“Grow Your Own Transplant” May be Possible for Men with Type 1 Diabetes”

Researchers turn human testes cells into insulin-producing islet cells; diabetic mice were “cured” for a week.
Men with type 1 diabetes may be able to grow their own insulin-producing cells from their testicular tissue, say Georgetown University Medical Center (GUMC) researchers who presented their findings today at the American Society of Cell Biology's 50th annual meeting in Philadelphia.
Their laboratory and animal study is a proof of principle that human spermatogonial stem cells (SSCs) extracted from testicular tissue can morph into insulin-secreting beta islet cells normally found in the pancreas. And the researchers say they accomplished this feat without use of any of the extra genes now employed in most labs to turn adult stem cells into a tissue of choice.
“No stem cells, adult or embryonic, have been induced to secrete enough insulin yet to cure diabetes in humans, but we know SSCs have the potential to do what we want them to do, and we know how to improve their yield,” says the study’s lead investigator, G. Ian Gallicano, Ph.D., an associate professor in the Department of Cell Biology and director of the Transgenic Core Facility at GUMC.
Given continuing progress, Gallicano says his strategy could provide a unique solution to treatment of individuals with type 1 diabetes (juvenile onset diabetes). Several novel therapies have been tried for these patients, but each has drawbacks.
1- Transplanting islet cells from deceased donors can result in rejection, plus few such donations are available.
2-Researchers have also cured diabetes in mice using induced pluripotent stem (IPS) cells – adult stem cells that have been reprogrammed with other genes to behave like embryonic stem cells – but this technique can produce teratomas, or tumors, in transfected tissue, as well as problems stemming from the external genes used to create IPS cells, Gallicano says.
3-Instead of using IPS cells, the researchers turned to a readily available source of stem cells, the SSCs that are the early precursors to sperm cells. They retrieved these cells from deceased human organ donors.
Because SSCs already have the genes necessary to become embryonic stem cells, it is not necessary to add any new genes to coax them to morph into these progenitor cells, Gallicano says. “These are male germ cells as well as adult stem cells.”
“We found that once you take these cells out of the testes niche, they get confused, and will form all three germ layers within several weeks,” he says. “These are true, pluripotent stem cells.”

The research team took 1 gram of tissue from human testes and produced about 1 million stem cells in the laboratory. These cells showed many of the biological markers that characterize normal beta islet cells. They then transplanted those cells into the back of immune deficient diabetic mice, and were able to decrease glucose levels in the mice for about a week – demonstrating the cells were producing enough insulin to reduce hyperglycemia.
While the effect lasted only week, Gallicano says newer research has shown the yield can be substantially increased.
The research was funded in part by the American Diabetes Association, patient contributions to the GUMC Office of Advancement, support from GUMC diabetes specialist Stephen Clement, M.D., and a grant from GUMC.
Co-authors include Anirudh Saraswathula, a student at Thomas Jefferson High School for Science and Technology in Alexandria, Va. GUMC researchers Shenglin Chen Ph.D., Stephen Clement, M.D., Martin Dym, Ph.D., and Asif Zakaria, Ph.D., also contributed to the research. The authors report having no personal financial interests related to the study.
 
http://explore.georgetown.edu/news/?ID=54742&PageTemplateID=295

Pfizer reaches out to academia—again

Nature Biotechnology, doi:10.1038/nbt0111-3

Pfizer is rolling out a grand plan to draw out drug-development-ready research from academia through a series of collaborations with leading medical centers worldwide. The first collaboration, announced in November, is with the University of California, San Francisco (UCSF), to which the pharma giant will commit $85 million. Coincidentally, London-based GlaxoSmithKline, is launching a similar outreach program, but with a very different approach. Like Pfizer, it wants to access leading academic researchers with targets ripe for translation into the clinic. Its scope, however, is more modest and targeted, focused on individual scientists.

For Pfizer, the overall aim in setting up these Global Centers for Therapeutic Innovation (CTIs) is to move novel bio-therapeutics rapidly into human clinical trials—each project will aim to deliver a drug through phase 1 testing in five years. Pfizer expects five such initiatives to be up and running in 2011 in the United States, Europe and Asia. Assuming eight projects per CTI, this could bring dozens of differentiated biologics against new targets into the clinical pipeline.

