Scientist Identifies Protein Molecule Used to Maintain Adult Stem Cells in Fruit Flies

Scientist Identifies Protein Molecule Used to Maintain Adult Stem Cells in Fruit Flies 
Tuesday, 23 April 2013

Understanding exactly how stem cells form into specific organs and tissues is the holy grail of regenerative medicine. Now a UC Santa Barbara researcher has added to that body of knowledge by determining how stem cells produce different types of "daughter" cells in Drosophila (fruit flies). The findings appear today in the Proceedings of the National Academy of Sciences.

Denise Montell, Duggan Professor of Molecular, Cellular and Developmental Biology at UCSB, and colleagues studied the ovaries of fruit flies in order to see stem cells in their natural environment. Because these organisms are excellent models for understanding stem cell biology, researchers were able to shed light on the earliest stages of follicle cell differentiation, a previously poorly understood area of developmental biology.

"It is clear that the fundamental principles that control cell behaviour in simple animals are conserved and control the behaviour of our cells as well," she said.

"There is so much we can learn by studying simple organisms."

This is a schematic drawing of a 

Drosophila ovariole and a magnified

germanium. Credit: UCSB. 

Using a nuclear protein expressed in follicle stem cells (FSCs), the researchers found that castor, which plays an important role in specifying which types of brain cells are produced during embryonic development, also helps maintain FSCs throughout the life of the animal.

"Having identified this important protein molecule in fruit flies, we can test whether the human version of the protein is important for stem cells and their daughters as well," said Montell.

"The more we know about the molecules that govern stem cell behaviour, the closer we will get to control these cells."

This image shows Denise Montell,

University of California, Santa Barbara.

Credit: George Foulsham, UCSB. 

Her research team placed the evolutionarily conserved castor (Cas) gene, which encodes a zinc finger protein, in a genetic circuit with two other evolutionarily conserved genes, hedgehog (Hh) and eyes absent (Eya), to determine the fates of specific cell progeny (daughters). What's more, they identified Cas as a critical, tissue-specific target of Hh signalling, which not only plays a key role in maintaining follicle stem cells but also assists in the diversification of their progeny.

The study also shows that complementary patterns of Cas and Eya reveal the gradual differentiation of polar and stalk precursor cells at the earliest stages of their development. In addition, it provides a marker for cell fates and insight into the molecular and cellular mechanisms by which FSC progeny diverge into distinct fates.

Follicle cells undergo a binary choice during early differentiation. Those that turn into specialized cells found at the poles of egg chambers go on to make two cell types: polar and stalk. The three genes, Cas, Eya and Hh, work in various combinations, sometimes repressively, to determine which types of cells are formed. Cas is required for polar and stalk cell fate specification, while Eya is a negative regulator of these cells' fate. Hh is necessary for Cas to be expressed, and Hh signalling is essential to repress Eya.

"If you just had one of these markers, it was hard to tell what's going on," explained Montell.

"All the cells looked the same and you had no idea when or how the process occurred. But now we can actually see how the cells acquire different identities."

Hh also plays many roles in embryonic development, adult homeostasis, birth defects, and cancer. Hh antagonists are currently in clinical trials for the treatment of several types of cancer. However, Hh signalling is important in so many different cell types and tissues that systemic delivery of such inhibitors may cause serious side effects. Therefore identifying the essential, tissue-specific effectors of Hh has the potential to lead to the identification of more specific therapeutic targets.

Someday, targeted inhibition of Hh signalling may be effective in the treatment and prevention of many types of human cancers.

