'CRISPR' science: Newer genome editing tool shows promise in engineering human stem cells

Johns Hopkins study could advance use of stem cells for treatment and disease research

A powerful "genome editing" technology known as CRISPR has been used by researchers since 2012 to trim, disrupt, replace or add to sequences of an organism's DNA. Now, scientists at Johns Hopkins Medicine have shown that the system also precisely and efficiently alters human stem cells.

In a recent online report on the work in Molecular Therapy, the Johns Hopkins team says the findings could streamline and speed efforts to modify and tailor human-induced pluripotent stem cells (iPSCs) for use as treatments or in the development of model systems to study diseases and test drugs.

"Stem cell technology is quickly advancing, and we think that the days when we can use iPSCs for human therapy aren't that far away," says Zhaohui Ye, Ph.D., an instructor of medicine at the Johns Hopkins University School of Medicine. "This is one of the first studies to detail the use of CRISPR in human iPSCs, showcasing its potential in these cells."

CRISPR originated from a microbial immune system that contains DNA segments known as clustered regularly interspaced short palindromic repeats. The engineered editing system makes use of an enzyme that nicks together DNA with a piece of small RNA that guides the tool to where researchers want to introduce cuts or other changes in the genome.

Previous research has shown that CRISPR can generate genomic changes or mutations through these interventions far more efficiently than other gene editing techniques, such as TALEN, short for transcription activator-like effector nuclease.

Despite CRISPR's advantages, a recent study suggested that it might also produce a large number of "off-target" effects in human cancer cell lines, specifically modification of genes that researchers didn't mean to change.

To see if this unwanted effect occurred in other human cell types, Ye; Linzhao Cheng, Ph.D., a professor of medicine and oncology in the Johns Hopkins University School of Medicine; and their colleagues pitted CRISPR against TALEN in human iPSCs, adult cells reprogrammed to act like embryonic stem cells. Human iPSCs have already shown enormous promise for treating and studying disease.

The researchers compared the ability of both genome editing systems to either cut out pieces of known genes in iPSCs or cut out a piece of these genes and replace it with another. As model genes, the researchers used JAK2, a gene that when mutated causes a bone marrow disorder known as polycythemia vera; SERPINA1, a gene that when mutated causes alpha1-antitrypsin deficiency, an inherited disorder that may cause lung and liver disease; and AAVS1, a gene that's been recently discovered to be a "safe harbor" in the human genome for inserting foreign genes.

Their comparison found that when simply cutting out portions of genes, the CRISPR system was significantly more efficient than TALEN in all three gene systems, inducing up to 100 times more cuts. However, when using these genome editing tools for replacing portions of the genes, such as the disease-causing mutations in JAK2 and SERPINA1 genes, CRISPR and TALEN showed about the same efficiency in patient-derived iPSCs, the researchers report.

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'CRISPR' science: Newer genome editing tool shows promise in engineering human stem cells

Two-thirds of adult cancers largely due to bad luck, study suggests

Lifestyle choices and genetics are big risk factors for certain cancers, but a new study concludes that the majority of cancer incidence is due mostly to bad luck when our cells divide.

The study comes from scientists at the Johns Hopkins Kimmel Cancer Center who created a statistical model to measure the proportion of cancer cases that are caused mainly by random DNA mutations during stem cell division.

By their calculations, two-thirds of adult cancer incidents can be explained by bad luck when stem cells divide.

All cancers are caused by a combination of bad luck, the environment and heredity, says lead researcher Dr. Bert Vogelstein, a professor of oncology at the Johns Hopkins University School of Medicine.

"Weve created a model that may help quantify how much of these three factors contribute to cancer development, he said in a statement.

Cancer occurs when stem cells in tissues make random mistakes, or mutations, during the replication process in cell division. The more that these mutations accumulate, the higher the risk that cells will begin to grow, unchecked, into tumours.

But Vogelstein says it's never been clearly understood how much of a contribution these random mistakes made to cancer incidence, compared to genetic inheritance, lifestyle, or environmental factors.

So they focused on 31 tissue types, looking at the number of stem cell divisions in each cancer. They then compared these rates with lifetime cancer risk among the same cancer types in the American population.

Significantly, they did not include breast cancer and prostate cancer in their study, even though these are two of the most commonly diagnosed cancers among adults. The researchers explained that they could not find reliable stem cell division rates on these cancer types.

Of the 31 cancer types they did look at, they found that 22 could be largely explained by the bad luck factor of random DNA mutations during cell division.

