Stem cell stories that caught our eye: brains, brains and more brains!

This week we bring you three separate stories about the brain. Two are exciting new advances that use stem cells to understand the brain and the third is plain creepy.

Bioengineering better brains. Lab grown mini-brains got an upgrade thanks to a study published this week in Nature Biotechnology. Mini-brains are tiny 3D organs that harbor similar cell types and structures found in the human brain. They are made from pluripotent stem cells cultured in laboratory bioreactors that allow these cells to mature into brain tissue in the span of a month.

The brain organoid technology was first published back in 2013 by Austrian scientists Jürgen Knoblich and Madeline Lancaster. They used mini-brains to study human brain development and a model a birth defect called microcephaly, which causes abnormally small heads in babies. Mini-brains filled a void for scientists desperate for better, more relevant models of human brain development. But the technology had issues with consistency and produced organoids that varied in size, structure and cell type.

Cross-section of a mini-brain. (Madeline Lancaster/MRC-LMB)

Fast forward four years and the same team of scientists has improved upon their original method by adding a bioengineering technique that will generate more consistent mini-brains. Instead of relying on the stem cells to organize themselves into the proper structures in the brain, the team developed a biological scaffold made of microfilaments that guides the growth and development of stem cells into organoids. They called these “engineered cerebral organoids” or enCORs for short.

In a news feature on IMBA, Jürgen Knoblich explained that enCORs are more reproducible and representative of the brain’s architecture, thus making them more effective models for neurological and neurodevelopmental disorders.

“An important hallmark of the bioengineered organoids is their increased surface to volume ratio. Because of their improved tissue architecture, enCORs can allow for the study of a broader array of neurological diseases where neuronal positioning is thought to be affected, including lissencephaly (smooth brain), epilepsy, and even autism and schizophrenia.”

Salk team finds genetic links between brain’s immune cells and neurological disorders. (Todd Dubnicoff)

Dysfunction of brain cells called microglia have been implicated in a wide range of neurologic disorders like Alzheimer’s, Parkinson’s, Huntington’s, autism and schizophrenia. But a detailed examination of these cells has proved difficult because they don’t grow well in lab dishes. And attempts to grow microglia from stem cells is hampered by the fact that the cell type hasn’t been characterized enough for researchers to know how to distinguish it from related cell types found in the blood.

By performing an extensive analysis of microglia gene activity, Salk Institute scientists have now pinpointed genetic links between these cells and neurological disease. These discoveries also demonstrate the importance of the microglia’s environment within the brain to maintain its identity. The study results were reported in Science.

Microglia are important immune cells in the brain. They are related to macrophages which are white blood cells that roam through the body via the circulatory system and gobble up damaged or dying cells as well as foreign invaders. Microglia also perform those duties in the brain and use their eating function to trim away faulty or damage nerve connections.

To study a direct source of microglia, the team worked with neurosurgeons to obtain small samples of brain tissue from patients undergoing surgery for epilepsy, a tumor or stroke. Microglia were isolated from healthy regions of brain tissue that were incidentally removed along with damaged or diseased brain tissue.

Salk and UC San Diego scientists conducted a vast survey of microglia (pictured here), revealing links to neurodegenerative diseases and psychiatric illnesses. (Image: Nicole Coufal)

A portion of the isolated microglia were immediately processed to take a snap shot of gene activity. The researchers found that hundreds of genes in the microglia had much higher activities compared to those same genes in macrophages. But when the microglia were transferred to petri dishes, gene activity in general dropped. In fact, within six hours of tissue collection, the activity of over 2000 genes in the cells had dropped significantly. This result suggests the microglial rely on signals in the brain to stimulate their gene activity and may explain why they don’t grow well once removed from that environment into lab dishes.

Of the hundreds of genes whose activity were boosted in microglia, the researchers tracked down several that were linked to several neurological disorders. Dr. Nicole Coufal summarized these results and their implications in a Salk press release:

“A really high proportion of genes linked to multiple sclerosis, Parkinson’s and schizophrenia are much more highly expressed in microglia than the rest of the brain. That suggests there’s some kind of link between microglia and the diseases.”

Future studies are needed to explain the exact nature of this link. But with these molecular descriptions of microglia gene activity now in hand, the researchers are in a better position to study microglia’s role in disease.

A stem cell trial to bring back the dead, brain-dead that is. A somewhat creepy stem cell story resurfaced in the news this week. A company called Bioquark in Philadelphia is attempting to bring brain-dead patients back to life by injecting adult stem cells into their spinal cords in combination with other treatments that include protein blend injections, electrical nerve stimulation and laser therapy. The hope is that this combination stem cell therapy will generate new neurons that can reestablish lost connections in the brain and bring it back to life.

Abstract image of a neuron. (Dom Smith/STAT)

You might wonder why the company is trying multiple different treatments simultaneously. In a conversation with STAT news, Bioquark CEO Ira Pastor explained,

“It’s our contention that there’s no single magic bullet for this, so to start with a single magic bullet makes no sense. Hence why we have to take a different approach.”

Bioquark is planning to relaunch a clinical trial testing its combination therapy in Latin America sometime this year. The company previously attempted to launch its first trial in India back in April of 2016, but it never got off the ground because it failed to get clearance from India’s Drug Controller General.

STATnews staff writer Kate Sheridan called the trial “controversial” and raised questions about how it would impact patients and their families.

“How do researchers complete trial paperwork when the person participating is, legally, dead? If the person did regain brain activity, what kind of functional abilities would he or she have? Are families getting their hopes up for an incredibly long-shot cure?”

Scientists also have questions mainly about whether this treatment will actually work or is just a shot in the dark. Adding to the uncertainty is the fact that Bioquark has no preclinical evidence that its combination treatment is effective in animal models. The STAT piece details how the treatments have been tested individually for other conditions such as stroke and coma, but not in brain-dead patients. To further complicate things, there is no consensus on how to define brain death in patients, so patient improvements observed during the trial could be unrelated to the treatment.