Source: University of California - Santa Barbara 

Contact: Julie Cohen

Reference:

Castor is required for Hedgehog-dependent cell-fate specification and follicle stem cell maintenance in Drosophila oogenesis

Yu-Chiuan Chang, Anna C.-C. Jang, Cheng-Han Lin, and Denise J. Montell

PNAS 2013 ;  April 22, 2013, doi:10.1073/pnas.1300725110

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Scientists Find Antibody that Transforms Bone Marrow Stem Cells Directly into Brain Cells

Scientists Find Antibody that Transforms Bone Marrow Stem Cells Directly into Brain Cells

Tuesday, 23 April 2013

In a serendipitous discovery, scientists at The Scripps Research Institute (TSRI) have found a way to turn bone marrow stem cells directly into brain cells.

Current techniques for turning patients' marrow cells into cells of some other desired type are relatively cumbersome, risky and effectively confined to the lab dish. The new finding points to the possibility of simpler and safer techniques. Cell therapies derived from patients' own cells are widely expected to be useful in treating spinal cord injuries, strokes and other conditions throughout the body, with little or no risk of immune rejection.

Scientists at the Scripps Research Institute

have found a simple way to turn bone marrow

stem cells directly into brain precursor cells,

such as those shown here. Credit: Image

courtesy of the Lerner lab, The Scripps Research

Institute. 

"These results highlight the potential of antibodies as versatile manipulators of cellular functions," said Richard A. Lerner, the Lita Annenberg Hazen Professor of Immunochemistry and institute professor in the Department of Cell and Molecular Biology at TSRI, and principal investigator for the new study.

"This is a far cry from the way antibodies used to be thought of — as molecules that were selected simply for binding and not function."

The researchers discovered the method, reported in the online Early Edition of the Proceedings of the National Academy of Sciences the week of April 22, 2013, while looking for lab-grown antibodies that can activate a growth-stimulating receptor on bone marrow cells. One antibody turned out to activate the receptor in a way that induces marrow stem cells — which normally develop into white blood cells — to become neural progenitor cells, a type of almost-mature brain cell.

Nature's Toolkit

Natural antibodies are large, Y-shaped proteins produced by immune cells. Collectively, they are diverse enough to recognize about 100 billion distinct shapes on viruses, bacteria and other targets. Since the 1980s, molecular biologists have known how to produce antibodies in cell cultures in the laboratory. That has allowed them to start using this vast, target-gripping toolkit to make scientific probes, as well as diagnostics and therapies for cancer, arthritis, transplant rejection, viral infections and other diseases.

In the late 1980s, Lerner and his TSRI colleagues helped invent the first techniques for generating large "libraries" of distinct antibodies and swiftly determining which of these could bind to a desired target. The anti-inflammatory antibody Humira®, now one of the world's top-selling drugs, was discovered with the benefit of this technology.

Hongkai Zhang, research associate in the

Lerner lab, which discovered a method for

rapidly finding antibodies that have a desired

effect on cells, not just a desired ability to

bind to a target. Credit: Photo by Cindy

Brauer. 

Last year, in a study spearheaded by TSRI Research Associate Hongkai Zhang, Lerner's laboratory devised a new antibody-discovery technique — in which antibodies is produced in mammalian cells along with receptors or other target molecules of interest. The technique enables researchers to determine rapidly not just which antibodies in a library bind to a given receptor, for example, but also which ones activate the receptor and thereby alter cell function.

Lab Dish in a Cell

For the new study, Lerner laboratory Research Associate Jia Xie and colleagues modified the new technique so that antibody proteins produced in a given cell are physically anchored to the cell's outer membrane, near its target receptors.

"Confining an antibody's activity to the cell in which it is produced effectively allows us to use larger antibody libraries and to screen these antibodies more quickly for a specific activity," said Xie. With the improved technique, scientists can sift through a library of tens of millions of antibodies in a few days.

In an early test, Xie used the new method to screen for antibodies that could activate the GCSF receptor, a growth-factor receptor found on bone marrow cells and other cell types. GCSF-mimicking drugs were among the first biotech bestsellers because of their ability to stimulate white blood cell growth — which counteracts the marrow-suppressing side effect of cancer chemotherapy.

