Central biobank for drug research

4 hours ago The biobank comprises three cryotanks, equipped with cooled protective hoods, and a transfer station from which the sample containers are transported via a rail system. There is enough space for approximately 60,000 samples. Credit: Fraunhofer IBMT

For the development of new drugs it is crucial to work with stem cells, as these allow scientists to study the effects of new active pharmaceutical ingredients. But it has always been difficult to derive enough stem cells of the right quality and in the right timeframe. A central biobank is about to remedy the situation.

Human stem cells allow scientists to assess how patients are likely to respond to new drugs and to examine how illnesses come about. For a few years now, it has been possible to take tissue samples from adults and use reverse programming to artificially produce stem cells, which have the potential to create any kind of cell found in the human body. Before this discovery, pharmaceutical researchers had to use adult stem cells or primary cells, which have a more limited potential. Another option is to use stem cells derived from human embryos, but quite apart from the ethical considerations these cells are available only in limited diversity. The new technique makes it possible for instance to reprogram adult skin or blood cells so that they behave in a similar way to embryonic stem cells and can become any type of cell. “These are known as induced pluripotent stem cells, or iPS cells for short,” says Dr. Julia Neubauer from the Fraunhofer Institute for Biomedical Engineering IBMT in St. Ingbert, Germany. Although an increasing number of local biobanks have emerged in recent years, none of them fulfills the requirements of the pharmaceutical industry and research institutions. What is needed is a supply of ‘ready-to-use’ stem cells, which means large numbers of consistently characterized, systematically catalogued cells of suitable quality.

At the beginning of 2014, the IBMT teamed up with 26 industry and research partners to launch a project aimed at establishing a central biobank the European Bank for induced pluripotent Stem Cells (EBiSC) to generate iPS cells from patients with specific diseases or genetic mutations (http://ebisc.org/). Six months into the project and the first cells are available for use in the development of new drugs. By its three-year mark, it is hoped the project will be in a position to offer over 1000 defined and characterized cell lines comprising a hundred million cells. Such quantities are needed because a single drug screening involves testing several million cells. The main biobank facility is being built in the English city of Cambridge and an identical “mirror site” will be set up at the IBMT’s Sulzbach location in Germany.

Gently freezing cells

The IBMT was brought on board for EBiSC by virtue of the comprehensive expertise it gained through participation in the EU’s “Hyperlab” and “CRYSTAL” projects. For EBiSC, IBMT scientists are responsible for freezing the cells and for automating cell cultivation and the biobank itself. For an efficient long-term storage of functional stem cells, they have to be cooled down to temperatures of below 130 degrees Celsius in a controlled way. The scientists have to prepare the cells so they can survive the cold shock of nitrogen gas. The IBMT has, for instance, developed technologies that allow cells to be frozen in an extremely gentle way. “Cells don’t like being removed from the surface they are grown on, but that’s what people used to do in order to freeze them. Our method allows the cells to stay adherent,” explains Neubauer.

Just as with foodstuffs, stem cells depend on an unbroken cold chain to preserve their functionality and viability. The scientists store the cells in special containers or cryotanks each measuring one by two meters. To remove a particular sample, the scientists have to open the cryotank. The problem is that this exposes all the other samples to warmer ambient air, causing them to begin to thaw out. “It’s just like when you go to your refrigerator at home it’s not a good idea to leave the door open too long,” says Neubauer. She and her colleagues at the IBMT and industry partner Askion GmbH have together developed a stem cell biobank complete with protective hoods that protect the other samples whenever the cryotank is opened. In addition to maintaining the temperature, the hoods help keep another key shelf-life criterion, humidity, at a constant level.

Flawless freezing is important, but it is just as important to automate the whole process. “That not only guarantees consistency, it’s what makes it possible to provide large quantities of cells of the required quality in the first place,” says Neubauer. And the scientists’ cooling process already boasts a finished technology. In their automated biobank, each cell sample is labelled with barcodes to allow them to be tracked. The samples travel along a conveyor belt to the individual cyrotanks, and a computer monitors the entire freezing and storage process.

Now the scientists are working on automating cell cultivation or the multiplying of the cells. There are essentially two possible approaches. One is to use robots that translate each preparation step into a mechanical one. The other is to use stirred bioreactors that provide free-moving cells with the ideal supply of nutrients and oxygen. Both technologies feature in the IBMT’s portfolio. “By the time the project is completed, we’ll know which is the better method for what we’re trying to do,” says Neubauer.

