05 Jun 2020

Astrocytes have been implicated in Alzheimer’s and other neurodegenerative diseases, but these beautiful, star-shaped cells are notoriously difficult to study. Animal models are little help, because their astrocytes are quite different from those in human brain. Induced pluripotent stem cells can generate human astrocytes, but most protocols for making them produce mixed cultures, and there is no efficient way to isolate astrocytes. In the May 22 Neuron, researchers led by Valentina Fossati at the New York Stem Cell Foundation Research Institute in New York City propose one.

The scientists identified a cell-surface marker, CD49f, that can be used to sort human astrocytes from cultured neurons, microglia, and oligodendrocytes. The sorted cells’ expression profile matched that of astrocytes in human brain, and they exhibited classic astrocyte behaviors in culture. When stimulated with pro-inflammatory cytokines, they became reactive and no longer supported neurons, suggesting that these cells could be useful for studies of neurodegeneration.

“These cells recapitulate the functions astrocytes have in vivo. They will give us the ability to do more in-depth studies on [disease] mechanisms,” Fossati told Alzforum.

Kerry O’Banion at the University of Rochester Medical Center in New York agreed. “Overall this approach provides an additional and potentially quite useful tool for investigating the roles of human astrocytes in cell model systems and neurologic disease,” he wrote to Alzforum (full comment below). “A strength of the work was the reproducibility of observed phenotypes across multiple human iPSC cultures.”

Astrocyte Diversity. Astrocytes generated from human iPSCs display a variety of shapes in culture, hinting at subtypes. [Courtesy of Barbar et al., Neuron.]

Previously, researchers led by the late Ben Barres at Stanford University found that astrocytes isolated from human brain express a set of genes distinct from mouse astrocytes, and also from cultured human astrocytes (Jan 2016 news). The finding suggested the need for better models. By now, many protocols exist for inducing iPS cells to differentiate into astrocytes, but they tend to generate immature astrocytes or a mixture of brain cells (e.g., May 2011 news; Aug 2017 news). In mixed mouse cultures, astrocytes can be purified using the cell adhesion protein HepaCAM, but cultured human astrocytes typically do not express this marker.

Fossati and colleagues set out to find a better marker. First author Lilianne Barbar differentiated human iPSCs into a mix of neurons, astrocytes, and oligodendrocyte precursor cells. She used a cocktail that included retinoic acid and sonic hedgehog, mimicking the environment of the developing spinal cord. After allowing the resulting cells to mature for three months, the authors dissociated the cultures into single-cell suspensions and incubated them with antibodies against various cell-surface markers. They separated labeled cells via fluorescence-activated cell sorting (FACS), then replated and stained them for the astrocyte marker GFAP.

Of a panel of 242 antibodies, anti-CD49f produced the purest astrocyte culture. A membrane protein, CD49f belongs to the family of integrin alpha chain proteins, and is encoded by the gene ITGA6.

Astrocyte Purity Test. All cells that express CD49f (aqua) also express the astrocyte marker aquaporin 4 (green), but not all express the reactive marker GFAP (purple; arrows). Nuclei are blue. [Courtesy of Barbar et al., Neuron.]

Were these sorted cells truly astrocytes? About 83 percent expressed GFAP, but this marker is known to not label all astrocytes. Transcriptome analysis painted a clearer picture. The expression profile of the CD49f-positive cells matched that previously reported for isolated human astrocytes, as well as that of astrocytes generated from iPSCs via a different protocol (Zhang et al., 2016; Tcw et al., 2017). Importantly, 97 percent of the cells expressed aquaporin 4, a key astrocyte marker, suggesting this was a fairly pure astrocyte population (see image above). Most of the cells were mature, with 10 percent expressing the immature astrocyte marker NUSAP1.

Nonetheless, the cells were not all identical (see black-and-white image). Expression analysis identified seven subclusters, in agreement with the finding that astrocytes in the brain can be quite heterogeneous.

Because the original differentiation protocol mimicked a spinal-cord-like environment, the authors investigated whether CD49f also marks brain astrocytes. In postmortem human brain sections, immunostaining with CD49f picked out the same cells as did staining with aquaporin 4 and GFAP, confirming its identity as an astrocyte marker. However, CD49f recognized endothelial cells, as well. This makes it less useful for isolating pure astrocyte cultures from brain than from mixed cell cultures, which typically do not contain this cell type.

Next, the authors tested whether CD49f-positive cells behaved like astrocytes in vitro. The cells passed this test with flying colors, taking up glutamate and producing spontaneous calcium transients. Astrocytes in the brain help stimulate neuronal synaptogenesis, and the CD49f cells did this, too. When neurons were co-cultured with them, the neurons fired bigger and more action potentials in response to stimulation, and were more spontaneously active as well. They also grew longer neurites than did neurons cultured without astrocytes.

Finally, the CD49f-positive cells were able to become reactive, as are astrocytes. The authors exposed the cultured cells to the cytokines IL-1α and TNF-α, along with the innate immunity complement factor C1q. This cocktail has been reported to induce a toxic “A1” phenotype in mouse astrocytes (Jan 2017 news). The treatment produced a similar gene-expression pattern in the human CD49f-positive astrocytes. They expressed less glutamate and fewer phagocytic receptors, in keeping with impairment in these processes seen in A1 cells, and changed shape to become less elongated (see image above). In addition, when these contracted A1-like human astrocytes were co-cultured with human neurons for 18 days, synaptic function weakened. Conditioned media collected from A1-like astrocyte cultures triggered apoptosis in cultured human neurons, hinting at a toxic effect of these “inflamed” astrocytes.