STAT asked expert doctors in the field whether Bioquark’s strategy was feasible. Orthopedic surgeon Dr. Ed Cooper said that there’s no way electric stimulation would work, pointing out that the technique requires a functioning brain stem which brain-dead patients don’t have. Pediatric surgeon Dr. Charles Cox, who works on a stem cell treatment for traumatic brain injury and is unrelated to Bioquark, commented, “it’s not the absolute craziest thing I’ve ever heard, but I think the probability of that working is next to zero.”

But Pastor seems immune to the skepticism and naysayers.

“I give us a pretty good chance. I just think it’s a matter of putting it all together and getting the right people and the right minds on it.”

Stem cell stories that caught our eye: developing the nervous system, aging stem cells and identical twins not so identical

Here are the stem cell stories that caught our eye this week. Enjoy!

New theory for how the nervous system develops.

There’s a new theory on the block for how the nervous system is formed thanks to a study published yesterday by UCLA stem cell scientists in the journal Neuron.

The theory centers around axons, thin extensions projecting from nerve cells that transmit electrical signals to other cells in the body. In the developing nervous system, nerve cells extend axons into the brain and spinal cord and into our muscles (a process called innervation). Axons are guided to their final destinations by different chemicals that tell axons when to grow, when to not grow, and where to go.

Previously, scientists believed that one of these important chemical signals, a protein called netrin 1, exerted its influence over long distances in a gradient-like fashion from a structure in the developing nervous system called the floor plate. You can think of it like a like a cell phone tower where the signal is strongest the closer you are to the tower but you can still get some signal even when you’re miles away.

The UCLA team, led by senior author and UCLA professor Dr. Samantha Butler, questioned this theory because they knew that neural progenitor cells, which are the precursors to nerve cells, produce netrin1 in the developing spinal cord. They believed that the netrin1 secreted from these progenitor cells also played a role in guiding axon growth in a localized manner.

To test their hypothesis, they studied neural progenitor cells in the developing spines of mouse embryos. When they eliminated netrin1 from the neural progenitor cells, the axons went haywire and there was no rhyme or reason to their growth patterns.

Left: axons (green, pink, blue) form organized patterns in the normal developing mouse spinal cord. Right: removing netrin1 results in highly disorganized axon growth. (UCLA Broad Stem Cell Research Center/Neuron)

A UCLA press release explained what the scientists discovered next,

“They found that neural progenitors organize axon growth by producing a pathway of netrin1 that directs axons only in their local environment and not over long distances. This pathway of netrin1 acts as a sticky surface that encourages axon growth in the directions that form a normal, functioning nervous system.”

Like how ants leave chemical trails for other ants in their colony to follow, neural progenitor cells leave trails of netrin1 in the spinal cord to direct where axons go. The UCLA team believes they can leverage this newfound knowledge about netrin1 to make more effective treatments for patients with nerve damage or severed nerves.

In future studies, the team will tease apart the finer details of how netrin1 impacts axon growth and how it can be potentially translated into the clinic as a new therapeutic for patients. And from the sounds of it, they already have an idea in mind:

“One promising approach is to implant artificial nerve channels into a person with a nerve injury to give regenerating axons a conduit to grow through. Coating such nerve channels with netrin1 could further encourage axon regrowth.”

Age could be written in our stem cells.

The Harvard Gazette is running an interesting series on how Harvard scientists are tackling issues of aging with research. This week, their story focused on stem cells and how they’re partly to blame for aging in humans.

Stem cells are well known for their regenerative properties. Adult stem cells can rejuvenate tissues and organs as we age and in response to damage or injury. However, like most house hold appliances, adult stem cells lose their regenerative abilities or effectiveness over time.

Dr. David Scadden, co-director of the Harvard Stem Cell Institute, explained,

“We do think that stem cells are a key player in at least some of the manifestations of age. The hypothesis is that stem cell function deteriorates with age, driving events we know occur with aging, like our limited ability to fully repair or regenerate healthy tissue following injury.”

Harvard scientists have evidence suggesting that certain tissues, such as nerve cells in the brain, age sooner than others, and they trigger other tissues to start the aging process in a domino-like effect. Instead of treating each tissue individually, the scientists believe that targeting these early-onset tissues and the stem cells within them is a better anti-aging strategy.

David Sadden, co-director of the Harvard Stem Cell Institute.
(Jon Chase/Harvard Staff Photographer)

Dr. Scadden is particularly interested in studying adult stem cell populations in aging tissues and has found that “instead of armies of similarly plastic stem cells, it appears there is diversity within populations, with different stem cells having different capabilities.”

If you lose the stem cell that’s the best at regenerating, that tissue might age more rapidly.  Dr. Scadden compares it to a game of chess, “If we’re graced and happen to have a queen and couple of bishops, we’re doing OK. But if we are left with pawns, we may lose resilience as we age.”

The Harvard Gazette piece also touches on a changing mindset around the potential of stem cells. When stem cell research took off two decades ago, scientists believed stem cells would grow replacement organs. But those days are still far off. In the immediate future, the potential of stem cells seems to be in disease modeling and drug screening.

“Much of stem cell medicine is ultimately going to be ‘medicine,’” Scadden said. “Even here, we thought stem cells would provide mostly replacement parts.  I think that’s clearly changed very dramatically. Now we think of them as contributing to our ability to make disease models for drug discovery.”

I encourage you to read the full feature as I only mentioned a few of the highlights. It’s a nice overview of the current state of aging research and how stem cells play an important role in understanding the biology of aging and in developing treatments for diseases of aging.

Identical twins not so identical (Todd Dubnicoff)

Ever since Takahashi and Yamanaka showed that adult cells could be reprogrammed into an embryonic stem cell-like state, researchers have been wrestling with a key question: exactly how alike are these induced pluripotent stem cells (iPSCs) to embryonic stem cells (ESCs)?

It’s an important question to settle because iPSCs have several advantages over ESCs. Unlike ESCs, iPSCs don’t require the destruction of an embryo so they’re mostly free from ethical concerns. And because they can be derived from a patient’s cells, if iPSC-derived cell therapies were given back to the same patient, they should be less likely to cause immune rejection. Despite these advantages, the fact that iPSCs are artificially generated by the forced activation of specific genes create lingering concerns that for treatments in humans, delivering iPSC-derived therapies may not be as safe as their ESC counterparts.

