Using skin cells to repair damaged hearts

heart-muscle

Heart muscle  cells derived from skin cells

When someone has a heart attack, getting treatment quickly can mean the difference between life and death. Every minute delay in getting help means more heart cells die, and that can have profound consequences. One study found that heart attack patients who underwent surgery to re-open blocked arteries within 60 minutes of arriving in the emergency room had a six times greater survival rate than people who had to wait more than 90 minutes for the same treatment.

Clearly a quick intervention can be life-saving, which means an approach that uses a patient’s own stem cells to treat a heart attack won’t work. It simply takes too long to harvest the healthy heart cells, grow them in the lab, and re-inject them into the patient. By then the damage is done.

Now a new study shows that an off-the-shelf approach, using donor stem cells, might be the most effective way to go. Scientists at Shinshu University in Japan, used heart muscle stem cells from one monkey, to repair the damaged hearts of five other monkeys.

In the study, published in the journal Nature, the researchers took skin cells from a macaque monkey, turned those cells into induced pluripotent stem cells (iPSCs), and then turned those cells into cardiomyocytes or heart muscle cells. They then transplanted those cardiomyocytes into five other monkeys who had experienced an induced heart attack.

After 3 months the transplanted monkeys showed no signs of rejection and their hearts showed improved ability to contract, meaning they were pumping blood around the body more powerfully and efficiently than before they got the cardiomyocytes.

It’s an encouraging sign but it comes with a few caveats. One is that the monkeys used were all chosen to be as close a genetic match to the donor monkey as possible. This reduced the risk that the animals would reject the transplanted cells. But when it comes to treating people, it may not be feasible to have a wide selection of heart stem cell therapies on hand at every emergency room to make sure they are a good genetic match to the patient.

The second caveat is that all the transplanted monkeys experienced an increase in arrhythmias or irregular heartbeats. However, Yuji Shiba, one of the researchers, told the website ResearchGate that he didn’t think this was a serious issue:

“Ventricular arrhythmia was induced by the transplantation, typically within the first four weeks. However, this post-transplant arrhythmia seems to be transient and non-lethal. All five recipients of [the stem cells] survived without any abnormal behaviour for 12 weeks, even during the arrhythmia. So I think we can manage this side effect in clinic.”

Even with the caveats, this study demonstrates the potential for a donor-based stem cell therapy to treat heart attacks. This supports an approach already being tested by Capricor in a CIRM-funded clinical trial. In this trial the company is using donor cells, derived from heart stem cells, to treat patients who developed heart failure after a heart attack. In early studies the cells appear to reduce scar tissue on the heart, promote blood vessel growth and improve heart function.

The study from Japan shows the possibilities of using a ready-made stem cell approach to helping repair damage caused by a heart attacks. We’re hoping Capricor will take it from a possibility, and turn it into a reality.

If you would like to read some recent blog posts about Capricor go here and here.

Stem cell stories that caught our eye: reality check on chimeras, iPS cells for drug discovery and cell family history

Here are some stem cell stories that caught our eye this past week. Some are groundbreaking science, others are of personal interest to us, and still others are just fun.

iPS cells becoming foot soldiers of drug discovery. Here at The Stem Cellar we write often about the power of iPS-type stem cells to model disease and accelerate drug development. This week provided a couple of strong reminders of the value of these induced pluripotent stem cells that researchers create by reprogramming any adult cell, usually skin or blood, into an embryonic stem cell-like state.

Researchers at Penn State University published work that used iPS cells from patients with Rett Syndrome to find a target for drug therapy for that severe form of autism spectrum disorder. After turning the stem cells into nerves they found those cells lacked a protein that is critical to the function of the neural transmitter GABA. That protein has now become a target for drug therapy. As a bonus for the field, the study, published in the Proceedings of the National Academy of Sciences, provided an explanation for why a drug already in clinical trials for Rett Syndrome might work. That drug is IGF1, insulin-like growth factor. The web site Medical News Today wrote up the research.