Rickie Patani, Francis Crick Institute, London, would like to see work on A1 astrocytes extended to the in vivo setting. “Further studies might build upon this important work by addressing whether such astrocyte reactive changes are largely uniform, or if they exhibit spatiotemporal heterogeneity within the nervous system and across different disease states,” he wrote (full comment below). The concept of A1 astrocytes is relatively new in the field, however, and other experts privately express doubt that these cells secrete a toxic factor.

A separate manuscript, currently posted to bioRxiv, also shines a light on astrocytes in neurodegenerative disease. Unlike Fossani’s experimental work, it represents a correlative study exploiting the data collected in the eight-year-old cohort study GENFI, aka Genetic FTD Initiative. Researchers led by Andre Altmann and Jonathan Rohrer at University College London investigated whether particular patterns of gene expression distinguish the brain regions that shrink in frontotemporal dementia. They used data from 47 symptomatic participants in GENFI, all of whom had familial forms of FTD. Ten of the 47 carried mutations in the MAPT gene, 12 in GRN, while 25 had the C9ORF72 expansion. Their MRI scans were compared to those of 144 healthy controls to identify patterns of brain atrophy.

Then the researchers compared this FTD atrophy pattern to human brain expression in cognitively normal people, as gathered by the Allen Institute for Brain Science. They identified 423 genes that were expressed more highly in brain areas that atrophy in people with FTD than in the rest of the brain, and 700 genes that were expressed less in these areas.

Curiously, the genes with high expression included a large number of astrocyte and oligodendrocyte genes. This implies that vulnerable regions could be enriched for these cells, or that something about the activity of glial cells renders these regions vulnerable. The high-expression set also included many vascular genes, suggesting potential involvement of endothelial cells and the blood-brain barrier in degeneration. Meanwhile, genes with lower expression in atrophied areas were primarily neuronal and microglial. Neuronal and microglial genes were relatively more highly expressed in brain regions spared in FTD.

Because the findings are correlative, it is unclear whether astrocyte activity in atrophied areas would be harmful or protective.—Madolyn Bowman Rogers

Comments

1. • M. Kerry O'Banion
     University of Rochester School of Medicine and Dentistry
   • Posted: 05 Jun 2020

   The Neuron paper has done a nice job of characterizing CD49f as a marker of human astrocytes and shown evidence that it can be used to isolate astrocytes from hIPSC cultures that mimic many classic features of astrocytes, including support of neurons, glutamate clearance, and responsiveness to inflammatory stimuli. They go on to show in vitro that these cells take on A1-like characteristics after inflammatory stimuli and become neurotoxic. A strength of the work was reproducibility of observed phenotypes across multiple hIPSC cultures. Overall, this approach provides an additional and potentially quite useful tool for investigating the roles of human astrocytes in cell model systems and neurologic disease. I find very little to criticize in their approach, or thoroughness of study, though an evaluation of developmental CD49f expression would have been a nice touch.

   View all comments by M. Kerry O'Banion
2. • Rickie Patani
     UCL Institute of Neurology and The Francis Crick Institute
   • Posted: 05 Jun 2020

   Astrocytes have increasingly recognized pathogenic roles in a range of neurodegenerative diseases. The ability to study enriched populations of human astrocytes is an important addition to our existing experimental repertoire. This study identifies a new marker to purify human induced pluripotent stem cell (hiPSC)-derived astrocytes and further demonstrates that these undergo deleterious reactive transformation in response to an established program of extrinsic pro-inflammatory cues.

   Importantly, the induction of this deleterious reactive state led to the disruption of key astrocyte homeostatic functions (that would otherwise support juxtaposed neurons in vivo). Further studies might build upon this important work by addressing whether such astrocyte reactive changes are largely uniform or if they exhibit spatiotemporal heterogeneity within the nervous system and across different disease states. Such information may help us to understand the scope of therapeutically targeting astrocytes in a range of neurodegenerative diseases.

   View all comments by Rickie Patani

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References

News Citations

1. Purification of Adult Human Astrocytes Shows: They Are Unique 15 Jan 2016
2. Stem Cell Recipe for Astrocytes 26 May 2011
3. Brain Spheroids Hatch Mature Astrocytes 18 Aug 2017
4. Microglia Give Astrocytes License to Kill 20 Jan 2017

Paper Citations

1. Zhang Y, Sloan SA, Clarke LE, Caneda C, Plaza CA, Blumenthal PD, Vogel H, Steinberg GK, Edwards MS, Li G, Duncan JA 3rd, Cheshier SH, Shuer LM, Chang EF, Grant GA, Gephart MG, Barres BA. Purification and Characterization of Progenitor and Mature Human Astrocytes Reveals Transcriptional and Functional Differences with Mouse. Neuron. 2016 Jan 6;89(1):37-53. Epub 2015 Dec 10 PubMed.
2. Tcw J, Wang M, Pimenova AA, Bowles KR, Hartley BJ, Lacin E, Machlovi SI, Abdelaal R, Karch CM, Phatnani H, Slesinger PA, Zhang B, Goate AM, Brennand KJ. An Efficient Platform for Astrocyte Differentiation from Human Induced Pluripotent Stem Cells. Stem Cell Reports. 2017 Aug 8;9(2):600-614. Epub 2017 Jul 27 PubMed.

External Citations

1. Genetic FTD Initiative

Further Reading

News

1. Does Taming Killer Astrocytes Spare Neurons in Parkinson’s Disease? 13 Jun 2018
2. Astrocytic IL-33 Signals Microglia to Engulf Synapses 7 Feb 2018
3. Aged Astrocytes Prime Brain for Neuroinflammation 21 Feb 2018
4. Do Tribes of Astrocytes Wage War on Synapses? 5 Apr 2019