Human ESC-Derived Chimeric Mouse Models of Huntington's Disease Reveal Cell-Intrinsic Defects in Glial Progenitor Cell Differentiation

Abstract

Huntington's disease (HD) is characterized by hypomyelination and neuronal loss. To assess the basis for myelin loss in HD, we generated bipotential glial progenitor cells (GPCs) from human embryonic stem cells (hESCs) derived from mutant Huntingtin (mHTT) embryos or normal controls and performed RNA sequencing (RNA-seq) to assess mHTT-dependent changes in gene expression. In human GPCs (hGPCs) derived from 3 mHTT hESC lines, transcription factors associated with glial differentiation and myelin synthesis were sharply downregulated relative to normal hESC GPCs; NKX2.2, OLIG2, SOX10, MYRF, and their downstream targets were all suppressed. Accordingly, when mHTT hGPCs were transplanted into hypomyelinated shiverer mice, the resultant glial chimeras were hypomyelinated; this defect could be rescued by forced expression of SOX10 and MYRF by mHTT hGPCs. The mHTT hGPCs also manifested impaired astrocytic differentiation and developed abnormal fiber architecture. White matter involution in HD is thus a product of the cell-autonomous, mHTT-dependent suppression of glial differentiation.

Keywords: Huntington’s disease; MYRF; astrocyte; chimera; chimeric mouse; embryonic stem cell; glia; myelin; neurodegenerative disease; oligodendrocyte.

Figure 1. HD hESC-derived hGPCs display profound mHTT-dependent changes in gene expression

A, Principal component analysis (PCA) based on expression of approximately 26,000 transcripts. The expression data are shown as transcripts per million (TPM), post-normalization to account for variance (Risso et al., 2014). The PCA plot shows the distinct transcriptome-wide expression signature of HD-derived hGPCs. B, Venn diagram shows intersections of lists of differentially expressed genes (DEGs) (green, down-regulated; red, up-regulated; fold change, FC > 2.0, FDR 1%), obtained by comparing hGPCs derived from 3 different HD patients to pooled control hGPCs from 2 donors. The list of DEGs shared by the 3 HD patients was then filtered by intersecting with those DEGs (FC > 2.0, FDR 1%) found in patient HD20 (GENEA20-derived) vs. its normal sibling CTR19 (GENEA19); this filtration step further increased the specificity of mHTT-associated DEGs. The gray-highlighted intersections together comprise the entire set of genes differentially expressed by all HD lines relative to their pooled controls. C, Expression heat-map based on transcripts per million (TPM) values for 429 DEGs highlighted in B, showing clustering of hGPCs by disease status. Dendrogram shows hierarchical clustering based on Euclidean distance calculated from log₂-TPM values from the three HD-ESCs lines (HD-17, HD-18, HD-20) and the two matched control lines (CTR19, CTR02). D, Network representation of functional annotations (Gene Ontology: Biological Process and Cellular Component, Bonferroni-corrected p<0.01) for the 429 intersection DEGs highlighted in B. Genes are round nodes with border colors representing their direction of dysregulation (green, down-regulated; red, up-regulated). Rounded rectangle nodes represent annotation terms. Nodes are sized by degree and colored by closely interconnected modules (M1 though M3) identified by community detection. For each module, 3 of the top annotations by significance and fold-enrichment are listed. Selected gene nodes are labeled, and include genes encoding key hGPC lineage transcription factors and stage-regulated proteins. E, Expression heat-map of 63 conserved DEGs identified in M1 (purple in D), annotations related to glial cell differentiation and myelination. F, Expression heat-map of 56 conserved DEGs identified in M2 (lilac in D), annotations related to axon guidance and axonogenesis. G, Expression heat-map of 68 conserved DEGs identified in M3 (yellow in D), annotations related to regulation of synapse structure and synaptic signaling. All DE results are 1% FDR and FC >2; GO annotation results are Bonferroni-corrected to p<0.01.