Later in the week an announcement popped up in my email for the two-day “inaugural” conference “Advances in iPS cell Technology for Drug Development Applications.” The field clearly has momentum. CIRM has funded a bank that will eventually house up to 3,000 cell lines relating to specific diseases. So far, 285 lines are available to researchers anywhere, 14 of them Autism spectrum lines, through the tissue banks at Coriell.

 

Tracking a cell’s family history. When cells divide their offspring can have a different identity from the mother cells. This occurs commonly in stem cells, as they mature into adult tissue, and in the immune system as cells respond to infections. Knowing the genetic details of how this happens could accelerate both stem cell science and our ability to understand and manipulate the immune system.

A team at MIT has taken us a step closer to this ability. They married a trendy new technique called single cell genetic analysis with a fluidic device that can isolate single cells in one chamber and daughter and grand daughter cells in subsequent chambers. In this case, they used single cell RNA-seq, the shorthand for sequencing. They wanted to know the differences between the cells in terms of genes that are actually active, and since the RNA representing a gene is only made when the gene is active, this provided a snapshot of each cell’s genetic identity.

Genetic Engineering News wrote about the work and quoted the lead author of the study Robert Kimmerling:

“Scientists have well-established methods for resolving diverse subsets of a population, but one thing that’s not very well worked out is how this diversity is generated. That’s the key question we were targeting: how a single founding cell gives rise to very diverse progeny.”

This new combined system should let researchers investigate how this happens. The MIT team started by looking at how one immune system cell can produce both the cells that attack and kill invaders and the cells that stick around and remember what the invaders looked like.

 

Human-animal chimeras, what are labs really doing. Antonio Regalado did a thorough piece in MIT Technology Review examining the work of the few labs around the country that are trying to grow human tissue in animal embryos—chimeras. He estimates that some 20 pig-human or sheep-human pregnancies have been established, but no one is letting those embryos grow more than a very few weeks. Their immediate goal is to better understand how the cells with different origins interact, not to breed chimeric animals.

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A pig at the UC Davis research center

One long-term goal is, for example, to grow a personalized new pancreas for diabetic patients who needs a new one of those insulin-producing organs. But no one in the field expects that to happen anytime soon. The process involves using modern genetic editing techniques to turn off the genes that would make a particular organ in the animal embryo, inserting human stem cells and hoping the growing embryo will hijack the genes for making the equivalent human organ, but not other human tissues.

The embryos examined so far have generally contained a very small amount of human DNA, less than one percent in a project at Stanford. So, probably not enough to give the animal human traits beyond the organ desired. Pablo Ross who has done some of the early work at the University of California, Davis explained the intent of those studies is “to determine the ideal conditions for generating human-animal chimeras.”

It is fascinating work and has great potential to alleviate organ shortages, but will require several more breakthroughs and much patience before that happens.

CIRM-funded team traces molecular basis for differences between human and chimp face

So similar yet so different
Whenever I go to the zoo, I could easily spend my entire visit hanging out with our not-so-distant relatives, the chimpanzees. To say we humans are similar to them is quite an understatement. Sharing 96% of our DNA, chimps are more closely related to us than they are to gorillas. And when you just compare our genes – that is, the segments of DNA that contain instructions for making proteins – we’re even more indistinguishable.

Chimps and Humans: So similar yet so different

Chimps and Humans: So similar yet so different

And yet you wouldn’t mistake a human for a chimp. I mean, I do have hairy arms, but they’re not that hairy. So what accounts for our very different appearance if our genes are so similar?

To seek out answers, a CIRM-funded team at Stanford University used both human and chimp induced pluripotent stem cells (iPSCs) to derive cranial neural crest cells (CNCCs). This cell type plays a key role in shaping the overall structure of the face during the early stages of embryo development. In a report published late last week in Cell, the team found differences, not in the genes themselves, but in gene activity between the human and chimp CNCCs.

