CIRM-funded scientists discover a new way to make stem cells using antibodies

Just as learning a new skill takes time to hone, scientific discoveries take time to perfect. Such is the case with induced pluripotent stem cells (iPSCs), the Nobel Prize winning technology that reprograms mature adult cells back into a pluripotent stem cell state. iPSCs are a powerful tool because they can develop into any cell found in the body. Scientists use iPSCs to model diseases in a dish, screen for new drugs, and to develop stem cell-based therapies for patients.

iPSCs grown in a cell culture dish.

The original iPSC technology, discovered by Dr. Shinya Yamanaka in 2006, requires viral delivery of four transcription factor genes, Oct4, Sox2, Klf4, and c-Myc, into the nucleus of an adult cell. These genes are inserted into the genome where they are activated to churn out their respective proteins. The combined expression of these four factors (OSKM) turns off the genetic programming of an adult cell and turns on the programming for a pluripotent stem cell.

The technology is pretty neat and allows scientists to make iPSCs from patients using a variety of different tissue sources including skin, blood, and even urine. However, there is a catch. Inserting reprogramming genes into a cell’s genome can be disruptive if the reprogramming genes fail to switch off or can cause cancer if nefarious oncogenes are turned on.

In response to this concern, scientists are developing alternative methods for making iPSCs using non-invasive methods. A CIRM-funded team from The Scripps Research Institute (TSRI) published such a study yesterday in the journal Nature Biotechnology.

Led by senior author and CIRM grantee Dr. Kristin Baldwin, the TSRI team screened a large library of antibodies – proteins that recognize and bind to specific molecules – to identify ones that could substitute for the OSKM reprogramming factors. The hope was that some of these antibodies would bind to proteins on the surface of cells and turn on a molecular signaling cascade from the outside that would turn on the appropriate reprogramming genes from the inside of the cell.

The scientists screened over 100 million antibodies and found ones that could replace three of the four reprogramming factors (Oct4, Sox2, and c-Myc) when reprogramming mouse skin cells into iPSCs. They were unable to find an antibody to replace Klf4 in the current study but have it on their to-do list for future studies.

Dr. Baldwin explained how her team’s findings improve upon previous reprogramming methods in a TSRI news release,

Kristen Baldwin

“This result suggests that ultimately we might be able to make IPSCs without putting anything in the cell nucleus, which potentially means that these stem cells will have fewer mutations and overall better properties.”

 

Other groups have published other non-invasive iPSC reprogramming methods using cocktails of chemicals, proteins or microRNAs in place of virally delivering genes to make iPSCs. However, Baldwin’s study is the first (to our knowledge) to use antibodies to achieve this feat.

An added benefit to antibody reprogramming is that the team was able to learn more about the signaling pathways that were naturally activated by the iPSC reprogramming antibodies.

“The scientists found that one of the Sox2-replacing antibodies binds to a protein on the cell membrane called Basp1. This binding event blocks Basp1’s normal activity and thus removes the restraints on WT1, a transcription factor protein that works in the cell nucleus. WT1, unleashed, then alters the activity of multiple genes, ultimately including Sox2’s, to promote the stem cell state using a different order of events than when using the original reprogramming factors.”

iPSCs made by antibody reprogramming could address some of the long-standing issues associated with more traditional reprogramming methods and could offer further insights into the complex signaling required to turn adult cells back into a pluripotent state. Baldwin and her team are now on the hunt for antibodies that will reprogram human (rather than mouse) cells into iPSCs. Stay tuned!

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Crossing the Grad School Bridge of Self and Scientific Discovery

Since 2010, the CIRM Bridges Program has provided paid stem cell research internships to students at California colleges and universities that don’t have major stem cell research programs. In order to keep in touch with these interns, The Stem Cellar has an ongoing CIRM Scholars blog series, inviting alumni from our training programs to reflect on the importance of their internships, to update readers on their career path and to give career advice to the current interns.

The blog below, written by Mimi Krutein from the 2011 Bridges program at Cal State University San Marcos, is based on a presentation she gave in late July at the 2017 Annual CIRM Bridges Trainee Meeting in San Diego. 

Mimi Krutein

The science graduate school experience is not at all what I was expecting. I imagined it as a mentally stimulating flurry of discoveries and training; before I started I pictured a cross between Harry Potter and The Magic School Bus.  What I got, and what most graduate students get, is a vaguely escorted slog into a land of uncertainty and imposter syndrome, sprinkled with fleeting moments of clarity and excitement.  But don’t get me wrong; it is worth it.

My personal road to graduate school was quite unorthodox.  I entered California State University San Marcos (CSUSM) as a nursing major, because I had a genuine interest in medicine and was fascinated by the complexity of the human body.

 It also didn’t require calculus level math, so I was sold.
I generally enjoyed my courses but everything changed for me when I took microbiology.  It was my first introduction to basic science.  Disease mechanisms of microorganisms blew my mind, sparked my curiosity, and catalyzed a shift in focus that never readjusted.

It was then I decided to add a biology minor to feed the beast, but didn’t have the confidence to switch majors completely.  The pre-nursing program actually advised me not to add the minor; my grades at that point were good but not stellar, and they thought that the new load would be too difficult.  That summer I formally applied to the CSUSM nursing program and was rejected, missing the cutoff by one point.  Chalking it up to fate, I turned gracefully on my heels and belly flopped into a molecular biology major with open arms, calculus and all.

