Scientists Create Stable, Self-Renewing Neural Stem Cells

In a paper published in the April 25 early online edition of the Proceedings of the National Academy of Sciences, researchers at the University of California, San Diego School of Medicine, the Gladstone Institutes in San Francisco and colleagues report a game-changing advance in stem cell science: the creation of long-term, self-renewing, primitive neural precursor cells from human embryonic stem cells (hESCs) that can be directed to become many types of neuron without increased risk of tumor formation.

"It's a big step forward," said Kang Zhang, MD, PhD, professor of ophthalmology and human genetics at Shiley Eye Center and director of the Institute for Genomic Medicine, both at UC San Diego. "It means we can generate stable, renewable neural stem cells or downstream products quickly, in great quantities and in a clinical grade millions in less than a week that can be used for clinical trials and, eventually, for clinical treatments. Until now, that has not been possible."

Human embryonic stem cells hold great promise in regenerative medicine due to their ability to become any kind of cell needed to repair and restore damaged tissues. But the potential of hESCs has been constrained by a number of practical problems, not least among them the difficulty of growing sufficient quantities of stable, usable cells and the risk that some of these cells might form tumors.

To produce the neural stem cells, Zhang, with co-senior author Sheng Ding, PhD, a former professor of chemistry at The Scripps Research Institute and now at the Gladstone Institutes, and colleagues added small molecules in a chemically defined culture condition that induces hESCs to become primitive neural precursor cells, but then halts the further differentiation process.

"And because it doesn't use any gene transfer technologies or exogenous cell products, there's minimal risk of introducing mutations or outside contamination," Zhang said. Assays of these neural precursor cells found no evidence of tumor formation when introduced into laboratory mice.

By adding other chemicals, the scientists are able to then direct the precursor cells to differentiate into different types of mature neurons, "which means you can explore potential clinical applications for a wide range of neurodegenerative diseases," said Zhang. "You can generate neurons for specific conditions like amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease), Parkinson's disease or, in the case of my particular research area, eye-specific neurons that are lost in macular degeneration, retinitis pigmentosa or glaucoma."

The new process promises to have broad applications in stem cell research. The same method can be used to push induce pluripotent stem cells (stem cells artificially derived from adult, differentiated mature cells) to become neural stem cells, Zhang said. "And in principle, by altering the combination of small molecules, you may be able to create other types of stem cells capable of becoming heart, pancreas, or muscle cells, to name a few."

The next step, according to Zhang, is to use these stem cells to treat different types of neurodegenerative diseases, such as macular degeneration or glaucoma in animal models.

Source: University of California

'Universal' Virus-Free Method Developed By Scientists To Turn Blood Cells Into 'Beating' Heart Cells

Johns Hopkins scientists have developed a simplified, cheaper, all-purpose method they say can be used by scientists around the globe to more safely turn blood cells into heart cells. The method is virus-free and produces heart cells that beat with nearly 100 percent efficiency, they claim.

"We took the recipe for this process from a complex minestrone to a simple miso soup," says Elias Zambidis, M.D., Ph.D., assistant professor of oncology and pediatrics at the Johns Hopkins Institute for Cell Engineering and the Kimmel Cancer Center.

Zambidis says, "many scientists previously thought that a nonviral method of inducing blood cells to turn into highly functioning cardiac cells was not within reach, but "we've found a way to do it very efficiently and we want other scientists to test the method in their own labs." However, he cautions that the cells are not yet ready for human testing.

To get stem cells taken from one source (such as blood) and develop them into a cell of another type (such as heart), scientists generally use viruses to deliver a package of genes into cells to, first, get them to turn into stem cells. However, viruses can mutate genes and initiate cancers in newly transformed cells. To insert the genes without using a virus, Zambidis' team turned to plasmids, rings of DNA that replicate briefly inside cells and eventually degrade.

Adding to the complexity of coaxing stem cells into other cell types is the expensive and varied recipe of growth factors, nutrients and conditions that bathe stem cells during their transformation. The recipe of this "broth" differs from lab to lab and cell line to cell line.

Reporting in the April 8 issue of Public Library of Science ONE (PLoS ONE), Zambidis' team described what he called a "painstaking, two-year process" to simplify the recipe and environmental conditions that house cells undergoing transformation into heart cells. They found that their recipe worked consistently for at least 11 different stem cell lines tested and worked equally well for the more controversial embryonic stem cells, as well as stem cell lines generated from adult blood stem cells, their main focus.

