Human Stem Cells Converted to Functional Lung Cells

Possibility of generating lung tissue for transplant using a patient's own cells

Monday, 02 December 2013

For the first time, scientists have succeeded in transforming human stem cells into functional lung and airway cells. The advance, reported by Columbia University Medical Center (CUMC) researchers, has significant potential for modelling lung disease, screening drugs, studying human lung development, and, ultimately, generating lung tissue for transplantation. The study was published today in the journal Nature Biotechnology.

Human skin cells can now be reprogrammed into

induced pluripotent stem cells, which are similar

to human embryonic stem cells, and then into

functional lung and airway cells. Such cells have

potential for use in disease modelling, drug

screening, the study of lung development, and the

generation of lung tissue for transplantation.

Credit: Hans-Willem Snoeck, M.D., Ph.D.,

Columbia University Medical Center.

"Researchers have had relative success in turning human stem cells into heart cells, pancreatic beta cells, intestinal cells, liver cells, and nerve cells, raising all sorts of possibilities for regenerative medicine," said study leader Hans-Willem Snoeck, MD, PhD, professor of medicine (in microbiology & immunology) and affiliated with the Columbia Center for Translational Immunology and the Columbia Stem Cell Initiative.

"Now, we are finally able to make lung and airway cells. This is important because lung transplants have a particularly poor prognosis. Although any clinical application is still many years away, we can begin thinking about making autologous lung transplants — that is, transplants that use a patient's own skin cells to generate functional lung tissue."

The research builds on Dr. Snoeck's 2011 discovery of a set of chemical factors that can turn human embryonic stem (ES) cells or human induced pluripotent stem (iPS) cells into anterior foregut endoderm — precursors of lung and airway cells. (Human iPS cells closely resemble human ES cells but are generated from skin cells, by coaxing them into taking a developmental step backwards. Human iPS cells can then be stimulated to differentiate into specialized cells — offering researchers an alternative to human ES cells.)

In the current study, Dr. Snoeck and his colleagues found new factors that can complete the transformation of human ES or iPS cells into functional lung epithelial cells (cells that cover the lung surface). The resultant cells were found to express markers of at least six types of lung and airway epithelial cells, particularly markers of type 2 alveolar epithelial cells. Type 2 cells are important because they produce surfactant, a substance critical to maintain the lung alveoli, where gas exchange takes place; they also participate in repair of the lung after injury and damage.

The findings have implications for the study of a number of lung diseases, including idiopathic pulmonary fibrosis (IPF), in which type 2 alveolar epithelial cells are thought to play a central role.

"No one knows what causes the disease, and there's no way to treat it," says Dr. Snoeck.

"Using this technology, researchers will finally be able to create laboratory models of IPF, study the disease at the molecular level, and screen drugs for possible treatments or cures."

"In the longer term, we hope to use this technology to make an autologous lung graft," Dr. Snoeck said.

"This would entail taking a lung from a donor; removing all the lung cells, leaving only the lung scaffold; and seeding the scaffold with new lung cells derived from the patient. In this way, rejection problems could be avoided." Dr. Snoeck is investigating this approach in collaboration with researchers in the Columbia University Department of Biomedical Engineering.

"I am excited about this collaboration with Hans Snoeck, integrating stem cell science with bioengineering in the search for new treatments for lung disease," said Gordana Vunjak-Novakovic, co-author of the paper and Mikati Foundation Professor of Biomedical Engineering at Columbia's Engineering School and professor of medical sciences at Columbia University College of Physicians and Surgeons.

Source: Columbia University Medical Center

Contact: Karin Eskenazi

Reference:

Highly efficient generation of airway and lung epithelial cells from human pluripotent stem cells

Sarah X L Huang, Mohammad Naimul Islam, John O'Neill, Zheng Hu, Yong-Guang Yang, Ya-Wen Chen, Melanie Mumau, Michael D Green, Gordana Vunjak-Novakovic, Jahar Bhattacharya & Hans-Willem Snoeck

Nature Biotechnology, 01 December 2013, doi:10.1038/nbt.2754

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Human Stem Cells Used to Elucidate Mechanisms of Beta-cell Failure in Diabetes

Mechanisms that impair insulin production in diabetes identified using a human stem cell model of Wolfram syndrome, a rare form of diabetes

Wednesday, 13 November 2013

Insulin-producing beta-cells. 

Scientists from the New York Stem Cell Foundation (NYSCF) Research Institute and Columbia University Medical Center (CUMC) have used stem cells created from the skin of patients with a rare form of diabetes — Wolfram syndrome — to elucidate an important biochemical pathway for beta-cell failure in diabetes. The findings by Linshan Shang and colleagues were published today in Diabetes.