A few semesters passed and I desperately craved more lab time so I applied to 12 summer undergraduate research programs and was swiftly rejected due to lack of experience.  The only position I was offered was a 100-hour, unpaid internship at a tiny biotech composed of 5 people, where we utilized bioluminescent phytoplankton to monitor water toxicity.  Then I joined the only research lab at CSUSM with an opening, and under Dr. Betsy Read I studied the metabolic pathways of the model organism Emiliania huxleyi, also a phytoplankton.

As much as I loved the lab and industry training I was receiving, I wanted to integrate my fascination of human medicine with my passion for laboratory science.  Betsy pulled me into her office one day and asked the very obtuse question “what do you want to do in science?”  To her surprise –and slight disappointment I’m sure- I told her that I didn’t want to stay in phytoplankton, but rather explore medically relevant research, and study human disease.  Happily she lit up and frantically told me about the CIRM Bridges internship that would be perfect, the caveat being that applications were due that very day.  I received a 24-hour extension, and was later accepted for the 2011 program.

I was equal parts inspired and terrified
For my CIRM internship I joined Tobin Dickerson’s lab in the department of chemistry at The Scripps Research Institute.  I received excellent one-on-one training in a small lab studying highly infectious agents, primarily botulinum toxin.  Now, botulinum toxin has an extremely simple mechanism of action, however, it is also the most potent neurotoxin known to man.  Approximately 1 gram of aerosolized toxin can kill 1 million people; and the bacteria that produces it, Clostridium botulinum, is relatively easy to propagate, making it a potential bioterrorist agent.

iPSC-derived motor neurons. Image courtesey of Mimi Kreitin/The Scripps Research Institute

For this reason, The Department of Defense gave us a grant to pursue high-throughput screening of small molecule inhibitors that could block the effects of this toxin.  I assisted in the screening and follow up tests on individual inhibitors.  At the same time, I established a robust method for generating motor neurons from human embryonic and induced pluripotent stem cells.  This work provided us with a virtually endless pool of boltulinum-sensitive cells for the use of cellular studies with prospective inhibitors found in our initial screens.  Deriving the neurons from stem cells also eliminated the need for expensive and tiresome motor neuron harvests from animals.  The cells I produced in the lab presented as bonafide motor neurons because they produced an appropriate dose response to live toxin.

I finally felt like a real scientist
After my internship, I was formally hired by the lab as a part time technician while I finished my last year of classes as CSUSM.  My two years of work in the lab resulted in three publications, one of which was accepted for the cover of ACS Combinatorial Science.  More importantly though, the years I spent in the Dickerson lab provided room for me to grow into myself as a scientist, receive unparalleled training, and gain perspective on what it meant to be in the thick of academic research.

After many discussions with my peers and mentors, I decided graduate school, ideally a PhD track, was the next step for my scientific career.  I knew I loved research, but I wanted to learn how to think, how to approach unanswered questions in a productive manner.  I wanted to be trained by everyone who could provide me with knowledge.

I was just plain hungry.
And like most 20-somethings on the edge of graduation, my passion was mixed in equal parts with indecisiveness.  I really didn’t know what I wanted to study, but I knew I wanted to utilize my stem cell training, and I knew what made my mind light up; I was -and still am- fascinated by how diseases work on a cellular and molecular level.  So, after months of searching, digging, and crosschecking, I applied to a dozen translational research programs across the US.

And then the news arrived
While running late to a class, I got the acceptance email from my dream school; the University of Washington. After reading the subject line I was frozen with disbelief, I called my mom, forgot where I was going and took a stroll the other direction until I realized I had a test waiting for me.  It never occurred to me that I could actually do this for real.

My first day of grad school was one I will never forget.  After a lukewarm five minutes of awkwardly chatting with my new postdoc lab members, we go out to get coffee and I proceed to faceplant in the middle of a puddle-filled crosswalk directly in front of a truck.  I skinned my knee and sliced my hand open, but magically managed to keep my coffee upright.  Understandably, my newly acquired lab members didn’t let me touch anything of real importance for 2 weeks.  Even after being considered a ‘seasoned’ graduate student I still knock over racks of pipette tips or spill liters of E. coli cultures on my new jeans.  Such is the grad school life.  Part of me hopes once I earn those fancy three letters after my name, I’ll evolve to the perfect scientist, but I won’t bet on it.

To those of you considering graduate school
I’ll end with these parting thoughts. Obviously, I’m still not on the other end of this whole grad school thing, but I can tell you from the four years I’ve spent doing this so far, there has been no experience more rewarding and humbling than pursuing a PhD.  If you find yourself interested in taking the leap in a similar direction, know that if you choose this path, it’s a marathon, not a sprint so take care of yourself through the process.  Maintain a strong support system, both for your personal and professional well-being.  Foster relationships with your peers to gain strength in numbers and build mentorships with individuals you admire to perpetuate curiosity.  Choose your home lab thoughtfully; the Principal Investigator to Student dynamic is the cornerstone of the graduate school experience; you can’t be on different pages with the lab’s leader and expect to write the same story.

Imposter syndrome is the greatest barrier to your success
I spent 22 years wholeheartedly believing I couldn’t do the thing I’m currently doing, and I’ll tell you guys a secret, some days I still feel that way. But it’s vital to recognize that you are worthy of success and not defined by your failures.  Lastly, find humor where you can and stay hungry for opportunities that you believe are just outside of your reach. And stay hungry for knowledge, it’s one of few things that doesn’t expire.

