggested to be worthless. USA Today ran a story summing up several years' worth of such news. (For those not up-to-speed on iPS cells, you can watch this video with UCLA's Jerome Zack talking about how the cells are made.)

The story goes something like this: One day, iPS cells reprogrammed from adult tissue are going to eliminate the need for embryonic stem cells. No destroying embryos!

Soon after, someone points out that the creation of iPS cells -- though cool -- requires inserting cancer-causing genes. Not good! They cause cancer! But then someone finds a better way, with no cancer genes. Good! But then iPS cells are shown to differ dramatically from embryonic stem cells. And they don't seem quite as willing to form all tissues. Confusing!

According to the USA Today story:

"Basically, we are looking at a lot of confusion," says Harvard stem cell scientist Alexander Meissner. "That's not to say one group is wrong and another is right. We have been making a lot of progress, but everyone is looking at the same problems from different sides."

The story mentioned last week's paper by Salk researchers showing a molecular memory in iPS cells and went on:

Combined with a September Nature paper showing similar memory signatures in mouse IPS cells and Scripps Research Institute researchers last month reporting more cancer genes in IPS cells compared to embryonic ones, things looked bad . "The finding suggests that (induced) cells may not be suitable substitutes for (embryonic) cells in modeling or treating disease," noted Nature science reporter Elie Dolgin.

Although iPS cells are clearly the source of some confusion in terms of their similarity to embryonic stem cells, they are still a great tool for mimicking disease. CIRM researchers at Salk have taken skin cells from people with ALS, matured those cells in a lab dish into the cells involved in the disease and learned details about the biology of that disease that would never have been possible without reprogrammed cells. (Here's a video about that work.)

Other grantees at the Parkinson's Research Institute are taking skin from people with Parkinson's disease, maturing those into the neurons involved in that disease, and using those cells that are genetically included to form Parkinson's disease to understand the disease and test drugs. (This video includes scientists at the Parkinson's Institute talking about that work.)

At Gladstone, CIRM grantees are generating heart tissue from the skin of people with genetic heart diseases and using those cells to screen drugs. (You can watch a video of Bruce Conklin talking about that work.)

In each case, it doesn't matter that iPS cells are not identical to embryonic stem cells. It matters that they are currently the only way to study mature disease-prone cells in a lab dish. Because those people with Parkinson's disease aren't giving up brain tissue and the heart disease patients aren't loaning out little chunks of their heart. But skin they can part with.

USA Today ends their story by instructing readers to hang on for a bumpy ride ahead as scientists resolve the meaning of the differences between iPS and embryonic stem cells. One day we'll know which cell type provides the best tool for treating and studying different diseases. In the mean time, USA Today is likely right that the ride won't be dull. 

- A.A.

iPS developments - faster creation, but questions raised

Two pieces of news came out today about reprogrammed iPS cells — one showing a new way of making them and the other suggesting that they may not be all they’re cracked up to be.

First, the new technique. A team at Sanford-Burnham Medical Research Institute in La Jolla figured out a way of removing barriers to reprogramming, in which skin or other adult cells are reprogrammed back to an embryonic-like state. Most techniques for reprogramming involving adding DNA or other factors to push the cells back in developmental time. But the process isn’t very efficient. This team identified two barriers to reprogramming and removed them using small inhibitory molecules called miRNAs.

The Sanford-Burnham press release quotes CIRM grantee Evan Snyder, director of Sanford-Burnham’s Stem Cells and Regenerative Biology program:

“Up until now, cellular differentiation and de-differentiation has focused principally on the expression of genes; this work indicates that the strategic non-expression of genes may be equally important. The work has demonstrated that miRNAs do function in the reprogramming process and that the generation of iPSCs can be greatly enhanced by modulating miRNA action. In addition to helping us generate better tools for the stem cell field, such findings inevitably facilitate our understanding of normal and abnormal stem cell behavior during development and in disease states.”

Ironically, on the same day the authors published the fruits of many years of labor, the news cycle delivered a blow. Researchers down the road at The Salk Institute for Biological Studies published yet another report showing critical differences between iPS and embryonic stem cells. There’s been a steady drumbeat over the past year of studies pointing out that iPS cells might not exactly mimic embryonic stem cells, and for that reason might not be ideal replacements in therapies.

William Lowry, a CIRM grantee at UCLA, is quoted in a Nature news story about the finding:

"The problem is that we don't know if any of these differences are going to be consequential."

Whether these differences between iPS and embryonic stem cells will turn out to be insurmountable in terms of future therapies is unknown. What is clear is that scientists have many hours in the lab ahead of them before we understand which cells are the safest and most effective for eventual therapies.