Enhancers: Volume controls for your genes
Pinpointing the differences in gene activity relied on a comparative analysis of so-called enhancer regions of human and chimp DNA. Unlike genes, the enhancer regions of DNA do not provide instructions for making proteins. Instead they dictate how much protein to make by acting like volume control knobs for specific genes. A particular volume level, or gene activity, is determined by specific combinations of chemical tags and DNA-binding proteins on an enhancer region of DNA.

Enhancers: DNA segments that act like a volume control know for gene activity (Image source: xxxx)

Enhancers: DNA segments that act like a volume control knobs for gene activity (Image source: FANTOM Project, University of Copenhagen)

The researchers used several sophisticated lab techniques to capture a snapshot of this enhancer tagging and binding in the CNCCSs. They mostly saw similarities between human and chimp enhancers but, as senior author Joanna Wysocka explains in a Stanford University press release, they did uncover some differences:

“In particular, we found about 1,000 enhancer regions that are what we termed species-biased, meaning they are more active in one species or the other. Interestingly, many of the genes with species-biased enhancers and expression have been previously shown to be important in craniofacial development.”

PAX Humana: A genetic basis for our smaller jawline and snout?
For example, their analysis revealed that the genes PAX3 and PAX7 are associated with chimp-biased enhancer regions, and they had higher levels of activity in chimp CNCCs. These results get really intriguing once you learn a bit more about the PAX genes: other studies in mice have shown that mutations interfering with PAX function lead to mice with smaller, lower jawbones and snouts. So the lower level of PAX3/PAX7 gene activity in humans would appear to correlate with our smaller jaws and snout (mouth and nose) compared to chimps. Did that just blow your mind? How about this:

The researchers also found a variation in the enhancer region for the gene BMP4. But in this case, BMP4 was highly related to human-biased enhancer regions and had higher activity in humans compared to chimps. Previous mouse studies have shown that forcing higher levels of BMP4 specifically in CNCCs leads to shorter lower and upper jawbones, rounder skulls, and eyes positioned more to the front of the face. These changes caused by BMP4 sound an awful lot like the differences in human and chimp facial structures. It appears the Stanford group has established a terrific strategy for tracing the genetic basis for differences in humans and chimps.

So what’s next? According to Wysocka, the team is digging deeper into their data:

“We are now following up on some of these more interesting species-biased enhancers to better understand how they impact morphological differences. It’s becoming clear that these cellular pathways can be used in many ways to affect facial shape.”

And in the bigger picture, the researchers also suggest that this “cellular anthropology” approach could also be applied to a human to human search for DNA enhancer regions that play a role in the variation between healthy and disease states.

Study Identifies Safer Stem Cell Therapies

To reject or not reject, that is the question facing the human immune system when new tissue or cells are transplanted into the body.

Stem cell-therapy promises hope for many debilitating diseases that currently have no cures. However, the issue of immune rejection has prompted scientists to carefully consider how to develop safe stem cell therapies that will be tolerated by the human immune system.

Before the dawn of induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs) were suggested as a potential source for transplantable cells and tissue. However, ESCs run into a couple of issues, including their origin, and the fact that ESC-derived cells likely would be rejected when transplanted into most areas of a human due to differences in genetic backgrounds.

The discovery of iPSCs in the early 2000’s gave new hope to the field of stem cell therapy. By generating donor cells and tissue from a patient’s own iPSCs, transplanting those cells/tissue back into the same individual shouldn’t – at least theoretically – cause an immune reaction. This type of transplantation is called “autologous” meaning that the stem cell-derived cells have the same genetic background as the person.

Unfortunately, scientists have run up against a roadblock in iPSC-derived stem cell therapy. They discovered that even cells derived from a patient’s own iPSCs can cause an immune reaction when transplanted into that patient. The answers as to why this occurs remained largely unanswered until recently.