Stories that caught our eye: stem cell transplants help put MS in remission; unlocking the cause of autism; and a day to discover what stem cells are all about

multiple-sclerosis

Motor neurons

Stem cell transplants help put MS in remission: A combination of high dose immunosuppressive therapy and transplant of a person’s own blood stem cells seems to be a powerful tool in helping people with relapsing-remitting multiple sclerosis (RRMS) go into sustained remission.

Multiple sclerosis (MS) is an autoimmune disorder where the body’s own immune system attacks the brain and spinal cord, causing a wide variety of symptoms including overwhelming fatigue, blurred vision and mobility problems. RRMS is the most common form of MS, affecting up to 85 percent of people, and is characterized by attacks followed by periods of remission.

The HALT-MS trial, which was sponsored by the National Institute of Allergy and Infectious Diseases (NIAID), took the patient’s own blood stem cells, gave the individual chemotherapy to deplete their immune system, then returned the blood stem cells to the patient. The stem cells created a new blood supply and seemed to help repair the immune system.

Five years after the treatment, most of the patients were still in remission, despite not taking any medications for MS. Some people even recovered some mobility or other capabilities that they had lost due to the disease.

In a news release, Dr. Anthony Fauci, Director of NIAID, said anything that holds the disease at bay and helps people avoid taking medications is important:

“These extended findings suggest that one-time treatment with HDIT/HCT may be substantially more effective than long-term treatment with the best available medications for people with a certain type of MS. These encouraging results support the development of a large, randomized trial to directly compare HDIT/HCT to standard of care for this often-debilitating disease.”

scripps-campus

Scripps Research Institute

Using stem cells to model brain development disorders. (Karen Ring) CIRM-funded scientists from the Scripps Research Institute are interested in understanding how the brain develops and what goes wrong to cause intellectual disabilities like Fragile X syndrome, a genetic disease that is a common cause of autism spectrum disorder.

Because studying developmental disorders in humans is very difficult, the Scripps team turned to stem cell models for answers. This week, in the journal Brain, they published a breakthrough in our understanding of the early stages of brain development. They took induced pluripotent stem cells (iPSCs), made from cells from Fragile X syndrome patients, and turned these cells into brain cells called neurons in a cell culture dish.

They noticed an obvious difference between Fragile X patient iPSCs and healthy iPSCs: the patient stem cells took longer to develop into neurons, a result that suggests a similar delay in fetal brain development. The neurons from Fragile X patients also had difficulty forming synaptic connections, which are bridges that allow for information to pass from one neuron to another.

Scripps Research professor Jeanne Loring said that their findings could help to identify new drug therapies to treat Fragile X syndrome. She explained in a press release;

“We’re the first to see that these changes happen very early in brain development. This may be the only way we’ll be able to identify possible drug treatments to minimize the effects of the disorder.”

Looking ahead, Loring and her team will apply their stem cell model to other developmental diseases. She said, “Now we have the tools to ask the questions to advance people’s health.”

A Day to Discover What Stem Cells Are All about.  (Karen Ring) Everyone is familiar with the word stem cells, but do they really know what these cells are and what they are capable of? Scientists are finding creative ways to educate the public and students about the power of stem cells and stem cell research. A great example is the University of Southern California (USC), which is hosting a Stem Cell Day of Discovery to educate middle and high school students and their families about stem cell research.

The event is this Saturday at the USC Health Sciences Campus and will feature science talks, lab tours, hands-on experiments, stem cell lab video games, and a resource fair. It’s a wonderful opportunity for families to engage in science and also to expose young students to science in a fun and engaging way.

Interest in Stem Cell Day has been so high that the event has already sold out. But don’t worry, there will be another stem cell day next year. And for those of you who don’t live in Southern California, mark your calendars for the 2017 Stem Cell Awareness Day on Wednesday, October 11th. There will be stem cell education events all over California and in other parts of the country during that week in honor of this important day.

 

 

Science and Improv: Spotlight on CIRM Bridges Scholar Jill Tsai

As part of our CIRM scholar series, we’re featuring the research and career accomplishments of CIRM funded students.

What do science and improv have in common? The answer is not a whole lot. However, I recently met a talented student from our CIRM Bridges master’s program who one day is going to change this.

Jill Tsai

Jill Tsai, CIRM Bridges scholar

Meet Jill Tsai. She recently graduated from the CIRM Bridges program at the Scripps Research Institute in San Diego and is now starting a PhD program in cancer biology at the City of Hope in Duarte California.

Jill received her Bachelors from UC Merced general biology and went to Cal Poly Pomona for a Master’s program in cancer research. While at Cal Poly Pomona, she successfully applied for a CIRM Bridges internship that allowed her to finish her Master’s degree at Scripps in the lab of Dr. Lazzerini Denchi.

I met Jill at the 2016 Bridges Conference in July and was immediately impressed by her passion for science and communications. I was also intrigued by her interest in improv and how she balances her time between two very different passions. I’m thrilled that Jill agreed to an interview for the Stem Cellar as I think it’s valuable to read about scientists who are pursuing multiple passions not necessarily related to science.

Enjoy!

Q: What did you study during your Bridges internship?