Explore further: Animal-free reprogramming of adult cells improves safety

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Japan Lab Fails to Replicate Stem Cell Findings

MOSCOW, August 27 (RIA Novosti) – Scientists at the RIKEN research institute in Japan have been unable to verify the discovery of a groundbreaking new method of creating “stem cells,” Nikkei Asian Review reported Wednesday.

The struggle to verify the results of research published by Haruko Obokata and colleagues earlier this year casts further doubt on the existence of stimulus-triggered acquisition of pluripotency, or STAP, the phenomenon they described. Exposing ordinary body cells to various stresses had made them pluripotent, or able to differentiate into any type of tissue, the authors had claimed, the newspaper writes.

The report states that scientists have so far been unable to recreate STAP cells. Researchers have conducted 22 experiments, none of which have been successful.

Using Obokatas methods, researchers have only been able to produce faint genetic markers of pluripotency, Nikkei reports, citing sources familiar with the experiments.

A study describing the creation of so-called STAP cells was initially published in the acclaimed scientific journal Nature in January this year. Amid falsification claims, the RIKEN institute, where Obokata is based, announced a month later that it would investigate her discoveries.

I am profoundly apologetic that the reports of STAP reprogramming have led to the current serious concerns about the integrity and reliability of this research, Masatoshi Takeichi, director of the RIKEN Center for Developmental Biology, wrote in a statement.

Takeichi urged the scientists to retract their publication in Nature. Obokata agreed to retract the paper in July. In August, co-author of the study, stem cell scientist Yoshiki Sasai, committed suicide at the institute.

RIKEN is expected to hold a press conference Wednesday, in which several leading officials, including Masatoshi Takeichi, are expected to be replaced. The institute is expected to be renamed and have its staff of about 400 researchers cut in half, Nikkei writes.

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How the zebrafish gets its stripes

23 hours ago The zebrafish (Danio rerio) owes its name to a repeating pattern of blue stripes alternating with golden stripes. Credit: MPI f. Developmental Biology/ P. Malhawar

The zebrafish, a small fresh water fish, owes its name to a striking pattern of blue stripes alternating with golden stripes. Three major pigment cell types, black cells, reflective silvery cells, and yellow cells emerge during growth in the skin of the tiny juvenile fish and arrange as a multilayered mosaic to compose the characteristic colour pattern.

While it was known that all three cell types have to interact to form proper stripes, the embryonic origin of the pigment cells that develop the stripes of the adult fish has remained a mystery up to now. Scientists of the Max Planck Institute for Developmental Biology in Tbingen have now discovered how these cells arise and behave to form the ‘zebra’ pattern. Their work may help to understand the development and evolution of the great diversity of striking patterns in the animal world.

Beauty in the living world amazes poets, philosophers and scientists alike. Nobel prize laureate Christiane Nsslein-Volhard, Director of the Department for Genetics at the Max Planck Institute for Developmental Biology, has long been fascinated by the biology behind the colour patterns displayed by animals. Her group uses zebrafish as a model organism to study the genetic basis of animal development.

New research by Nsslein-Volhard’s laboratory published in Science shows that the yellow cells undergo dramatic changes in cell shape to tint the stripe pattern of zebrafish. “We were surprised to observe such cell behaviours, as these were totally unexpected from what we knew about colour pattern formation”, says Prateek Mahalwar, first author of the study. The study builds on a previous work from the laboratory, which was published in June this year in Nature Cell Biology (NCB), tracing the cell behaviour of silvery and black cells. Both studies describe diligent experiments to uncover the cellular events during stripe pattern formation. Individual juvenile fish carrying fluorescently labelled pigment cell precursors were imaged every day for up to three weeks to chart out the cellular behaviours. This enabled the scientists to trace the multiplication, migration and spreading of individual cells and their progeny over the entire patterning process of stripe formation in the living and growing animal. “We had to develop a very gentle procedure to be able to observe individual fish repeatedly over long periods of time. So we used a state of the art microscope which allowed us to reduce the adverse effects of fluorescence illumination to a minimum,” says Ajeet Singh, first author of the earlier NCB study.