Careful comparisons of DNA between iPSCs and ESCs have shown that they are indeed differences in chemical tags found on specific spots on the cell’s DNA. These tags, called epigenetic (“epi”, meaning “in addition”) modifications can affect the activity of genes independent of the underlying genetic sequence. These variations in epigenetic tags also show up when you compare two different preparations, or cell lines, of iPSCs. So, it’s been difficult for researchers to tease out the source of these differences. Are these differences due to the small variations in DNA sequence that are naturally seen from one cell line to the other? Or is there some non-genetic reason for the differences in the iPSCs’ epigenetic modifications?

Marian and Vivian Brown, were San Francisco’s most famous identical twins. Photo: Christopher Michel

A recent CIRM-funded study by a Salk Institute team took a clever approach to tackle this question. They compared epigenetic modifications between iPSCs derived from three sets of identical twins. They still found several epigenetic variations between each set of twins. And since the twins have identical DNA sequences, the researchers could conclude that not all differences seen between iPSC cell lines are due to genetics. Athanasia Panopoulos, a co-first author on the Cell Stem Cell article, summed up the results in a press release:

“In the past, researchers had found lots of sites with variations in methylation status [specific term for the epigenetic tag], but it was hard to figure out which of those sites had variation due to genetics. Here, we could focus more specifically on the sites we know have nothing to do with genetics. The twins enabled us to ask questions we couldn’t ask before. You’re able to see what happens when you reprogram cells with identical genomes but divergent epigenomes, and figure out what is happening because of genetics, and what is happening due to other mechanisms.”

With these new insights in hand, the researchers will have a better handle on interpreting differences between individual iPSC cell lines as well as their differences with ESC cell lines. This knowledge will be important for understanding how these variations may affect the development of future iPSC-based cell therapies.

Stem Cell Stories That Caught Our Eye: Plasticity in the pancreas and two cool stem cell tools added to the research toolbox

There’s more plasticity in the pancreas than we thought. You’re taught a lot of things about the world when you’re young. As you get older, you realize that not everything you’re told holds true and it’s your own responsibility to determine fact from fiction. This evolution in understanding happens in science too. Scientists do research that leads them to believe that biological processes happen a certain way, only to sometimes find, a few years later, that things are different or not exactly what they had originally thought.

There’s a great example of this in a study published this week in Cell Metabolism about the pancreas. Scientists from UC Davis found that the pancreas, which secretes a hormone called insulin that helps regulate the levels of sugar in your blood, has more “plasticity” than was originally believed. In this case, plasticity refers to the ability of a tissue or organ to regenerate itself by replacing lost or damaged cells.

The long-standing belief in this field was that the insulin producing cells, called beta cells, are replenished when beta cells actively divide to create more copies of themselves. In patients with type 1 diabetes, these cells are specifically targeted and killed off by the immune system. As a result, the beta cell population is dramatically reduced, and patients have to go on life-long insulin treatment.

UC Davis researchers have identified another type of insulin-producing cell in the islets, which appears to be an immature beta cell shown in red. (UC Davis)

But it turns out there is another cell type in the pancreas that is capable of making beta cells and they look like a teenage, less mature version of beta cells. The UC Davis team identified these cells in mice and in samples of human pancreas tissue. These cells hangout at the edges of structures called islets, which are clusters of beta cells within the pancreas. Upon further inspection, the scientists found that these immature beta cells can secrete insulin but cannot detect blood glucose like mature beta cells. They also found their point of origin: the immature beta cells developed from another type of pancreatic cell called the alpha cell.

Diagram of immature beta cells from Cell Metabolism.

In coverage by EurekAlert, Dr Andrew Rakeman, the director of discovery research at the Juvenile Diabetes Research Foundation, commented on the importance of this study’s findings and how it could be translated into a new approach for treating type 1 diabetes patients:

“The concept of harnessing the plasticity in the islet to regenerate beta cells has emerged as an intriguing possibility in recent years. The work from Dr. Huising and his team is showing us not only the degree of plasticity in islet cells, but the paths these cells take when changing identity. Adding to that the observations that the same processes appear to be occurring in human islets raises the possibility that these mechanistic insights may be able to be turned into therapeutic approaches for treating diabetes.”

 

Say hello to iPSCORE, new and improved tools for stem cell research. Stem cells are powerful tools to model human disease and their power got a significant boost this week from a new study published in Stem Cell Reports, led by scientists at UC San Diego School of Medicine.

The team developed a collection of over 200 induced pluripotent stem cell (iPS cell) lines derived from people of diverse ethnic backgrounds. They call this stem cell tool kit “iPSCORE”, which stands for iPSC Collection for Omic Research (omics refers to a field of study in biology ending in -omics, such as genomics or proteomics). The goal of iPSCORE is to identify particular genetic variants (unique differences in DNA sequence between people’s genomes) that are associated with specific diseases and to understand why they cause disease at the molecular level.

In an interview with Phys.org, lead scientist on the study, Dr. Kelly Frazer, further explained the power of iPSCORE:

“The iPSCORE collection contains 75 lines from people of non-European ancestry, including East Asian, South Asian, African American, Mexican American, and Multiracial. It includes multigenerational families and monozygotic twins. This collection will enable us to study how genetic variation influences traits, both at a molecular and physiological level, in appropriate human cell types, such as heart muscle cells. It will help researchers investigate not only common but also rare, and even family-specific variations.”

This research is a great example of scientists identifying a limitation in stem cell research and expanding the stem cell tool kit to model diseases in a diverse human population.

A false color scanning electron micrograph of cultured human neuron from induced pluripotent stem cell. Credit: Mark Ellisman and Thomas Deerinck, UC San Diego.

Stem cells that can grow into ANY type of tissue. Embryonic stem cells can develop into any cell type in the body, earning them the classification of pluripotent. But there is one type of tissue that embryonic stem cells can’t make and it’s called extra-embryonic tissue. This tissue forms the supportive tissue like the placenta that allows an embryo to develop into a healthy baby in the womb.