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Two-thirds of adult cancers largely due to bad luck, study suggests

Two-thirds of adult cancers largely down to bad luck rather than genes

Colon cancer cell. Colon tissue undergoes four times more stem cell divisions than small intestine tissue in humans, and is much more prevalent. Photograph: Micro Discovery/Corbis

Good luck, rather than good genes, may be the key reason why some people are protected from certain cancers while others develop the disease, according to a new study.

Two-thirds of adult cancers, say the researchers from the Johns Hopkins Kimmel Cancer Center in the United States, are caused by random mutation in the tissue cells during the ordinary process of stem cell division. In the other third, our genetic inheritance and lifestyles are the main factors.

The scientists have created a mathematical model which, they say, shows it is wrong to assume that there are such things as good genes that may prevent us getting cancer even though we smoke, drink heavily and carry excessive weight.

All cancers are caused by a combination of bad luck, the environment and heredity, and weve created a model that may help quantify how much of these three factors contribute to cancer development, says Bert Vogelstein, the Clayton professor of oncology at the Johns Hopkins University school of medicine and one of the authors of the paper published in the journal Science. Cancer-free longevity in people exposed to cancer-causing agents, such as tobacco, is often attributed to their good genes, but the truth is that most of them simply had good luck.

The scientists looked at how often stem cell division, the normal process of cell renewal, takes place in 31 different tissue types, to find out whether the sheer number of divisions can lead to more mistakes or DNA mutations occurring. They did not look at tissues from two of the commonest forms of cancer breast and prostate which are known to have particular environmental triggers, such as obesity. These were not included because they could not find reliable data on the normal division rate of stem cells in these tissues.

Our study shows, in general, that a change in the number of stem cell divisions in a tissue type is highly correlated with a change in the incidence of cancer in that same tissue, said Vogelstein. One example, he says, is in colon tissue, which undergoes four times more stem cell divisions than small intestine tissue in humans. Likewise, colon cancer is much more prevalent than small intestinal cancer.

It could be argued, they say, that the colon is exposed to more environmental factors than the small intestine but they point out that the opposite is true for mice, which have more stem cell divisions and a higher rate of cancer in their small intestines than in their colon.

The scientists say that bad luck plays a stronger role in some cancers than in others. In two-thirds of the cancers 22 cancer types random mutations in genes that drive cancer could explain why the disease occurred. The other nine cancers occurred more often than the random mutation rate would predict, suggesting that inherited genes or lifestyle factors were the main cause. They included lung cancer, where smoking is the major cause, and skin cancer, which can be triggered by sun exposure.

Speaking on the BBC Radio 4 Today programme on Friday, co-author biomathematician Dr Cristian Tomasetti, also from Johns Hopkins University, said: Im not claiming any cancers, overall across the population, are the result of pure chance, but what I am claiming is there are some tissues for example blood cancer where there is very little evidence of any hereditary or environmental factor.

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Two-thirds of adult cancers largely down to bad luck rather than genes

Stem cells to transplant in the brain: Stealth UCSF spinout Neurona Therapeutics raises $7.6M

A UCSF spinout is growing neuronal stemcells to transplant into the brain, for potential use in treating epilepsy, spinal cord injury, Parkinsons and Alzheimers disease and investors are listening. Because one thing thatdifferentiatesNeurona Therapeutics is that its stem cells turn exclusively intointerneuron cells which are less likely to be tumorigenic than other IPS cells.

The companyhasraised $7.6 million of a proposed $24.3 million round, according to a regulatory filing. But the companys staying a touch under the radar it lacks a website, and tis the season for calls to the company to remain unanswered.

But funding for the six-year-old company comes from 11 investors. Listed on the documents contact pages areTim Kutzkeyand David Goeddel, both partners at early stage healthcare venture firm The Column Group giving some insight into who the startupsinvestors are.

Also listed is Leo Guthart, a managing partner at New York private equity firm TopSpin Partner, and Arnold Kriegstein, director of the UCSF developmental and stem cell biology program.

Kriegsteinand his UCSF colleagues filed a patentfor the in vitro production of medial ganglionic eminence (MGE) precursor cells which are, in essence, immature cells that morphinto nerve cells. The work that led to the patent was funded bythe California Institute of Regenerative Medicine, the NIH and the Osher Foundation.

We think this one type of cell may be useful in treating several types of neurodevelopmental and neurodegenerative disorders in a targeted way,Kriegstein said in a UCSF statement last year.