Surprisingly, the analysis revealed that the three cell types reach the skin by completely different routes: A pluripotent cell population situated at the dorsal side of the embryo gives rise to larval yellow cells, which cover the skin of the embryo. These cells begin to multiply at the onset of metamorphosis when the fish is about two to three weeks old. However, the black and silvery cells come from a small set of stem cells associated with nerve nodes located close to the spinal cord in each segment. The black cells reach the skin migrating along the segmental nerves to appear in the stripe region, whereas the silvery cells pass through the longitudinal cleft that separates the musculature and then multiply and spread in the skin.

Brigitte Walderich, a co-author of the Science paper, who performed cell transplantations to trace the origin of yellow cells, explains: “My attempt was to create small clusters of fluorescently labelled cells in the embryo which could be followed during larval and juvenile stages to unravel growth and behaviour of the yellow cells. We were surprised to discover that they divide and multiply as differentiated cells to cover the skin of the fish before the silvery and black cells arrive to form the stripes.”

A striking observation is that both the silvery and yellow cells are able to switch cell shape and colour, depending on their location. The yellow cells compact to closely cover the dense silvery cells forming the light stripe, colouring it golden, and acquire a loose stellate shape over the black cells of the stripes. The silvery cells thinly spread over the stripe region, giving it a blue tint. They switch shape again at a distance into the dense form to aggregate, forming a new light stripe. These cell behaviours create a series of alternating light and dark stripes. The precise superposition of the dense form of silvery and yellow cells in the light stripe, and the loose silvery and yellow cells superimposed over the black cells in the stripe cause the striking contrast between the golden and blue coloration of the pattern.

The authors speculate that variations on these cell behaviours could be at play in generating the great diversity of colour patterns in fish. “These findings inform our way of thinking about colour pattern formation in other fish, but also in animals which are not accessible to direct observation during development such as peacocks, tigers and zebras”, says Nsslein-Volhard.

Explore further: Study of zebrafish skin patterns shows cells chasing other cells around (w/ video)

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Japan lab unable to replicate 'stem cell' findings

Researchers in Japan said Wednesday they have been unable to replicate experiments that were hailed earlier this year as a "game-changer" in the quest to grow transplant tissue, amid claims evidence was faked. In a scandal that rocked Japan's scientific establishment, Riken — the research institute that sponsored the study — launched an independent experiment in April to verify research …

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Japan lab unable to replicate 'stem cell' findings: report

Researchers in Japan have been unable to replicate experiments that were hailed earlier this year as a "game-changer" in the quest to grow transplant tissue, amid claims evidence was faked, a report said Wednesday. In a scandal that rocked Japan's scientific establishment, Riken — a research institute that sponsored the study — launched an independent experiment in April to verify research …

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Japan lab fails to replicate 'stem cell' findings: report

(08-27 12:21)

Researchers in Japan have been unable to replicate experiments that were hailed earlier this year as a “game-changer” in the quest to grow transplant tissue, amid claims evidence was faked, a report said Wednesday. In a scandal that rocked Japan’s scientific establishment, Riken — a research institute that sponsored the study — launched an independent experiment in April to verify research published by scientist Haruko Obokata and her colleagues earlier this year, AFP reports. But the struggle to replicate the experiment casts further doubt on the existence of stem cell-like cells, what the researchers called Stimulus-Triggered Acquisition of Pluripotency (STAP) cells, Japan’s Nikkei daily reported. Obokata was feted after unveiling findings that appeared to show a straightforward way to re-program adult cells to become stem cells — precursors that are capable of developing into any other cell in the human body. Identifying a readily manufacturable supply of stem cells could one day help meet a need for transplant tissues, or even whole organs, meaning that any advance in the field is met with excitement in the scientific community. But after being accused of fabricating results, she agreed to retract papers published in the respected journal Nature. Earlier this month, Obokata’s co-author, stemcell scientist Yoshiki Sasai, hanged himself. Researchers have been trying to replicate results appearing to show that exposing ordinary cells to various stresses had made them pluripotent, or able to differentiate into any type of tissue. Riken had planned to implant these cells into mouse embryos to test whether they really were pluripotent. But the experiments have been fraught with difficulty from the outset, with researchers unable to reproduce such cells, the Nikkei said, citing unnamed sources. Obokata has been trying in tandem to reproduce her own results since July, but the existence of the STAP cells at this point looks highly doubtful, the Nikkei said. Riken is to release an interim report on the follow-up study and announce a shake-up of the developmental biology centre in a Wednesday news conference, where it is expected to slash its staff of around 400 researchers by half.

