A New Way to Replace Damaged or Missing Cells

When certain cells in our bodies are missing or nonfunctional, the only current options are to treat the symptoms with drugs or try to acquire transplants. But what if cells in our own bodies could be transformed to take on the missing functions? What if we could convert cells from other organs to function as neurons after a stroke; cardiomyocytes to address heart disease; gland cells to address endocrine diseases, or cartilaginous cells to address joint deterioration? This may be a real possibility thanks to funding from the Caltech Innovation Initiative, a philanthropically funded internal grant program designed to provide research funds to high-risk but potentially high-reward projects that could produce disruptive technologies with practical applications in the marketplace.

Working with Caltech colleague Isabelle Peter and Yong Zhu of the start-up Vivoscript, Caltech biologist Eric Davidson applied seed funds from the Caltech Innovation Initiative to test experimentally a new theory of cell-fate transformation that he and Peter codeveloped. The theory posits that cells in a living patient can be converted to work as completely different types of cells. Davidson and colleagues have transformed liver cells into cells that function like pancreatic cells, at least to the extent that they produce insulin.

The work, inspired by the group's studies of gene regulatory networks in sea urchin embryos, begins with putting suites of protein molecules called transcription factors near the cells targeted for transformation. These proteins have been engineered in a proprietary way, in collaboration with Vivoscript, so that they will be taken up by cells and will travel into the nuclei. The new proteins control cellular activity by regulating gene expression. Researchers want to be able to predict the complete suite of proteins needed to durably reproduce the gene expression — and resulting normal functions — of one kind of cell in a different original cell.

These new possibilities for therapies based on cell transformation avoid the expense and hazards associated with use of genetically transformed cells or in vitro differentiated stem cells.

If the approach is generalizable and predictable, it could answer the urgent need for a safe, inexpensive way to replace damaged or missing cells. Caltech has applied for patent protection. Davidson's next step is to demonstrate the degree to which the conversion of liver cells into pancreatic cells works.  

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Friday, September 27, 2013 to Sunday, September 29, 2013

Biology & Biological Engineering Annual Retreat

Caltech to Offer Online Courses through edX

To expand its involvement in online learning, the California Institute of Technology will offer courses through the online education platform edX beginning this October.

The edX course platform is an online learning initiative launched in 2012 by founding partners Harvard University and the Massachusetts Institute of Technology (MIT). Caltech's rigorous online course offerings will join those of 28 other prestigious colleges and universities in the edX platform's "xConsortium."

This new partnership with edX comes one year after Caltech offered three courses through the online learning platform Coursera in fall 2012. The Institute will now offer courses through both platforms.

"Coursera and edX have some foundational differences which are of interest to the faculty," says Cassandra Horii, director of teaching and learning programs at Caltech. Both organizations offer their courses at no cost to participating students; edX, however, operates as a nonprofit and plans to partner with only a small number of institutions, whereas Coursera—a for-profit, self-described "social entrepreneurship company"—partners with many institutions and state university systems.

The two platforms also emphasize different learning strategies, says Horii. "Coursera has a strong organizational principle built around lectures, so a lot of the interactivity is tied right into the video," she says. Though edX still enables the use of video lectures, a student can customize when he or she would like to take quizzes and use learning resources. In addition, edX allows faculty to embed a variety of learning materials—like textbook chapters, discussions, diagrams, and tables—directly into the platform's layout.

In the future, data collected from both platforms could provide valuable information about how students best learn certain material, especially in the sciences. "Caltech occupies this advanced, really rigorous scientific education space, and in general our interest in these online courses is to maintain that rigor and quality," Horii says. "So, with these learning data, we have some potential contributions to make to the general understanding of learning in this niche that we occupy."

Even before joining edX and Coursera, Caltech had already become an example in the growing trend of Massive Open Online Courses (MOOCs). Yaser Abu-Mostafa, professor of electrical engineering and computer science, developed his own MOOC on machine learning, called "Learning from Data," and offered it on YouTube and iTunes U beginning in April 2012.

Since its debut, Abu-Mostafa's MOOC has reached more than 200,000 participants, and it received mention in the NMC Horizon Report: 2013 Higher Education Edition—the latest edition of an annual report highlighting important trends in higher education. The course will be offered again in fall 2013 on iTunes U, and is now also open for enrollment in edX.