JT: I was a research intern in the lab of Dr. Lazzerini Denchi. In his lab, we study telomeres, which are the pieces of DNA at the end of chromosomes that help protect them from being degraded. We’re specifically looking at proteins that help maintain telomere function in mouse stem cells. We do big protein pull downs to try to figure out what new and novel proteins are surrounding the mechanisms that maintain telomere function, and then we do functional assays to figure out what these proteins do.

Lazzerini Denchi’s lab focuses on basic research and how certain proteins affect telomere length and also the telomere deprotection response. One function of telomeres is that they suppress the double and single stranded DNA repair mechanism. If you don’t suppress those mechanisms, then the ends of those linear chromosomes look exactly like double stranded DNA breaks and repair proteins try to fix them by fusing those chromosomes together.

There are great pictures from Lazzerini Denchi’s first author publication showing chromosomes hooked end to end to end like long strings of spaghetti as a result of telomere deprotection. We are studying novel proteins that assist telomeres with the deprotection response and determining whether these proteins have some other kind of function as well.

Telomere deprotection results in chromosomes that are linked together (right) instead of separate (left). (Source Denchi et al. Nature)

Telomere deprotection results in chromosomes that are linked together (right) instead of separate (left). (Source Nature: Denchi et al., 2007)

Our larger focus in the lab is being able to understand cancer and specific telomere related genetic disorders that are associated with cancer.

Q: What was your CIRM Bridges experience like?

JT: CIRM was really amazing, and I credit it a lot for being able to start a PhD this fall. I’d been working in my lab at Cal Poly Pomona for five years, and my research unfortunately wasn’t working out. I was probably going to have to quit the program or take an out with an easier project. When I applied to CIRM, I was hoping to get the internship because if I didn’t get it, I was going to go down a completely different career path.

The CIRM internship was very valuable to me. It provided training through stem cell classes and lectures and allowed me to immerse myself in a real lab that had real equipment and personnel. The experience took my research knowledge to the next level and then some. And I knew for sure it had when I was at the poster session during the Bridges conference. I was walking around and asking students about their research, and I understood clearly the path of their research. I knew what questions were good to ask and what the graphs meant without having to take them home and dissect them. It was extremely satisfying to be able to understand other’s scientific research by just listening to them.

I am so excited to start my PhD in the fall. For the first time, I feel confident about my foundational biology and research skills. I also have a better understanding of myself and where I need to improve in comprehension and technique. I am ready to jump into grad school and improve as a scientist.

Q: What are your future career steps?

JT: I want to do something that involves teaching or being able to educate people. I’ve worked as a TA in my master’s program for a few years, and I really enjoy that experience of clarifying complex subjects for people. But to be honest, I also don’t know what I want to do right now so I’m keeping my options open.

Q: What’s your favorite thing about being scientist?

JT: Being a scientist forces you to never be complacent in what you understand. If I had never gotten my master’s, there would be this whole level of critical thinking that I wouldn’t have right now. Learning more is one of the biggest reasons why I want to get my PhD even if I don’t know exactly what I want to do yet.

I want to be able to think at a higher level because I think it’s valuable. And I see my Professor at Scripps: he has all these publications under his belt, but he’s always tinkering with things and he’s always learning new software and he’s always reading new papers. As a scientist, you can’t be stagnant in your learning, and I think because of that you’re always pushing yourself to your best potential.
Q: Do you have advice for future Bridges students?

JT: For anyone who is interested in doing a PhD, this is the world’s best preparatory program. After you start a PhD, you hit the ground running. If I were to give advice, I’d say to not be too hard on yourself. There’s going to be expectations put on you that you might not be ready for and you might not do the best job. But you should try your best and know it’s going to help you grow.

Usually people who go into PhD programs are people that have always done well in school. But it’s important to know that learning in grad school is very different than how we are taught to learn elsewhere. Every other time it’s just like show up, listen, take the test you’re done. A PhD relies on a little bit of luck, getting the right project, and doing everything meticulously.

Q: What are your hobbies?

JT: My favorite hobby is improv comedy. What I really like about improv is that it is so different from science and it helps me to relax after work.

Improv is performing comedic scenes on stage with a bunch of people without a script. Skills that it requires are not being stuck in your own head and really paying attention to what’s going on around you. You also need to take big risks and not worry so much about what the end result is going to be, which is very different from research. It’s a nice break to be able to make big giant mistakes and know that after that day it doesn’t matter.

As a researcher, it’s hard to make friends, and even if you have friends, it’s hard to find the time to hang out with them. I love improv because it’s a built in activity. All of my friends outside of work are in improv. We show up and we play make believe together on stage – it’s just a really nice atmosphere. In improv we teach a philosophy that everything you have is enough. Everything you come in with is enough. It’s really nice, because being an adult is hard and life is hard. So it’s a nice thing to hear.

Jill's Improv team.

Flyspace Improv team.

Q: Do you see yourself combining your passions for science and improve in the future?

JT: I do. I don’t know what I want to do yet as a career, but improv is such a big part of my identity that it will always play a role in my life. Improv is so important in communication and interpersonal connections. I believe everyone in science could benefit from it. Ideally, I will find a career that allows me to use both of these passions to help people.

Stem Cells May Help Endangered Species Live Long and Prosper

It’s the year 2286. The transmission signal of an alien space probe is wreaking havoc on Earth, knocking out the worldwide power grid and causing massive storms. It turns out the mysterious orbiting probe is trying to communicate with humpback whales through whale song and the devastation won’t stop until contact is made. But there’s a tiny problem: in that future, the humpback has long since become extinct. So the captain and crew travel back in time to snag two whales and save 23rd century civilization. Phew!