Figure 2. Increasing CAG lengths correlate with diminished oligodendroglial gene expression

Correlation analysis between the differential expression fold-change (FC), and the number of CAG repeats, indicates a strong inverse relationship between glial gene expression and the CAG repeat number. A, Expression heat map based on transcripts per million (TPM) values calculated from raw counts of 429 differentially expressed genes (DEGs) (1% FDR, FC>2.0) found in the intersection of DEGs by comparisons of hGPCs derived from each of the three different HD patients against pooled control hGPCs from two different donors. Row side colors show the Pearson’s R correlation coefficient between fold change of that gene in each HD-derived hGPC line against pooled controls and the corresponding CAG repeat number in that HD line (HD17 = 40x CAG, HD18 = 46x CAG, and HD20 = 48x CAG). Selected genes encoding transcription factors and stage-regulated proteins involved in glial differentiation and myelination are listed. B, Combined scatterplot with linear fit lines, obtained by regression of fold-changes of each of the 429 DEGs shown in heat map in A, against the CAG repeat number in the corresponding hGPC line. C, Histogram showing the distribution of Pearson’s R coefficients for correlation between fold changes of DEGs in 3 HD lines, to corresponding CAG length. For 255 of the 429 genes (|Pearson’s R| > 0.75), the correlation analysis indicated that the absolute magnitude of the fold-change increased with CAG repeat number; 228 of these genes displayed an inverse correlation of gene expression level to the CAG repeat number, with longer repeats associated with diminished glial gene expression.

Figure 3. Myelination was impaired in mice chimerized with mHTT-expressing human GPCs

Human glial chimeric mice were established by neonatal injection of hGPCs into shiverer x rag2 hosts, which were sacrificed at 8, 13 and 18 weeks. A-B Whereas myelin basic protein (MBP) expression by control hGPCs (GENEA19) was evident by 8 weeks after neonatal graft (A), mice engrafted with HD-derived, mHTT-expressing hGPCs (GENEA20) manifested little or no MBP immunolabeling by that point (B). C-D, By 13 weeks, by which time mice engrafted with control GPCs exhibited robust myelin production (C), only scattered islands of MBP expression were noted in matched recipients of HD-derived GPCs (D). E-F, Control GPC-derived myelination was increasingly robust by 18 weeks (E), relative to mHTT GPC chimeric mice (F). G-I, The density of engrafted human GPCs did not differ between control and mHTT GPCs at any timepoint assessed (G), but the fraction of these GPCs that differentiated as transferrin (TF)⁺ oligodendrocytes was significantly lower among mHTT-expressing GPCs (H), resulting in fewer TF-defined oligodendrocytes in chimeras engrafted with mHTT GPCs (I). J-L, Among donor-derived oligodendrocytes, the proportion that became myelinogenic, as defined by MBP co-expression of human TF and MBP, was significantly lower in mHTT- than control GPC-engrafted chimeric brains (J). Similarly, the fraction of all donor cells that developed MBP expression was significantly higher in mice engrafted with control compared to HD-derived GPCs (K). Accordingly, myelin luminance, as assessed on MBP-immunostained sections, was significantly higher in control-engrafted corpus callosa than in corresponding mHTT GPC-engrafted white matter (L). Neither the density (G) nor the distribution of engrafted human GPCs (M-N, dot maps) differed significantly between control and HD-derived GPCs, indicating that the myelination defect in mHTT GPC-engrafted brains was due to impaired oligodendroglial differentiation and myelinogenesis, rather than to differential engraftment. Scale: 50 μm. Values are represented as mean ± SEM. **p<0.01 and ***p<0.001 by two-way ANOVA with Bonferroni post-hoc tests.