Although Caltech is now actively exploring several outlets for online learning, the Institute's commitment to educational outreach is not a recent phenomenon. In the early 1960s, Caltech physicist Richard Feynman reorganized the Institute's introductory physics course, incorporating contemporary research topics and making the course more engaging for students. His lectures were recorded and eventually incorporated into a widely popular physics book, The Feynman Lectures on Physics, which has sold millions of copies in a dozen languages.

Continuing in the tradition set by Feynman, the MOOCs at Caltech seek to provide a high-quality learning environment that is rigorous but accessible. "No dumbing down of courses for popular consumption . . . no talking over people's heads either; at Caltech, we explain things well because we understand them well," adds Abu-Mostafa.

More information on Caltech's online learning opportunities is available on the Online Education website.

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Friday, October 4, 2013

Undergraduate Teaching Assistant Orientation

A Home for the Microbiome

Caltech biologists identify, for the first time, a mechanism by which beneficial bacteria reside and thrive in the gastrointestinal tract

The human body is full of tiny microorganisms—hundreds to thousands of species of bacteria collectively called the microbiome, which are believed to contribute to a healthy existence. The gastrointestinal (GI) tract—and the colon in particular—is home to the largest concentration and highest diversity of bacterial species. But how do these organisms persist and thrive in a system that is constantly in flux due to foods and fluids moving through it? A team led by California Institute of Technology (Caltech) biologist Sarkis Mazmanian believes it has found the answer, at least in one common group of bacteria: a set of genes that promotes stable microbial colonization of the gut.

A study describing the researchers' findings was published as an advance online publication of the journal Nature on August 18.    

"By understanding how these microbes colonize, we may someday be able to devise ways to correct for abnormal changes in bacterial communities—changes that are thought to be connected to disorders like obesity, inflammatory bowel disease and autism," says Mazmanian, a professor of biology at Caltech whose work explores the link between human gut bacteria and health.

The researchers began their study by running a series of experiments to introduce a genus of microbes called Bacteriodes to sterile, or germ-free, mice. Bacteriodes, a group of bacteria that has several dozen species, was chosen because it is one of the most abundant genuses in the human microbiome, can be cultured in the lab (unlike most gut bacteria), and can be genetically modified to introduce specific mutations.

"Bacteriodes are the only genus in the microbiome that fit these three criteria," Mazmanian says.

Lead author S. Melanie Lee (PhD '13), who was an MD/PhD student in Mazmanian's lab at the time of the research, first added a few different species of the bacteria to one mouse to see if they would compete with each other to colonize the gut. They appeared to peacefully coexist. Then, Lee colonized a mouse with one particular species, Bacteroides fragilis, and inoculated the mouse with the same exact species, to see if they would co-colonize the same host. To the researchers' surprise, the newly introduced bacteria could not maintain residence in the mouse's gut, despite the fact that the animal was already populated by the identical species.

"We know that this environment can house hundreds of species, so why the competition within the same species?" Lee says. "There certainly isn't a lack of space or nutrients, but this was an extremely robust and consistent finding when we tried to essentially 'super-colonize' the mice with one species."

To explain the results, Lee and the team developed what they called the "saturable niche hypothesis." The idea is that by saturating a specific habitat, the organism will effectively exclude others of the same species from occupying that niche. It will not, however, prevent other closely related species from colonizing the gut, because they have their own particular niches. A genetic screen revealed a set of previously uncharacterized genes—a system that the researchers dubbed commensal colonization factors (CCF)—that were both required and sufficient for species-specific colonization by B. fragilis.

But what exactly is the saturable niche? The colon, after all, is filled with a flowing mass of food, fecal matter and bacteria, which doesn't offer much for organisms to grab onto and occupy.

"Melanie hypothesized that this saturable niche was part of the host tissue"—that is, of the gut itself—Mazmanian says. "When she postulated this three to four years ago, it was absolute heresy, because other researchers in the field believed that all bacteria in our intestines lived in the lumen—the center of the gut—and made zero contact with the host…our bodies. The rationale behind this thinking was if bacteria did make contact, it would cause some sort of immune response."

Nonetheless, when the researchers used advanced imaging approaches to survey colonic tissue in mice colonized with B. fragilis, they found a small population of microbes living in miniscule pockets—or crypts—in the colon. Nestled within the crypts, the bacteria are protected from the constant flow of material that passes through the GI tract. To test whether or not the CCF system regulated bacterial colonization within the crypts, the team injected mutant bacteria—without the CCF system—into the colons of sterile mice. Those bacteria were unable to colonize the crypts.