Scientists from NYSCF produced induced pluripotent stem (iPS) cells from skin samples from individuals with a rare form of diabetes, Wolfram syndrome. They then derived insulin-producing cells (beta cells) from these iPS cells, creating a human diabetes model in vitro. Next, they showed that the beta cells failed to normally secrete insulin because of protein-folding — or endoplasmic reticulum (ER) — stress. They found that a chemical, 4-phenyl butyric acid, that relieves this stress, prevents the cells from failing, suggesting a potential target for clinical intervention.

"These cells represent an important mechanism that causes beta-cell failure in diabetes. This human iPS cell model represents a significant step forward in enabling the study of this debilitating disease and the development of new treatments," said Dieter Egli, PhD, principal investigator of the study, and Senior Research Fellow at NYSCF and NYSCF–Robertson Stem Cell Investigator.

Wolfram syndrome is a rare, often fatal genetic disorder characterized by the development of insulin-dependent diabetes, vision loss, and deafness. Since all forms of diabetes are ultimately the result of an inability of pancreatic beta cells to provide sufficient insulin in response to blood sugar concentrations, this Wolfram patient stem cell model enables analysis of a specific pathway leading to beta-cell failure in more prevalent forms of diabetes. It also enables the testing of strategies to restore beta-cell function that may be applicable to all types of diabetes.

"Utilizing stem cell technology, we were able to study a devastating condition to better understand what causes the diabetes symptoms as well as discover possible new drug targets," said Susan L. Solomon, Co-Founder and Chief Executive Officer of The New York Stem Cell Foundation.

"This report highlights again the utility of close examination of rare human disorders as a path to elucidating more common ones," said co-author Rudolph L. Leibel, MD, the Christopher J. Murphy Professor of Diabetes Research and co-director of the Naomi Berrie Diabetes Center at CUMC.

"Our ability to create functional insulin-producing cells using stem cell techniques on skin cells from patients with Wolfram's syndrome has helped to uncover the role of ER stress in the pathogenesis of diabetes. The use of drugs that reduce such stress may prove useful in the prevention and treatment of diabetes."

Clinicians from the Naomi Berrie Diabetes Center recruited Wolfram syndrome patients to donate a skin sample. All Wolfram patients had childhood-onset diabetes requiring treatment with injected insulin, and all had vision loss. Additional cell lines were obtained from Coriell Institute for Medical Research. The researchers at NYSCF "reprogrammed," or reverted, the skin cells to an embryonic-like state to become iPS cells. An iPS cell line generated from a healthy individual was used as a normal control.

The researchers differentiated the iPS cells from the Wolfram subjects and the controls into beta cells, an intricate process that took several weeks. They implanted both Wolfram and control iPS cell-derived beta cells under the kidney capsule of immuno-compromised mice. Beta cells from the Wolfram subjects produced less insulin in the culture dish and secreted less insulin into the bloodstream of the mice when they were challenged with high blood-sugar levels.

A key finding was that these beta cells showed elevated markers of ER stress. Treatment with 4-phenyl butyric acid reduced the ER stress and increased the amount of insulin produced by the beta cells, thereby increasing the ability to secrete insulin in response to glucose.

Direct evidence in mice, as well as circumstantial evidence in humans with both type 1 and type 2 diabetes, highlights the role of the ER stress response mechanism in the survival of insulin-producing beta cells. The ER stress response mechanisms oppose both the stress of immune assault in type 1 diabetes and the metabolic stress of high blood glucose in both types of diabetes. When the ER stress response fails cell death occurs, potentially reducing the number of insulin-producing cells.

Source: New York Stem Cell Foundation 

Contact: David McKeon

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Human Muscle Stem Cell Therapy Gets Help from Zebrafish

HSCI researchers improve therapy prospects

Thursday, 07 November 2013

Zebrafish in Research Lab for Animal Testing.

Harvard Stem Cell scientists have discovered that the same chemicals that stimulate muscle development in zebrafish can also be used to differentiate human stem cells into muscle cells in the laboratory, a historically challenging task that, now overcome, makes muscle cell therapy a more realistic clinical possibility.

The work, published this week in the journal Cell, began with a discovery by Boston Children's Hospital researchers, led by Leonard Zon, MD, and graduate student Cong (Tony) Xu, who tested 2,400 different chemicals in cultures of zebrafish embryo cells to determine if any could increase the numbers of muscle cells formed. Using fluorescent reporter fish in which muscle cells were visible during their creation, the researchers found six chemicals that were very effective at promoting muscle formation.

Zon shared his results with Harvard Department of Stem Cell and Regenerative Biology professor Amy Wagers, PhD, and Mohammadsharif Tabebordbar, a graduate student in her laboratory, who tested the six chemicals in mice. One of the six, called forskolin, was found to increase the numbers of muscle stem cells from mice that could be obtained when these cells were grown in laboratory dishes. Moreover, the cultured cells successfully integrated into muscle when transplanted back into mice.