My fellow science fiction nerds will recognize that plot line from 1986’s Star Trek IV: A Voyage Home. It’s pure fantasy and yet there is a real lesson for our present day world: you shouldn’t underestimate how the extinction of a species will impact our world. For instance, the collapse and potential extinction of the bee population and other pollinators threatens to destabilize our global food supply.

Northern White Rhinos: At the Brink of Extinction
Beyond how it may affect us humans, I think there’s also a moral obligation to save endangered species that have dwindled in number directly due to human actions. It may be too late for the northern white rhino though. Because their horns are highly sought after as a status symbol and for use in traditional medicine, poachers have wiped out the population and now only three – Sudan, Najin and Fatu (grandfather, mother and daughter) – exist in the world. Sadly, none of them can breed naturally so they quietly graze in a Kenyan conservation park as their species heads towards extinction.

whiterhino

One of the three remaining northern white rhinos in the world (Image source: The Guardian)

Jeanne Loring, a CIRM grantee and professor at The Scripps Research Institute, still sees a glimmer of hope in the form of stem cells. In an essay published yesterday in Genetic Engineering and Biotechnology News, Loring describes her research team’s efforts to apply stem cell technology toward saving the Northern White Rhino and other endangered species.

Their efforts began about ten years ago in 2007, the same year that Shinya Yamanaka’s lab first reported that human fibroblasts, collected from a skin sample, can be reprogrammed into an embryonic stem cell-like state with the capacity to indefinitely make copies of themselves and to specialize into almost every cell type of the body. The properties of these induced pluripotent stem (iPS) cells have provided an important means for studying all sorts of human diseases in a lab dish and for deriving potential cell therapies.

FrozenZoo® and iPS Cells: A Modern Day Noah’s Ark?

But it was a free tour at the San Diego Safari Park just two months after Yamanka’s discovery which inspired the Loring lab to chart this additional research path using iPS cells. In exchange for the free safari ride, the team reciprocated by chatting with Oliver Ryder, director of the San Diego Zoo Institute for Conservation Research, about using stem cells to help save endangered species. Ryder’s institute runs the FrozenZoo® a cell and tissue bank containing thousands of frozen samples from a diverse set of species. In her essay, Loring recounts what happened after the visit:

“It was obvious to us: why not try to reprogram fibroblasts from the FrozenZoo®? When my group returned to the lab from the safari, I asked them: who would like to try to reprogram fibroblasts from an endangered species? It was far from a safe bet, but a young postdoctoral researcher who had recently joined my lab from Israel said that she’d love to give it a try. Inbar Friedrich Ben-Nun spent the next couple of years trying out methods in parallel on human cells and fibroblasts from the zoo. We chose fibroblasts from the drill because it is [an endangered] primate, making it more likely that the technology used for humans would work.

Oliver [Ryder] chose the northern white rhino, a particular favorite of his, and one of the world’s most endangered mammals.  Through hard work and insight, Inbar reprogrammed both species, and in 2011, we published the first report of making iPSCs from endangered species (Ben-Nun, et al., 2011). Nature Methods featured our work, with a cover illustration of an ark stuffed with endangered animals.”

 

 

 

So how exactly would these iPS cells be used to save the northern white rhino and other animals from the brink of extinction? Last December, Ben-Nun along with 20 other scientists and zoologists from four continents met in Vienna to map out a strategy. They published their plan on May 3rd in Zoo Biology.

The Stem Cell-Based Plan to Save the Northern White
In the first phase, an in vitro fertilization (IVF) procedure for the rhino – never before attempted – will be worked out. Frozen sperm samples from four now-deceased rhinos plus one sample from Sudan are ready for IVF. Researchers then hope to collect eggs from Najin and Fatu and implant embryos in surrogates of a related species, the southern white rhino. However, even if IVF is successful, the offspring would not represent enough genetic diversity to ultimately thrive as a species in the wild. So in the second phase, iPS cells will be generated using tissue fibroblast samples from several more northern whites that were banked in The FrozenZoo®. Those iPS cells will be specialized into sperm and eggs to provide a larger, more diverse set of embryos which again will be implanted in surrogate rhinos. Breeding animals using iPS-derived sperm and eggs has only been successful in mice so much work remains.

“Does this plan have any chance of succeeding?” Loring asks. Her response is cautiously optimistic:

“I know it will be difficult, but I think it’s not impossible. Perhaps the most important advance is that such a diverse group agreed on a plan—it wasn’t just a stem cell biologist like me imagining how the cells might be used, but rather a whole chain of experts who can imagine how to accomplish each step.”

 

Not all experts agree with this strategy. In a Nature News interview back in May, Michael Knight, chair of the International Union for Conservation Nature’s African Rhino Specialist Group, expressed concerns that the effort is misdirected:

“It’s Star Trek-type science. They should not be pushing this idea that they’re saving a species. If you want to save a [rhino] species, put your money into southern white conservation.”

IMHO
Knight’s point is well-taken that conventional conservation approaches are critical to ensure that the southern white rhino doesn’t meet the same disastrous fate as the northern white. But if the funding is available, it seems worth the effort to also attempt this innovative iPS strategy, a technology that’s deep in development now and not awaiting Captain Kirk’s distant Star Trek future.