Stem cells that can develop into both extra-embryonic and embryonic tissue are called totipotent, and they are extremely hard to isolate and study in the lab because scientists lack the methods to maintain them in their totipotent state. Having the ability to study these special stem cells will allow scientists to answer questions about early embryonic development and fertility issues in women.

Reporting this week in the journal Cell, scientists from the Salk Institute in San Diego and Peking University in China identified a cocktail of chemicals that can stabilize human stem cells in a totipotent state where they can give rise to either tissue type. They called these more primitive stem cells extended pluripotent stem cells or EPS cells.

Salk Professor Juan Carlos Izpisua Bemonte, co–senior author of the paper, explained the problem their study addressed and the solution it revealed in a Salk news release:

“During embryonic development, both the fertilized egg and its initial cells are considered totipotent, as they can give rise to all embryonic and extra-embryonic lineages. However, the capture of stem cells with such developmental potential in vitro has been a major challenge in stem cell biology. This is the first study reporting the derivation of a stable stem cell type that shows totipotent-like bi-developmental potential towards both embryonic and extra-embryonic lineages.”

Human EPS cells (green) can be detected in both the embryonic part (left) and extra-embryonic parts (placenta and yolk sac, right) of a mouse embryo. (Salk Institute)

Using this new method, the scientists discovered that human EPS stem cells were able to develop chimeric embryos with mouse stem cells more easily than regular embryonic stem cells. First author on the study, Jun Wu, explained why this ability is important:

“The superior chimeric competency of both human and mouse EPS cells is advantageous in applications such as the generation of transgenic animal models and the production of replacement organs. We are now testing to see whether human EPS cells are more efficient in chimeric contribution to pigs, whose organ size and physiology are closer to humans.”

The Salk team reported on advancements in generating interspecies chimeras earlier this year. In one study, they were able to grow rat organs – including the pancreas, heart and eyes – in a mouse. In another study, they grew human tissue in early-stage pig and cattle embryos with the goal of eventually developing ways to generate transplantable organs for humans. You can read more about their research in this Salk news release.

Rhythmic brain circuits built from stem cells

The TV commercial is nearly 20 years old but I remember it vividly: a couple is driving down a street when they suddenly realize the music on their tape deck is in sync with the repetitive activity on the street. From the guy casually dribbling a basketball to people walking along the sidewalk to the delivery people passing packages out of their truck, everything and everyone is moving rhythmically to the beat.

The ending tag line was, “Sometimes things just come together,” which is quite true. Many of our basic daily activities like breathing and walking just come together as a result of repetitive movement. It’s easy to take them for granted but those rhythmic patterns ultimately rely on very intricate, interconnected signals between nerve cells, also called neurons, in the brain and spinal cord.

Circuitoids: a neural network in a lab dish

A CIRM-funded study published yesterday in eLife by Salk Institute scientists reports on a method to mimic these repetitive signals in a lab dish using neurons grown from embryonic stem cells. This novel cell circuitry system gives the researchers a tool for gaining new insights into neurodegenerative diseases, like Parkinson’s and ALS, and may even provide a means to fix neurons damaged by injury or disease.

The researchers changed or specialized mouse embryonic stem cells into neurons that either stimulate nerve signals, called excitatory neurons, or neurons that block nerve signals, called inhibitory neurons. Growing these groups of cells together led to spontaneous rhythmic nerve signals. These clumps of cells containing about 50,000 neurons each were dubbed circuitoids by the team.

pfaff-circutoid-cropped

Confocal microscope immunofluorescent image of a spinal cord neural circuit made entirely from stem cells and termed a “circuitoid.” Credit: Salk Institute.

Making neural networks dance to a different beat

A video produced by the Salk Institute (see below), shows some fascinating microscopy visualizations of these circuitoids’ repetitive signals. In the video, team leader Samuel Pfaff explains that changing the ratio of excitatory vs inhibitory neurons had noticeable effects on the rhythm of the nerve impulses:

“What we were able to do is combine different ratios of cell types and study properties of the rhythmicity of the circuitoid. And that rhythmicity could be very tightly control depending on the cellular composition of the neural networks that we were forming. So we could regulate the speed [of the rhythmicity] which is kind of equivalent to how fast you’re walking.”

It’s possible that the actual neural networks in our brains have the flexibility to vary the ratio of the active excitatory to inhibitory neurons as a way to allow adjustments in the body’s repetitive movements. But the complexity of those networks in the human brain are staggering which is why these circuitoids could help:

Samuel Pfaff. (Salk Institute)

Samuel Pfaff. (Salk Institute)

“It’s still very difficult to contemplate how large groups of neurons with literally billions if not trillions of connections take information and process it,” says Pfaff in a press release. “But we think that developing this kind of simple circuitry in a dish will allow us to extract some of the principles of how real brain circuits operate. With that basic information maybe we can begin to understand how things go awry in disease.”

Growing a rat pancreas in a mouse with stem cells & CRISPR: a solution for the organ shortage crisis?

Right now, about 120,000 Americans are on a waiting list for an organ transplant and 22 will die today before any organs become available. The plain truth is there aren’t enough organ donors to meet the demand. And according to the U.S. Department of Health and Human Services, the number of available organ donors has remained static over the past decade. How can we overcome this crisis?

chimera_chart

The need for organ transplants is growing but the number of donors is stagnant. Image: U.S. Dept. Human Health Services

One answer may be stem cells. These “blank slate” cells can specialize into virtually any cell type in the body which has many scientists pursuing the holy grail of stem cell research: creating an unlimited supply of human organs. Today, a team of Salk Institute scientists report in Cell that they’ve taken an early but important step toward that goal by showing it’s possible to grow rat organs within a mouse. The results bode well for not only organ transplants but also for the study of human development and disease.