In a paper published last week in Cell Stem Cell, scientists from the University of California, San Diego (UCSD) reported that different mature cell types derived from human iPSCs have varying immunogenic effects (the ability to cause an immune reaction) when transplanted into “humanized” mice that have a human immune system. This study along with the research conducted to generate the humanized mice was funded by CIRM grants (here, here).

In this study, retinal pigment epithelial cells (RPE) and skeletal muscle cells (SMC) derived from human iPSCs were transplanted into humanized mice. RPEs were tolerated by the immune system while SMCs were rejected. (Adapted from Zhao et al. 2015)

Scientists took normal mice and replaced their immune system with a human one. They then took human iPSCs generated from the same human tissue used to generate the humanized mice and transplanted different cell types derived from the iPSCs cells into these mice.

Because they were introducing cells derived from the same source of human tissue that the mouse’s immune system was derived from, in theory, the mice should not reject the transplant. However, they found that many of the transplants did indeed cause an immune reaction.

Interestingly, they found that certain mature cell types derived from human iPSCs created a substantial immune reaction while other cell types did not. The authors focused on two specific cell types, smooth muscle cells (SMC) and retinal pigment epithelial cells (RPE), to get a closer look at what was going on.

iPSC-derived smooth muscle cells created a large immune response when transplanted into humanized mice. However, when they transplanted iPSC-derived retinal epithelial cells (found in the retina of the eye), they didn’t see the same immune reaction. As a control, they transplanted RPE cells made from human ESCs, and as expected, they saw an immune response to the foreign ESC-derived RPE cells.

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iPSC derived RPE cells (green) do not cause an immune reaction (red) after transplantation into humanized mice while H9 embryonic stem cell derived RPE cells do. (Zhao et al. 2015)

When they looked further to determine why the humanized mice rejected the muscle cells but accepted the retinal cells, they found that SMCs had a different gene expression profile and higher expression of immunogenic molecules. The iPSC-derived RPE cells had low expression of these same immunogenic molecules, which is why they were well tolerated in the humanized mice.

Results from this study suggest that some cell types generated from human iPSCs are safer for transplantation than others, an issue which can be addressed by improving the differentiation techniques used to produce mature cells from iPSCs. This study also suggests that iPSC-derived RPE cells could be a safe and promising stem cell therapy for the treatment of eye disorders such as age-related macular degeneration (AMD). AMD is a degenerative eye disease that can cause vision impairment or blindness and usually affects older people over the age of 50. Currently there is no treatment for AMD, a disease that affects approximately 50 million people around the world. (However there is a human iPSC clinical trial for AMD out of the RIKEN Center for Developmental Biology in Japan that has treated one patient but is currently on hold due to safety issues.)

The senior author on this study, Dr. Yang Xu, commented on the importance of this study in relation to AMD in a UCSD press release:

Dr. Yang Xu

Dr. Yang Xu

Immune rejection is a major challenge for stem cell therapy. Our finding of the lack of immune rejection of human iPSC-derived retinal pigment epithelium cells supports the feasibility of using these cells for treating macular degeneration. However, the inflammatory environment associated with macular degeneration could be an additional hurdle to be overcome for the stem cell therapy to be successful.

Xu makes an important point by acknowledging that iPSC-derived RPE cells aren’t a sure bet for curing AMD just yet. More research needs to be done to address other issues that occur during AMD in order for this type of stem cell therapy to be successful.

 


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More than Meets the Eye: Stem Cells Generated using Different Methods Produce Different Types of Cells

What’s the best way to make a fully versatile, ‘pluripotent,’ stem cell? Three different methods each have their pluses and minuses. But now new research has found that the stem cells created by each method, while similar on the surface, show vast differences.

The findings, published online today in the journal Nature, reveal new insights into stem cells’ underlying cellular machinery—which is of utmost importance as researchers transform their discoveries from the lab and into much-needed therapies for patients.

