Stem Cells from Amniotic Fluid: Reprogrammed Amniotic Fluid Cells Can Generate All Types of Body Cells

Stem Cells from Amniotic Fluid: Reprogrammed Amniotic Fluid Cells Can Generate All Types of Body Cells

  10:06:00 AM    Science Lover

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Reprogrammed amniotic fluid cells can generate all types of body cells. High hopes rest on stem cells: one day, they may be used to treat many diseases. To date, embryos are the main source of these cells, but this raises ethical problems. Scientists at the Max Planck Institute for Molecular Genetics in Berlin have now managed to convert amniotic fluid cells into pluripotent stem cells. These amniotic fluid-derived iPS cells are hardly distinguishable from embryonic stem cells. However, they "remember" where they came from.

Top: Before their reprogramming into amniotic fluid iPS cells, 
human amniotic fluid cells are outwardly distinguishable from 
embryonic stem cells. Bottom left: Amniotic fluid iPS cells 
produce OCT4 (green), one of the most important marker 
proteins for embryonic stem cells. Bottom right: Starting from 
this embryonic stem cell phase, the amniotic fluid iPS cells can 
form hepatocyte-like cells and others. They produce the plasma
protein alpha-fetoprotein (red), which is abundant in fetal 
liver. (Credit: Max Planck Institute for Molecular Genetics)

The research appears in the online journal PLoS ONE, published by the Public Library of Science.

The special abilities of embryonic stem cells can today be used in multiple "grown-up" cells (e.g. skin and hair cells). This is done by reprogramming the cells and converting them to "induced pluripotent stem cells" (iPS cells). These then possess the typical properties of embryonic stem cells, meaning they can generate any of the cell types of the human body (pluripotency), and they can multiply endlessly.

Stem cells with memory
 
The scientists have shown that the amniotic fluid iPS cells can form different human cell types. They have also discovered that induced pluripotent stem cells can remember the original cell type from which they were generated. During cellular reprogramming, various genes that control the development of stem cells are apparently switched on or remain active. This confirms other current research results, which show that iPS cells derived from distinct tissues are prone to follow their pre-destined developmental path upon spontaneous differentiation. "We don't know just yet whether this donor-cell type memory will have an impact on possible medical treatment, or which type of somatic cell-derived iPS cell will be most suitable for treatment," cautions Katharina Wolfrum of the Max Planck Institute for Molecular Genetics.

Amniotic fluid cells have a number of advantages over other cell types. For one thing, amniotic fluid cells are routinely harvested in antenatal examinations to enable the early detection of disease. In most cases, more cells are obtained than are actually needed. In addition, the amniotic fluid mixture contains different types of cells from the unborn child, including stem-cell-like cells. As they are not very old, they have fewer environmentally-induced mutations, making them genetically more stable. "This may mean that it is possible to reprogram these amniotic fluid cells faster and more easily than other cell types, making amniotic fluid-derived iPS cells an interesting complement to embryonic stem cells," explains James Adjaye of the Max Planck Institute in Berlin.

Moreover, amniotic fluid cells could be extracted for cellular reprogramming before the birth of a child and be prepared for their intended use while the pregnancy is still ongoing. "This would make it possible to test which drugs work for a baby and whether they are tolerated, before that baby is born. Moreover, in the future, sick newborns can be treated with cells from their own body," says Adjaye.

Admin's Note: This article is not intended to provide medical advice, diagnosis or treatment.

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A New Way to Make Stem Cells Using RNA instead of DNA could avoid the health risks--and the political pitfalls--of stem-cell treatments.

A New Way to Make Stem Cells Using RNA instead of DNA could avoid the health risks--and the political pitfalls--of stem-cell treatments.

  1:27:00 AM    Science Lover

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A Harvard researcher has developed a way to make pluripotent stem cells that solves several of the major impediments to using them to treat human diseases.

New cell: This microscope image shows neurons
(colored green) created from pluripotent stem
cells using modified RNA.
Credit: Phil Manos, Cell Press

Derrick Rossi, an assistant professor at Harvard Medical School, created pluripotent stem cells--which can turn into virtually any other type of cell in the body--from non-stem cells without using viruses to tinker with a cell's genome, as conventional methods do. This means that Rossi's method could be substantially safer for treating disease. The work is published today in the journal Cell Stem Cell.

"Rossi has figured out how to turn a skin cell into a stem cell without genetic modifications, and to do it efficiently," said Doug Melton, codirector of the Harvard Stem Cell Institute, where Rossi is a principal faculty member, at a press conference.