Chimera – monster or medical marvel?
Our regular Stem Cellar readers will be familiar with several fascinating studies using stem cell-based 3D bioprinters or bioscaffolds which aim to one day enable the manufacturing of human tissues and organs. Instead of taking this engineering approach, the Salk team seeks a strategy in which chimeric animals are bred to grow human organs. The term “chimeric” is borrowed from Greek mythology that told tales of the chimera, a monster with a lion’s heads, a goat’s body and a serpent’s tail.

chimera_859px-chimera_di_arezzo

The chimera of Greek Mythology: part lion, goat and snake. Image: Wikimedia Commons

The team’s first set of experiments explored the feasibility of this method by first focusing on rat-mouse chimeras. Reprogramming skin cells collected from rat tails, the scientists generated induced pluripotent stem cells (iPSCs) – cells with the embryonic stem cell-like ability to become any cell type – and injected them into very early stage mouse embryos. The embryos were then implanted into surrogate female mice and successfully carried to term. Examination of the resulting mouse pups showed that their tissues and organs contained a patchwork of both rat and mouse cells.

And for my next trick, I will make a rat pancreas in a mouse
Now, if the ultimate goal is to grow organs that are 100% human in a host animal, an organ that merely has a random patchwork human cells would miss the mark. To show there’s a way around this problem, the Salk team used the CRISPR gene-editing technique to generate mouse embryos that lacked a gene that’s critical for the development of the pancreas. Without the gene, no pancreas forms and the mice died shortly after birth. But when the rat iPSCs were integrated into the gene edited mice embryos, the rat cells picked up the slack as the embryo developed, resulting in chimeric mice with rat pancreases.

Using the same CRISPR gene editing strategy, the researchers also grew rat hearts, and if you can believe it, rat eyes in the chimeric mice. On top of that, the mice in these experiments were healthy with most reaching adulthood and one living two years, an elderly age for mice.

A first step toward growing patient-specific human organs in large animals
One small, actually big, problem is that mice are much too little to serve as chimeric hosts for human organs. So the team repeated these mixed species experiments in pigs which are much better matched to humans. In this case, they added human iPSCs to the pig embryos, implanted them into female pigs and let the embryos develop for four weeks. Although it wasn’t as efficient as the rat-mouse chimeras, the researchers did indeed observe human cells that had incorporated into the chimera and were showing the early signs of specializing in different cell types within the implanted pig embryos.

This work is the first time human iPSCs have been incorporated into large animal species (they also got it to work with cattle) and many years of lab work remain before this approach can help solves the organ shortage crisis. But the potential applications are spellbinding. Imagine a patient in need of an organ transplant: a small skin biopsy is collected to make iPSCs and, using this chimeric animal approach, a patient-derived organ could be grown.

Juan Carlos Izpisua Belmonte, the study’s team leader, talked about this possibility and more in a press release:

“Of course, the ultimate goal of chimeric research is to learn whether we can use stem-cell and gene-editing technologies to generate genetically-matched human tissues and organs, and we are very optimistic that continued work will lead to eventual success. But in the process we are gaining a better understanding of species evolution as well as human embryogenesis and disease that is difficult to get in other ways.”

Ethical concerns
Now, if the idea of breeding pigs or cows with human organs make you a little uneasy, you aren’t alone.  In fact, the National Institutes of Health announced in 2015 that they had halted funding research that introduces human stem cells into other animals. They want more time “to evaluate the state of the science in this area, the ethical issues that should be considered, and the relevant animal welfare concerns associated with these types of studies.”  To read more discussion on this topic, read this MIT Technology Review article from a year ago.

 

Brain Models Get an Upgrade: 3D Mini-Brains

Every year, companies like Apple, Microsoft and Google work tirelessly to upgrade their computer, software and smartphone technologies to satisfy growing demands for more functionality. Much like these companies, biomedical scientists work tirelessly to improve the research techniques and models they use to understand and treat human disease.

Today, I’ll be talking about a cool stem cell technology that is an upgrade of current models of neurological diseases. It involves growing stem cells in a 3D environment and turning them into miniature organs called organoids that have similar structures and functions compared to real organs. Scientists have developed techniques to create organoids for many different parts of the body including the brain, gut, lungs and kidneys. These tiny 3D models are useful for understanding how organs are formed and how viruses or genetic mutations can affect their development and ability to function.

Brain Models Get an Upgrade

Organoids are especially useful for modeling complex neurological diseases where current animal and 2D cell-based models lack the ability to fully represent the cause, nature and symptoms of a disease. The first cerebral, or brain, organoids were generated in 2013 by Dr. Madeline Lancaster in Austria. These “mini-brains” contained nerve cells and structures found in the cortex, the outermost layer of the human brain.

Since their inception, mini-brains have been studied to understand brain development, test new drugs and dissect diseases like microcephaly – a disease that causes abnormal brain development and is characterized by very small skulls. Mini-brains are still a new technology, and the question of whether these organoids are representative of real human brains in their anatomy and behavior has remained unanswered until now.

Published today in Cell Reports, scientists from the Salk Institute reported that mini-brains are more like human brains compared to 2D cell-based models where brain cells are grown in a single layer on a petri dish. To generate mini-brains, they collaborated with a European team that included the Lancaster lab. They grew human embryonic stem cells in a 3D environment with a cocktail of chemicals that prompted them to develop into brain tissue over a two-month period.

Cross-section of a mini-brain. (Madeline Lancaster/MRC-LMB)

Cross-section of a mini-brain. (Madeline Lancaster/MRC-LMB)

After generating the mini-brains, the next step was to prove that these organoids were an upgrade for modeling brain development. The teams found that the cells and structures formed in the mini-brains were more like human brain tissue at the same stage of early brain development than the 2D models.

Dr. Juergen Knoblich, co-senior author of the new paper and head of the European lab explained in a Salk News Release, “Our work demonstrates the remarkable degree to which human brain development can be recapitulated in a dish in cerebral organoids.”

Are Mini-Brains the Real Thing?

The next question the teams asked was whether mini-brains had similar functions and behaviors to real brains. To answer that question, the scientists turned to epigenetics. This is a fancy word for the study of chemical modifications that influence gene expression without altering the DNA sequence in your genome. The epigenome can be thought of as a set of chemical tags that help coordinate which genes are turned on and which are turned off in a cell. Epigenetics plays important roles in human development and in causing certain diseases.