Scanning electron micrograph of cultured human neuron from induced pluripotent stem cell.  [Credit: Mark Ellisman and Thomas Deerinck, National Center for Microscopy and Imaging Research, UC San Diego]

Scanning electron micrograph of cultured human neuron from induced pluripotent stem cell. [Credit: Mark Ellisman and Thomas Deerinck, National Center for Microscopy and Imaging Research, UC San Diego]

Stem cells have held promise for regenerating tissues, or even organs, lost or damaged by injury or disease. This is due to stem cells’ ‘pluripotency’—their ability to transform into virtually any cell in the body. Initially, scientists used stem cells extracted from unused embryos that consenting couples had donated to research. But the use of these so-called embryonic stem cells, or ES cells, has since been limited due to ethical considerations and early limits to federal funding.

So scientists have been on the hunt for an alternative method of creating pluripotent cells. And so far, they have come up with two.

One, called somatic cell nuclear transfer (SCNT) takes the genetic material of an adult cell and transplants it into an unfertilized egg. The second method transforms adult cells, such as skin or blood, back into embryonic-like stem cells—called induced pluripotent stem cells, or iPS cells—by manipulating various genes.

Each of the newer methods has its pluses and minuses—but which produces cells that most closely resemble ES cells, still considered the “Gold Standard” in stem cell biology? Since the success of the SCNT technique is so recent, no one had taken a close look until now. So a collaboration of researchers from the University of California, San Diego (UCSD), The Salk Institute for Biological Sciences and Oregon Health & Science University (OHSU), compared the two methods side by side. And what they found was surprising.

Dr. Louise Laurent, co-senior author from UCSD, explained in today’s news release:

“The nuclear transfer ES cells are much more similar to real ES cells than the iPS cells. They are more completely reprogrammed and have fewer alterations in gene expression and DNA methylation levels that are attributable to the reprogramming process itself.”

iPS cell technology, which was pioneered in 2006 by Shinya Yamanaka, offers a series of advantages over traditional ES cells. As Laurent continued:

“The ability to make personalized iPS cells from a patient that could be transplanted back into that patient has generated excitement because it would eliminate the need for immunosuppression.”

iPS cells have generated so much excitement, in fact, that Yamanaka was awarded the 2012 Nobel Prize in Physiology or Medicine for developing this technique.

The SCNT method was developed more recently by OHSU’s Dr. Shoukhrat Mitalipov. The current researchers generated lines of cells using both methods. After confirming that each line was, in fact, pluripotent, they used advanced genomics techniques to examine the biochemical process called ‘DNA methylation’ in each line.

DNA methylation is a fundamental chemical process within each cell. It’s responsible for switching key genes on and off at precise intervals. In recent years, researchers have discovered that the order and timing of this process is vital for the correct development of the cell. As Dr. Joseph Ecker, co-senior author from the Salk Institute, explained:

“If you believe that gene expression and DNA methylation are important, which we do, the closer you get to the patterns of embryonic cells, the better. Right now, nuclear transfer cells look closer to the embryonic stem cells than do the iPS cells.”

However, while the scientists confirmed that SCNT cells more closely resemble ES cells, the process of producing them is far from ideal. First, the SCNT method is technically difficult. And second, federal funds still cannot be used in this procedure—representing a significant hurdle to being widely adopted.

On the other hand, iPS cell generation is, by comparison, a much easier process technically. So perhaps these findings can spur the development of an improved method, taking the technological ease of iPS cell generation and marrying it with the accuracy of the SCNT method. Laurent argues that this could yield a new and improved approach:

“Our results have shown that widely used iPS cell reprogramming methods make cells that are similar to standard ES cells in broad strokes, but there are important differences when you look really closely. By using the egg cell to do the job, we can get much closer to the real thing. If we can figure out what factors in the egg drive the reprogramming process, maybe we can design a better iPS cell reprogramming method.”