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. 2017 Mar 23;8(3):e2696.
doi: 10.1038/cddis.2017.89.

Aberrant iPSC-derived human astrocytes in Alzheimer's disease

V C Jones  1 , R Atkinson-Dell  2 , A Verkhratsky  2   3 , L Mohamet  2
Affiliations

Aberrant iPSC-derived human astrocytes in Alzheimer's disease

V C Jones et al. Cell Death Dis. .

Erratum in

Abstract

The pathological potential of human astroglia in Alzheimer's disease (AD) was analysed in vitro using induced pluripotent stem cell (iPSC) technology. Here, we report development of a human iPSC-derived astrocyte model created from healthy individuals and patients with either early-onset familial AD (FAD) or the late-onset sporadic form of AD (SAD). Our chemically defined and highly efficient model provides >95% homogeneous populations of human astrocytes within 30 days of differentiation from cortical neural progenitor cells (NPCs). All astrocytes expressed functional markers including glial fibrillary acidic protein (GFAP), excitatory amino acid transporter-1 (EAAT1), S100B and glutamine synthetase (GS) comparable to that of adult astrocytes in vivo. However, induced astrocytes derived from both SAD and FAD patients exhibit a pronounced pathological phenotype, with a significantly less complex morphological appearance, overall atrophic profiles and abnormal localisation of key functional astroglial markers. Furthermore, NPCs derived from identical patients did not show any differences, therefore, validating that remodelled astroglia are not as a result of defective neural intermediates. This work not only presents a novel model to study the mechanisms of human astrocytes in vitro, but also provides an ideal platform for further interrogation of early astroglial cell autonomous events in AD and the possibility of identification of novel therapeutic targets for the treatment of AD.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Directed differentiation of healthy and AD-neural progenitor cells into cortical neurones. (a–c) NPCs were seeded at 1 × 105 per well and propagated in monolayer culture for 6/7 days. FAD and SAD cortical NPCs showed indistinguishable culture morphology with healthy (control) NPCs (N = 5 per cell line). (d and e) No significant differences in NPC growth rates were identified (N=4 per cell line, two-way Kruskal–Wallis P=NS). (f–n) IPS-derived NPCs from healthy (control), FAD and SAD patients were assessed for canonical marker expression. Progenitor cells formed polarised rosettes expressing nestin (green; f–h), PAX6 (red; I–k) and SOX1 (green, l–n). (o) No significant difference in nestin+ cells was observed between healthy and AD cell lines (N=4 per cell line, ANOVA, F(2,9)=0.022, P=NS). (p–r) Under terminal neuronal differentiation conditions for 35–40 days, all patient samples showed positive expression of the neural marker β-III-tubulin (green). (s) No significant difference in the proportion of β-III-tubulin+ neurones between any individual (N=4 per group, ANOVA, F(2,9)=0.128, P=NS). (t–v) Expression of the mature deep-layer cortical neuronal marker, CTIP2 was observed throughout cultures from each patient (green). Scale bars, 50 μm
Figure 2
Figure 2
Induced astrocytes derived from healthy patient fibroblasts express mature astrocyte markers and exhibit varied morphologies. Induced astrocytes were generated in 30 days and confirmed by positive immunoreactivity to the functional markers: GFAP (a), S100B (b), EAAT1 (c) and GS (d). Immunostaining is shown in green, DAPI counterstained nuclei are shown in blue. Heterogeneity of morphology within the population of induced astrocytes was evident, with cells falling into three broad categories when stained for GFAP: arborised cells (e); polarised cells (f, arrowhead); or fibroblast-like, process-devoid cells (f, arrow); summarised in (g). **P<0.005, *P<0.05. Scale bars, 50 μm (inset, 20 μm)
Figure 3
Figure 3
Induced astrocytes derived from PSEN1 M146L FAD patient fibroblasts express normal astrocyte markers but show reduced morphological heterogeneity compared with healthy cells. Induced astrocytes were immunostained for GFAP (a), S100B (b), EAAT1 (c) and GS (d). Immunostaining is shown in green, DAPI counterstained nuclei are shown in blue; >95% of observed cells were positive for all markers tested. The significant majority of FAD astrocytes display a fibroblast-like, process-devoid appearance (summarised in e). **P<0.005, *P<0.05. Scale bars, 50 μm (inset, 20 μm)
Figure 4
Figure 4
Induced astrocytes derived from ApoE4+/+ SAD patient fibroblasts express classical astrocyte markers but show reduced morphological heterogeneity compared with healthy cells. Induced astrocytes were immunostained for GFAP (a), S100B (b), EAAT1 (c) and GS (d). Immunostaining is shown in green, DAPI counterstained nuclei are shown in blue. All observed cells were positive for all markers. A significant majority of SAD astrocytes displayed a fibroblast-like appearance, indicating limited heterogeneity of morphology compared with healthy controls; summarised in (e). **P<0.005. Scale bars, 50 μm (inset, 20 μm)
Figure 5
Figure 5
Astrocytes derived from PSEN1 M146L FAD and ApoE4+/+ SAD patients exhibit significant atrophy when compared with those from healthy patients as revealed by visual binning according to overall morphology (a). Exemplar 3D IsoSurface renders constructed from serial confocal z-stacks display clear differences in cell size and overall morphology (b). Scale bar, 10 μm. Quantification of cells using these renders by way of surface area (c), cell volume (d) and SA:Vol ratio (e) reveal significant differences in all aspects of cellular morphology between healthy and diseased astrocytes. Quantification of mean fluorescence intensity per immunoreactive cell reveals no significant difference in GFAP staining intensities between AD and control astrocytes (f) but S100B, EAAT1 and GS intensities are reduced in both FAD and SAD cells (g–i, respectively). ***P<0.001, **P<0.005, *P<0.05
Figure 6
Figure 6
S100B localisation within astrocytes is dramatically altered in FAD and SAD Healthy astrocytes were immunostained for S100B and visualised using deconvolution fluorescence microscopy (a) revealing S100B to be distributed throughout the cytoplasm (top panel). In contrast, in FAD and SAD cells, S100B appeared to be localised exclusively at the nucleus (middle and bottom panels, respectively). Scale bar, 20 μm. IsoSurface renders of S100B (red) and of DAPI-stained nuclei (blue; with a clipping plane set to permit visualisation of the inside of the nucleus) were constructed from serial confocal z-sections to further investigate the subcellular localisation of S100B (b; scale bars, 10 μm). GFAP staining (green) is shown for comparison for healthy cells to reveal the overall cell morphology. In healthy cells, as expected, S100B displayed a cytosolic distribution, similar to that of GFAP. In both FAD and SAD astrocytes, S100B is localised exclusively to discrete foci within the nucleus. Nuclei images have been zoomed in × 2 for clarity. Movies of these renders can be viewed in Supplementary Movies S1–S3
Figure 7
Figure 7
Comparison of healthy control, FAD and SAD patient-derived βIII-tubulin immunoreactive neurones and GFAP immunoreactive astrocytes. Early neuronal appearance is indistinguishable across the groups, whereas AD astrocytes show markedly reduced heterogeneity of morphology and striking atrophy compared with healthy cells. Scale bar, 50 μm
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