The Salk team studied the epigenomes of cells in the mini-brains to see whether their patterns were similar to cells found in human brain tissue. Interestingly, they found that the epigenetic patterns in the 3D mini-brains were not like those of real brain tissue at the same developmental stage. Instead they shared a commonality with the 2D brain models and had random epigenetic patterns. While the reason for these results is still unknown, the authors explained that it is common for cells and tissues grown in a lab dish to have these differences.

In a Salk news release, senior author and Salk professor Dr. Joseph Ecker said that even though the current mini-brain models aren’t perfect yet, scientists can still gather valuable information from them in the meantime.

“Our findings show that cerebral organoids as a 3D model of brain function are getting closer to a real brain than 2D models, so perhaps by using the epigenetic pattern as a gauge we can get even closer.”

And while the world eagerly waits for the next release of the iPhone 7, neuroscientists will be eagerly waiting for a new and improved version of mini-brains. Hopefully the next upgrade will produce organoids that behave more like the real thing and can model complex neurological diseases, such as Alzheimer’s, where so many questions remain unanswered.

Stem Cell Stories that caught our eye: a womb with a view, reversing aging and stabilizing stem cells

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.

Today we bring you a trifecta of stem cell stories that were partially funded by grants from CIRM.

A womb with a view: using 3D imaging to observe embryo implantation. Scientists have a good understanding of how the beginning stages of pregnancy happen. An egg cell from a woman is fertilized by a sperm cell from a man and the result is a single cell called a zygote. Over the next week, the zygote divides into multiple cells that form the developing embryo. At the end of that period, the embryo hatches out of its protective membrane and begins implanting itself into the lining of the mother’s uterus.

It’s possible to visualize the early stages of embryo development in culture dishes, which has helped scientists understand the biological steps required for an embryo to survive and develop into a healthy fetus. However, something that is not easy to observe is the implantation stage of the embryo in the uterus. This process is complex and involves a restructuring of the uterine wall to accommodate the developing embryo. As you can imagine, replicating these events would be extremely complicated and difficult to do in a culture dish, and current imaging techniques aren’t adequate either.

That’s where new CIRM-funded research from a team at UCSF comes to the rescue. They developed a 3D imaging technology and combined it with a previously developed “tissue clearing” method, which uses chemicals to turn tissues translucent, to provide clear images of the uterine wall during embryo implantation in mice. Their work was published this week in the journal Development.

According to a UCSF news release,

“Using their new approach, the team observed that the uterine lining becomes extensively folded as it approaches its window of receptivity for an embryo to implant. The geometry of the folds in which the incoming embryos dwell is important, the team found, as genetic mutants with defects in implantation have improper patterns of folding.”

Ultimately, the team aims to use their new imaging technology to get an inside scoop on how to prevent or treat pregnancy disorders and also how to improve the outcome of pregnancies by in vitro fertilization.

Senior author on the study, UCSF professor Diana Laird concluded:

“This new view of early pregnancy lets us ask fundamentally new questions about how the embryo finds its home within the uterus and what factors are needed for it to implant successfully. Once we can understand how these processes happen normally, we can also ask why certain genetic mutations cause pregnancies to fail, to study the potential dangers of environmental toxins such as the chemicals in common household products, and even why metabolic disease and obesity appears to compromise implantation.”

If you want to see this womb with a view, check out the video below.

Watch these two videos for more information:

Salk scientists reverse signs of aging in mice. For our next scintillating stem cell story, we’re turning back the clock – the aging clock that is. Scientists from the Salk Institute in La Jolla, reported an interesting method in the journal Cell  that reverses some signs of aging in mice. They found that periodic expression of embryonic stem cell genes in skin cells and mice could reverse some signs of aging.

The Salk team made use of cellular reprogramming tools developed by the Nobel Prize winning scientist Shinya Yamanaka. He found that four genes normally expressed in embryonic stem cells could revert adult cells back to a pluripotent stem cell state – a process called cellular reprogramming. Instead of turning adult cells back into stem cells, the Salk scientists asked whether the Yamanaka factors could instead turn back the clock on older, aging cells – making them healthier without turning them back into stem cells or cancer-forming cells.

The team found that they could rejuvenate skin cells from mice without turning them back into stem cells if they turned on the Yamanaka genes on for a short period of time. These skin cells were taken from mice that had progeria – a disease that causes them to age rapidly. Not only did their skin cells look and act younger after the treatment, but when the scientists used a similar technique to turn on the Yamanaka genes in progeria mice, they saw rejuvenating effects in the mice including a more rapid healing and regeneration of muscle and pancreas tissue.

(Left) impaired muscle repair in aged mice; (right) improved muscle regeneration in aged mice subjected to reprogramming. (Salk Institute)

(Left) impaired muscle repair in aged mice; (right) improved muscle regeneration in aged mice subjected to reprogramming. (Salk Institute)

The senior author on the study, Salk Professor Juan Carlos Izpisua Belmonte, acknowledged in a Salk news release that this is early stage work that focuses on animal models, not humans:

“Obviously, mice are not humans and we know it will be much more complex to rejuvenate a person. But this study shows that aging is a very dynamic and plastic process, and therefore will be more amenable to therapeutic interventions than what we previously thought.”

This story was very popular, which is not surprising as aging research is particularly fascinating to people who want to live longer lives. It was covered by many news outlets including STATnews, Scientific American and Science Magazine. I also recommend reading Paul Knoepfler’s journal club-style blog on the study for an objective take on the findings and implications of the study. Lastly, you can learn more about the science of this work by watching the movie below by the Salk.

Movie:

Stabilizing unstable stem cells. Our final stem cell story is brought to you by scientists from the UCLA Broad Stem Cell Research Center. They found that embryonic stem cells can harbor genetic instabilities that can be passed on to their offspring and cause complications, or even disease, later in life. Their work was published in two separate studies in Cell Stem Cell and Cell Reports.

The science behind the genetic instabilities is too complicated to explain in this blog, so I’ll refer you to the UCLA news release for more details. In brief, the UCLA team found a way to reverse the genetic instability in the stem cells such that the mature cells that they developed into turned out healthy.