The process began with Johns Hopkins postdoctoral scientist Paul Burridge, Ph.D., who studied some 30 papers on techniques to create cardiac cells. He drew charts of 48 different variables used to create heart cells, including buffers, enzymes, growth factors, timing, and the size of compartments in cell culture plates. After testing hundreds of combinations of these variables, Burridge narrowed the choices down to between four to nine essential ingredients at each of three stages of cardiac development.

Beyond simplification, an added benefit is reduced cost. Burridge used a cheaper growth media that is one-tenth the price of standard media for these cells at $250 per bottle lasting about one week.

Zambidis says that he wants other scientists to test the method on their stem cell lines, but also notes that the growth "soup" is still a work in progress. "We have recently optimized the conditions for complete removal of the fetal bovine serum from one brief step of the procedure - it's made from an animal product and could introduce unwanted viruses," he says.

In their experiments with the new growth medium, the Hopkins team began with cord blood stem cells and a plasmid to transfer seven genes into the stem cells. They delivered an electric pulse to the cells, making tiny holes in the surface through which plasmids can slip inside. Once inside, the plasmids trigger the cells to revert to a more primitive cell state that can be coaxed into various cell types. At this stage, the cells are called induced pluripotent stem cells (iPSC).

Burridge then bathed the newly formed iPSCs in the now simplified recipe of growth media, which they named "universal cardiac differentiation system." The growth media recipe is specific to creating cardiac cells from any iPSC line.

Finally, they incubated the cells in containers that removed oxygen down to a quarter of ordinary atmospheric levels. "The idea is to recreate conditions experienced by an embryo when these primitive cells are developing into different cell types," says Burridge. They also added a chemical called PVA, which works like glue to make cells stick together.

Nine days later, the nonviral iPSCs turned into functional, beating cardiac cells, each the size of a needlepoint.

Burridge manually counted how often iPSCs formed into cardiac cells in petri dishes by peering into a microscope and identifying each beating cluster of cells. In each of 11 cell lines tested, each plate of cells had an average of 94.5 percent beating heart cells. "Most scientists get 10 percent efficiency for IPSC lines if they're lucky," says Zambidis.

Zambidis and Burridge also worked with Johns Hopkins University bioengineering experts to apply a miniversion of an electrocardiograph to the cells, which tests how cardiac cells use calcium and transmit a voltage. The resulting rhythm showed characteristic pulses seen in a normal human heart.

Virus-free, iPSC-derived cardiac cells could be used in laboratories to test drugs that treat arrhythmia and other conditions. Eventually, bioengineers could develop grafts of the cells that are implanted into patients who suffered heart attacks.

Zambidis' team has recently developed similar techniques for turning these blood-derived iPSC lines into retinal, neural and vascular cells.

Notes:

The research was funded by the Maryland Stem Cell Research Fund and the National Institutes of Health.

Research participants include Susan Thompson, Michal Millrod, Seth Weinberg, Xuan Yuan, Ann Peters, Vasiliki Mahairaki, Vassilis E. Koliatsos, and Leslie Tung at Johns Hopkins.

Source:
Vanessa Wasta
Johns Hopkins Medical Institutions

 

Scientists Identify A Surprising New Source Of Cancer Stem Cells

Whitehead Institute researchers have discovered that a differentiated cell type found in breast tissue can spontaneously convert to a stem-cell-like state, the first time such behavior has been observed in mammalian cells. These results refute scientific dogma, which states that differentiation is a one-way path; once cells specialize, they cannot return to the flexible stem-cell state on their own.

This surprising finding, published online this week in the Proceedings of the National Academy of Sciences (PNAS), may have implications for the development of cancer therapeutics, particularly those aimed at eradicating cancer stem cells.

"It may be that if one eliminates the cancer stem cells within a tumor through some targeted agent, some of the surviving non-stem tumor cells will generate new cancer stem cells through spontaneous de-differentiation," says Whitehead Founding Member Robert Weinberg. Cancer stem cells are uniquely capable of reseeding tumors at both primary and distant sites in the body.

During differentiation, less-specialized stem cells mature into many different cell types with defined functions. These differentiated cells work together to form tissues and organs. In breast tissue, for example, differentiated basal cells and luminal cells combine to form milk ducts.

