Posts tagged induced pluripotent stem cells
Articles and news from the latest research reports.
Brain repair after injury and Alzheimer’s disease
Researchers at Penn State University have developed an innovative technology to regenerate functional neurons after brain injury, and also in model systems used for research on Alzheimer’s disease. The scientists have used supporting cells of the central nervous system, glial cells, to regenerate healthy, functional neurons, which are critical for transmitting signals in the brain.
Gong Chen, a professor of biology, the Verne M. Willaman Chair in Life Sciences at Penn State, and the leader of the research team, calls the method a breakthrough in the long journey toward brain repair. “This technology may be developed into a new therapeutic treatment for traumatic brain and spinal cord injuries, stroke, Alzheimer’s disease, Parkinson’s disease, and other neurological disorders,” Chen said. The research will be posted online by the journal Cell Stem Cell on 19 December 2013.
When the brain is harmed by injury or disease, neurons often die or degenerate, but glial cells become more branched and numerous. These “reactive glial cells” initially build a defense system to prevent bacteria and toxins from invading healthy tissues, but this process eventually forms glial scars that limit the growth of healthy neurons. “A brain-injury site is like a car-crash site,” Chen explained. “Reactive glial cells are like police vehicles, ambulances, and fire trucks immediately rushing in to help — but these rescue vehicles can cause problems if too many of them get stuck at the scene. The problem with reactive glial cells is that they often stay at the injury site, forming a glial scar and preventing neurons from growing back into the injured areas,” he explained.
So several years ago, Chen’s lab tested new ways to transform glial scar tissue back to normal neural tissue. “There are more reactive glial cells and fewer functional neurons in the injury site,” Chen said, “so we hypothesized that we might be able to convert glial cells in the scar into functional neurons at the site of injury in the brain. This research was inspired by the Nobel prize-winning technology of induced pluripotent stem cells (iPSCs) developed in Shinya Yamanaka’s group, which showed how to reprogram skin cells into stem cells,” Chen recalled.
Chen and his team began by studying how reactive glial cells respond to a specific protein, NeuroD1, which is known to be important in the formation of nerve cells in the hippocampus area of adult brains. They hypothesized that expressing NeuroD1 protein into the reactive glial cells at the injury site might help to generate new neurons — just as it does in the hippocampus. To test this hypothesis, his team infected reactive glial cells with a retrovirus that specifies the genetic code for the NeuroD1 protein. “The retrovirus we used is replication-deficient and thus cannot kill infected cells like other viruses found in the wild,” Chen said. “More importantly, a retrovirus can infect only dividing cells such as reactive glial cells, but it does not affect neurons, which makes it ideal for therapeutic use with minimal side effect on normal brain functions.”
In a first test, Chen and his team investigated whether reactive glial cells can be converted into functional neurons after injecting NeuroD1 retrovirus into the cortex area of adult mice. The scientists found that two types of reactive glial cells — star-shaped astroglial cells and NG2 glial cells — were reprogrammed into neurons within one week after being infected with the NeuroD1 retrovirus. “Interestingly, the reactive astroglial cells were reprogrammed into excitatory neurons, whereas the NG2 cells were reprogrammed into both excitatory and inhibitory neurons, making it possible to achieve an excitation-inhibition balance in the brain after reprogramming,” Chen said. His lab also performed electrophysiological tests, which demonstrated that the new neurons converted by the NeuroD1 retrovirus could receive neurotransmitter signals from other nerve cells, suggesting that the newly converted neurons had successfully integrated into local neural circuits.
In a second test, Chen and his team used a transgenic-mouse model for Alzheimer’s disease, and demonstrated that reactive glial cells in the mouse’s diseased brain also can be converted into functional neurons. Furthermore, the team demonstrated that even in 14-month-old mice with Alzheimer’s disease — an age roughly equivalent to 60 years old for humans — injection of the NeuroD1 retrovirus into a mouse cortex can still induce a large number of newborn neurons reprogrammed from reactive glial cells. “Therefore, the conversion technology that we have demonstrated in the brains of mice potentially may be used to regenerate functional neurons in people with Alzheimer’s disease,” Chen said.
To ensure that the glial cell-to-neuron conversion method is not limited to rodent animals, Chen and his team further tested the method on cultured human glial cells. “Within 3 weeks after expression of the NeuroD1 protein, we saw in the microscope that human glial cells were reinventing themselves: they changed their shape from flat sheet-like glial cells into normal-looking neurons with axon and dendritic branches,” Chen said. The scientists further tested the function of these newly converted human neurons and found that, indeed, they were capable of both releasing and responding to neurotransmitters.
