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Japan carries out first iPS stem cell implant surgery

Japanese researchers Friday conducted the world's first surgery to implant "iPS" stem cells in a human body in a major boost to regenerative medicine, two institutions involved said. A female patient in her 70s with age-related macular degeneration (AMD), a common medical condition that can lead to blindness in older people, had a sheet of retina cells that had been created from iPS cells …

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Japan carries out first iPS stem cell retina surgery

TOKYO: Japanese researchers on Friday (Sep 12) conducted the world’s first surgery to implant “iPS” stem cells in a human body in a major boost to regenerative medicine, two institutions involved said.

A female patient in her 70s with age-related macular degeneration (AMD), a common medical condition that can lead to blindness in older people, had a sheet of retina cells that had been created from iPS cells implanted. “It is the first time in the world that iPS cells have been transplanted into a human body,” a spokeswoman for Riken, one of the research institutions, told AFP.

The research team used induced Pluripotent Stem (iPS) cells – which have the potential to develop into any cell in the body – that had originally come from the skin of the patient. Until the discovery of iPS several years ago, the only way to obtain stem cells was to harvest them from human embryos.

“We feel very much relieved,” ophthalmologist Masayo Takahashi, the leader of the project at Riken, told a news conference after the surgery in Kobe. “We want to take it as a big step forward. But we must go on and on from here.”

In a statement, the institution said that “no serious adverse phenomena such as excessive bleeding occurred” during the two-hour procedure. The surgery is still at an experimental stage, but if it is successful, doctors hope it will stop the deterioration in vision that comes with AMD.

The patient – one of six expected to take part in the trial – will be monitored over the next four years to determine how well the implants have performed, whether the body has accepted them and if they have become cancerous.

AMD, a condition that is incurable at present, affects mostly middle-aged and older people and can lead to blindness. It afflicts around 700,000 people in Japan alone.

The study was being carried out by researchers from government-backed research institution Riken and the Institute of Biomedical Research and Innovation Hospital.

Stem cell research is a pioneering field that has excited many in the scientific community with the potential they believe it offers. Stem cells are infant cells that can develop into any part of the body. Harvesting from human embryos is controversial because it requires the destruction of the embryo, a process to which religious conservatives, among others, object.

Groundbreaking work done in 2006 by Shinya Yamanaka at Kyoto University, a Nobel Laureate in medicine last year, succeeded in generating stem cells from adult skin tissue.

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STAP co-author offers yet another recipe for stem cells

A senior co-author on controversial, and now retracted, stem cell papers has quietly posted new tips on how the research can be replicated.

Two papers claiming that stressing the bodys cell could produce embryonic-like stem cells, a process called stimulus triggered acquisition of pluripotency (STAP), were heralded when published in Nature in January but thrashed soon after when problematic images and figures were soon found.

That might not have been so worrisome if the experiments, which the authors called easy to do, were replicated, but various external groups tried and failed to do so. Co-authors in Japan responded with a tipsheet. Soon after that, the lead author on the paper laying out the fundamental STAP technology, Charles Vacanti of the Brigham and Womens Hospital in Boston, Massachusetts, released his own, quite different, list of tips for reproducing STAP.Still no one succeeded in replicating the findings.

Since April, Hitoshi Niwa, awell-respected mouse stem cell specialist atthe Center for Developmental Biology (CDB) in Kobe and a co-author on the papers, has been giving a focused, last-ditch effort to replicate the experiment; on August 27, he reported no luck so far and suggested that light emission from dying cells, known as autofluorescence, might have been confused with fluorescent tags meant to signal conversion to the embryonic-like state.

During that period, the lead author on both papers, the CDBs Haruko Obokata, was found guilty of misconductandboth papers were retracted. Obokatas supervisor at the CDB, Yoshiki Sasai, committed suicide, andVacanti stepped down as chairman of Brigham and Womens Department of Anesthesiology, Preoperative and Pain Medicine. The CDB itself has halved in size.

One might have thought that STAP was finished.But Vacanti is not one to give up so easily.

Even when he finally agreed to retract the papers, he maintained, in a post on his departments website, that there has been no information that cast doubt on the existence of the stimulus-triggered acquisition of pluripotency (STAP) cell phenomenon itself. Vacanti said that he was confident that Niwa would replicate the core STAP cell concept that my brother Martin and I originally hypothesized, and trust that it will be verified by the RIKEN as well as independently by others.

Now, in a note posted without fanfare on Vacantis departments websiteand dated September 3 one week after Niwa announced failure to replicate the findings Vacanti has offered his second revision to the STAP protocol.

In comparison his first revised protocol in March (Refined protocol for generating STAP cells from mature somatic cells), the new one (REVISED STAP CELL PROTOCOL. 09.03.14:) highlights the use of ATP in the solution, in combination with two stresses exposure to acid and physical pressure on the cell membranes that he used in the previous recipe. In recentmonths, our lab decided to re-explore the utility of a low pH solution containing ATP ingenerating STAP cells, Vacanti writes in the revised protocol. We found that while pH alone resulted in the generation of STAP cells, the use of a low pH solution containing ATP, dramatically increased the efficacy of this conversion. When this acidic ATP solution was used in combination with mechanical trituration of maturecells, the results were even more profound (emphasis original).

