18 Aug 2017

Astrocytes are more than bystanders in neurotransmissionthey take an active role in synaptic activity. However, their functions are hard to study because the cells are difficult to grow in vitro and its hard to coax them to mature from progenitors. Now, researchers from the labs of Sergiu Paca and Ben Barres, both at Stanford University School of Medicine, California, report that astrocytes come of age in spherical balls of human brain cells cultured in a dish for almost two years. As reported in the August 16 Neuron, these astrocytes develop much like those from real brains, undergoing similar transcriptomic, morphologic, and functional changes. Studying the processes involved in this astrocyte maturation will help researchers understand neurodevelopmental disorders such as autism and schizophrenia, researchers say, and might even shed light on problems in adultbrains.

That these 3D cultures can be maintained for such a long time allows us to capture an interesting transition in astrocytes, said Paca. We are starting to appreciate aspects of human brain development to which we would not otherwise haveaccess.

The breakthrough is that they can develop human astrocytes very close to maturity in their 3D culture models, said Doo Yeon Kim, Massachusetts General Hospital, Charlestown, who uses 3D culture models to study pathological process that occur in Alzheimers disease. Some researchers are using 3D cultures to model other neurodegenerative disorders, such as ALS, and still others are planning to use cultured astrocytes for cell therapy. If astrocytes are not mature enough in culture, patterns [we see] may not be the same as in the diseased brain, saidKim.

This developing human astrocyte (red), which comes from a 350-day-old cortical spheroid, is taking shape as a mature cell. [Image courtesy of Sloan et al.Neuron]

A few years back, Pacas group developed a method for differentiating human induced pluripotent stem cells (hiPSCs) into a 3D culture of brain cells. They used special dishes that the cells could not easily attach to, coaxing them to stick to each other instead. Under these conditions the iPSCs balled up into neural spheroids that grew to about 4 mm in diameter. A cocktail of growth factors early on encouraged them to form excitatory pyramidal cells like those in the cortex, and the cells spontaneously organized into layers. These cortical spheroids survived a year or more and spontaneously grew astrocytes in addition to neurons (Paca et al., 2015). Not long after, the Barres lab reported that astrocytes in the adult human brain look different from those isolated from fetuses. They called the latter astrocyte progenitor cells (APCs). Each had their own transcriptional patterns and functions (Jan 2016 news). Together, Barres and Paca wondered if it was possible to see the APCs morph into mature astrocytes in these long-lived corticalspheroids.

To find out, first author Steven Sloan and colleagues examined spheroids generated from iPSCs derived from healthy human fibroblasts. Sloan grew the spheroids for about 20 months. Along the way, he took samples, isolated the astrocytes, and compared them to those isolated from fetal and postnatal humanbrain.

At about 100 days in culture, astrocytes began to sprout spontaneously from within the mostly neuronal milieu of the cortical spheroids. At first, these cells were simple, adorned by few branches and expressing genes akin to those active in APCs. But as the spheroids reached about 250 days, the astrocytes therein looked more mature, having numerous processes. After this point, APC gene expression tapered off and the astrocytes started producing proteins typical of matureastrocytes.

Astrocytes also underwent functional changes as they matured. Early versions divided in fast and furious fashion, much like their counterparts from the fetal tissue. That division slowed as the spheroids aged. Dividing APCs dropped from 35 percent of all astrocytes at day 167 to 3 percent at day 590. Taken from the spheroids at day 150 and cultured in a 2D layer, immature astrocytes also harbored a voracious appetite for added synaptosomes, much like immature astrocytes recently characterized in mice (see image below; Dec 2013 conference news on Chung et al., 2013). However, that hunger waned as astrocytes approached the 590-daymark.

At the older end of the spectrum, mature astrocytes seemed to take on a supportive role, strengthening calcium signaling in nearbyneurons.

Studying the neurons and astrocytes in these cortical spheroids could be useful for addressing certain unanswered questions about human biology, said other researchers. This could be a very strong opportunity to understand what goes wrong in human genetic disorders that affect astrocyte function, said M. Kerry OBanion, University of Rochester Medical Center, New York. Its also possible that such cultures could reveal as yet unknown facets of familial mutations that cause Alzheimers disease, he suggested. However, given that these cultures take a long time to grow and develop, they are unlikely to completely supplant other types of cultures or faster-maturing animal models, hesaid.

Kim agreed, saying, The results are very exciting, but not practical yet for disease modeling." However, Kim hopes that researchers will make progress on accelerating the maturationprocess.

The Barres and Paca labs are trying just that with the spheroid. They will also analyze what they secrete to support neuronal signaling. In addition, they are exploring how to make the astrocytes reactive, as they often are in neurodegenerative diseases, such as Alzheimers. Doing so might reveal how such astrocytes interact withneurons.

An immature astrocyte taken from a 150-day-old spheroid gobbles up added synaptosomes (red). [Neuron, Sloan et al.2017]

To Pacas knowledge, these cortical spheroids are some of the longest human cell cultures ever reported. His group has continued to cultivate these clumps, with the oldest still going strong at day 850. Granted, these systems are missing many cell types: endothelial cells, oligodendrocytes, and microglia to name a few, he said. However, his lab has introduced new ways to add in other cells. Earlier this year, he reported 3D cultures of cortical glutamatergic neurons and GABAergic interneurons that fused together when they were placed side-by-side (Birey et al., 2017).

Clive Svendsen, Cedars-Sinai Medical Center in Los Angeles, California, saw clinical implications for this paper. It shows iPSC derived astrocytes can mature to an adult phenotype, he said. This further supports their use in clinical transplantation, as we are planning to do. His group has begun a Phase 1 clinical trial that implants human fetal astrocytes into the spinal cords of ALS patients.Gwyneth DickeyZakaib

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