Inspired by the successful application of these chemicals in mice, Salvatore Iovino, PhD, a joint postdoctoral fellow in the Wagers lab and the lab of C. Ronald Kahn, MD, at the Joslin Diabetes Center, investigated whether the chemicals would also affect human cells and found that a combination of three chemicals, including forskolin, could induce differentiation of human induced pluripotent stem (iPS) cells, made by reprogramming skin cells. Exposure of iPS cells to these chemicals converted them into skeletal muscle, an outcome the Wagers and Kahn labs had been striving to achieve for years using conventional methods. When transplanted into a mouse, the human iPS-derived muscle cells also contributed to muscle repair, offering early promise that this protocol could provide a route to muscle stem cell therapy in humans.

The interdisciplinary, cross-laboratory collaboration between Zon, Wagers, and Kahn highlights the advantage of open exchange between researchers.

"If we had done this screen directly on human iPS cells, it would have taken at least 10 times as long and cost 100 times as much," said Wagers.

"The zebrafish gave us a big advantage here because it has a fast generation time, rapid development, and can be easily and relatively cheaply screened in a culture dish."

"This research demonstrates that over 300 million years of evolution, the pathways used in the fish are conserved through vertebrates all the way up to the human," said Wagers' fellow HSCRB professor Leonard Zon, chair of the Harvard Stem Cell Institute Executive Committee and director of the stem cell program at Boston Children's Hospital.

"We can now make enough human muscle progenitors in a dish to allow us to model diseases of the muscle lineage, like Duchenne muscular dystrophy, conduct drug screens to find chemicals that correct that disease, and in the long term, efficiently transplant muscle stem cells into a patient."

In a similar biomedical application, Kahn, who is chief academic officer at the Joslin, plans to apply the new ability to quickly produce muscle stem cells for diabetes research. His lab will generate iPS-derived muscle cells from people who are at risk for diabetes and people who have diabetes to identify alterations that lead to insulin resistance in the muscle.

Going forward, Zon plans to apply this platform of cross-species discovery to other stem cell lines, including those involved in blood and eye development.

"We have a new system to use to study tissue development, and it's not just muscle that can be studied, every single organ can be studied in the zebrafish system," he said.

Source: Harvard University 

Contact: B. D. Colen

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Induced Pluripotent Stem Cells Reveal Differences between Humans and Great Apes

Key differences in the regulation of jumping genes may have arisen relatively recently in evolution

Thursday, 24 October 2013

Researchers at the Salk Institute for Biological Studies have, for the first time, taken chimpanzee and bonobo skin cells and turned them into induced pluripotent stem cells (iPSCs), a type of cell that has the ability to form any other cell or tissue in the body.

This microscope image shows induced pluripotent

stem cells (iPSC) from our closest living relatives.

Skin cells from bonobos (pigmy chimps) were

reprogrammed to pluripotent stem cells, an

advance that allows scientists to study the

differences between the neurons of humans and

chimps. The colours show different aspects of the

cells' molecular components. Credit: Courtesy of

Carol Marchetto, Salk Institute for Biological

Studies. 

Mouse iPSCs were created in 2006 by Kazutoshi Takahashi and Shinya Yamanaka at Kyoto University in Japan, and human iPSCs soon followed – feats which earned Yamanaka the Nobel Prize in Physiology or Medicine last year. Now scientists regularly use iPSCs to model diseases using cells that would be otherwise difficult to obtain from a living person or animal. By adding a combination of four key factors, a skin cell can be made into an iPSC, which can then be coaxed into forming liver, lung and brain cells in a culture dish.

It's now possible to not only model disease using the cells, but also to compare iPSCs from humans to those of our closest living relatives – great apes, with which we share a majority of genes – for insight into what molecular and cellular features make us human.

"Comparing human, chimpanzee and bonobo cells can give us clues to understand biological processes, such as infection, diseases, brain evolution, adaptation or genetic diversity," says senior research associate Iñigo Narvaiza, who led the study with senior staff scientist Carol Marchetto at the Salk Institute in La Jolla.

"Until now, the sources for chimpanzee and bonobo cells were limited to post-mortem tissue or blood. Now you could generate neurons, for example, from the three different species and compare them to test hypotheses."

In the new study, published online October 23 in the journal Nature, scientists found disparities in the regulation of jumping genes or transposons – DNA elements that can copy and paste themselves into spots throughout the genome – between humans and non-human primate cells. Jumping genes provide a means to rapidly shuffle DNA and might be shaping the evolution of our genomes, the scientists say.

From left are Salk scientists Ahmet Denli, Carol

Marchetto, Iñigo Narvaiza and Fred Gage.

Credit: Courtesy of the Salk Institute for
Biological Studies.

Working in the lab of Salk's Fred Gage, the Vi and John Adler Chair for Research on Age-Related Neurodegenerative Disease, Narvaiza, Marchetto and their colleagues identified genes that are differentially expressed between iPSCs from humans and both chimpanzees and bonobos.

To the group's surprise, two of those genes code for proteins that restrict a jumping gene called long interspersed element-1or L1, for short. Compared with non-human primate cells, human iPSCs expressed higher levels of these restrictors, called APOBEC3B and PIWIL2.