The New York–based pharma will set up shop on each campus, contributing proprietary phage display libraries, peptide libraries and associated technologies for rapidly generating antibodies to be used as probes against the novel targets flagged by university researchers. Each CTI will be staffed with 20–25 Pfizer employees with expertise in cell-line generation, protein characterization and purification—the skill sets needed to rapidly identify and advance molecules into the clinic. All decision making, from the initial acceptance of proposals through the determination to start clinical testing, will be made by a joint steering committee. “The concept is to make a transition away from the vertically integrated R&D model into smaller, decentralized groups of a truly global nature,” says Pfizer's Anthony Coyle, who is heading up the program out of the company's Cambridge, Massachusetts, facilities.

As important, the CTI model creates a 50-50 joint relationship where the goals of the investigators and the company are aligned and both sides are empowered to succeed. “There has got to be a change in the mindset from 'We own this, you do this for us,'” says Coyle. The CTIs will seek out investigators who have already developed a hypothesis around a novel disease mechanism and are keen to translate their discoveries into drugs. “These will be projects where we can articulate very clearly at the beginning what the first-in-man study will be,” says Coyle. The strategy will be to define the mechanism of action and in parallel develop the appropriate drug to hit the target and also determine the right patient population to target with it.

The model “allows us to leverage all of the drug discovery capability in our organization—the ability to make clinical grade material, the finances to perform the right enabling toxicology studies, and the regulatory support to allow the investigator to realize the ambition and see the concept translated,” says Coyle. He also hopes to bypass animal modeling. “What's becoming clear to me is that the time you spend on in vivo validation has zero impact, in most cases, on whether you will be successful going into the clinic. Here we propose to define the mechanism based on a human in vitro system, very quickly, which is again aided by having our phage library right there with the individuals doing the research.”

Funding for CTI initiatives will follow a pre-negotiated template Pfizer will put down at each institution. The company will pay for one to three post docs for each participating laboratory and the steering committee will have access to a flexible fund used either for additional biology or to allow the joint project team to move a compound into trials. There will be two clinical milestone payments, at proof of mechanism and successful proof of concept. All joint inventions will be jointly owned, with Pfizer holding an exclusive option to license a drug after proof of mechanism. In the event Pfizer exercises its option, any jointly developed enabling intellectual property (IP) would be licensed from the institution. If Pfizer declines, IP and other joint assets revert to the institution, which could then partner with someone else.

“There's going to be less of an establishment of value going into this, and more of it saved for the negotiation about the IP, which is downstream,” says S. Claiborne Johnston, director of the UCSF Clinical and Translational Science Institute.

In the past, most collaborations, however, have failed to lead to new drugs. “I think they generally have failed because of the misalignment of the interests of the academic investigators and the industrial partners,” says David Mack of the venture firm Alta Partners, in San Francisco, either because the academics were driven by other basic research questions or because of a lack of appreciation for the cost, risk and time that drug development takes. “They see that they've created an asset that is worth a lot, but actually it's not worth a lot because all of the risk is ahead of us—investment capital, development, technical risk.”

But as grant funding proves ever harder to find, it's an opportune time for exploring new models. Plus, the venture capital industry is contracting significantly and is also shifting its focus, where possible, to more late-stage, downstream investments. The absence of an initial public offering market has made some of the investigators more realistic. “It's the right time for that kind of approach—getting them involved on a risk-sharing basis and setting some realistic near- to midterm milestones to achieve some value creation, even if it means then passing it on to Pfizer in exchange for a royalty,” says Mack. The ability to hit the group running with a program and have immediate access to Pfizer's development resources may also be attractive to academics who are either uncomfortable or impatient with the venture capital process, where initial fund-raising could take years.

But more experienced academic entrepreneurs might not want to trade control or more potential upside in exchange for expediency. Paul Schimmel of the Scripps Research Institute in La Jolla, California, believes that “To preserve their freedom and work in an academic-like way, they'll probably want to turn to do that in the venture community and startups rather than the pharmaceutical industry, where it can get buried and disappear.”

A tendency for people within companies to move is another ongoing issue. Regis Kelly, director of the California Institute for Quantitative Biosciences (QB3), a nonprofit institute spanning three University of California campuses in the San Francisco Bay Area, points to pharma's frequent management changes as a potential snag in making the partnerships thrive. For instance, in 2008, soon after Pfizer merged with Wyeth, it dissolved the Bio-therapeutics and Bio-innovation Center (BBC) on UCSF's Mission Bay campus—set up in 2007 as a hybrid between academia and industry, to work on translational projects (Nat. Biotechnol. 27, 308, 2009). For about a year, Kelly recalls, “there was a hiatus, where we couldn't start any new programs together.”

