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Investigators Induce Human Pluripotent Stem Cells into Astrocytes

ARTICLE IN BRIEF

Astrocyte PROGENITORS AND IMMATURE ASTROCYTES

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Investigators induced in the lab dish both embryonic and human stem cells to become astrocytes.

Scientists are able to make neurons and other brain cells types from human pluripotent stem cells (hPSC) but it has been challenging to make a pure population of astrocytes. Su-Chun Zhang, MD, PhD, and his colleagues at the University of Wisconsin have figured out a technique that does just that and they believe that the cells will be invaluable for the study of neurological diseases and testing of new therapeutics. The findings were published in May 22 in the online edition of Nature Biotechnology.

The investigators were able to create large numbers of cells from hPSCs, including human embryonic stem cells (hESCs) and induced pluripotent stem (iPS) cells. The cells were differentiated into neuroepithelial cells and then directed to become regional progenitors. The population was expanded in the presence of growth factors. Before this new approach, the existing technology made it virtually impossible to grow large stores of pure astrocytes. This cell population, according to the scientists, is free of immune cells (microglia) and they figured out a way to grow regionally specific astroglia, which could be important when planning future strategies to repopulate damaged brain tissue.

Dr. Zhang and his colleagues tested their approach on three different hPSC cell lines and the progenitors generated continued to expand in the culture and produce cells that when tested continued to show all of the properties of astroglial cells.

According to Dr. Zhang, a professor of neuroscience and neurology, the astroglial progenitors that they created from human pluripotent stem cells differentiate into immature astrocytes. He added that abnormalities in astroglial cells are linked to a growing number of neurodegenerative conditions. Ultimately, the cells will help investigators figure out how these glia become damaged in certain diseases and what can be done to stop the process and prevent the damage or repopulate the area with healthy cells.

Most importantly, their study also provides the recipe for making pure populations of astrocytes. They performed a series of studies to test the structure and function of the cells and they showed different characteristics of astrocytes. The iPS-induced immature astrocytes have similar genetic patterns as primary astrocytes and in the test tube the cells exhibit properties of glutamate uptake and promote synaptogenesis, the investigators wrote. When the cells were transplanted into mouse brain they became mature astrocytes that formed connections with blood vessels. “This is very exciting,” said Dr. Zhang. “We can begin to think about how we can use these cells as treatments for neurological diseases.”

The group is also making iPS cell lines from skin fibroblasts culled from patients with amyotrophic lateral sclerosis to test how the cells differentiate into motor neurons and astrocytes. The hope is to understand how the disease process affects discrete populations of cells. They have also made iPS cell lines from patients with Alexander disease, a condition marked by protein aggregation in the astrocytes. In preliminary tests, they can see the protein aggregate in the astrocytes. They are just beginning to grow iPS cell lines from patients with fragile X, autism, and Down syndrome as well.

Dr. Zhang said that it was patience that finally led to the ability to make astrocytes. They allowed the iPS cells to first develop into neural stem cells. These cells normally grow into ‘neurospheres’ but the scientists separated the cells and allowed them to continue growing and they gradually lost their ability to generate into neurons. Instead, they shift to a glial fate and became astrocytes. Once they become a uniform population of glial cells the scientists can keep expanding them to create billions of cells.

Another way that they got a large population of glial cells was to take progenitors and make them differentiate into astrocytes and transplant them into hippocampal tissue in the mouse. Their cell fate is fixed and they never become anything but glial cells, Dr. Zhang said.

When exposed to specific neural environments the cells grow into regionally and functionally distinct subtypes of astrocytes, just like there are different types of neurons. What's more, they maintain their identities when transplanted into ectopic mouse brain regions.

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“This is an important study,” said Ben Barres, MD, PhD, professor of neurobiology and developmental biology at Stanford University. “Now there is a way to make and study astrocytes from humans to see if they are abnormal in neurological diseases and also perhaps (and this is a long shot at present) to use them to treat disease.” For instance, Dr. Barres' lab has shown that an important function of astrocytes is to control synapse formation and function. There is evidence that synaptic defects occur in autism, as well as neurodegenerative and psychiatric disease, and that this is caused by abnormalities in the astrocytes rather than neurons.

DR. SU-CHUN ZHANG: “This is very exciting. We can begin to think about how we can use these cells as treatments for neurological diseases.”

Dr. Barres, a glial expert, and his colleagues have developed a new method that allows purification of mature mouse brain astrocytes and they are now adapting it to purify human brain astrocytes. “We are about to use the same method to purify astrocytes from patient generated iPS neural cells” in collaboration with Ricardo Dolmetsch, PhD, an assistant professor of neurobiology. “The goal is to determine whether astrocytes from patients with autism are defective in their synaptic functions,” said Dr. Barres.

Others have shown that defective astrocytes can contribute in a large way to brain disease, For example, Jeffrey D. Rothstein, MD, PhD, the John W. Griffin director of the Brain Science Institute and professor of neurology and neuroscience at Johns Hopkins University School of Medicine, first demonstrated defective astroglia contribute to human ALS. His group later showed that transplantation of rodent astroglia can ameliorate, at least focally, ALS neurodegeneration.

“Dr. Zhang and his colleagues did a beautiful job,” said Dr. Rothstein. “The paper is valuable because it teaches us a method of creating these very important human cells. The difficulty has been the fact that we are rebuilding what happens in fetal development and astrocytes take time to develop. The challenge in making astrocytes is that they develop three months later than neurons. This could prove to be a real limitation in planning for large studies of the cells or for their use in more commercial applications,” Dr. Rothstein said.

Dr. Rothstein, who is also director of the Robert Packard Center for ALS Research, is leading a consortium of investigators from Columbia University and Harvard in making human iPS cell lines from ALS patients. These lines will be part of a federally funded library that will make them available to investigators. The cells are being converted into motor neurons and astrocytes so scientists can unravel the properties that lead to the death of the motor neurons.

The hope of the current research is to be able to grow and transplant human astroglia in an attempt to test whether or not it could ultimately be used as a treatment for neurological disease.

THE PROCESS FOR CREATING ASTROCYTES

In 2007, scientists discovered that a handful of genes — Sox-2, Oct-4, c-MYC, Nanog, Lin28, Klf4 — are responsible for creating pluripotent stem cells. This discovery meant that scientists could develop human pluripotent stem cells without an egg. The induced pluripotent stem (iPS) cells are made by putting extra copies of the genes (delivered through viruses) into normal adult skin cells in culture. These human pluripotent stem cells (embryonic or induced pluirpotent stem cells) are then converted to neural stem cells in a chemically defined culture condition in the first two weeks.

In the second week of differentiation, specific morphogens or growth factors — for example, sonic hedgehog, Wnts, retinoic acid, and others — may be added to pattern neural progenitors with particular regional identities such as the forebrain, midbrain, spinal cord, or cerebral cortex vs. basal ganglia. These regionalized neural progenitors usually expand for two weeks before they produce neurons from the fourth week, which can last for several months. Some of the progenitors will gradually turn into a glial fate from the second month of differentiation and begin to generate astrocytes in the third month, which will last for at least several months.

REFERENCE:

Krencik R, Weick JP, Zhang SC,et al. Specification of transplantable astroglial subtypes from human pluripotent stem cells. Nat Biotechnol 2011; E-pub 2011 May 22