"There is something in that crypt—and we don't know what it is yet—that normal B. fragilis can use to get a foothold via the CCF system," Mazmanian explains. "Finding the crypts is a huge advance in the field because it shows that bacteria do physically contact the host. And during all of the experiments that Melanie did, homeostasis, or a steady state, was maintained. So, contrary to popular belief, there was no evidence of inflammation as a result of the bacteria contacting the host. In fact, we believe these crypts are the permanent home of Bacteroides, and perhaps other classes of microbes."

He says that by pinpointing the CCF system as a mechanism for bacterial colonization and resilience, in addition to the discovery of crypts in the colon that are species specific, the current paper has solved longstanding mysteries in the field about how microbes establish and maintain long-term colonization.

"We've studied only a handful of organisms, and though they are numerically abundant, they are clearly not representative of all the organisms in the gut," Lee says. "A lot of those other bacteria don't have CCF genes, so the question now is: Do those organisms somehow rely on interactions with Bacteroides for their own colonization, or their replication rates, or their localization?"

Suspecting that Bacteroides are keystone species—a necessary factor for building the gut ecosystem—the researchers next plan to investigate whether or not functional abnormalities, such as the inability to adhere to crypts, could affect the entire microbiome and potentially lead to a diseased state in the body.

"This research highlights the notion that we are not alone. We knew that bacteria are in our gut, but this study shows that specific microbes are very intimately associated with our bodies," Mazmanian says. "They are living in very close proximity to our tissues, and we can't ignore microbial contributions to our biology or our health. They are a part of us."

Funding for the research outlined in the Nature paper, titled "Bacterial colonization factors control specificity and stability of the gut microbiota," was provided by the National Institutes of Health and the Crohn's and Colitis Foundation of America. Additional coauthors were Gregory Donaldson and Silva Boyajian from Caltech and Zbigniew Mikulski and Klaus Ley from the La Jolla Institute for Allergy and Immunology in La Jolla, California.

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Katie Neith
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Arnold Appointed New Director of Rosen Bioengineering Center

Now in its sixth year of exploring the intersection between biology and engineering, the Donna and Benjamin M. Rosen Bioengineering Center has chosen Caltech professor Frances Arnold as its new director. Arnold, the Dick and Barbara Dickinson Professor of Chemical Engineering, Bioengineering and Biochemistry began her tenure as director on June 1.

A recipient of the 2011 National Medal of Technology and Innovation, Arnold pioneered methods of "directed evolution" – processes now widely used to create biological catalysts that are important in the production of fuels from renewable resources. She was selected for the directorship because "of her demonstrated leadership in the field of bioengineering," says Stephen Mayo, William K. Bowes Jr. Foundation Chair of the Division of Biology and Biological Engineering.

The Rosen Center supports bioengineering research through the funding of fellows and faculty from many disciplines, including applied physics, chemical engineering, synthetic biology, and computer science.

"Bioengineering is an incredibly exciting field right now," Arnold says. "Solutions to some of the biggest problems in science, medicine, and sustainability will come from the interface between biology and engineering, and Caltech is well positioned to be at the forefront. The Rosen Center will help make that happen with innovative programs for bioengineering research and education."

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A Secret to Making Macrophages

Caltech researchers find a key in cell-cycle duration

Biologists at the California Institute of Technology (Caltech) have worked out the details of a mechanism that leads undifferentiated blood stem cells to become macrophages—immune cells that attack bacteria and other foreign pathogens. The process involves an unexpected cycle in which cell division slows, leading to an increased accumulation of a particular regulatory protein that in turn slows cell division further. The finding provides new insight into how stem cells are guided to generate one cell type as opposed to another.

Previous research has shown that different levels of a key regulatory protein called PU.1, which is involved in the new cycle, are important for the production of at least four different kinds of differentiated blood cells. For example, levels of PU.1 need to increase in order for macrophages to form, but must decrease during the development of another type of white blood cell known as the B cell. Precisely how such PU.1-level changes occur and are maintained in the cells has been unclear. But by observing differentiation in both macrophages and B cells, the Caltech team discovered something unusual in the feedback loop that produces macrophages. Their findings appear in the current issue of Science Express.

"Our results explain how blood stem cells and related progenitor cells can differentiate into macrophages and slow down their cell cycle, coordinating these two processes at the same time," says lead author Hao Yuan Kueh, a postdoctoral scholar at Caltech who works with biologists Michael Elowitz and Ellen Rothenberg, who were both principal investigators on the study. "We are excited about this because it means other systems could also use this mechanism to coordinate cell proliferation with differentiation."