We made a significant mistake in our original declaration that the protocol was easy to repeat, the protocol continues. This was our belief at the time, but it turned out to be incorrect. Many of the steps described appear to be a function of the technique of the individual investigator. Consequently, the revised protocol below should increase the likelihood of success.

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First iPS stem cell retina surgery gives hope for AMD patients

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Scientists revert human stem cells to pristine state

Researchers at EMBL-EBI have resolved a long-standing challenge in stem cell biology by successfully ‘resetting’ human pluripotent stem cells to a fully pristine state, at point of their greatest developmental potential. The study, published in Cell, involved scientists from the UK, Germany and Japan and was led jointly by EMBL-EBI and the University of Cambridge.

Embryonic stem (ES) cells, which originate in early development, are capable of differentiating into any type of cell. Until now, scientists have only been able to revert ‘adult’ human cells (for example, liver, lung or skin) into pluripotent stem cells with slightly different properties that predispose them to becoming cells of certain types. Authentic ES cells have only been derived from mice and rats.

“Reverting mouse cells to a completely ‘blank slate’ has become routine, but generating equivalent nave human cell lines has proven far more challenging,” says Dr Paul Bertone, Research Group Leader at EMBL-EBI and a senior author on the study. “Human pluripotent cells resemble a cell type that appears slightly later in mammalian development, after the embryo has implanted in the uterus.”

At this point, subtle changes in gene expression begin to influence the cells, which are then considered ‘primed’ towards a particular lineage. Although pluripotent human cells can be cultured from in vitro fertilised (IVF) embryos, until now there have been no human cells comparable to those obtained from the mouse.

Wiping cell memory

“For years, it was thought that we could be missing the developmental window when nave human cells could be captured, or that the right growth conditions hadn’t been found,” Paul explains. “But with the advent of iPS cell technologies, it should have been possible to drive specialised human cells back to an earlier state, regardless of their origin — if that state existed in primates.”

Taking a new approach, the scientists used reprogramming methods to express two different genes, NANOG and KLF2, which reset the cells. They then maintained the cells indefinitely by inhibiting specific biological pathways. The resulting cells are capable of differentiating into any adult cell type, and are genetically normal.

The experimental work was conducted hand-in-hand with computational analysis.

“We needed to understand where these cells lie in the spectrum of the human and mouse pluripotent cells that have already been produced,” explains Paul. “We worked with the EMBL Genomics Core Facility to produce comprehensive transcriptional data for all the conditions we explored. We could then compare reset human cells to genuine mouse ES cells, and indeed we found they shared many similarities.”

Together with Professor Wolf Reik at the Babraham Institute, the researchers also showed that DNA methylation (biochemical marks that influence gene expression) was erased over much of the genome, indicating that reset cells are not restricted in the cell types they can produce. In this more permissive state, the cells no longer retain the memory of their previous lineages and revert to a blank slate with unrestricted potential to become any adult cell.

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New details emerge on retracted STAP papers

Posted on behalf of David Cyranoski.

New leaked emails showing the comments of referees for Science and Nature provide additional insight into the saga of the STAP papers, which Nature published in January and retracted in July.

The papers had promised new, simpler ways to produce stem cells by applying stress to cells taken from a patients tissues. But no other lab was able to reproduce the results, and experts pointed to several problems and inconsistencies in the papers.In April,first author Haruko Obokata of the RIKEN Center for Developmental Biology in Kobe, Japan, was declared guilty of scientific misconduct; the controversylater took a tragic turn as another co-author, Yoshiki Sasai, committed suicide on 5 August.

An investigative report into the papers, released in May, revealed that a previous version of the work had been rejected by Nature, Cell, and Science in 2012, before being resubmitted and accepted by Nature. (Natures news and comment team is editorially independent of its research editorial team.)

That report gave details from the Science referees who pointed out that one figure had been reconstructed in a way at odds with normal scientific practice and another one had a suspiciously sharp band (see Misconduct verdict stands for Japanese stem-cell researcher).

The blog Retraction Watchposted the full comments of three referees who reviewed the paper for Science on 10 September.

The reviewsinclude a modicum of support, but overall the paper is panned by all three. Reviewer number 2 notes, Unfortunately, the paper presents only a superficial description of many critical aspects of the work, before launching into 21 points that need to be addressed, ranging from seemingly sloppy mistakes to fundamental problems with the data.

Reviewer 3 noted, If these results are repeatable, a paradigm of developmental biology would be changed.

The manuscript itself is not available, so it is impossible to know exactly how similar the rejected Science manuscript is to the version that was eventually published in Nature.

When the committee initially brought the problems in the Science paper to her attention, Obokata defended herself by saying that the published Nature paper had main conclusions that differed from those in the rejected Science manuscript, and she refused to show the latter to the investigative committee.

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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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