"We weren't expecting that," Marchetto says.

"Those genes caught our eyes, so they were the first targets we focused on."

L1 and a handful of other jumping genes are abundant throughout our genomes. Where these bits of DNA insert themselves is hard to predict, and they can produce variable effects. For example, they might completely disrupt genes, modulate them, or cause them to be processed into entirely new proteins.

Using L1 tagged with a fluorescent marker, the group observed higher numbers of fluorescent iPSCs from non-human primates compared with humans. In separate experiments, they produced iPSCs with too much or too little APOBEC3B and PIWIL2, finding – as expected – that an excess of the two proteins dampened the mobility and reduced the appearance of newly inserted DNA in the non-human primate cells.

These results suggested that L1 elements insert themselves less often throughout our genomes. Indeed, looking at genomes of humans and chimpanzees that had already been sequenced, the researchers found that the primates had more copies of L1 sequences than did humans.

The question that remains is what would be the impact of differences in L1 regulation?

"It could mean that we have gone, as humans, through one or more bottlenecks in evolution, that decrease the variability present in our genome," says Marchetto, though the hypothesis is admittedly hard to prove. It is known, however, that humans' genomes are less variable than chimpanzees'.

The new study provides proof of concept that the iPSC technology can be used to understand some of the evolutionary differences between humans and non-human primates, says Narvaiza. The group plans to make technology, and all the data, available to the broader research community – which is especially helpful now that great ape research is severely restricted in the United States and abroad – so that other scientists can learn about primates using non-invasive, ethically sound methods.

The team plans to differentiate the stem cells into other tissues, such as neurons, and comparing how the cells from different species behave. They will also use the iPSC technology to investigate how chimpanzees might differ from people in susceptibility to cancer, genetic diseases and viral infection.

Source: Salk Institute for Biological Studies

Contact: Kat Kearney

Reference:

Differential L1 regulation in pluripotent stem cells of humans and apes

Maria C. N. Marchetto, Iñigo Narvaiza, Ahmet M. Denli, Christopher Benner, Thomas A. Lazzarini, Jason L. Nathanson, Apuã C. M. Paquola, Keval N. Desai, Roberto H. Herai, Matthew D. Weitzman, Gene W. Yeo, Alysson R. Muotri, Fred H. Gage 

Nature (2013), doi:10.1038/nature12686

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Autologous Transplantation Shows Promising Results for iPS Cell Therapy in Parkinson's Disease

First confirmation in primates

Sunday, 29 September 2013

Autologous and allogeneic transplantation of

iPSC-derived neural cells. Credit: Stem Cell

Reports, Morizane et al. 

A research team led by Professor Jun Takahashi and Assistant Professor Asuka Morizane at the Center for iPS Cell Research and Application (CiRA) at Kyoto University, Japan, has carried out a study to compare the impact of immune response in autologous transplantation (transplantation of cells from the subject's own body) and allogeneic transplantation (transplantation of cells from a different individual of the same species). The researchers used cynomolgus monkeys to carry out transplantation into the brain of neural cells derived from iPS cells. Autologous transplantation was found to produce almost no immune reaction and to result in viable neural cells. By contrast, allogeneic transplantation provoked immune reaction by microglia and lymphocytes.

Parkinson's disease is a progressive and intractable disease of the nervous system in which the loss of dopaminergic neurons in the brain leads to reduced dopamine production, resulting in limb tremor, stiffness causing difficulty in movement, and other symptoms. The therapies applied up till now, based on drugs or electrode treatment, may improve symptoms but have proved unable to halt the depletion of dopaminergic neurons. Hopes have therefore become focused on a therapy with the more radical approach of replacing the lost neural cells through cell transplantation, thereby promoting the formation of new neural pathways to restore brain function. Human iPS cells are looked to as a potential source of the transplant cells.

This is an immunostaining of primate iPSC-

derived neurons on day 39. Green colors shows

dopaminergic neural cells. Credit: Courtesy of

Dr. Asuka Morizane. 

It is hoped that iPS cells will make it possible to use cells derived from the transplant patients themselves to perform autologous transplantation. If autologous transplantation could allow immune reaction to be avoided, it would also make unnecessary the use of immunosuppressant drugs and avert the risk of side-effects caused by immunosuppression. However, the studies of iPS cell-based autologous transplantation carried out so far, which have used a mouse model, have produced no firm conclusion, with immune reaction observed in some studies but not in others. Moreover, these studies did not involve transplantation of differentiated cells derived from iPS cells in a way that mimicked clinical application. There had thus been no studies directly investigating the effect of autologous transplantation and allogeneic transplantation in primates. This study by Dr. Takahashi's group sought to clarify this area by transplanting dopaminergic neurons prepared from iPS cells into the brains of cynomolgus monkeys and comparing the extent of immune reaction between autologous and allogeneic transplantation.

iPS cells prepared from four cynomolgus monkeys were differentiated into dopaminergic neural cells over a period of 28 days and transplanted into the monkeys' brains, which were observed over a period of approximately three months during which no immunosuppressant were used. The study data show that, in primates, autologous transplantation of iPS cell-derived neural cells produces almost no immune reaction and is superior to allogeneic transplantation in terms of immune reaction control and cell viability.