Rossi's innovation, which has not yet been tested in people, was to use messenger RNA instead of DNA to produce the four proteins needed to reprogram the cell. He has started a company called ModeRNA to commercialize this use of messenger RNA. He said the approach may also have potential in gene therapy, which also relies on viruses to deliver treatment, but he declined to talk further about the company or possible gene therapy applications because the work is at such an early stage.

Improving the usability of man-made stem cells is key to helping patients and ending the political morass that has slowed stem-cell research. On Tuesday, a U.S. federal appeals court allowed federal funding of embryonic stem-cell research to continue while a legal case against such funding proceeds.

The human embryonic stem cells used in research were mostly derived from embryonic tissue grown a decade ago. These are the most versatile cells in the body, and the gold standard by which man-made cells are judged.

Four years ago, Japanese researcher Shinya Yamanaka showed that regular cells could be turned into embryonic-like stem cells--called induced pluripotent stem (iPS) cells--through the introduction of four specific proteins. Theoretically, this meant that doctors could take skin cells from a sick or disabled person, transform them into stem cells, and then into a specialized cell to treat them--an insulin-producing islet cell for someone with diabetes or a nerve cell for someone who is paralyzed, for instance. Using iPS cells avoids the need to destroy embryos and, because they can be derived from the patient's own cells, means less risk of rejection.

But only one in 1,000 or one in 10,000 skin cells could be transformed into a stem cell using Yamanaka's method. It also changes a cell's genes in ways that might trigger cancer or other problems.

Rossi 's idea was to produce Yamanaka's four proteins in a different way. Instead of using the DNA that holds the instructions for making proteins, he wanted to use RNA, which carries those instructions to the place in a cell where proteins are made.

His first several attempts were miserable failures. When he tried to change the messages the RNA carried, he triggered a serious immune response and most of the cells shut down or self-destructed. Rossi then tried modifying the RNA chemically and eventually figured out a way to allow his changes to escape immune detection while delivering the message. "This was key to our success," said Rossi, who is also a researcher at Children's Hospital Boston. "We could encode RNA for any protein we wanted to express and insert it into a cell."

Rossi said it was a happy coincidence that using RNA instead of changing the DNA was as much as 100-fold more efficient. He said the effect was possibly because the process more closely reflects how cells themselves transform.

Rossi successfully differentiated his stem cells into muscle cells using RNA, a process that may offer promise in gene therapy and other treatments. His method does not alter the cell's underlying genome, though Rossi admits that he does not yet understand what it does to the cell's epigenome, which controls expression of genes.

Rossi said that his cells, which he's named RiPS, for "RNA induced Pluripotent Stem" cells, are more like embryonic stem cells than traditional iPS cells because they have not been genetically altered.

Melton said the Harvard Stem Cell Institute, which includes several hundred stem-cell researchers from across Harvard University and its affiliated hospitals, will now be making its standard iPS cells with Rossi's method.

In a prepared statement, Yamanaka, now at the University of California, San Francisco, said Rossi's approach to generating stem cells seems promising, and he would like to have someone in his lab try it.

"The quality of the induced pluripotent stem cells generated by this method should be carefully examined because their characteristics vary depending on the induction methods and the origins of the resulting cells," he said. "The standard method to generate iPSCs for clinical applications has yet to be established. I think this method has the potential for it."

Jacob Hanna, a postdoctoral fellow at the Whitehead Institute for Biomedical Research in Cambridge, said he's also eager to begin working with the cells.

"I think it's a very exciting paper with a very promising method," said Hanna, who was not involved in the research. When asked if he was jealous that Rossi had developed the method first, Hanna said, "Yes, of course! It's a very nice paper," quickly adding, "Jealous in a very positive and supporting way."

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Ancient Viral Invasion Shaped Human Genome

Ancient Viral Invasion Shaped Human Genome

  2:14:00 AM    Science Lover

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Scientists at the Genome Institute of Singapore (GIS), a biomedical research institute of the Agency for Science, Technology and Research (A*STAR), and their colleagues from the National University of Singapore, Nanyang Technological University, Duke-NUS Graduate Medical School and Princeton University have recently discovered that viruses that 'invaded' the human genome millions of years ago have changed the way genes get turned on and off in human embryonic stem (ES) cells.