As for the future impact of this research, “The research team, led by Kathrin Plath, found a way to correct the instability by resetting the stem cells from a later stage of development to an earlier stage of development. This fundamental discovery could have great impact on the creation of healthy tissues to cure disease.”

Stem cell stories that caught our eye: insights into stem cell biology through telomeres, reprogramming and lung disease

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.

Telomeres and stem cell stability: too much of a good thing

Just like those plastic tips at the end of shoelaces (fun fact: they’re called aglets), telomeres form a protective cap on the end of chromosomes. Because of the way DNA replication works, the telomeres shorten each time a cell divides. Trim away enough of the telomere over time and, like a frayed shoelace, the chromosomes become unstable and an easy target for damage which eventually leads to cell death.

telomere_caps

Telomeres (white dots) form a protective cap on chromsomes (gray). (Wikimedia) 

Stem cells are unique in that they contain an enzyme called telomerase that lengthens telomeres. Telomerase activity and telomere lengthening are critical for a stem cell’s ability to maintain virtually limitless cell divisions. So you’d assume the longer the telomere, the more stable the cell. But Salk Institute scientists reported this week that too much telomere can be just as bad, if not worse, than too little.

The CIRM-funded work, which was published in Nature Structural & Molecular Biology, used genetic engineering to artificially vary telomerase activity in human embryonic stem cells. Cells with low telomerase activity had shorter telomeres and died. This result wasn’t a surprise since the short telomeres-cell death observation has been well documented. Based on those results, the team was expecting cells with boosted telomerase activity and, in turn, extended telomeres would be especially stable. But that’s not what happened as senior author Jan Karlseder mentioned in a Salk press release:

“We were surprised to find that forcing cells to generate really long telomeres caused telomeric fragility, which can lead to initiation of cancer. These experiments question the generally accepted notion that artificially increasing telomeres could lengthen life or improve the health of an organism.”

The researchers also examined induced pluripotent stem (iPS) cells in the study and found that the cells contain “footprints” of telomere trimming. So the team is in a position to study how a cell’s telomere history relates to how well it can be reprogrammed into iPS cells. First author Teresa Rivera pointed out the big picture significance of this finding:

“Stem cell reprogramming is a major scientific breakthrough, but the methods are still being perfected. Understanding how telomere length is regulated is an important step toward realizing the promise of stem cell therapies and regenerative medicine.”

jan-karlseder_teresa-rivera-garcia0x8c7144w

Jan Karlseder and Teresa Rivera

Lego set of gene activators takes trial and error out of cellular reprogramming

To convert one cell type into another, stem cell researchers rely on educated guesses and a lot of trial and error. In fact, that’s how Shinya Yamanaka identified the four Yamanaka Factors which, when inserted into a skin cell, reprogram it into the embryonic stem cell-like state of an iPS cell. That ground-breaking discovery ten years ago has opened the way for researchers worldwide to specialize iPS cells into all sorts of cell types from nerve cells to liver cells. While some cell types are easy to generate this way, others are much more difficult.

Reporting this week in PNAS, a University of Wisconsin–Madison research team has developed a nifty systematic, high-throughput method for identifying the factors necessary to convert a cell from one type to another. Their strategy promises to free researchers from the costly and time consuming trial and error approach still in use today.

The centerpiece of their method is artificial transcription factors (ATFs). Now, natural transcription factors – Yamanaka’s Factors are examples – are proteins that bind DNA and activate or silence genes. Their impact on gene activity, in turn, can have a cascading effects on other genes and proteins ultimately causing, say a stem cell, to start making muscle proteins and turn into a muscle cell.

Transcription factors are very modular proteins – one part is responsible for binding DNA, another part for affecting gene activity and other parts that bind to other proteins. The ATFs generated in this study are like lego versions of natural transcription factors – each are constructed from combinations of different transcription factor parts. The team made nearly 3 million different ATFs.

As a proof of principle, the researchers tried reproducing Yamanaka’s original, groundbreaking iPS cell experiment. They inserted the ATFs into skin cells that already had 3 of the 4 Yamanaka factors, they left out Oct4. They successfully generated iPS with this approach and then went back and studied the makeup of the ATFs that had caused cells to reprogram into iPS cells. Senior author Aseem Ansari gave a great analogy in a university press release:

“Imagine you have millions of keys and only a unique key or combination of keys can turn a motor on. We test all those keys in parallel and when we see the motor fire up, we go back to see exactly which key switched it on.”

atf_ips_cells

Micrograph of induced pluripotent stem cells generated from artificial transcription factors. The cells express green fluorescent protein after a key gene known as Oct4 is activated. (ASUKA EGUCHI/UW-MADISON)

The analysis showed that these ATFs had stimulated gene activity cascades which didn’t directly involve Oct4 but yet ultimately activated it. This finding is important because it suggests that future cell conversion experiments could uncover some not so obvious cell fate pathways. Ansari explains this point further:

“It’s a way to induce cell fate conversions without having to know what genes might be important because we are able to test so many by using an unbiased library of molecules that can search nearly every corner of the genome.”

This sort of brute force method to accelerate research discoveries is music to our ears at CIRM because it ultimately could lead to therapies faster.

Search for clues to treat deadly lung disease

When researchers don’t understand what causes a particular disease, a typical strategy is to compare gene activity in diseased vs healthy cells and identify important differences. Those differences may lead to potential paths to developing a therapy. That’s the approach a collaborative team from Cincinnati Children’s Hospital and Cedars-Sinai Medical took to tackle idiopathic pulmonary fibrosis (IPF).

IPF is a chronic lung disease which causes scarring, or fibrosis, in the air sacs of the lung. This is the spot where oxygen is taken up by tiny blood vessels that surround the air sacs. With fibrosis, the air sacs stiffen and thicken and as a result less oxygen gets diffused into the blood and starves the body of oxygen.  IPF can lead to death within 2 to 5 years after diagnosis. Unfortunately, no cures exist and the cause is unknown, or idiopathic.