In the study, the researchers captured movies of blood stem cells taken from transgenic mice. The cells expressed a green fluorescent protein that serves as an indicator of PU.1 levels in the cell: the brighter the cells appeared in the movies, the more PU.1 was present. By measuring PU.1 levels over time using this indicator, the scientists were able to monitor changes in the rate of PU.1's synthesis.

PU.1 can work through a positive feedback loop, binding to its own DNA regulatory sequence to stimulate its own production in a self-reinforcing manner. This type of loop is thought to be a general mechanism that allows a stem cell to switch into a differentiated state. In the case of PU.1, the process cranks up to produce macrophages, for example, and turns down to produce B cells.

And, indeed, when the researchers looked at B cell development, they saw what they expected: developing B cells decreased PU.1 levels by putting the brakes on the production of the protein.

The surprise came when they observed macrophages. Although the amount of PU.1 in the cells increased when the stem cells became macrophages, the researchers saw no change in the rate of PU.1 synthesis.

So where was the increase coming from? Upon investigation, the researchers observed that cells increased their PU.1 levels simply by slowing down their rate of division. With fewer cells being produced as the rate of PU.1 production marched steadily on, higher levels of the PU.1 protein were able to accumulate in the cells. Indeed, by slowing down the cell cycle, the researchers found that they could raise PU.1 levels enough to prompt the generation of macrophages. This result suggested that a different type of positive feedback loop might be responsible for the decisive final increase in PU.1 levels during macrophage differentiation.

"This work shows the amazing power of movies of individual cells in deciphering the dynamics of gene circuits," says Elowitz, who is a professor of biology and bioengineering at Caltech and an investigator with the Howard Hughes Medical Institute. "Just by following how the amount of PU.1 protein changed over time in a single cell, one can see directly that cells use a very different kind of feedback architecture than we usually associate with cellular differentiation."

Time-lapse movie of blood progenitor cells dividing and differentiating in culture. The brightness of green fluorescence indicates the amount of the regulatory protein PU.1 present in each cell. Green fluorescence images are acquired at a lower frame rate compared to the gray bright-field images of the cells. Time is given in hours.

To test what kind of positive feedback loop might control these events, the researchers forced cells to express extra PU.1, and measured its effect on the cells' own PU.1. They found that the extra PU.1 did not boost the cell's own PU.1 synthesis rate any further, but instead slowed the rate of cell division, causing PU.1 to accumulate to higher levels in the cells—an effect that slowed the cell cycle further.

"The key to this mechanism is that PU.1 is a very stable protein," says Rothenberg, the Albert Billings Ruddock Professor of Biology at Caltech. "Its central role in blood cell development has come from the fact that it collaborates with different regulatory protein partners to guide stem cells to make different cell types. We've known for some time that the exact ratios between PU.1 and its partners are important in these decisions, but it has been hard to see how the cells can manage to control the balance between so many of these different regulators with such precision. The beauty of this mechanism is that this ratio can be controlled simply by altering cell-cycle length. This shows us a new tool that factors like PU.1 and its collaborators can use to guide stem cells into precise developmental paths."

The team also used mathematical modeling to test the properties of a feedback loop that relies on the length of the cell cycle. They were able to show that a system that incorporated both the new loop and the PU.1-production feedback loop was able to account for three distinct levels of PU.1—one corresponding to B cells, one to progenitor cells, and one to macrophages.

"That was a proof-of-principle that this type of architecture can work," Kueh says. "The modeling will also help us to generate predictions for future studies."

In addition to Kueh, Elowitz, and Rothenberg, the paper, titled "Positive feedback between PU.1 and the cell cycle controls myeloid differentiation," is also coauthored by Ameya Champhekar, a postdoctoral scholar at Caltech, and Stephen Nutt, head of the Division of Molecular Immunology at the Walter and Eliza Hall Institute of Medical Research in Parkville, Victoria, Australia. The work was supported by a CRI Irvington Postdoctoral Fellowship, an Australian Research Council Future Fellowship, the Victorian State Government Operational Infrastructure Support, the National Health and Medical Research Council of Australia, the National Institutes of Health, the Albert Billings Ruddock Professorship, the Al Sherman Foundation, and the Louis A. Garfinkle Memorial Laboratory Fund.

