27 May 2011. Researchers at the University of Wisconsin-Madison have cooked up a method to nudge pluripotent stem cells all the way to astrocytes. The new protocol, published online May 22 in Nature Biotechnology, means researchers can better model the astrocyte contributions to neurodegenerative diseases. Mimicking the natural developmental recipe for these star-shaped neuron supporters, the scientists created astrocytes that swallow glutamate, encourage synapse formation, and whip up a blood-brain barrier, just like the nature-made versions do.
Senior author Su-Chun Zhang and first author Robert Krencik, who studied in the Zhang lab but has since moved to the University of California, San Francisco, led the effort. Zhang, along with others, figured out how to turn stem cells into neurons nearly a decade ago (Zhang et al., 2001). But astrocytes have proved more elusive. With several studies pointing to astrocytes as participants in neurodegenerative diseases such as amyotrophic lateral sclerosis, researchers needed a way to grow their own.
Krencik used human starting material, either embryonic stem cell (ESC) or induced pluripotent stem cell (iPSC) lines. He applied standard protocols to shepherd them through the neuroepithelial and into the neural progenitor stage, then figured out how to make them into astrocytes. Three key factors allowed the group to succeed, Zhang told ARF: patience, chemically defined media, and regular disruption of clumped-together cells.
During human development, Zhang pointed out, astrocytes appear three months after neurons. Hence, the researchers waited out two months of culture, and at that point the progenitors started making more astrocytes than neurons.
Although serum works to feed and produce mouse astrocytes, it impedes the process in human cells, Zhang said. Again, natural biology provided the clue: “In the brain, you do not need serum,” he said, adding that because of the blood-brain barrier, the brain actually “hates” serum. If the culture is contaminated with any cells that do prefer serum, he said, those cells could take over the population. Thus, carefully defined chemical media proved to be the way to go.
The progenitors tend to make neurons when clustered together, but are more likely to switch to glia when single cells float alone in suspension. Therefore, Krencik dissociated the cells in the growing cultures every week. Finally, he plated individual cells and added growth factors to help them continue developing as astrocytes. Adding particular growth factors produced different astrocyte subtypes.
To measure his success, Krencik looked for, and found, every astrocyte marker he could think of. Further, he checked that the cells can carry a current but do not produce action potentials as neurons do. Their glutamate receptors and transporters were operable, and they propagated calcium waves like proper glia do. When co-cultured with neural progenitors, the astrocytes nursed the formation of synapses between developing neurites.
In a final experiment, Krencik implanted the immature astrocytes into the lateral ventricles of neonatal mice. The human cells not only survived, but also reached out to encircle blood vessels, suggesting they helped form the blood-brain barrier. The implanted cells lasted for at least six months, with no evidence of overproliferation or tumor formation, Zhang said.
François Berthod of the University of Laval in Québec, Canada, wrote in an e-mail to ARF that the functional test results were convincing. However, he noted, the researchers started with standard cell lines. What many researchers would like to do, and are already doing with neurons, is to use patient-derived iPSCs to make disease-specific cell lines for drug screening and other experiments (see ARF related news story). It remains to be shown that a person’s cells, turned from fibroblasts to iPSCs to astrocytes, will still exhibit pathology, he wrote.
The study also shows that making these kinds of cell lines will be a slow process, said Nicholas Maragakis, who called the timeline “sobering.” Maragakis works at the Johns Hopkins School of Medicine in Baltimore, Maryland. Obtaining statistically significant results will require hundreds of patient-specific cell lines. “It takes a long time to generate a cell line; that is something as a community we are going to have to face,” he said.
Some scientists are already testing transplantation of stem cell-derived lines as a therapeutic for amyotrophic lateral sclerosis (see ARF related news story). Although he was careful to call astrocyte transplants “purely speculative,” Berthod noted that the protocol might, in the future, allow doctors to collect a person’s cells and create customized astrocytes for grafts.—Amber Dance.
Krencik R, Weick JP, Liu Y, Zhang ZJ, Zhang SC. Specification of transplantable astroglial subtypes from human pluripotent stem cells. Nat Biotechnol. 2011 May 22. Abstract