Scientists have discovered that viruses that "invaded" 
the human genome millions of years ago have changed the 
way genes get turned on and off in human embryonic
stem cells. (Credit: iStockphoto/Martin McCarthy)

The study provides definitive proof of a theory that was first proposed in the 1950s by Nobel Laureate in physiology and medicine, Barbara McClintock, who hypothesized that transposable elements, mobile pieces of the genetic material (DNA), such as viral sequences, could be "control elements" that affect gene regulation once inserted in the genome.

This finding is an important contribution to the advancement of stem cell research and to its potential for regenerative medicine. Led by GIS Senior Group Leader Dr Guillaume Bourque, the study was published in Nature Genetics on June 6, 2010.

Through the use of new sequencing technologies, the scientists studied the genomic locations of three regulatory proteins (OCT4, NANOG and CTCF) in human and mouse embryonic stem (ES) cells. Interestingly, while the scientists found a lot of similarities, they also found many differences in the methods and the types of genes that are being regulated in humans. In particular, it was discovered that specific types of viruses that inserted themselves in the human genomes millions of years ago have dramatically changed the gene regulatory network in human stem cells.

"This study is a computational and experimental tour de force. It provides undeniable evidence that some transposable elements, which are too often dismissed as merely junk DNA, are key components of a regulatory code underlying human development," said Dr Cedric Feschotte, Associate Professor of the University of Texas Arlington.

The comparisons between the human and mouse model system in the study of gene regulatory networks help to advance the understanding of how stem cells differentiate into various cell types of the body. "This understanding is crucial in the improved development of regenerative medicine for diseases such as Parkinson's disease and leukaemia," said Dr Bourque. "Despite the advantages of using mouse ES cells in the study of gene regulatory networks, further research must focus more directly on human stem cells. This is due to the inherent challenges of converting the results of studies done from one species to that of the next. More research will need to be done in both human and non-human primate stem cells for findings on stem cells to be used in clinical application."

Prof Raymond L. White, PhD, Rudi Schmid Distinguished Professor of Neurology, University of California said, "The paper reports very exciting new findings that establish a new and fundamentally distinct mechanism for the regulation of gene expression. By comparing the genomes of mouse with human, the scientists were able to show that the binding sites for gene regulatory factors are very often not in the same place between the two species. This by itself would be very surprising, but the investigators go further and demonstrate that many of the sites are imbedded within a class of DNA sequences called "transposable" elements because of their ability to move to new places in the genome. There are a number of such elements believed to be the evolutionary remnants of viral genomes, but it was very surprising to learn that they were carrying binding sites for regulatory elements to new locations. These changes in regulation would be expected to create major changes in the organisms which carry them. Indeed, many think that regulatory changes are at the heart of speciation and may have played a large role in the evolution of humans from their predecessors. This is likely to be a landmark paper in the field."

Dr Eddy Rubin, Director of the U.S. Department of Energy Joint Genome Institute and Director of the Genomics Division at Lawrence Berkeley National Laboratory in Berkeley added, "This study using a comparative genomics strategy discovered important human specific properties of the regulatory network in human ES cells. This information is significant and should contribute to helping move the regenerative medicine field forward."

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Better Way to Grow Stem Cells Developed

Better Way to Grow Stem Cells Developed

  10:59:00 PM    Science Lover

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Human pluripotent stem cells, which can become any other kind of body cell, hold great potential to treat a wide range of ailments, including Parkinson's disease, multiple sclerosis and spinal cord injuries. However, scientists who work with such cells have had trouble growing large enough quantities to perform experiments -- in particular, to be used in human studies. Furthermore, most materials now used to grow human stem cells include cells or proteins that come from mice embryos, which help stimulate stem-cell growth but would likely cause an immune reaction if injected into a human patient.

This image shows human embryonic stem cells grown on a synthetic surface developed by MIT researchers. The cells at top (blue) are stained to reveal their nuclei, while the cells in the middle and bottom are stained for proteins that are known to be present when cells are pluripotent. Green cells are stained for Oct4 (using green fluorescent protein) and red cells are stained for SSEA-4. (Credit: Ying Mei, Krishanu Saha, Robert Langer, Rudolf Jaenisch, and Daniel G. Anderson)

To overcome those issues, MIT chemical engineers, materials scientists and biologists have devised a synthetic surface that includes no foreign animal material and allows stem cells to stay alive and continue reproducing themselves for at least three months. It's also the first synthetic material that allows single cells to form colonies of identical cells, which is necessary to identify cells with desired traits and has been difficult to achieve with existing materials.

The research team, led by Professors Robert Langer, Rudolf Jaenisch and Daniel G. Anderson, describes the new material in the Aug. 22 issue of Nature Materials. First authors of the paper are postdoctoral associates Ying Mei and Krishanu Saha.