- A.A.

Better, faster stem cell reprogramming

A group at the Harvard Stem Cell Institute led by Derrick Rossi has taken a big step toward a holy grail in stem cell science — reprogramming skin cells to resemble embryonic stem cells without viruses (Cell Stem Cell paper). The new technique uses transient RNA to reprogram the cells into what they are calling RiPS cells (for RNA induced Pluripotent Stem Cells)

Reprogrammed human iPS cells were first generated in 2007 by Shinya Yamanaka of the Gladstone Institutes and Kyoto University, but his approach involved using a virus to insert four genes permanently into the cells. A Harvard press release describes the problems with the initial approach:

First, the use of the integrating viruses raised the very real possibility that cancers might inadvertently be triggered; and second, inserting the genes into the genome could lead to changes that would alter the properties of the resulting iPS cells so that they would not be identical to human embryonic stem cells.

Since that first discovery, scientists around the globe and in California have been working toward techniques that avoid viruses and don’t permanently alter cell's DNA. (Here is a list of CIRM grants focused on new ways of generating iPS cells.) Rossi describes the benefits of his approach:

“Gene therapy trials unfortunately taught us the danger in leaving viruses in the genome as some patients developed cancers that were driven by the integrated viruses. So when one thinks about strategies for regenerative medicine, you need to envisage utilizing cells whose genome has not been breached. We believe that utilizing RNA to generate transplantable cells and tissues is a ideal solution because, to the best of our knowledge, RNA is completely non-integrative.”

What’s still unclear is how similar these iPS cells are to their embryonic counterparts. Several recent papers have found that iPS cells can differ significantly from embryonic stem cells in their ability to generate all tissues. (We have blog entries on those papers here, here and here.) 

A.A.

iPS cells from women create model for muscular dystrophy, X-linked diseases

Reprogrammed skin cells showing inactivated X in red

CIRM grantees at the University of California, Los Angeles have uncovered a feature of reprogrammed iPS cells that make them uniquely excellent for understanding diseases that arise from mutations on the X chromosome.

First some background. Men inherit an X chromosome from their mother, which contains many thousands of genes, and a Y from the father, which does little except confer manhood. Women inherit one X chromosome from each parent. Those female cells overcome their genetic overabundance by shutting down, at random, one of the two X chromosomes, putting the cells at genetic par with male cells.

But the two aren’t really equal. If men inherit a mutation on an X chromosome, it is present in every cell of the body and can cause muscular dystrophy, Rett Syndrome, color-blindness and other disorders. Women who inherit a mutation on an X chromosome from one parent will only show that mutation in half their cells. The other half of the body's cells, with the non-mutated chromosome active, can generally compensate.

So what does this have to do with reprogrammed cells and disease modeling? It turns out that the process of reprogramming skin cells into embryonic-like induced pluripotent stem cells doesn’t overturn the inactivated X. Reprogramming cells from a woman’s skin sample will produce two distinct types of iPS cell lines; half with one X active, and half of with the other X active. If one of those two chromosomes carries a mutation, say, for muscular dystrophy, some of those iPS lines will also display that mutation.

In a press release from UCLA, senior author Kathrin Plath said:

“This non-random pattern of X chromosome inactivation found in iPS cell lines has critical implications for clinical applications and disease modeling and could be exploited for a unique form of gene therapy for X-linked diseases.”

In a publication in Cell Stem Cell, Plath and her colleagues report that they created iPS cell lines from a woman who had inherited one X chromosome carrying a mutation that can cause muscular dystrophy. The other X chromosome had a normal copy of the gene. Scientists can now mature both groups of cells into skeletal muscle and compare the resulting tissue as a way of understanding—and perhaps one day treating—the devastating disease.

Cell Stem Cell: September 3, 2010
CIRM funding: Sean Sherman (TG2-01169), Kathrin Plath (RN1-00564), William Lowry (RS1-00259), Jerome Zack. (RL1-00681)

iPS and embryonic stem cells -- similar but not the same

Two papers in Nature publications have raised questions about whether reprogrammed adult cells, called iPS cells, are truly interchangeable with embryonic stem cells as many have been assuming. The papers found that iPS cells created from different adult tissues still bear some hallmarks of those starting blocks.

In a press release, George Daley, who was senior author on the Nature paper and is Director of the Stem Cell Transplantation Program at Children's Hospital Boston, said:

"iPS cells made from blood are easier to turn back into blood than, say, iPS cells made from skin cells or brain cells."