Neurona Therapeutics scientific backers collaborated on a paper on these MGE cells inCell Stem Cell,finding that mouse models closely mimicked human cells inneural cell development and that human cells can successfully be transplanted into mouse brains. UCSF writes:

Kriegstein sees MGE cells as a potential treatment to better control nerve circuits that become overactive in certain neurological disorders. Unlike other neural stem cells that can form many cell types and that may potentially be less controllable as a consequence most MGE cells are restricted to producing a type of cell called an interneuron. Interneurons integrate into the brain and provide controlled inhibition to balance the activity of nerve circuits.

To generate MGE cells in the lab, the researchers reliably directed the differentiation of human pluripotent stem cells either human embryonic stem cells or induced pluripotent stem cells derived from human skin. These two kinds of stem cells have virtually unlimited potential to become any human cell type. When transplanted into a strain of mice that does not reject human tissue, the human MGE-like cells survived within the rodent forebrain, integrated into the brain by forming connections with rodent nerve cells, and matured into specialized subtypes of interneurons.

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Stem cells to transplant in the brain: Stealth UCSF spinout Neurona Therapeutics raises $7.6M

High-fat diet, obesity during pregnancy harms stem cells in developing fetus

Findings may provide broad context for the rise in immune disease and allergic disposition in children

PORTLAND, Ore. -- Physician-scientists at OHSU Doernbecher Children's Hospital reveal a high-fat diet and obesity during pregnancy compromise the blood-forming, or hematopoietic, stem cell system in the fetal liver responsible for creating and sustaining lifelong blood and immune system function.

The life-long burden of a western-style diet on the heart and circulatory system have long been appreciated. However, prior to this study, no one had considered whether the developing blood stem cells might be similarly vulnerable to prenatal high-fat diet and/or maternal obesity. The findings are published in the journal Molecular Metabolism.

"Our results offer a model for testing whether the effects of a high-fat diet and obesity can be repaired through dietary intervention, a key question when extrapolating this data to human populations," said Daniel L. Marks, M.D., Ph.D., co-investigator and professor of pediatric endocrinology in the OHSU School of Medicine and Pap Family Pediatric Research Institute at OHSU Doernbecher Children's Hospital.

Several years ago, Marks and colleagues developed a mouse model that closely mimics the high-fat, high-simple-sugar diet currently consumed by many young women of childbearing age. Their subsequent research demonstrated that maternal overnutrition in mice significantly reduced the size of the fetal liver.

Armed with this information, Marks partnered with another stem cell expert, Peter Kurre, M.D., co-investigator on the current study and professor of pediatric oncology in the OHSU School of Medicine and the Pap Family Pediatric Research Institute at OHSU Doernbecher Children's Hospital.

Together, they discovered that the complex changes that occur as a result of maternal high-fat diet and obesity put significant constraints on the growth and expansion of blood stem cells in the fetal liver, which ultimately compromises the developing immune system.

"In light of the spreading western-style, high-fat diet and accompanying obesity epidemic, this study highlights the need to better understand the previous unrecognized susceptibility of the stem and progenitor cell system," Kurre said. "These findings may provide broad context for the rise in immune disease and allergic disposition in children."

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The study, "Maternal high-fat diet and obesity compromise fetal hematopoiesis," was funded by Friends of Doernbecher and by the Oregon Clinical Translational Research Institute at OHSU. Research reported in this press release] was supported by National Center for Advancing Translational Sciences of the National Institutes of Health under award number UL1TR000128

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High-fat diet, obesity during pregnancy harms stem cells in developing fetus

Stem Cell Scandal Scientist Haruko Obokata Resigns

A Japanese Stem Cell Scientist At The Heart Of A Scandal Over False Claims And Fabricated Research Has Resigned.

Dr Haruko Obokata published supposedly groundbreaking research showing stem cells could be made quickly and cheaply.

There were irregularities in data, no other group in the world could repeat her findings and her own university concluded it could not be done.

In a statement Dr Obokata said: "I even can't find the words for an apology."

Stem cells can become any other type of tissue and hold great potential in medicine.

They are already being investigated to heal the damage caused by a heart attack and to restore sight.

But they are expensive and difficult to produce and one source - embryos - raises serious ethical questions.

'Major discovery'

Dr Obokata's scientific paper published in the prestigious journal Nature claimed that stem cells could be produced from normal adult cells by dipping them into acid for a 30-minute shock period.

The announcement of the creation of these "Stap" cells (stimulus-triggered acquisition of pluripotency) sent shockwaves around the world.