Outsmarting cancer’s deadly tricks

Cancer cells are devious monsters that kill people by sabotaging normal cell functions toward a path of uncontrolled cell growth. Without an effective treatment, aggressive cancers can crowd out healthy tissue and ultimately cause organ failure and death. This devastation by design makes it seem as though a cancer cell has a mind of its own but in reality it’s all due to mindless mutations in DNA. Gaining a deep understanding of those mutations provides scientists with insights into the molecular mechanisms of cancer which can help pinpoint targets for potential cancer treatments.

A team at The Scripps Research Institute (TSRI) followed the trail of such a mutation in a gene called POT1. Today in Cell Reports the researchers, funded in part by CIRM, describe their identification of a novel mechanism for cancer progression in cells carrying the POT1 mutation and they also speculate on the development on a unique therapeutic strategy.

Chromosomes go to pot without POT1
The POT1 protein is one component of shelterin, a multi-protein structure that binds to and protects telomeres, a region of DNA found at the ends of chromosomes. The team found that when POT1’s function is disrupted by mutation, the telomeres become vulnerable to damage which leads to chromosome instability. As a result, many regions of DNA on the chromosomes get rearranged leading to further gene mutations that in turn can accelerate the process of cancerous growth.

Telomere_caps

Human chromosomes (grey) capped by telomeres (white) Wikipedia

However, in the case of POT1 mutations, the DNA damage in the unstable chromosomes stimulates an enzyme called ATR that’s known to shut down cell division and initiate apoptosis, or programmed cell death. Now, unless I’m missing something, cells that have either stopped dividing or even died would seem to be the opposite of cancer progression. So why then are POT1 mutations found in a number of cancers such as leukemia, melanoma (skin cancer) and glioma (brain cancer)? As TSRI Associate Professor Eros Lazzerini Denchi, a co-leader on the publication, mentions in a press release, this conundrum presented an opportunity to better understand POT1 related cancers:

lazzerini_denchi

Eros Lazzerini Denchi

“Somehow those cells found a way to survive—and thrive. We thought that if we could understand how that happens, maybe we could find a way to kill those cells.”

 

Mutant POT1 and p53: diabolical partners in cancer progression
The team looked for answers by studying the POT1 mutation in the presence of a mutated form of the p53 tumor suppressor gene, found in over 50% of all human cancers. Mice bred with the POT1 mutation alone formed no cancers while those animals with the p53 mutation alone developed T cell lymphomas, a type of immune system cancer, by 20 weeks and survived 24 weeks. Mice with both mutations fared much worse with median survival times of just 17 weeks. So somehow the p53 mutation was bringing out the potential of the POT1 mutation to cause aggressive cancer growth.

Further experiments revealed that the p53 mutation quashed the ATR enyzme’s programmed cell death signal which the team had shown was stimulated by the POT1 mutation. As a result, the cells avoided programmed cell death. Because the cells had no mechanism to die, more cancer-causing mutations had the opportunity to develop from the chromosome instability caused by the POT1 mutation.

The bright side to this diabolical cooperation between mutant POT1 and p53 is that it presents a possible opening for new treatment strategies. It turns out that no cell, not even a cancerous one, can survive in the complete absence of ATF. Since cells with the POT1 mutations already have a reduced level of ATF, the authors suggest that delivery of low doses of ATF inhibitors, which have already been developed for the clinic, could kill cancer cells without affecting healthy cells. No doubt the team is eager to follow up on this hypothesis.

It’s comforting to know that there are crafty scientists out there who are closing in on ways to outsmart the sneaky tactics of cancer cells. And it wouldn’t be possible without this fundamental research, as Lazzerini Denchi points out:

“This study shows that by looking at basic biological questions, we can potentially find new ways to treat cancer.”

 

Unlocking the brain’s secrets: scientists find over 100 unique mutations in brain cells

Your brain is made up of approximately 100 billion neurons. These are the cells that process information and pass along electrical and chemical signals to their other neuron buddies throughout the body to coordinate thoughts, movement, and many other functions. It’s no small task to create the intricate neuronal network that is the backbone of the central nervous system. If any of these neurons or a group of neurons acquire genetic mutations that alter their function, a lot can go wrong.

The genetic makeup of neurons is particularly interesting because it appears that each neuron has its own unique genome. That means 100 billion different genomes in a single cell type in the brain. Scientists suggest that this “individuality” could explain why monozygotic, or identical, twins have different personalities and susceptibilities to neurological disorders or mental illnesses and why humans develop brain diseases or cancer over time.

To understand what a genome of a cell looks like, you need to sequence its genetic material, or the DNA, that’s housed in a cell’s nucleus. Sequencing the genome of an individual cell is hard to do accurately with our current technology, so scientists have developed clever alternatives to get a front-row view into the workings of neuronal genomes.

Cloning mouse neurons reveals 100+ unique genetic mutations

One such method was published recently in the journal Neuron by a CIRM-funded team from The Scripps Research Institute (TSRI). Led by senior author and Associate Professor at TSRI, Kristen Baldwin, the team took on the challenge of cloning individual mouse neurons to unlock the secrets of neuronal genomes. (For those who aren’t familiar with the term, cloning is a process that produces new cells or organisms that harbor identical genetic information from the originating cell.)