Source: Center for iPS Cell Research and Application - Kyoto University

Contact: Akemi Nakamura

Reference:

Direct Comparison of Autologous and Allogeneic Transplantation of iPSC-Derived Neural Cells in the Brain of a Nonhuman Primate

Asuka Morizane, Daisuke Doi, Tetsuhiro Kikuchi, Keisuke Okita, Akitsu Hotta, Toshiyuki Kawasaki, Takuya Hayashi, Hirotaka Onoe, Takashi Shiina, Shinya Yamanaka and Jun Takahashi

Stem Cell Reports, 26 September 2013, 10.1016/j.stemcr.2013.08.007

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Stem Cell Reprogramming Made Easier

Weizmann Institute scientists show that removing 1 protein from adult cells enables them to efficiently turn back the clock to a stem-cell-like state

Wednesday, 18 September 2013

This is Dr. Yaqub Hanna. Credit: Weizmann

Institute of Science. 

Embryonic stem cells have the enormous potential to treat and cure many medical problems. That is why the discovery that induced embryonic-like stem cells can be created from skin cells (iPS cells) was rewarded with a Nobel Prize in 2012. But the process has remained frustratingly slow and inefficient, and the resulting stem cells are not yet ready for medical use. Research in the lab of the Weizmann Institute's Dr. Yaqub Hanna, which appears today in Nature, dramatically changes that. He and his group revealed the "brake" that holds back the production of stem cells, and found that releasing this brake can both synchronize the process and increase its efficiency from around 1% or less today to 100%. These findings may help facilitate the production of stem cells for medical use, as well as advancing our understanding of the mysterious process by which adult cells can revert back into their original, embryonic state.

Embryonic stem cells are those that have not undergone any "specialization"; thus they can give rise to any type of cell in the body. This is what makes them so valuable: They can be used, among other things, to repair damaged tissue, treat autoimmune disease and even grow transplant organs. Using stem cells taken from embryos is problematic because of availability and ethical concerns, but the hopes for their use were renewed in 2006, when a team led by Shinya Yamanaka of Kyoto University discovered that it is possible to "reprogram" adult cells. The resulting cells, called "induced pluripotent stem cells" (iPSCs), are created by inserting four genes into their DNA. Despite this breakthrough, the reprograming process is fraught with difficulty: It can take up to four weeks; the timing is not coordinated among the cells; and less than one percent of the treated cells actually end up becoming stem cells.

Hanna and his team asked: What is the main obstacle – or obstacles – that prevent successful reprograming in the majority of cells? In his postdoctoral research, Hanna had employed mathematical models to show that a single obstacle was responsible. Of course in biology, Hanna is the first to admit, experimental proof is required to back up the models. The present study not only provides the proof, it reveals the identity of that single obstacle and shows that removing it can dramatically improve reprograming.

Left column: This is the previous method

for creating induced pluripotent stem cells

(iPSCs); right column: These are iPSCs

produced with the new method developed

by Dr. Hanna. Top: Skin cells (red); center:

iPSCs from skin cells (green); bottom: super-

imposed top and center images. Skin cells

that have been reprogrammed into iPSCs

appear light yellow. Only a small percentage

of the cells on the left have been

reprogrammed, in contrast with the high

success rate seen with the new method on

the right. Credit: Weizmann Institute of

Science. 

Hanna's group, led by Dr. Noa Novershtern, Yoach Rais, Asaf Zviran and Shay Geula of the Molecular Genetics Department, together with members of the genomics unit of the Institute's Israel Structural Proteomics Center, looked at a certain protein, called MBD3, whose function was unknown. MBD3 had caught their attention because it is expressed in every cell in the body, at every stage of development. This is quite rare: In general, most types of proteins are produced in specific cells, at specific times, for specific functions. The team found that there is one exception to the rule of universal expression of this protein: the first three days after conception. These are exactly the three days in which the fertilized egg begins dividing, and the nascent embryo is a growing ball of pluripotent stem cells that will eventually supply all the cell types in the body. Starting on the fourth day, differentiation begins and the cells already start to lose their pluripotent status. And that is just when the MBD3 proteins first appear.

This finding has significant implications for the producing iPSCs for medical use. Yamanaka used viruses to insert the four genes but, for safety reasons, these are not used in reprograming cells to be used in patients. This gives the process an even lower success rate of only around a tenth of a percent. The researchers showed that removing MBD3 from the adult cells can improve efficiency and speed the process by several orders of magnitude. The time needed to produce the stem cells was shortened from four weeks to eight days. As an added bonus, since the cells all underwent the reprograming at the same rate, the scientists will now be able, for the first time, to actually follow it step by step and reveal its mechanisms of operation. Hanna points out that his team's achievement was based on research into the natural pathways of embryonic development.