(Wikimedia)

(Wikimedia)

The transfer of oxygen from air sacs to blood vessels is an intricate one with many cell types involved. So pinpointing what goes wrong in IPF at a cellular and molecular level has proved difficult. In the current study, the scientists, for the first time, collected gene sequencing data from single cells from healthy and diseased lungs. This way, a precise cell by cell analysis of gene activity was possible.

One set of gene activity patterns found in healthy sample were connected to proper formation of a particular type of air sac cell called the aveolar type 2 lung cell. Other gene patterns were linked to abnormal IPF cell types. With this data in hand, the researchers can further investigate the role of these genes in IPF which may open up new therapy approaches to this deadly disease.

The study funded in part by CIRM was published this week in Journal of Clinical Investigation Insight and a press release about the study was picked up by PR Newswire.

Advancements in gene editing make blind rats see light

Gene editing is a rapidly advancing technology that scientists are using to manipulate the genomes of cells with precision and accuracy. Many of these experiments are being conducted on stem cells to genetic mutations in an attempt to find cures for various diseases like cancer, HIV and blindness.

Speaking of blindness, researchers from the Salk Institute reported today that they’ve improved upon the current CRISPR/Cas9 gene editing technology and found a more efficient way to edit the genomes of cells in living animals. They used their technology on blind rats that had a genetic disease called retinitis pigmentosa (RP) and found that the rats were able to see light following the treatment.

The really exciting part about their findings is that their CRISPR technology works well on dividing cells like stem cells and progenitor cells, which is typically how scientists use the CRISPR technology, but it also works on adult cells that do not divide – a feat that hasn’t been accomplished before.

Their results, which were published today in the journal Nature, offer a new tool that scientists can use to target cells that no longer divide in tissues and organs like the eye, brain, pancreas and heart.

According to a Salk news release:

“The new Salk technology is ten times more efficient than other methods at incorporating new DNA into cultures of dividing cells, making it a promising tool for both research and medicine. But, more importantly, the Salk technique represents the first time scientists have managed to insert a new gene into a precise DNA location in adult cells that no longer divide, such as those of the eye, brain, pancreas or heart, offering new possibilities for therapeutic applications in these cells.”

CRISPR gene edited neurons, which are non-dividing brain cells, are shown in green while cell nuclei are shown in blue. (Salk)

CRISPR gene edited neurons, which are non-dividing brain cells, are shown in green while cell nuclei are shown in blue. (Salk)

Salk Professor and senior author on the study, Juan Carlos Izpisua Belmonte, explained the big picture of their findings:

“We are very excited by the technology we discovered because it’s something that could not be done before. For the first time, we now have a technology that allows us to modify the DNA of non-dividing cells, to fix broken genes in the brain, heart and liver. It allows us for the first time to be able to dream of curing diseases that we couldn’t before, which is exciting.”

If you want to learn more about the science behind their new CRISPR gene editing technology, check out the Salk news release and coverage in Genetic Engineering & Biotechnology News. You can also watch this short three minute video about the study made by the Salk Institute.

Salk scientists explain why brain cells are genetically diverse

twin_boys

I’ve always wondered why some sets of genetically identical twins become not so identical later in life. Sometimes they differ in appearance. Other times, one twin is healthy while the other is plagued with a serious disease. These differences can be explained by exposure to different environmental factors over time, but there could also be a genetic explanation involving our brains.

The brain is composed of approximately 100 billion cells called neurons, each with a DNA blueprint that contains instructions that determine the function of these neurons in the brain. Originally it was thought that all cells, including neurons, have the same DNA. But more recently, scientists discovered that the brain is genetically diverse and that neurons within the same brain can have slightly different DNA blueprints, which could give them slightly different functions.

Jumping genes and genetic diversity

gage-web

Fred “Rusty” Gage: Photo courtesy Salk Institute

Why and how neurons have differences in their DNA are questions that Salk Institute professor Fred Gage has pursued for more than a decade. In 2005, his lab discovered a mechanism during neural development that causes differences in the DNA of neurons. As a brain stem cell develops into a neuron, long interspersed nuclear elements (L1s), which are small pieces of DNA, copy and paste themselves, seemingly at random, throughout a neuron’s genome.

These elements were originally dubbed “jumping genes” because of their ability to hop around and insert themselves into DNA. It turns out that L1s do more than copy and paste themselves to create changes in DNA, they also can delete chunks of DNA. In a CIRM-funded study published this week in the journal Nature Neuroscience, Gage and colleagues at the Salk Institute reported new insights into L1 activity and how it creates genetic diversity in the brain.

Copy, paste, delete

Gage and his team had clues that L1s can cause DNA deletions in neurons back in 2013. They used a technique called single-cell sequencing to record the sequence of individual neuronal genomes and saw that some of their genomes had large sections of DNA added or missing.

They thought that L1s could be the reason for these insertions and deletions, but didn’t have proof until their current study, which used an improved method to identify areas of the neuronal genome modified by L1s. This method, combined with a computer algorithm that can easily tell the difference between various types of L1 modifications, revealed that areas of the genome with L1s were susceptible to DNA cutting caused by enzymes that home in on the L1 sequences. These breaks in the DNA then cause the observed deletions.

Gage explained their findings in a news release:

“In 2013, we discovered that different neurons within the same brain have various complements of DNA, suggesting that they function slightly differently from each other even within the same person. This recent study reveals a new and surprising form of variation that will help us understand the role of L1s, not only in healthy brains but in those affected by schizophrenia and autism.”

Jennifer Erwin, first author on the study, further elaborated:

“The surprising part was that we thought all L1s could do was insert into new places. But the fact that they’re causing deletions means that they’re affecting the genome in a more significant way,” says Erwin, a staff scientist in Gage’s group.”

Insights into brain disorders

It’s now known that L1s are important for the brain’s genetic diversity, but Gage also believes that L1s could play a role in causing brain disorders like schizophrenia and autism where there is heightened L1 activity in the neurons of these patients. In future work, Gage and his team will study how L1s can cause changes in genes associated with schizophrenia and autism and how these changes can effect brain function and cause disease.