What they found from their cloning experiment was surprising: each neuron they sequenced had an average of more than 100 unique genetic mutations, and these mutations tended to appear in genes that were heavily used by neurons, something that is uncommon in cell types of other organs that tend to protect their frequently used genes. Their findings could help unravel the mystery behind some of the causes for diseases like Alzheimer’s and autism.

In a TSRI news release, Kristen Baldwin explained:

Kristen Baldwin

Kristen Baldwin

“Neuronal genomes have remained a mystery for a long time. The findings in this study and the extensive validation of genome sequencing-based mutation discovery that this method permits, open the door to additional studies of brain mutations in aging and disease, which may help us understand or treat cognitive decline in aging, neurodegeneration and neurodevelopmental diseases such as autism.”

Making mice with neuronal genomes

To clone individual neurons, the team took the nucleus of a single neuron and transplanted it into a mouse egg cell that lacked its own nucleus. The egg developed and matured all while copying and passing on the genetic information of the original mouse neuron. The team generated cloned embryonic stem cell lines from these eggs and were able to expand the stem cell lines to generate millions of stem cells that contained the same genetic material.

TSRI Research Assistant Alberto Rodriguez uses a tiny straw-like micropipette to pick up red fluorescent neurons and transfer their genomes into an egg.

TSRI scientists extract the nuclei of neurons and transfer their genomes into an egg. (Image courtesy of TSRI)

They made several different cloned stem cell lines representing different neuronal genomes and sequenced these lines to identify unique genetic mutations. They also were able to generate cloned stem cell lines from the neurons of older mice, and thus were able to track the accumulation of genetic mutations over time. Even more impressive, they made living mice that contained the cloned genomes of individual neurons in all of their cells, proving that neuronal genomes are compatible with development.

The team did report that not all neurons could be developed into cloned stem cell lines for reasons that they couldn’t fully explain, but they decided to focus on studying the cloned stem cell lines that were successful.

What does this mean for humans?

Baldwin explained that what was most surprising about their study was “that every neuron we looked at was unique – carrying more than 100 DNA changes or mutations that were not present in other cells.”

The next steps for their research are to explore why this diversity among neuronal genomes exists and how this could contribute to neurological disease in humans.

Co-first authors Jennifer Hazen and Gregory Faust.

Co-first authors Jennifer Hazen and Gregory Faust.

Co-first author Jennifer Hazen explains, “We need to know more about mutations in the brain and how they might impact cell function.”

Also mentioned in the news release, the team plans “to study neuronal genomes of very old mice and those with neurological diseases. They hope this work will lead to new insights and therapeutic strategies for treating brain aging and neurologic diseases caused by neuronal mutations.”


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A TWIST in mesenchymal stem cell trials: protein predicts therapy’s potential

Mesenchymal stem cells are adult stem cells with the potential to specialize into a somewhat limited number of cell types – those responsible for making fat, bone and cartilage. But MSCs are also known for their anti-inflammatory properties which are carried out via the release of protein factors.  This ability to dampen the immune system makes the MSC an extremely attractive source material for cell therapies. In fact, there are over 500  mesenchymal stem cell-based clinical trials testing treatments for diseases that target a wide range of tissues including spinal cord injury, diabetes, multiple sclerosis, respiratory disorders and graft versus host disease, just to name a few.

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Human mesenchymal stem cells grown in a single layer on the bottom of a flask; 4x magnification Image source: EuroStemCell

 MSCs and the Variability Problem
While some MSC-based human trials have had promising results in patients, other studies haven’t been as successful. A key culprit of these mixed results is the lack of standardization on what exactly is a MSC. It’s well documented that preparations of MSC vary significantly from one patient to the next. Even the composition of MSCs from one patient is far from a pure population of cells. And few of the cell surface markers used to define MSCs provide a measure of the cells’ function. This is a real problem for demonstrating the effectiveness and the marketability of MSC-based cell therapies which rely on the delivery of cell product with a consistent, well-defined composition and functional activity.

Help is now on the way based on research reported this week in EBioMedicine by a research team led by Professor Donald Phinney at the Florida campus of The Scripps Research Institute. In the study, the team found that the amount of TWIST1, a protein that regulates gene activity, in a given batch of MSCs could reliably predict the therapeutic effectiveness of those cells.

Meet TWIST1: predictor of a MSC therapy’s potential
They set their sights on TWIST1 because previous research described its important role in driving a MSC fate during human development. The team examined the natural variability of TWIST1 levels in human MSCs from several donors. They showed that lower levels of TWIST1 correlated to MSCs with stronger anti-inflammatory properties. Higher levels of TWIST1, on the other hand, were consistent with MSCs that induced angiogenesis, or blood vessel growth, another known ability of this versatile cell type. In another set of experiments, TWIST1 production was silenced using genetic tools. As predicted by the earlier results, these MSCs showed increased anti-inflammatory properties.

Move over Ritcher, Say Hello to the CLIP Scale

CLIPscale

The Clinical Indication Prediction (CLIP) scale. Image: Boregowda et al. EBioMedicine, Volume 4 , 62-73

Putting this data together, the team devised a scale they call Clinical Indication Prediction, or CLIP for short. The scale gives a clinical researcher an indication of the therapeutic potential of a given batch of donor MSCs based on the TWIST1 protein levels. This information could have a major impact on a clinical trial’s fate. Depending on the goal of a MSC-based cell therapy, a clinical team could set themselves up for failure before the trial even gets underway if they don’t take TWIST1 levels into account. First author Siddaraju V. Boregowda explains this scenario in a press release:

boregowda_siddaraju copy

Siddaraju V. Boregowda

“There are a number of clinical trials testing mesenchymal stem cells to treat arthritis. Since angiogenesis is a key part of the disease process, stem cells with high levels of TWIST1 (indicating they are more angiogenic) would not be beneficial. These cells might be helpful instead for indications such as peripheral vascular disease where new vascularization is beneficial. The proposed CLIP scale accurately predicts these indications and contra-indications.”