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Japan's Riken to Shake Up Research Center

By Dow Jones Business News, August 27, 2014, 12:55:00 AM EDT

TOKYO–A scandal that started with a few suspicious images has led Japan’s most prestigious research institute to slash its stem-cell unit by half and acknowledge deeper flaws in its ethics.

The move by the Riken institute came seven months after the publication of papers that it initially hailed as equal in importance to the Copernican revolution in astronomy. Since then, the papers have been retracted, and one of the co- authors committed suicide.

On Wednesday, Riken said it would scale down to half its size the Center for Developmental Biology, rename the center and choose a new director with input from non-Japanese scientists, an indication of how the scandal has damaged the reputation of Japanese science.

“We believe it is important to move forward with the restructuring to improve the quality and promote honest research,” said Ryoji Noyori, the Nobel Prize winner who leads Riken.

Riken’s overhaul could also sway the field of stem-cell science, which has received billions of dollars in research funds in the hopes of cures for ailments such as diabetes and heart disease.

Some details of the overhaul, including whether anyone beside the director would indeed lose their job, remained murky. Nevertheless, science writer Shinya Midori said, “This could trigger scaling down in the field of regenerative medicine.”

The scandal at Riken has deeply shaken the country’s science establishment and the wider stem-cell world and sparked a debate about research ethics in Japan amid “results-first” pressure.

The drama has focused on the institute’s 14-year-old developmental-biology center and erupted after one of its scientists, Haruko Obokata, was found guilty of manipulating data in a pair of papers published in the journal Nature. The studies, which claimed to show a groundbreaking method of making stem cells by dipping cells in a mild acid solution, were quickly challenged and Nature retracted the papers in July, saying they contained inaccurate data.

Riken initially stood by the 31-year-old Dr. Obokata, who had been hailed as a national hero after her research was first published, but later distanced itself from what it called her “sloppy data management” and poor research ethics.

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Japan lab unable to replicate 'stem cell' findings (Update)

6 hours ago A photo released by Japan’s national institute Riken on January 28, 2014 shows what is meant to be Stimulus Triggered Acquisition of Pluripotency (STAP) cells. The research has now been called into question.

Researchers in Japan said Wednesday they have been unable to replicate experiments that were hailed earlier this year as a “game-changer” in the quest to grow transplant tissue, amid claims evidence was faked.

In a scandal that rocked Japan’s scientific establishment, Rikenthe research institute that sponsored the studylaunched an independent experiment in April to verify research published by scientist Haruko Obokata and her colleagues earlier this year.

But the failure to replicate the experiment casts further doubt on the existence of stem cell-like cells, what the researchers called Stimulus-Triggered Acquisition of Pluripotency (STAP) cells.

“Researchers have conducted 22 experiments thus far, but we could not confirm the emergence of cells in the conditions described in (Obokata’s) papers,” Riken said in an interim report issued Wednesday.

Obokata since July has been trying in tandem with independent teams to reproduce her own results.

The researchers will continue their experiments under more diverse conditions while also considering data obtained by Obokata herself, Shinichi Aizawa, a special adviser at Riken, told a lengthy press conference.

Obokata was feted after unveiling findings that appeared to show a straightforward way to re-programme adult cells to become stem cellsprecursors that are capable of developing into any other cell in the human body.

Identifying a readily manufacturable supply of stem cells could one day help meet a need for transplant tissues, or even whole organs, meaning that any advance in the field is met with excitement in the scientific community.

But suspicions began to emerge in the weeks and months after the research was published, building into one of the biggest controversies in scientific publishing for a decade.

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Stem Cells Reveal How Illness-Linked Genetic Variation Affects Neurons

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Newswise A genetic variation linked to schizophrenia, bipolar disorder and severe depression wreaks havoc on connections among neurons in the developing brain, a team of researchers reports. The study, led by Guo-li Ming, M.D., Ph.D., and Hongjun Song, Ph.D., of the Johns Hopkins University School of Medicine and described online Aug. 17 in the journal Nature, used stem cells generated from people with and without mental illness to observe the effects of a rare and pernicious genetic variation on young brain cells. The results add to evidence that several major mental illnesses have common roots in faulty wiring during early brain development.

This was the next best thing to going back in time to see what happened while a person was in the womb to later cause mental illness, says Ming. We found the most convincing evidence yet that the answer lies in the synapses that connect brain cells to one another.

Previous evidence for the relationship came from autopsies and from studies suggesting that some genetic variants that affect synapses also increase the chance of mental illness. But those studies could not show a direct cause-and-effect relationship, Ming says.