"Scientists investigating reprograming can benefit from a deeper understanding of how embryonic stem cells are produced in nature. After all, nature still makes them best, in the most efficient manner."

Source: Weizmann Institute of Science 

Contact: Yivsam Azgad

Reference:

Deterministic direct reprogramming of somatic cells to pluripotency

Yoach Rais, Asaf Zviran, Shay Geula, Ohad Gafni, Elad Chomsky, Sergey Viukov, Abed AlFatah Mansour, Inbal Caspi, Vladislav Krupalnik, Mirie Zerbib, Itay Maza, Nofar Mor, Dror Baran, Leehee Weinberger, Diego A. Jaitin, David Lara-Astiaso, Ronnie Blecher-Gonen, Zohar Shipony, Zohar Mukamel, Tzachi Hagai, Shlomit Gilad, Daniela Amann-Zalcenstein, Amos Tanay, Ido Amit, Noa Novershtern & Jacob H. Hanna

Nature (2013), doi:10.1038/nature12587

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Reprogramming in situ: Spanish Team is First to Produce Embryonic Stem Cells in Living Adult Organisms

The characteristics of the obtained stem cells correspond to a primitive totipotent state that has never before been obtained

Wednesday, 11 September 2013

A team from the Spanish National Cancer Research Centre (CNIO) has become the first to make adult cells from a living organism retreat in their evolutionary development to recover the characteristics of embryonic stem cells.

Pictured are Manuel Serrano and Maria Abad

in his laboratory at the CNIO. Credit: Spanish

National Cancer Research Center (CNIO).

Researchers have also discovered that these embryonic stem cells, obtained directly from the inside of the organism, have a broader capacity for differentiation than those obtained via in vitro culture. Specifically, they have the characteristics of totipotent cells: a primitive state never before obtained in a laboratory.

The study, carried out by CNIO, was led by Manuel Serrano, the director of the Molecular Oncology Programme and head of the Tumoural Suppression Laboratory. The study was supported by Manuel Manzanares's team from the Spanish National Cardiovascular Research Centre (CNIC).

Embryonic stem cells are the main focus for the future of regenerative medicine. They are the only ones capable of generating any cell type from the hundreds of cell types that make up an adult organism, so they are the first step towards curing illnesses such as Alzheimer, Parkinson's disease or diabetes. Nevertheless, this type of cell has a very short lifespan, limited to the first days of embryonic development, and they do not exist in any part of an adult organism.

One of the greatest achievements in recent biomedical research was in 2006 when Shinya Yamanaka managed to create embryonic stem cells (pluripotent stem cells, induced in vitro or in vitro iPSCs) in a laboratory from adult cells, via a cocktail of just four genes. Yamanaka's discovery, for which he was awarded the Nobel Prize in Medicine in 2012, opened a new horizon in regenerative medicine.

CNIO researchers have taken another step forward, by achieving the same as Yamanaka, but this time within the same organism, in mice, without the need to pass through in vitro culture dishes. Generating these cells within an organism brings this technology even closer to regenerative medicine.

The first challenge for CNIO researchers was to reproduce the Yamanaka experiment in a living being. They chose a mouse as a model organism. Using genetic manipulation techniques, researchers created mice in which Yamanaka's four genes could be activated at will. When these genes were activated, they observed that the adult cells were able to retreat in their evolutionary development to become embryonic stem cells in multiple tissues and organs.

María Abad, the lead author of the article and a researcher in Serrano's group, said:

"This change of direction in development has never been observed in nature. We have demonstrated that we can also obtain embryonic stem cells in adult organisms and not only in the laboratory".

Manuel Serrano added that:

"We can now start to think about methods for inducing regeneration locally and in a transitory manner for a particular damaged tissue".

Stem cells obtained in mice also show totipotent characteristics never generated in a laboratory, equivalent to those present in human embryos at the 72-hour stage of development, when they are composed of just 16 cells.

In comparison with the cells obtained with the technique developed by Yamanaka, the stem cells obtained by CNIO therefore represent an even earlier embryonic state, with greater capacity for differentiation.

The authors were even able to induce the formation of pseudo-embryonic structures (teratomas) in the thoracic and abdominal cavities of the mice. These pseudo-embryos displayed the three layers typical of embryos (ectoderm, mesoderm and endoderm), and extra-embryonic structures such as the Vitelline membrane and even signs of blood cell formation.

"This data tell us that our stem cells are much more versatile than Yamanaka's in vitro iPSCs, whose potency generates the different layers of the embryo but never tissues that sustain the development of a new embryo, like the placenta", said the CNIO researcher.