Human stem cells can come from two sources -- embryonic cells or body cells that have been reprogrammed to an immature state. That state, known as pluripotency, allows the cells to develop into any kind of specialized body cells.

It also allows the possibility of treating nearly any kind of disease that involves injuries to cells. Scientists could grow new neurons for patients with spinal cord injuries, for example, or new insulin-producing cells for people with type 1 diabetes.

To engineer such treatments, scientists would need to be able to grow stem cells in the lab for an extended period of time, manipulate their genes, and grow colonies of identical cells after they have been genetically modified. Current growth surfaces, consisting of a plastic dish coated with a layer of gelatin and then a layer of mouse cells or proteins, are notoriously inefficient, says Saha, who works in Jaenisch's lab at the Whitehead Institute for Biomedical Research.

"For therapeutics, you need millions and millions of cells," says Saha. "If we can make it easier for the cells to divide and grow, that will really help to get the number of cells you need to do all of the disease studies that people are excited about."

Previous studies had suggested that several chemical and physical properties of surfaces -- including roughness, stiffness and affinity for water -- might play a role in stem-cell growth. The researchers created about 500 polymers (long chains of repeating molecules) that varied in those traits, grew stem cells on them and analyzed each polymer's performance. After correlating surface characteristics with performance, they found that there was an optimal range of surface hydrophobicity (water-repelling behavior), but varying roughness and stiffness did not have much effect on cell growth.

They also adjusted the composition of the materials, including proteins embedded in the polymer. They found that the best polymers contained a high percentage of acrylates, a common ingredient in plastics, and were coated with a protein called vitronectin, which encourages cells to attach to surfaces.

Using their best-performing material, the researchers got stem cells (both embryonic and induced pluripotent) to continue growing and dividing for up to three months. They were also able to generate large quantities of cells -- in the millions.

The MIT researchers hope to refine their knowledge to help them build materials suited to other types of cells, says Anderson, from the MIT Department of Chemical Engineering, the Harvard-MIT Division of Health Sciences and Technology, and the David H. Koch Institute for Integrative Cancer Research. "We want to better understand the interactions between the cell, the surface and the proteins, and define more clearly what it takes to get the cells to grow," he says.

Other MIT authors of the paper are Said Bogatyrev, Z. Ilke Kalcioglu, Maisam Mitalipova, Neena Pyzocha, Fredrick Rojas and Krystyn Van Vliet. Jing Yang, Andrew Hook, Martyn Davies and Morgan Alexander of the University of Nottingham (United Kingdom) and Seung-Woo Cho of Yonsei University (Korea) are also authors of the paper.

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Toward Deafness Cure: Inner-Ear Cells Created

Toward Deafness Cure: Inner-Ear Cells Created

  9:47:00 PM    Science Lover

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Deep inside the ear, specialized cells called hair cells detect vibrations in the air and translate them into sound. Ten years ago, Stefan Heller, PhD, professor of otolaryngology at the Stanford University School of Medicine, came up with the idea that if you could create these cells in the laboratory from stem cells, it would go a long way toward helping scientists understand the molecular basis of hearing in order to develop better treatments for deafness.

Section through the organ of corti, 
showing inner and outer hair cells. 
(Credit: Courtesy of Wikimedia Commons)

After years of lab work, researchers in Heller's lab will report in the May 14 issue of Cell that they have found a way to develop mouse cells that look and act just like the animal's inner-ear hair cells -- the linchpin to our sense of hearing and balance -- in a petri dish.

If they can further perfect the recipe to generate hair cells in the millions, it could lead to significant scientific and clinical advances along the path to curing deafness in the future, they said.

"This gives us real hope that there might be some kind of therapy for regenerating hair cells," said David Corey, PhD, professor of neurobiology at Harvard University who was not involved in the study. "It could take a decade or more, but it's a possibility."

Using both embryonic stem cells from mice as well as reprogrammed mouse fibroblasts (a type of relatively undifferentiated cell found in many parts of the body), the researchers present a step-by-step guide on how to coax these cells into the sensory cells that normally reside in the inner ear.

"We knew it was really working when we saw them in the electron microscope," Heller said. "They really looked like they were more or less taken out of the ear."

Humans are born with 30,000 cochlear and vestibular hair cells per ear. (By contrast, one retina harbors about 120 million photoreceptors.) When a significant number of these cells are lost or damaged, hearing loss occurs. The major reason for hearing loss and certain balance disorders is that -- unlike other species such as birds -- humans and other mammals are unable to spontaneously regenerate these hearing cells.