We’ll be keeping our eye on this exciting discovery to see if CLIP becomes an integral step in developing MSC-based cell therapies. If it pans out, the CLIP scale could help accelerate the development of new therapies by providing scientists with more clarity and confidence around classifying the identity of a MSC cell product. Stay tuned!

CIRM-funded study suggests methods to make pluripotent stem cells are safe

We live in an era where stem cell treatments are already being tested in human clinical trials for eye disease, spinal cord injury, and type 1 diabetes. The hope is that transplanting stem cells or their cell derivatives will replace diseased tissue, restore function, and cure patients – all while being safe and without causing negative side effects.

Safety will be the key to the future success of stem cell replacement therapies. We’ve learned our lesson from early failed gene therapy experiments where genetically altered stem cells that were supposed to help patients actually caused them to get cancer. Science has since developed methods of gene therapy that appear safe, but new concerns have cropped up around the safety of the methods used to generate pluripotent stem cells, which are considered a potential starting material for cell replacement therapies.

Stem cell reprogramming can cause problems

Induced pluripotent stem cells (iPS cells) cultured in a dish.

Induced pluripotent stem cells (iPS cells) cultured in a dish.

Induced pluripotent stem cells, or iPS cells, are a potential source of pluripotent stem cells for cell therapy. These cells are equivalent to embryonic stem cells but can be generated from adult tissue (such as skin or even blood) by reprogramming cells back to a pluripotent state. During cellular reprogramming, one set of genes is turned off and another set is turned on through a process called epigenetic remodeling. We don’t have time to explain epigenetics in this blog, but to be brief, it involves chromatin remodeling (chromatin is the complex of DNA and protein that make up chromosomes) and is essential for controlling gene expression.

To make healthy iPS cells, the intricate steps involved in cellular reprogramming and epigenetic remodeling have to be coordinated perfectly. Scientists worry that these processes aren’t always perfect and that cancer-causing mutations could be introduced that could cause tumors when transplanted into patients.

A CIRM-funded study published Friday in Nature Communications offers some relief to this potential roadblock to using reprogrammed iPS cells for cell therapy. Scientists from The Scripps Research Institute (TSRI) and the J. Craig Venter Institute (JCVI) collaborated on a study that assessed the safety of three common methods for generating iPS cells. Their findings suggest that these reprogramming methods are relatively safe and unlikely to give cancer-causing mutations to patients.

Comparing three reprogramming methods

In case you didn’t know, iPS cells are typically made by turning on expression of four genes – OCT4, SOX2, KLF4, and c-MYC – that maintain stem cells in a pluripotent state. Scientists can force an adult cell to express these genes by delivering extra copies into the cell. In this study, the scientists conducted a comparative genomic analysis of three commonly used iPS cell reprogramming methods (integrating retroviral vectors, non-integrating Sendai virus, and synthetic mRNAs) to search for potential cancer-causing mutations in the DNA of the iPS cells.

Unlike previous studies that focused on finding a single type of genetic mutation in reprogrammed iPS cells, the group looked at multiple types of genetic mutations – from single nucleotide changes in DNA to large structural variations – by comparing whole-genome sequencing data of the starting parental cells (skin cells) to iPS cells.

They concluded that the three reprogramming methods generally do not cause serious problems and hypothesized that cancer-causing mutations likely happen at a later step after the iPS cells are already made, an issue the team is addressing in ongoing work.

They explained in their publication:

“We detected subtle differences in the numbers of [genetic] variants depending on the method, but rarely found mutations in genes that have any known association with increased cancer risk. We conclude that mutations that have been reported in iPS cell cultures are unlikely to be caused by their reprogramming, but instead are probably due to the well-known selective pressures that occur when hPSCs [human pluripotent stem cells] are expanded in culture.”

The safety of patients comes first

Senior authors on the study, Dr. Jeanne Loring from TSRI and Dr. Nicholas Schork from JCVI, explained in a TSRI News Release that the goal of this study was to make sure that the reprogramming methods used to make iPS cells were safe for patients.

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Jeanne Loring

“We wanted to know whether reprogramming cells would make the cells prone to mutations,” said Jeanne Loring, “The answer is ‘no.’ The methods we’re using to make pluripotent stem cells are safe.”

 

Nicholas Schork added:

Nicholas Schork

Nicholas Schork

“The safety of patients comes first, and our study is one of the first to address the safety concerns about iPSC-based cell replacement strategies and hopefully will spark further interest.”

 

 

Moving from bench to clinic

It’s good news that reprogramming methods are relatively safe, but the fact that maintaining and expanding iPS cells in culture causes cancerous mutations is still a major issue that scientists need to address.

Jeanne Loring recognizes this important issue and says that the next steps are to use similar genomic analyses to assess the safety of reprogrammed iPS cells before they are used in patients.

“We need to move on to developing these cells for clinical applications,” said Loring. “The quality control we’re recommending is to use genomic methods to thoroughly characterize the cells before you put them into people.”