The team soon isolated one antibody type or "clone" that could activate the GCSF receptor and stimulate growth in test cells. The researchers then tested an unanchored, soluble version of this antibody on cultures of bone marrow stem cells from human volunteers. Whereas the GCSF protein, as expected, stimulated such stem cells to proliferate and start maturing towards adult white blood cells, the GCSF-mimicking antibody had a markedly different effect.

"The cells proliferated, but also started becoming long and thin and attaching to the bottom of the dish," remembered Xie.

To Lerner, the cells were reminiscent of neural progenitor cells — which further tests for neural cell markers confirmed they were.

A New Direction

Changing cells of marrow lineage into cells of neural lineage — a direct identity switch termed "transdifferentiation" — just by activating a single receptor is a noteworthy achievement. Scientists do have methods for turning marrow stem cells into other adult cell types, but these methods typically require a radical and risky deprogramming of marrow cells to an embryonic-like stem-cell state, followed by a complex series of molecular nudges toward a given adult cell fate. Relatively few laboratories have reported direct transdifferentiation techniques.

"As far as I know, no one has ever achieved transdifferentiation by using a single protein — a protein that potentially could be used as a therapeutic," said Lerner.

Current cell-therapy methods typically assume that a patient's cells will be harvested, then reprogrammed and multiplied in a lab dish before being re-introduced into the patient. In principle, according to Lerner, an antibody such as the one they have discovered could be injected directly into the bloodstream of a sick patient. From the bloodstream it would find its way to the marrow, and, for example, convert some marrow stem cells into neural progenitor cells.

"Those neural progenitors would infiltrate the brain, find areas of damage and help repair them," he said.

While the researchers still aren't sure why the new antibody has such an odd effect on the GCSF receptor, they suspect it binds the receptor for longer than the natural GCSF protein can achieve, and this lengthier interaction alters the receptor's signalling pattern. Drug-development researchers are increasingly recognizing that subtle differences in the way a cell-surface receptor is bound and activated can result in very different biological effects. That adds complexity to their task, but in principle expands the scope of what they can achieve.

"If you can use the same receptor in different ways, then the potential of the genome is bigger," said Lerner.

Source: Scripps Research Institute 

Contact: Mika Ono

Reference:

Autocrine signaling based selection of combinatorial antibodies that transdifferentiate human stem cells

Jia Xie, Hongkai Zhang, Kyungmoo Yea, and Richard A. Lerner

PNAS April 23, 2013, doi:10.1073/pnas.1306263110 

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For more on stem cells and cloning, go to CellNEWS at

http://cellnews-blog.blogspot.com/

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Reinventing Drug Discovery using Stem Cells

Promising drug target for ALS

Thursday, 18 April 2013

Using a new stem-cell based drug screening technology with the potential to reinvent and greatly reduce the cost of the way new pharmaceuticals are developed, Harvard Stem Cell Institute (HSCI) researchers have found a compound more effective in protecting the neurons killed in amyotrophic lateral sclerosis (ALS) – Lou Gehrig's disease – than two drugs that failed in human clinical trials after hundreds of millions of dollars had been invested in them.

The new stem cell screening technique developed by Lee Rubin, a member of HSCI's Executive Committee and a professor in Harvard's Department of Stem Cell and Regenerative Biology, successfully predicted that the two drugs that eventually failed in the third and final stage of human testing would, in fact, fail.

"It's a deep, dark secret of drug discovery that very few drugs have been tested on human-diseased cells before being tested in a live person," said Rubin, who heads HSCI's program in translational medicine.

"We were interested in the notion that we can use stem cells to correct that situation."

Rubin's model is built on an earlier proof-of-concept developed by HSCI Principal Faculty member Kevin Eggan, who demonstrated that it was possible to move a neuron-based disease into a laboratory dish using stem cells carrying the genes of patients with the disease.