"Our dream is to develop this in vivo conversion method into a useful therapy to treat people suffering from neural injury or neurological disorders," Chen said. "Our passionate motivation for this research is the idea that an Alzheimer’s patient, who for a long time was not able to remember things, could start to have new memories after regenerating new neurons as a result of our in vivo conversion method, and that a stroke victim who could not even move his legs might start to walk again."
Study finds a patchwork of genetic variation in the brain
It was once thought that each cell in a person’s body possesses the same DNA code and that the particular way the genome is read imparts cell function and defines the individual. For many cell types in our bodies, however, that is an oversimplification. Studies of neuronal genomes published in the past decade have turned up extra or missing chromosomes, or pieces of DNA that can copy and paste themselves throughout the genomes.
The only way to know for sure that neurons from the same person harbor unique DNA is by profiling the genomes of single cells instead of bulk cell populations, the latter of which produce an average. Now, using single-cell sequencing, Salk Institute researchers and their collaborators have shown that the genomic structures of individual neurons differ from each other even more than expected. The findings were published November 1, 2013, in Science.
"Contrary to what we once thought, the genetic makeup of neurons in the brain aren’t identical, but are made up of a patchwork of DNA," says corresponding author Fred Gage, Salk’s Vi and John Adler Chair for Research on Age-Related Neurodegenerative Disease.
In the study, led by Mike McConnell, a former junior fellow in the Crick-Jacobs Center for Theoretical and Computational Biology at the Salk, researchers isolated about 100 neurons from three people posthumously. The scientists took a high-level view of the entire genome—looking for large deletions and duplications of DNA called copy number variations or CNVs—and found that as many as 41 percent of neurons had at least one unique, massive CNV that arose spontaneously, meaning it wasn’t passed down from a parent. The CNVs are spread throughout the genome, the team found.
The miniscule amount of DNA in a single cell has to be chemically amplified many times before it can be sequenced. This process is technically challenging, so the team spent a year ruling out potential sources of error in the process.
"A good bit of our study was doing control experiments to show that this is not an artifact," says Gage. "We had to do that because this was such a surprise—finding out that individual neurons in your brain have different DNA content."
The group found a similar amount of variability in CNVs within individual neurons derived from the skin cells of three healthy people. Scientists routinely use such induced pluripotent stem cells (iPSCs) to study living neurons in a culture dish. Because iPSCs are derived from single skin cells, one might expect their genomes to be the same.
"The surprising thing is that they’re not," says Gage. "There are quite a few unique deletions and amplifications in the genomes of neurons derived from one iPSC line."
Interestingly, the skin cells themselves are genetically different, though not nearly as much as the neurons. This finding, along with the fact that the neurons had unique CNVs, suggests that the genetic changes occur later in development and are not inherited from parents or passed to offspring.
It makes sense that neurons have more diverse genomes than skin cells do, says McConnell, who is now an assistant professor of biochemistry and molecular genetics at the University of Virginia School of Medicine in Charlottesville. “The thing about neurons is that, unlike skin cells, they don’t turn over, and they interact with each other,” he says. “They form these big complex circuits, where one cell that has CNVs that make it different can potentially have network-wide influence in a brain.”
Spontaneously occurring CNVs have also been linked to risk for brain disorders such as schizophrenia and autism, but those studies usually pool many blood cells. As a result, the CNVs uncovered in those studies affect many if not all cells, which suggests that they arise early in development.
The purpose of CNVs in the healthy brain is still unclear, but researchers have some ideas. The modifications might help people adapt to new surroundings encountered over a lifetime, or they might help us survive a massive viral infection. The scientists are working out ways to alter genomic variability in iPSC-derived neurons and challenge them in specific ways in the culture dish.
Cells with different genomes probably produce unique RNA and then proteins. However, for now, only one sequencing technology can be applied to a single cell.
"If and when more than one method can be applied to a cell, we will be able to see whether cells with different genomes have different transcriptomes (the collection of all the RNA in a cell) in predictable ways," says McConnell.
In addition, it will be necessary to sequence many more cells, and in particular, more cell types, notes corresponding author Ira Hall, an associate professor of biochemistry and molecular genetics at the University of Virginia. “There’s a lot more work to do to really understand to what level we think the things we’ve found are neuron-specific or associated with different parameters like age or genotype,” he says.