Even as Pfizer focuses on decentralizing industry-academic partnerships, London-based GlaxoSmithKline (GSK) will soon adopt a virtual approach. GSK aims to create up to ten relationships with individual researchers throughout the world, forming a virtual project team with each of them in order to, like Pfizer, provide immediate access to GSK resources. “We're not talking about giving lots of money across to academia,” says GSK's Patrick Vallance, who is leading the program. An experienced drug discoverer will work in tandem with the research group. “At the beginning it's very focused, with access to the whole of GSK's expertise,” he says.

GSK is set to announce the first of its collaborations under the program, with Mark Pepys at University College, London (UCL), and Pepys' UCL spinout, Pentraxin Therapeutics, for the development of a small molecule to treat amyloidosis. GSK and Pentraxin are already working together to develop an antibody to treat the disease.

To some extent, Pfizer's CTI programs echo the spirit of Eli Lilly's Chorus initiative, started in 2007, in which a venture firm supplies the Indianapolis-based pharma with compounds for Lilly to rapidly advance through phase 1. But whereas both emphasize speed to the clinic from a similar preclinical starting point, the CTIs will also explore the biology around its targets in depth, at greater cost, but also presumably to its benefit. Indeed, although Pfizer is aware of the importance of targeted therapeutics and personalized medicine, “It's not an area we have invested a significant amount of time in,” says Coyle. By focusing on translational medicine up front, “We're going to have a broader impact in the organization,” he says.

p21 Activated Kinases 1 and 3 Control Brain Size through Coordinating Neuronal Complexity and Synaptic Properties

doi:10.1128/MCB.00969-10

Abstract: The molecular mechanisms that coordinate postnatal brain enlargement, synaptic properties and cognition remain an enigma. Here we demonstrate that neuronal complexity controlled by p21 activated kinases (PAKs) is a key determinant for postnatal brain enlargement and synaptic properties. We showed that double knockout (DK) mice lacking both PAK1 and PAK3 were born healthy, with normal brain size and structure, but severely impaired in postnatal brain growth, resulting in a dramatic reduction in brain volume. Remarkably, the reduced brain size was accompanied by minimal changes in total cell count, due to a significant increase in cell density. However, the DK neurons have smaller soma, markedly simplified responses due to enhanced individual synaptic potency but were severely impaired in bidirectional synaptic plasticity. The actions of PAK1 and PAK3 are possibly mediated by cofilin dependent actin regulation, because the activity of cofilin and the properties of actin filaments were altered in the DK mice. These results reveal an essential in vivo role of PAK1 and PAK3 in coordinating neuronal complexity and synaptic properties and highlight the critical importance of dendrite/ axon growth in dictating postnatal brain growth and attainment of normal brain size and function.

Getting Smart about p21-Activated Kinases

Mollier L. Kelly and Jonathan Chernoff; Molecular and Cellular Biology; doi:10.1128/MCB.01267-10

p21-activated kinases (PAK) are a family of Cdc42/Rac-activated serine/threonine kinases involved in a variety of cellular processes, such as motility, migration and cytoskeletal reorganization. Various members of the PAK family are known to influence brain development and cognitive function, but there remain many unanswered questions regarding PAK functions in neurons, the significance of individual PAK isoforms and their molecular mechanisms of action. A new genetic study by Huang and colleagues provides insight into how murine PAKs, specifically PAK1 and PAK3 are involved in brain development through morphogenesis of dendritic spines as well as development of synaptic networks. As PAK3 mutations in humans cause mental retardation, the findings in this new study may lead to new approaches to treat disorders of cognitive function.

On structural and biochemical grounds, the PAK family of kinases can be divided into two groups, group I(PAK1, –2 and –3) and group II (PAK4,-5 and –6). All PAKs contain a highly conserved N-terminal Cdc42/Rac-binding domain and C-terminal protein kinase domain but differ substantially between these two domains. Within the group I PAKs, PAK1 and PAK3 are nearly identical in amino acid sequences (81% over all and >95% in the Cdc42/Rac-binding and kinase domains). and both are highly expressed in the brain. These features suggest that PAK1 and PAK3 share redundant functions. However, despite these close similarities only PAK1 has a binding site for LC8, which is required for its nuclear entry. Since nuclear functions of PAK1 play an important role in development in zebrafish as well as in signaling in mammalian cells one might suppose on these grounds that PAK1 and PAK3 functions are nonredundant. Thus, both the similarities and the one known difference between PAK1 and PAK3 have made it difficult to determine the unique functions, if any. of each of these two isoforms.