As the population has aged and noise pollution has grown more severe, health experts now estimate that one in three adults over the age of 65 has developed a handicapping hearing loss due to the destruction of these limited number of hair cells.

One of the roadblocks to understanding the molecular basis of hearing is the paucity of hair cells available for study, Heller said. While researchers will ultimately need human hair cells, the mouse version is a good model for the initial phases of experimentation, he said. In addition to using mouse embryonic stem cells, the researchers used fibroblasts that had been reprogrammed to behave like stem cells: These are known as induced pluripotent stem cells, or iPS cells.

"Our study offers a protocol to generate millions of functional hair cells from a renewable source," Heller said. "We can now generate these cells and don't have to go through dozens of mice for a single experiment. This allows us to do molecular studies with much higher efficiency."

The study details how the researchers succeeded in coaxing the mouse embryonic stem cells and the iPS cells through different phases of development that occur in the womb. According to lead author Kazuo Oshima, MD, PhD, a research instructor at Stanford who works in Heller's lab, they started by turning the stem and iPS cells into the type of cells that form a young embryo's ectoderm -- the embryo's outer layer of cells that eventually differentiate into many tissues and structures, such as skin and nerve cells. Next they used specific growth factors to transform them into "otic-progenitor" cells (otic means ear). And after that, they varied the chemical soup in the dish, so that the cells clustered in a manner similar to hair cells and developed stereociliary bundles, which are also characteristic of hair cells.

"We looked at how the ear develops in an embryo, at the developmental steps, and mimicked these steps in a culture dish," Heller said.

Hair cells in the inner ear contain tiny clumps of hair-like projections, known as stereocilia. Sound vibrations cause the stereocilia to bend slightly, causing mechanical vibrations that are then converted into an electrochemical signal that the brain interprets as sound.

The cells in the petri dish, under close examination, had this same structure.

"These cells have a very intriguing structure," Heller said. "They look like they have hair tufts of stereocilia."

More importantly, further study showed that the cells also responded to mechanical stimulation by producing currents just like hair cells. Using a probe, researchers stimulated the bundles and recorded the currents that were evoked. Co-author Anthony Ricci, PhD, associate professor of otolaryngology, was responsible for this step of the work.

Heller, a leader in stem-cell based research on the inner ear, has recently been focused on two paths for possible cures for deafness: drug therapy -- which could be as simple as an application of ear drops -- and stem cell transplantation into the inner ear.

Both paths could be further advanced by the ability to develop hair-cell-like cells, he said. "We could now test thousands of drugs in a culture dish," he explained. "It is impossible to achieve such a scale in animals. Within a decade or so we could reap the benefits of this type of screening."

The lab's research into the regeneration of hair cells for transplantation into the inner ear to cure deafness will also continue.

"We made hair-cell-like cells in a petri dish," said Oshima. "This is an important step toward development of future therapies."

The study was funded by grants from the National Institute of Health, the California Institute for Regenerative Medicine and by a Neuroscience of Brain Disorders Award from the McKnight Endowment Fund for Neuroscience.

Other Stanford co-authors include postdoctoral scholars Kunyoo Shin, PhD; Mark Diensthuber, MD; and Anthony Peng, PhD.

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Major Step Forward In Cell Reprogramming, Researchers Report

Major Step Forward In Cell Reprogramming, Researchers Report

  3:02:00 AM    Science Lover

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A team of Harvard Stem Cell Institute (HSCI) researchers has made a major advance toward producing induced pluripotent stem cells, or iPS cells, that are safe enough to use in treating diseases in patients.

Lee Rubin, director of translational medicine at HSCI and the other senior author on the research team, said that "our goals were to try to as discretely and specifically as possible guide the cells through the deprogramming process" from the adult state to the embryonic-like state. (Credit: Photograph by Kris Snibbe/Harvard Staff Photographer)

“This demonstrates that we’re halfway home, and remarkably we got halfway home with just one chemical,” said Kevin Eggan, an HSCI principal faculty member who is the senior author of the paper being published online today by the journal Cell Stem Cell.

“There are four genes that do this, and with just one chemical we replaced half the genes,” said Eggan, who is also an assistant professor in Harvard’s Department of Stem Cell and Regenerative Biology. “The one chemical replaces those two genes in different ways at different times in the experiment. The experiments we performed not only led to discovery of the chemical, but they also explained how it works,” he said.

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