One difficulty in studying the genetics of common mental illnesses is that they are generally caused by environmental factors in combination with multiple gene variants, any one of which usually could not by itself cause disease. A rare exception is the gene known as disrupted in schizophrenia 1 (DISC1), in which some mutations have a strong effect. Two families have been found in which many members with the DISC1 mutations have mental illness.

To find out how a DISC1 variation with a few deleted DNA letters affects the developing brain, the research team collected skin cells from a mother and daughter in one of these families who have neither the variation nor mental illness, as well as the father, who has the variation and severe depression, and another daughter, who carries the variation and has schizophrenia. For comparison, they also collected samples from an unrelated healthy person. Postdoctoral fellow Zhexing Wen, Ph.D., coaxed the skin cells to form five lines of stem cells and to mature into very pure populations of synapse-forming neurons.

After growing the neurons in a dish for six weeks, collaborators at Pennsylvania State University measured their electrical activity and found that neurons with the DISC1 variation had about half the number of synapses as those without the variation. To make sure that the differences were really due to the DISC1 variation and not to other genetic differences, graduate student Ha Nam Nguyen spent two years making targeted genetic changes to three of the stem cell lines.

In one of the cell lines with the variation, he swapped out the DISC1 gene for a healthy version. He also inserted the disease-causing variation into one healthy cell line from a family member, as well as the cell line from the unrelated control. Sure enough, the researchers report, the cells without the variation now grew the normal amount of synapses, while those with the inserted mutation had half as many.

We had our definitive answer to whether this DISC1 variation is responsible for the reduced synapse growth, Ming says.

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New Blood: Tracing the Beginnings of Hematopoietic Stem Cells

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Newswise Hematopoietic stem cells (HSCs) give rise to all other blood cell types, but their development and how their fate is determined has long remained a mystery. In a paper published online this week in Nature, researchers at the University of California, San Diego School of Medicine elaborate upon a crucial signaling pathway and the role of key proteins, which may help clear the way to generate HSCs from human pluripotent precursors, similar to advances with other kinds of tissue stem cells.

Principal investigator David Traver, PhD, professor in the Department of Cellular and Molecular Medicine, and colleagues focused on the Notch signaling pathway, a system found in all animals and known to be critical to the generation of HSCs in vertebrates. Notch signaling between emitting and receiving cells is key to establishing HSC fate during development, said Traver. What has not been known is where, when and how Notch signal transduction is mediated.

Traver and colleagues discovered that the Notch signal is transduced into HSC precursor cells from signal emitting cells in the somite embryologic tissues that eventually contribute to development of major body structures, such as skeleton, muscle and connective tissues much earlier in the process than previously anticipated.

More specifically, they found that JAM proteins, best known for helping maintain tight junctions between endothelial cells to prevent vascular leakage, were key mediators of Notch signaling. When the researchers caused loss of function in JAM proteins in a zebrafish model, Notch signaling and HSCs were also lost. When they enforced Notch signaling through other means, HSC development was rescued.

To date, it has not been possible to generate HSCs de novo from human pluripotent precursors, like induced pluripotent stem cells, said Traver. This has been due in part to a lack of understanding of the complete set of factors that the embryo uses to make HSCs in vivo. It has also likely been due to not knowing in what order each required factor is needed.

Our studies demonstrate that Notch signaling is required much earlier than previously thought. In fact, it may be one of the earliest determinants of HSC fate. This finding strongly suggests that in vitro approaches to instruct HSC fate from induced pluripotent stem cells must focus on the Notch pathway at early time-points in the process. Our findings have also shown that JAM proteins serve as a sort of co-receptor for Notch signaling in that they are required to maintain close contact between signal-emitting and signal-receiving cells to permit strong activation of Notch in the precursors of HSCs.

The findings may have far-reaching implications for eventual development of hematopoietic stem cell-based therapies for diseases like leukemia and congenital blood disorders. Currently, it is not possible to create HSCs from differentiation of embryonic stem cells or induced pluripotent stem cells pluripotent cells artificially derived from non-pluripotent cells, such as skin cells that are being used in other therapeutic research efforts.

Co-authors include Isao Kobayashi, Jingjing Kobayashi-Sun, Albert D. Kim and Claire Pouget, UC San Diego Department of Cellular and Molecular Medicine; Naonobu Fujita, UC San Diego Section of Cell and Developmental Biology; and Toshio Suda, Keio University, Japan.

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