Due to their high levels of expressions in the brain both PAK1 and PAK3 have been closely examined for their role in nerve cell function. A number of studies suggest a role for PAK1 in regulating dendritic spine morphology although the underlying mechanism remains unclear. PAK3 has also been implicated in having a role in neuron development and plasticity. The most powerful evidence in this regard has come from studies of human mental retardation which revealed a causal association between certain X-linked nonsyndromic forms of mental retardation and PAK3 loss-of-function mutations. To date, five distinct point mutations have been found in various kindreds:three in the kinase domain (abolishing kinase activity), one in the Cdc42/Rac- binding domain (abrogating binding to these GTPases) and one in an intron (causing a premature stop). Patients with these mutations display hyperactivity, excessive anxiety, restlessness and impaired memory. Such behavioral and cognitive impairments are often associated with abnormal neuron plasticity, suggesting a plausible link to PAK1/PAK3 cellular functions in neurons. However, the exact roles of these proteins in brain development and their levels of redundancy are unknown. Additionally, the role of these PAKs in the adult brain and their contributions to a stable neuron network have yet to be elucidated.

To understand the mechanism by which PAK1 and PAK3 contribute to brain function, single-knockout mouse models have been produced. However, mouse knockouts of PAK1 and PAK3 have been notable for their lack of dramatic neuronal phenotypes. For example, loss of PAK1 alone gives rise to modest defects in long-term potentiation in hippocampal CA1 synapses, as does loss of PAK3 alone but neither are associated with neuroanatomic defects or abnormalities in cellular actin structures. Not so the double knockout. When Huange et al. crossed PAK1 and PAK3 null mice, the resulting double knockout mice were normal at birth but soon showed major loss of brain volume compared to that of wild type mice, despite normal brain organization. PAK1/PAK3 double knockout mice also had severely impaired learning and memory and hyperactive behavior, a phenotype that echoes that seen in human patients with mutations in PAK3. For these reasons, the PAK1/PAK3 double knockout mouse represents a useful model to study the role of PAK function in postnatal brain development and a plausible platform for testing therapeutic agents.

What is the cellular basis for the brain defects in PAK1/PAK3 knockout mice? To answer this question, Huang et al. evaluated morphogenesis and maturity of the neurons. As PAK1 and PAK3 are both expressed in neurons after mitosis and in differentiated neurons, the authors hypothesized that neuron maturation is affected in the double knockout. The group found that the morphology of neurons was much less complex with reduced dendrite length and number of dendritic tips in the double knockout mice, showing that PAK1 and PAK3 are involved in branch formation. Surprisingly, however, the double knockout mice displayed enhanced synaptic transmission.This is likely related to the fact that the few synapses that were present on neurons in double knockout mice were functionally more potent, leading to the apparent enhanced synaptic transmission. This finding shows that PAK1 and PAK3 are essential for normal spine morphology and synaptic properties. Such defects in dendritic spine structure are likely to explain their further finding that long-term potentiation and depression were significantly reduced in the double- knockout mice. These are noteworthy observations, as long-term potentiation and depression are important in learning and memory and may explain the diminished cognitive function in the double knockout mice.

Given these cellular and physiological defects other questions remain: what molecular pathways do these kinases regulates and why is deletion of both required to obtain a phenotype? To assess the biochemical mechanisms by which PAKs exerts their effects in neurons, Huang et al. examined the activity of a number of well known PAK regulated pathways . Surprisingly, the combined loss of PAK1/PAK3 had no effect on extracellular signal regulated kinase (ERK) activity or on other well studied PAK substrates, such as myosin light chain kinase. In fact, of the seven substrates tested, phosphorylation was significantly reduced in only one, cofilin, which is a target of PAK substrate LIM kinase (LIMK). That cofilin should be involved makes perfect sense, as the major phenotype observed in the double-knockout neurons, namely, altered dendritic spine morphology, is consistent with the loss of F-actin and a natural consequence of cofilin activation, which is associated with the unphosphorylated state. An interesting corollary that emerges from these data is that the remaining group I PAK, PAK2, which is also highly expressed in neurons, must lack the ability to phosphorylate LIMK. Perhaps the lack of effect on other PAK-activated pathways in the double knockouts is due to redundancy among the three group I PAKs in most signaling circuits in neurons, with LIMK/cofilin representing an important exception.

The findings of Huang et al. could have implications beyond mental retardation, as synaptic dysfunction associated with underphosphorylated cofilin underlies the cognitive impairments accompanying a wide range of neurological disorders and normal aging. Thus, the PAK1/PAK3 double knockout model, in revealing what had been obscured by redundant group I PAK functions, enlightens us regarding broader issues of brain development and function.