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Stem Cell Scandal Scientist Haruko Obokata Resigns

Embattled Stem Cell Researchers Sue Harvard And Brigham And Women's Hospital

Two embattled and highly controversial stem cell researchers are suing the Brigham and Womens Hospital and Harvard Medical School for an ongoing investigation into their research. The investigation has already resulted in the retraction of one paper inCirculationand anexpression of concern about another paper in theLancet.

The suit was filed by Piero Anversa, the highly prominent stem cell researcher who is a Harvard professor and the head of a large lab at the Brigham, and his longtime colleague,Annarosa Leri, an associate professor of medicine at Harvard who has coauthored many papers with Anversa. The suit places the blame for any scientific misconduct relating to the two papers on a third colleague and coauthor,Jan Kajstura, their longtime collaborator. In an explanation of the problems relating to the Circulation paper,Anversa and Leri accuse Kajstura of doctoring data in a spreadsheet in such a way that they could not have detected it. For theLancet paper the two scientists say thatKajstura and another unnamed scientist in the lab altered two images. Kastura is no longer at the Brigham.

The news was first reported byCarolyn Johnson in theBoston GlobeandJessica Bartlett in the Boston Business Journal. The story has also been reported in depth by Ivan Oransky onRetraction Watch.

The lawsuit accuses Elizabeth Nabel, the president of the Brigham, and the individual members of the investigating panel, of inappropriate and illegal behavior and conflicts of interest.At one point the complaint alleges that the scientists on the panel lack substantial expertise in the relevant scientific areas, including cardiac stem cells. But then, when another member was added to the panel, Ulrich von Andrian, the complaint states that he suffers from serious conflicts of interest that impede his ability to participate in the investigation in an impartial manner. Nabel and von Andrian, along with other Harvard and Brigham figures, serve on the scientific advisory board of Moderna Therapeutics, a stem cell company pursuing an alternative modality for regenerative treatment of cardiac disease.

Anversa and Leri further allege that the investigation caused the withdrawal of a multimillion dollar offer to purchase their company, Autologous/Progenital. The investigation also ended efforts to recruit Anversa and Leri to the University of Miami and the Mt. Sinai School of Medicine in New York.

The complaint discloses that as a result of the ongoing investigation Anversa and Leri were subject to embarrassing questions from other prominent stem cell researchers, including Joshua Hare, Steven Houser, and Eduardo Marban. These researchers, the complaint states, had no need to know of the inquiry.

Anversa and Leri also criticizethe panel for expanding its investigation to at least 15 papers from the research group. There is no justification for expanding the investigation to encompass these additional papers at this late stage. Most were published before the inquiry process began in January 2013, and all were published before the investigation began in February 2014. It is unclear from the complaint why these papers should not have been subject to scrutiny.

As I reported in 2011, the Lancet paper reporting the results of theSCIPIO trial was the subject of considerable hype at the time of its original publication. ABC News, CBS News and other media outlets used phrases like medical breakthrough and heart failure cure. ABC News correspondent Richard Besser was so enthusiastic that anchor Diane Sawyer commented that she had never seen him so excited. The first author of SCIPIO, Roberto Bolli, said the work could represent the biggest advance in cardiology in my lifetime.

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Embattled Stem Cell Researchers Sue Harvard And Brigham And Women's Hospital

University of Toronto cell biologists discover on-off switch for key stem cell gene – Discovery may propel advances in …

TORONTO, ON Consider the relationship between an air traffic controller and a pilot. The pilot gets the passengers to their destination, but the air traffic controller decides when the plane can take off and when it must wait. The same relationship plays out at the cellular level in animals, including humans. A region of an animals genome the controller directs when a particular gene the pilot can perform its prescribed function.

A new study by cell and systems biologists at the University of Toronto (U of T) investigating stem cells in mice shows, for the first time, an instance of such a relationship between the Sox2 gene which is critical for early development, and a region elsewhere on the genome that effectively regulates its activity. The discovery could mean a significant advance in the emerging field of human regenerative medicine, as the Sox2 gene is essential for maintaining embryonic stem cells that can develop into any cell type of a mature animal.

We studied how the Sox2 gene is turned on in mice, and found the region of the genome that is needed to turn the gene on in embryonic stem cells, said Professor Jennifer Mitchell of U of Ts Department of Cell and Systems Biology, lead investigator of a study published in the December 15 issue of Genes & Development.

Like the gene itself, this region of the genome enables these stem cells to maintain their ability to become any type of cell, a property known as pluripotency. We named the region of the genome that we discovered the Sox2 control region, or SCR, said Mitchell.