The c-Jun-mediated cell death pathway (marked

by red nuclei that are positive for phospho-cjun) is

activated in stem cell-derived motor neurons (green)

exposed to trophic factor withdrawal (upper left panel).

C-Jun activation and cell death are blocked by

kenpaullone, an inhibitor GSK-3 and HGK (MAP4K4)

kinases (lower right panel; kenpaullone structure

superimposed). Credit: Cell Stem Cell, Yang et al..

In a paper published today in the journal Cell Stem Cell, Rubin lays out how he and his colleagues applied their new method of stem cell-based drug discovery to ALS. The disease is associated with the progressive death of motor neurons, which pass information between the brain and the muscles. As cells die, people with ALS experience weakness in their limbs followed by rapid paralysis and respiratory failure. The disease typically strikes later in life. Ten percent of cases are genetically predisposed, but for most patients there is no known trigger.

Rubin's lab began by first studying the disease in mice, growing billions of motor neurons from mouse embryonic stem cells, half normal and half with a genetic mutation known to cause ALS.

Investigators starved the cells of nutrients and then screened five thousand drug-like molecules to find any that would keep the motor neurons alive.

Several hits were identified, but the molecule that best prolonged the life of both normal and ALS motor neurons was kenpaullone, previously known for blocking the action of an enzyme (GSK-3) that switches on and off several cellular processes, including cell growth and death.

"Shockingly, this molecule keeps cells alive better than the standard culture medium that everybody keeps motor neurons in," Rubin said.

Kenpaullone proved effective in several follow-up experiments that put mouse motor neurons in situations of certain death. Neuron survival increased in the presence of the molecule whether the cells were programmed to die or placed in a toxic environment.

After further investigation, Rubin's lab discovered kenpaullone's potency comes from its ability to also inhibit HGK – an enzyme that sets off a chain of reactions that leads to motor neuron death. This enzyme was not previously known to be important in motor neurons or associated with ALS, marking the discovery of a new drug target for the disease.

"I think that stem-cell screens will discover new compounds that have never been discovered before by other methods," Rubin said.

"I'm excited to think that someday one of them might actually be good enough to go into the clinic."

To find out if kenpaullone works in diseased human cells, Rubin's lab exposed patient motor neurons and motor neurons grown from human embryonic stem cells to the molecule, as well as two drugs that did well in mice but failed in phase III human clinical trials for ALS. Once again, kenpaullone increased the rate of neuron survival, while one drug saw little response, and the other drug failed to keep any cells alive.

According to Rubin, before kenpaullone could be used as a drug, it would need a substantial molecular makeover to make it better able to target cells and find its way into the spinal cord so it can access motor neurons.

"This is kind of a proof of principle on the do-ability of the whole thing," he said.

"I think it's possible to use this method to discover new drug targets and to pre-validate compounds on real human disease cells before putting them in the clinic."

In the meantime, Rubin's next steps will be to continue searching for better drug-like compounds that can inhibit HGK and thus enhance motor neuron survival. He believes that the new information that comes out of this research will be useful to academia and the pharmaceutical industry.

"These kinds of exploratory screens are hard to fund, so being part of the HSCI" – which provided some of the funding – "has been absolutely essential," Rubin said.

Source: Harvard University 

Contact: B. D. Colen

Reference:

A Small Molecule Screen in Stem-Cell-Derived Motor Neurons Identifies a Kinase Inhibitor as a Candidate Therapeutic for ALS

Yin M. Yang, Shailesh K. Gupta, Kevin J. Kim, Berit E. Powers, Antonio Cerqueira, Brian J. Wainger, Hien D. Ngo, Kathryn A. Rosowski, Pamela A. Schein, Courtney A. Ackeifi, Anthony C. Arvanites, Lance S. Davidow, Clifford J. Woolf, and Lee L. Rubin
Cell Stem Cell 18 April 2013, 10.1016/j.stem.2013.04.003

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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at

http://cellnews-blog.blogspot.com/

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