While analyzing cells from human breast tissue, Christine Chaffer, who is a postdoctoral researcher in the Weinberg lab and first author of the PNAS paper, observed a small number of living basal cells floating freely in the tissue culture medium.

Intrigued by the cells' unusual behavior, Chaffer conducted further targeted investigations, including injection of the floating basal cells into mice. After 12 weeks she found that the injected basal cells gave rise to milk duct-like structures containing both basal and luminal cells a clear indication that the floating cells had de-differentiated into stem-like cells.

Until now, no one has shown that differentiated mammalian cells, like these basal cells, have the ability to spontaneously revert to the stem-like state (a behavior described as plasticity).

To see if basal cells could become cancer stem cells, Chaffer inserted cancer-causing genes into the cells. When these transformed cells were injected into mice, the resulting tumors were found to include a cancer stem cell population that descended from the original injected basal (more differentiated) cells. These results indicate that basal cells in breast cancer tumors can serve as a previously unidentified source of cancer stem cells.

As research for new cancer therapies has recently focused on eliminating cancer stem cells, Weinberg cautions that the plasticity seen in these basal cells suggests a more complicated scenario than previously thought.

"Future drug therapies that are targeted against cancer will need to eliminate the cancer stem cells and, in addition, get rid of the non-stem cells in tumors both populations must be removed," says Weinberg, who is also a professor of biology at MIT. "Knocking out one or the other is unlikely to suffice to generate a durable clinical response."

Chaffer is now focusing on what actually prompts these flexible cells to de-differentiate, and in the case of cancer cells, how to stop the cells from converting into cancer stem cells.

"This plasticity can occur naturally, and it seems that the trigger may be a physiological mechanism for restoring a pool of stem cells," says Chaffer. "We believe that certain cells are more susceptible to such a trigger and therefore to conversion from a differentiated to a stem-like state, and that this process occurs more frequently in cancerous cells."

In the case of normal epithelial cells, the observed behavior may also allow patient specific adult stem cells to be derived without genetic manipulation, holding promise for degenerative disease therapy.

This research was supported by the National Health and Medical Research Council of Australia, National Institutes of Health, MIT's Ludwig Center for Molecular Oncology, the Breast Cancer Research Foundation, and a Department of Defense Breast Cancer Research Program Idea Award.

Source: Whitehead Institute for Biomedical Research

 

GIS Scientists Propose A New Paradigm For Embryonic Stem Cells, Potentially Speeding Up Development Of Disease Therapies

Scientists from the Genome Institute of Singapore (GIS) have put forward a novel explanation for the pluripotency[1] of embryonic stem (ES) cells. Their groundbreaking explanation opens new doors for understanding how stem cells create specific cell types, fundamental knowledge that will drive changes and improvements in the therapeutic and translational usage of stem cells. A better understanding of ES cells could help advance the development of treatments for diseases such as diabetes, Parkinson's disease, and Huntington's disease. The work, published in the journal Cell Stem Cell, was led by Dr Bing Lim, Senior Group Leader of the Stem Cell and Developmental Biology department at the GIS, and Kyle Loh, GIS student from Dr Lim's lab.

By re-examining current data with a fresh eye, Lim and Loh were able to suggest a novel paradigm that may resolve the 30-year-old mystery behind pluripotency. The prevailing model of stem cell pluripotency suggests that stem cell genes active in ES cells prevent these stem cells from turning into specific cell types. This model accounts for how ES cells can remain undifferentiated, but is unable to explain convincingly the ability of stem cells to create any bodily cell type. Lim and Loh suggest that, contrary to current thinking, individual stem cell genes do not completely suppress differentiation, but instead actively direct ES cells to produce particular bodily cell types. In their new paradigm, Lim and Loh propose that the activation of a combination of such stem cell genes within ES cells is what enables ES cells to create any bodily cell type.

[1] Pluripotency refers to the ability of ES cells to differentiate into all bodily cell types. ES cells can potentially create, on demand, any cell type that clinicians or scientists need for therapeutic, biotechnological, or research purposes. Hence, the cells are currently used as a source of specialized cell types used in cell replacement therapies. An understanding of how ES cells are able to produce all these cell types is of intense pragmatic and theoretical interest.

Source
Genome Institute of Singapore