CIRM grantee Mahendra Rao at Life Technologies said in a story in The Scientist that iPS cells:

"are not truly similar to [embryonic stem cells] when examined at a high resolution.”

This work is generating such a stir because people have tended to think of iPS cells as the less controversial equivalent to embryonic stem cells. The same press release quotes the author of the Nature Biotechnology paper, Konrad Hochedlinger from the Massachusetts General Hospital Center for Regenerative Medicine, as saying that iPS cells do become more similar to embryonic stem cells the more times they divide in a lab dish.

On their blog, the Australian Stem Cell Centre wrote:

But what does this all mean for stem cell science? Ultimately findings such as these will help to improve reprogramming technologies. It also means that scientists need to be able to continue to work with and explore all of the different types of stem cells – iPS and embryonic stem cells derived from both donated IVF embryos and SCNT embryos. Limiting research by restricting access to certain cell types at this stage would severely impact progress towards using these cells to understand and ultimately treat disease.

This work comes after several papers showing some consistent differences between embryonic and iPS cells. We’ve blogged about that work here and here.

Nature, July 19, 2010
Nature Biotechnology, July 19, 2010
CIRM Funding: Jun Seita (T1-00001)

A.A.

Cancer genes also involved in embryogenesis, stem cell maintenance

CIRM grantee Paul Knoepfler at UC Davis just published an interesting paper. He also publishes a blog, so we'll let him describe this findings in his own words:

We just published a paper supported by CIRM funding showing that knocking out c- and N-myc in mESC leads to a wave of differentiation-associated gene expression, decreased cell cycling, and a moderate elevation of apoptosis.  The myc-deficient mESC also fail to contribute to early embryogenesis. This is the first analysis of a role for myc genes in early embryogenesis.

We think that in part that Myc contributes to iPS formation by repressing differentiation-associated gene expression (ala Sridharan, et al).

So to induce pluripotency Myc appears to be doing what much the same job as it does to maintain pluripotency in ESC.  A role in cell cycle is also involved.

Differentiation, May 26, 2010
CIRM Funding: Paul Knoepfler (RN2-00922)

A.A.

iPS cells and embryonic stem cells -- similar but not the same

In the most recent face-off between iPS and embryonic stem cells, the ES cells came out ahead -- turns out iPS cells aren't the same as ES cells even when they carry the same mutation. That's according to work published in the May 7 issue of Cell Stem Cell.

(The image shows colonies of embryonic and iPS cells, taken taken in the lab of Jeanne Loring at The Scripps Research institute.)


First some background. Embryonic stem cells come from 5-6 day old embryos left over after in vitro fertilization. These primitive cells can turn into all cells of the body. iPS cells come from adult cells -- most often the skin -- that are reprogrammed to act like embryonic stem cells. They can also form all cells of the body. When iPS cells were first created in 2007 by Shinya Yamanaka, the prevailing wisdom was that they might one day completely replace ES cells, but only if they are safe and are truly equivalent to their embryonic counterparts.

That "but" has been the source of ongoing experiments, many by CIRM-funded researchers, who have found that the two cell types have different genes active (see this blog entry). In this latest work, the European and Israeli researchers found that when they created iPS and ES cells both containing a mutation that causes fragile X syndrome, the two resulting groups of cells behaved very differently, with the iPS cells not even activating the mutated gene.

This finding raises questions about whether iPS cells will be the best choice for mimicking diseases in a dish.

In an article in Medical News Today, Nissim Benvenisty, director of the Stem Cell Unit at the Hebrew University of Jerusalem and a leading author of the study, says:

"Until we understand better the differences between these two types of cells, the optimal approach might be to model human genetic disorders using both systems, whenever possible".

That said, when Stefan Heller of Stanford University created ear hair cells from both ES and iPS cells he found no difference in their functionality.

A.A.

Questions About iPS Cells

In his blog, CIRM grantee Paul Knoepfler at UC Davis posted a response to the journal Stem Cells, which had published a list of the most pressing questions about iPS cells:

“What I found most striking is that not one of their 10 questions had anything to do with safety or tumorigenicity, the question I rank #1, but otherwise my top 5 most important questions about IPS cells are similar to theirs. I know they think safety is a crucial issue, which is why I'm so surprised it wasn't on their list.”

Knoepfler's focus on tumorigenicity stems from his lab's work, which he discusses in this video about the safety of stem cell-based therapies (embryonic or iPS).



Knoepfler is taking suggestions for additional top 5 lists.

A.A.