(Source: salk.edu)
Microglia Can Be Derived From Patient-Specific Human Induced Pluripotent Stem Cells and May Help Modulate the Course of Central Nervous System Diseases
Today, during the 81st American Association of Neurological Surgeons (AANS) Annual Scientific Meeting, researchers announced new findings regarding the development of methods to turn human induced pluripotent stem cells (iPSC) into microglia, which could be used for not only research but potentially in treatments for various diseases of the central nervous system (CNS).
Microglia are the resident inflammatory cells of the CNS and can modulate the outcomes of a wide range of disorders including trauma, infections, stroke, brain tumors, and various degenerative, inflammatory and psychiatric diseases. However, the effective therapeutic use of microglia demonstrated in various animal CNS disease models currently cannot be translated to patients due to the lack of methods for procuring high-purity patient-specific microglia. Developing a method for obtaining these cells would be highly valuable.
In the study Differentiation of Induced Pluripotent Stem Cells to Microglia for Treatment of CNS Diseases, mouse and human iPSCs were generated and sequentially co-cultured on various cell monolayers and in the presence of added growth factors. The microglial identity of the resulting cells was confirmed using fluorescence activated cell sorting analyses, functional assays, gene expression analyses and brain engraftment ability. The study results will be shared by presenting author John K. Park, MD, PhD, FAANS, from 3:34-3:42 p.m. on Monday, April 29. Co-authors are Michael Shen, BS; Yong Choi, PhD; and Hetal Pandya, PhD.
In the results, researchers found mouse and human iPSCs co-cultured with OP9 cells differentiate into hematopoietic progenitor cells (HPCs). HPCs in turn co-cultured with astrocytes, generate cells that express CD11b, Iba-1 and CX3CR1; secrete the cytokines IL-6, IL-1ß and TNF-a; generate reactive oxygen species; and phagocytose fluorescent particles, all consistent with a microglial phenotype. Gene expression clustering using self-organizing maps indicates that iPSC-derived microglia more closely resemble normal microglia than other inflammatory cell types. The iPSC-derived microglia engraft and migrate to areas of injury within the brain. These finding have led researchers to conclude that iPSC-derived microglia may one day be useful as gene and protein delivery vehicles to the CNS.
“The actual results of our research were not surprising to us, but the overall importance of microglia in a wide variety of brain and spinal cord diseases was surprising. Microglia likely have a role in improving or worsening diseases such as multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, obsessive compulsive disorder and Rett’s syndrome, just to name a few,” said John K. Park, MD, PhD, FAANS. “Microglia are the principal immune system cells of the brain and spinal cord, and help fight infections as well as help the healing process after injuries such as trauma and strokes. They also play a poorly understood role in many neurodegenerative and psychiatric diseases. We have developed methods to turn iPSCs into microglia. Because human iPSC can easily be obtained in large numbers, we can now generate large numbers of human microglia not only for use in experiments, but also potentially for use in treatments. The ability to study normal and diseased human microglia will lead to a greater understanding of their roles in healthy brains and various diseases. Diseases that are caused or exacerbated by defective microglia or a paucity of normal microglia may potentially be treated by microglia generated from a patient’s iPSC.”
(Source: newswise.com)
Yamanaka invented cell time machine
Dr. Shinya Yamanaka invented a time machine.
In the simplest of terms, that’s how he and his colleagues sometimes describe their work. They take full-grown cells from humans and they regress them - they send them back in time, to their earliest, embryonic state - and then they coax them into the future, into totally new types of cells.
Last week, Yamanaka was awarded the Nobel Prize in physiology or medicine for his work creating induced pluripotent stem (IPS) cells - cells that are genetically engineered into blank slates, allowing them to be transformed into any type of cell in the body.
His technique could allow scientists to explore human diseases like they never have before, or help doctors regenerate tissue lost to injury or illness. Using his technology, scientists can now take a skin cell and transform it into a heart cell that will actually beat in a lab dish.
"I was here, at Gladstone, the moment I learned we got human IPS cells," said Yamanaka last month, in an interview from his part-time office at San Francisco’s Gladstone Institutes. Yamanaka did most of the IPS cell work at his main lab in Japan.
"My colleague sent me the image, and it was, wow," Yamanaka said, offering a brief, modest smile. "We had beating human heart cells, made from IPS cells."