The authors emphasise that the possible therapeutic applications of their work are still distant, but they admit that, without doubt, it might mean a change of direction for stem cell research, for regenerative medicine or for tissue engineering.

"Our stem cells also survive outside of mice, in a culture, so we can also manipulate them in a laboratory", said Abad.

"The next step is studying if these new stem cells are capable of efficiently generating different tissues such as that of the pancreas, liver or kidney".

Source: Centro Nacional de Investigaciones Oncologicas (CNIO) 

Contact: Juan J. Gomez

Reference:

Reprogramming in vivo produces teratomas and iPSCs with totipotency features

María Abad, Lluc Mosteiro, Cristina Pantoja, Marta Cañamero, Teresa Rayón, Inmaculada Ors, Osvaldo Graña, Diego Megías, Orlando Domínguez, Dolores Martínez, Miguel Manzanares, Sagrario Ortega, Manuel Serrano

Nature (2013), DOI: 10.1038/nature12586

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Researchers Develop Efficient Model for Generating Human iPSCs

Approach has potential to simplify generation of iPSCs for use in human stem cell therapies

Thursday, 01 August 2013

Researchers at the University of California, San Diego School of Medicine report a simple, easily reproducible RNA-based method of generating human induced pluripotent stem cells (iPSCs) in the August 1 edition of Cell Stem Cell. Their approach has broad applicability for the successful production of iPSCs for use in human stem cell studies and eventual cell therapies.

This graphic depicts single RNA generation

of human iPS cells. Credit: Peter Allen, UC

Santa Barbara. 

Partially funded by grants from the California Institute for Regenerative Medicine (CIRM) and the National Institutes of Health (NIH), the methods developed by the UC San Diego researchers dramatically improve upon existing DNA-based approaches – avoiding potential integration problems and providing what appears to be a safer and simpler method for future clinical applications.

The generation of human iPSCs has opened the potential for regenerative medicine therapies based on patient-specific, personalized stem cells. Pluripotent means that these cells have the ability to give rise to any of the body's cell types. The human iPSCs are typically artificially derived from a non-pluripotent adult cell, such as a skin cell. They retain the characteristics of the body's natural pluripotent stem cells, commonly known as embryonic stem cells. Because iPSCs are developed from a patient's own cells, it was first thought that treatment using them would avoid any immunogenic responses. However, depending on methods used to generate such iPSCs, they may pose significant risks that limit their use. For example, using viruses to alter the cell's genome could promote cancer in the host cell.

Methods previously developed to generate integration-free iPSCs were not easily and efficiently reproducible. Therefore, the UC San Diego researchers focused their approach on developing a self-replicating, RNA-based method (one that doesn't integrate into the DNA) with the ability to be retained and degraded in a controlled fashion, and that would only need to be introduced once into the cell.

Using a Venezuelan equine virus (VEE) with structural proteins deleted, but non-structural proteins still present, the scientists added four reprogramming factors (OCT4, KLF4, SOX2 with either c-MYC or GLIS1). They made a single transfection of the VEE replicative form (RF) RNA into new-born or adult human fibroblasts, connective tissue cells that provide a structural framework for many other tissues.

"This resulted in efficient generation of iPSCs with all the hallmarks of stem cells," said principal investigator Steven Dowdy, PhD, professor in the UC San Diego Department of Cellular & Molecular Medicine.

"The method is highly reproducible, efficient, non-integrative – and it works."

Dowdy added that it worked on both young and old human cells. He explained that this is important since – in order to be used therapeutically in fighting disease or to create disease models for research – iPSCs will need to be derived from the cells of middle-aged to old adults who are more prone to the diseases scientists are attempting to treat. In addition, reprogramming factors can be easily changed.

Source: University of California - San Diego 

Contact: Debra Kain

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ZenMaster

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

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

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Epilepsy in a Dish

Stem cell research reveals clues to disease's origins and possible treatment

Thursday, 25 July 2013

A new stem cell-based approach to studying epilepsy has yielded a surprising discovery about what causes one form of the disease, and may help in the search for better medicines to treat all kinds of seizure disorders.

This diagram shows the process by which

scientists can take skin cells from patients

with epilepsy, convert them to stem cells, and

then create neurons (brain nerve cells) from

them. The induced neurons contain the same

genetic mutation(s) carried by the patients.

Credit: Parent lab, University of Michigan

Medical School. 

The findings, reported by a team of scientists from the University of Michigan Medical School and colleagues, use a technique that could be called "epilepsy in a dish".

By turning skin cells of epilepsy patients into stem cells, and then turning those stem cells into neurons, or brain nerve cells, the team created a miniature testing ground for epilepsy. They could even measure the signals that the cells were sending to one another, through tiny portals called sodium channels.

In neurons derived from the cells of children who have a severe, rare genetic form of epilepsy called Dravet syndrome, the researchers report abnormally high levels of sodium current activity. They saw spontaneous bursts of communication and "hyper excitability" that could potentially set off seizures. Neurons made from the skin cells of people without epilepsy showed none of this abnormal activity.