Virus-free Technique Yields Pluripotent Stem Cells

Stem cells in fat hold intrigue for scientists because most of us have excess to spare, and the cells seem to be quite versatile. Now a team at Stanford has found a way to transform them into induced pluripotent stem (iPS) cells without using potentially dangerous viruses to carry the reprogramming genes into the cells.

This paper marks another step toward the holy grail of reprogramming, which is to find a safe, efficient way of returning adult cells to their embryonic-like state, called pluripotency. So far, most techniques are either not efficient or require inserting genes that may make the cells unsafe for therapeutic use.

The team used so-called minicircles of DNA to reprogram the cells into pluripotency. These minicircles contain just the four genes needed to transform the cells along with a fluorescence gene that allows the cells to be tracked. The minicricles are about half the size of naturally occurring plasmid rings that have been used in some other iPS transformations, and unlike integrating viruses, the minicircles do not get replicated as the cells multiply so the extra genes are lost over time, making the cells safer for therapy.

A press release from Stanford University quoted co-author Michael Longaker saying:

“This technique is not only safer, it’s relatively simple. It will be a relatively straightforward process for labs around the world to begin using this technique. We are moving toward clinically applicable regenerative medicine.”

Another co-author, Mark Kay, developed the minicircle technology a few years ago for use in gene therapy trials. This paper provides a great example of discoveries in one field impacting another, and moving them both forward.

Nature Methods, February 7, 2010
CIRM funding: Michael Longaker (RL1-00662-1); (T1-00001)

DG,

Visual Function Rescued in Rats Using Cells derived from iPS Cells

Induced pluripotent stem (iPS) cells have created excitement and head scratching ever since they were first created a little over two years ago. The excitement arises from their creation through reprogramming adult cells by manipulating their gene function, which does not require a human embryo and could potentially give a patient personalized replacement cells. But determining just how identical they are to embryonic stem cells in function has caused much consternation.

Now, a team at UC Santa Barbara and University College London has provided some pro and con information on the functionality question. Working in a rat model for age-related macular degeneration in which defects in retinal pigmented epithelial (RPE) cells lead to death of photoreceptors, they showed that RPE cells grown from iPS cells inserted into the retina prior to photoreceptor death were able to rescue the receptors and the rats retained vision.

A press release from UCSB quoted Sherry Hikita, an author on the paper saying:

“Although much work remains to be done, we believe our results underscore the potential for stem-cell based therapies in the treatment of age-related macular degeneration.”

However, the team also saw a difference between the iPS derived RPE cells and embryonic stem cell-derived RPE cells used in earlier experiments. The ESC-derived cells survived after transplant long-term, where as the iPS-derived RPE cells suffered rejection by the immune system.  This would not occur if the cells were derived from the patient receiving the therapy, but many leaders in the field have hoped that banks of iPS cells could be developed that would be less expensive than deriving new cells for each patient. Also, these banked cells could avoid transplanting cells with the same genetic mutation that caused the problem in the first place.

In the December 3 PLoS  ONE the authors speculate:

“The embryonic origin of hESC-derived RPE may reflect a more immune privileged cell type in comparison to iPS-RPE.”

To further complicate the equation, the rats in this model retained long-term visual function despite rejection of the transplanted cells suggesting the transplanted cells induced some sort of protective response for RPE cells in the surrounding tissue.

PLOS ONE, December 3, 2010

CIRM funding: David Buchholz (T3-00009)


DG

Genetic differences found between adult cell and embryonic-derived stem cells

Researchers at the University of California, Los Angeles have found genetic differences that distinguish induced pluripotent stem (iPS) cells from embryonic stem cells. These differences diminish over time, but never disappear entirely. iPS cells are created when adult cells, such as those from the skin, are reprogrammed to look and behave like embryonic stem cells. But until now, scientists didn’t know if the two types of stem cells were actually identical at a molecular level. This latest research shows that iPS and embryonic stem cells differ in which genes they have turned on or off. All early iPS cells share these genetic traits, regardless of what animal they come from, the type of adult cells the iPS cells start as, or what method was used to reprogram those adult cells. However, later cultures of iPS cells show that most, but not all, of these differences disappear over time, making later cultures of iPS cells more similar to embryonic stem cells. If scientists want to use iPS cells in medical therapies, this research will give them a better idea of how similar they are to embryonic stem cells.

Cell Stem Cell: July 2, 2009
CIRM funding: Mike Teitell (RS1-00313), Kathrin Plath (RN1-00564-1), William Lowry (RS1-00259-1, RL1-00681-1)

Related Information: Press Release, University of California, Los Angeles