Since the sequencing of the human genome was completed in 2003, researchers have been trying to figure out which parts of the genome made some people more likely to develop certain diseases. They have found that the answers are more often in the regions of the human genome that turn genes on and off.

If we want to understand how genes are turned on and off, we need to know where the sequences that perform this function are located in the genome, said Mitchell. The parts of the human genome linked to complex diseases such as heart disease, cancer and neurological disorders can often be far away from the genes they regulate, so it can be difficult to figure out which gene is being affected and ultimately causing the disease.

It was previously thought that regions much closer to the Sox2 gene were the ones that turned it on in embryonic stem cells. Mitchell and her colleagues eliminated this possibility when they deleted these nearby regions in the genome of mice and found there was no impact on the genes ability to be turned on in embryonic stem cells.

We then focused on the region weve since named the SCR as my work had shown that it can contact the Sox2 gene from its location 100,000 base pairs away, said study lead author Harry Zhou, a former graduate student in Mitchells lab, now a student at U of Ts Faculty of Medicine. To contact the gene, the DNA makes a loop that brings the SCR close to the gene itself only in embryonic stem cells. Once we had a good idea that this region could be acting on the Sox2 gene, we removed the region from the genome and monitored the effect on Sox2.

The researchers discovered that this region is required to both turn Sox2 on, and for the embryonic stem cells to maintain their characteristic appearance and ability to differentiate into all the cell types of the adult organism.

Just as deletion of the Sox2 gene causes the very early embryo to die, it is likely that an abnormality in the regulatory region would also cause early embryonic death before any of the organs have even formed, said Mitchell. It is possible that the formation of the loop needed to make contact with the Sox2 gene is an important final step in the process by which researchers practicing regenerative medicine can generate pluripotent cells from adult cells.

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University of Toronto cell biologists discover on-off switch for key stem cell gene - Discovery may propel advances in ...

Scientists make stem cell breakthrough

Sydney, Dec 11 (IANS): An Australian research team together with international scientists has discovered a new stem cell that can be programmed to become any part of the body.

The ramifications of the find mean that a transplant can be conducted by using the patient's own cells, which can be made into organs and tissue.

The discovery, published in the journal Nature Thursday, is a breakthrough in stem cell research.

"These are remarkably useful cells, because you can apply them to several different areas of medicine," Xinhua quoted molecular biologist Thomas Preiss, from the Australian National University, as telling Fairfax Media.

More than 50 researchers from Australia, Canada, the Netherlands and South Korea worked in the study, known as Project Grandiose, which identified the pluripotent stem cell.

The new cell is considered a potential prototype for the mass production of therapeutic stem cells to treat a huge range of illnesses and injuries.

Medical conditions such as blindness, Parkinson's, Alzheimer's, stroke and spinal cord injury will be major beneficiaries of the new find.

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Scientists make stem cell breakthrough

Unlocking the secrets of stem cell generation

14 hours ago Professor Thomas Preiss discusses gene networks during stem cell reprogramming with his JCSMR colleagues Dr Jen Clancy and Dr Hardip Patel. Credit: Stuart Hay.

International scientists have carried out the most detailed study of how specialised body cells can be reprogrammed to be like cells from the early embryo.

The findings are a major advance in stem cell science and could help usher in a new era of regenerative medicine, where a small sample of a patient's cells could be used to grow new tissues and organs for transplant.

"This kind of work will speed up the development of treatments for many illnesses that currently have no cure," said Professor Thomas Preiss from The John Curtin School of Medical Research.

"It could one day lead to treatments for age-related macular degeneration, Parkinson's, Alzheimer's, spinal cord injury, stroke, diabetes, blood and kidney diseases, and many others which are associated with tissue damage and cell loss."

Professor Preiss and the team at ANU were part of the international consortium known as Project Grandiose, which mapped the detailed molecular process involved in the generation of induced pluripotent stem cells (iPS).

The discovery that body cells can in principle be coaxed to become iPS cells led to the award of the Nobel Prize for Physiology or Medicine in 2012. Since then there has been a surge in global research to better understand iPS cell reprogramming, as it might help avoid the ethically-sensitive use of embryo-derived cells.

"The race is on to make reprogramming a safe and efficient process so that the resulting stem cells can be broadly applied in therapies," Professor Preiss said.

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"We have described in unprecedented detail the molecular changes that cells undergo as they reprogram into stem cells and also discovered a new kind of pluripotent cell that can be seen as a prototype for therapeutic cell production."

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Unlocking the secrets of stem cell generation