They report their results online in the Annals of Neurology, and have further work in progress to create induced pluripotent stem cell lines from the cells of patients with other genetic forms of epilepsy. The work is funded by the National Institutes of Health, the American Epilepsy Society, the Epilepsy Foundation and U-M.

The new findings differs from what other scientists have seen in mice – demonstrating the importance of studying cells made from human epilepsy patients. Because the cells came from patients, they contained the hallmark seen in most patients with Dravet syndrome: a new mutation in SCN1A, the gene that encodes the crucial sodium channel protein called Nav1.1. That mutation reduces the number of channels to half the normal number in patients' brains.

"With this technique, we can study cells that closely resemble the patient's own brain cells, without doing a brain biopsy," says senior author and team leader Jack M. Parent, M.D., professor of neurology at U-M and a researcher at the VA Ann Arbor Healthcare System.

"It appears that the cells are overcompensating for the loss of channels due to the mutation. These patient-specific induced neurons hold great promise for modelling seizure disorders, and potentially screening medications."

With the new paper, Parent, postdoctoral fellow Yu Liu, Ph.D. and their collaborators Lori Isom, Ph.D., professor of Pharmacology and of Molecular and Integrative Physiology at U-M, and Miriam Meisler, Ph.D., Distinguished University Professor of Human Genetics at U-M, report striking discoveries about what is happening at the cell level in the neurons of Dravet syndrome patients with a mutated SCN1A gene.

They also demonstrated that the effect is rooted in something that happens after function of the gene is reduced due to the mutation, though they don't yet know how or why the nerve cells overcompensate for partial loss of this channel.

And, they found that the neurons didn't show the tell-tale signs of hyper excitability in the first few weeks after they were made – consistent with the fact that children with Dravet syndrome often don't suffer their first seizures until they are several months old.

"In addition, reproduction of the hyperactivity of epileptic neurons in these cell cultures demonstrates that there is an intrinsic change in the neurons that does not depend on input from circuits in the brain," says co-author Meisler.

A platform for testing medications

Many Dravet patients don't respond to current epilepsy medications, making the search for new options urgent. Their lives are constantly under threat by the risk of SUDEP, sudden unexplained death in epilepsy – and they never outgrow their condition, which delays their development and often requires round-the-clock care.

"Working with patient families, and translating our sodium channel research to a paediatric disease, has made our basic science work much more immediate and critical," says Isom, who serves on the scientific advisory board of the Dravet Syndrome Foundation along with Meisler. Parent, who co-directs U-M's Comprehensive Epilepsy Program, was recently honoured by the foundation.

The team is now working toward screening specific compounds for seizure-calming potential in Dravet syndrome, by testing their impact on the cells in the "epilepsy in a dish" model. The National Institutes of Health has made a library of drugs that have been approved by the U.S. Food and Drug Administration available for researchers to use – potentially allowing older drugs to have a second life treating an entirely different disease from what they were initially intended.

Parent and his colleagues hope to identify drugs that affect certain aspects of sodium channels, to see if they can dampen the sodium currents and calm hyper excitability. The team is exploring new techniques that can make this process faster, using microelectrodes and calcium-sensitive dyes. They also hope to use the model to study potential drugs for non-genetic forms of epilepsy.

Having a U-M team that includes experts in induced pluripotent stem cell biology, sodium channel physiology and epilepsy genetics expertise helps the research progress, Parent notes.

"Epilepsy is a complicated brain network disease," he says.

"It takes team-based science to address it."

Patients as part of the research team

The U-M team's research wouldn't be possible without the participation of patients with Dravet syndrome and other genetic forms of epilepsy, and their parents.

More than 100 of them have joined the International Ion Channel Epilepsy Patient Registry, which is based at U-M and Miami Children's Hospital and co-funded by the Dravet Syndrome Foundation and the ICE Epilepsy Alliance. The researchers hope to be able to conduct clinical trials of potential drugs with participation by these patients and others.

Meanwhile, patients with other genetically based neurological diseases can also help U-M scientists discover more about their conditions, by taking part in other efforts to create induced neurons from skin cells. Parent and his team have worked with several other U-M faculty to create stem cell lines from skin cells provided by patients with other diseases including forms of ataxia and lysosomal storage disease.

Source: University of Michigan Health System 

Contact: Kara Gavin

Reference:

Dravet syndrome patient-derived neurons suggest a novel epilepsy mechanism

Liu, Y., Lopez-Santiago, L. F., Yuan, Y., Jones, J. M., Zhang, H., O'Malley, H. A., Patino, G. A., O'Brien, J. E., Rusconi, R., Gupta, A., Thompson, R. C., Natowicz, M. R., Meisler, M. H., Isom, L. L. and Parent, J. M.

Ann Neurol. (2013), doi: 10.1002/ana.23897

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ZenMaster

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

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

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