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Kimm Fesenmaier
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Thursday, September 26, 2013
Ramo Auditorium

Graduate TA Orientation & Teaching Conference

New Research Sheds Light on M.O. of Unusual RNA Molecules

The genes that code for proteins—more than 20,000 in total—make up only about 1 percent of the complete human genome. That entire thing—not just the genes, but also genetic junk and all the rest—is coiled and folded up in any number of ways within the nucleus of each of our cells. Think, then, of the challenge that a protein or other molecule, like RNA, faces when searching through that material to locate a target gene.

Now a team of researchers led by newly arrived biologist Mitchell Guttman of the California Institute of Technology (Caltech) and Kathrin Plath of UCLA, has figured out how some RNA molecules take advantage of their position within the three-dimensional mishmash of genomic material to home in on targets. The research appears in the current issue of Science Express.

The findings suggest a unique role for a class of RNAs, called lncRNAs, which Guttman and his colleagues at the Broad Institute of MIT and Harvard first characterized in 2009. Until then, these lncRNAs—short for long, noncoding RNAs and pronounced "link RNAs"—had been largely overlooked because they lie in between the genes that code for proteins. Guttman and others have since shown that lncRNAs scaffold, or bring together and organize, key proteins involved in the packaging of genetic information to regulate gene expression—controlling cell fate in some stem cells, for example.

In the new work, the researchers found that lncRNAs can easily locate and bind to nearby genes. Then, with the help of proteins that reorganize genetic material, the molecules can pull in additional related genes and move to new sites, building up a "compartment" where many genes can be regulated all at once.

"You can now think about these lncRNAs as a way to bring together genes that are needed for common function into a single physical region and then regulate them as a set, rather than individually," Guttman says. "They are not just scaffolds of proteins but actual organizers of genes."

The new work focused on Xist, a lncRNA molecule that has long been known to be involved in turning off one of the two X chromosomes in female mammals (something that must happen in order for the genome to function properly). Quite a bit has been uncovered about how Xist achieves this silencing act. We know, for example, that it binds to the X chromosome; that it recruits a chromatin regulator to help it organize and modify the structure of the chromatin; and that certain distinct regions of the RNA are necessary to do all of this work. Despite this knowledge, it had been unknown at the molecular level how Xist actually finds its targets and spreads across the X chromosome.

To gain insight into that process, Guttman and his colleagues at the Broad Institute developed a method called RNA Antisense Purification (RAP) that, by sequencing DNA at high resolution, gave them a way to map out exactly where different lncRNAs go. Then, working with Plath's group at UCLA, they used their method to watch in high resolution as Xist was activated in undifferentiated mouse stem cells, and the process of X-chromosome silencing proceeded.

"That's where this got really surprising," Guttman says. "It wasn't that somehow this RNA just went everywhere, searching for its target. There was some method to its madness. It was clear that this RNA actually used its positional information to find things that were very far away from it in genome space, but all of those genes that it went to were really close to it in three-dimensional space."

Before Xist is activated, X-chromosome genes are all spread out. But, the researchers found, once Xist is turned on, it quickly pulls in genes, forming a cloud. "And it's not just that the expression levels of Xist get higher and higher," Guttman says. "It's that Xist brings in all of these related genes into a physical nuclear structure. All of these genes then occupy a single territory."

The researchers found that a specific region of Xist, known as the A-repeat domain, that is known to be vital for the lncRNA's ability to silence X-chromosome genes is also needed to pull in all the genes that it needs to silence. When the researchers deleted the domain, the X chromosome did not become inactivated, because the silencing compartment did not form.

One of the most exciting aspects of the new research, Guttman says, is that it has implications beyond just explaining how Xist works. "In our paper, we talk a lot about Xist, but these results are likely to be general to other lncRNAs," he says. He adds that the work provides one of the first direct pieces of evidence to explain what makes lncRNAs special. "LncRNAs, unlike proteins, really can use their genomic information—their context, their location—to act, to bring together targets," he says. "That makes them quite unique."  

The new paper is titled "The Xist lncRNA exploits three-dimensional genome architecture to spread across the X-chromosome." Along with Guttman and Plath, additional coauthors are Jesse M. Engreitz, Patrick McDonel, Alexander Shishkin, Klara Sirokman, Christine Surka, Sabah Kadri, Jeffrey Xing, Along Goren, and Eric Lander of the Broad Institute of Harvard and MIT; as well as Amy Pandya-Jones of UCLA. The work was funded by an NIH Director's Early Independence Award, the National Human Genome Research Institute Centers of Excellence in Genomic Sciences, the California Institute for Regenerative Medicine, and funds from the Broad Institute and from UCLA's Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research. 

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Kimm Fesenmaier
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Biology Commencement 2013

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Julie Boucher
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