Figure 4. mHTT GPC-engrafted brains exhibited diminished and delayed axonal myelination

A-F, Confocal images of hGPC-engrafted shiverer corpus callosum show the greater MBP expression and higher proportion of ensheathed axons in mice engrafted with GENEA19 control hGPCs (A-C), compared to mice engrafted with GENEA20-derived mHTT-expressing hGPCs (D-F). A’ and B’, Confocal z-stacks with orthogonal views of donor-derived MBP⁺ oligodendrocytes. C’, Higher magnification of C, showing MBP immunoreactivity surrounding ensheathed axons. G-H, The proportion of MBP-ensheathed NF⁺ host axons overall (G), and per MBP⁺ donor-derived oligodendrocyte (H). Scale: A-F, 20 μm; A’,B’,C’, 5 μm. Values represented as mean ± SEM. **p<0.01 and ***p<0.001 by 2-way ANOVA with Bonferroni post-hoc tests.

Figure 5. SOX10 and MYRF rescued oligodendrocyte differentiation and myelinogenesis by mHTT GPCs

A, A doxycycline-regulated dual vector lentiviral (LV) transduction strategy allowed the DOX-triggered, inter-dependent over-expression of SOX10 and MYRF, with concurrent expression of CD4 to permit FACS-based immunoisolation of SOX10-MYRF-transduced hGPCs. B-D. The effects of SOX10 and MYRF over-expression in mHTT-expressing hGPCs were assessed by transducing matched sets of 180 DIV Genea20-derived hGPCs with doxycycline-regulated lentiviral SOX10/MYRF, and exposing some cultures to doxycycline, while leaving matched control cultures untreated. After an additional week in vitro, the cells were immunostained using mAb O4, which recognizes oligodendrocytic sulfatide. B, Without DOX, the mHTT GPCs were stably maintained and expressed no detectable O4. C, In contrast, those mHTT GPCs raised in DOX, with up-regulated SOX10 and MYRF expression, exhibited a sharp and significant increment in oligodendrocyte differentiation (D). E, This schematic outlines the experimental design used to assess the in vivo myelinogenic competence of HD-derived hGPCs, with and without rescue of SOX10 and MYRF expression. All cells were exposed transiently to doxycycline in vitro, so as to initiate CD4 expression and permit FACS isolation before transplant into neonatal immunodeficient shiverer mice. When nine weeks of age, the engrafted mice were either given doxycycline for another 4 weeks to initiate SOX10 and MYRF expression (+DOX), ornot so treated (-DOX, controls). F, Shiverer mice engrafted neonatally with hGPCs derived from normal HTT-expressing hESCs (Genea 19) developed abundant myelin basic protein (MBP) expression and oligodendrocytic morphologies by 13 weeks in vivo. G, In contrast, mice engrafted with mHTT-expressing hGPCs produced from HD hESCs (Genea 20, or G20) developed little detectable MBP by that point. H-I, At 9 weeks of age, some Genea 20 mHTT hGPC-engrafted mice were given oral doxycycline to trigger SOX10 and MYRF expression, while matched controls were not given dox. H, The DOX(+) mice, in whose donor-derived hGPCs SOX10 and MYRF were induced, exhibited significant numbers of MBP⁺ myelinating oligodendrocytes in the engrafted white matter. I-J, By that same time point, no donor cells in the DOX(−) control mice had developed MBP expression, despite analogous donor cell engraftment (K). In the DOX(+) mice engrafted with SOX10/MYRF-transduced Genea20 GPCs, the donor-derived oligodendrocytes induced the robust formation of nodes of Ranvier, evidenced by the clustering of ßIV-spectrin flanked by Caspr protein that typifies nodal architecture, and which is otherwise absent in untreated shiverer brain (Figures 5L–M). Scale: B-C, F-I, 50 μm. L, 1 μm; M, 0.5 μm. Means ± SEM. ***p<0.001, t-tests.

Figure 6. Astrocytic differentiation is delayed in mHTT glial progenitor cells

Astrocytic differentiation was significantly delayed in mHTT glial chimeras. Mice neonatally transplanted with normal HTT GENEA19-derived GPCs began to develop significant donor-derived GFAP+ astrocytes by 8 weeks (A), robustly so by 13 weeks (B), with dense astrocytic colonization of the callosal white matter by 18 weeks (C). In contrast, mHTT-expressing hGPCs derived from GENEA20 sibling hESCs developed astrocytic phenotype more slowly, with little evident GFAP expression at 8 and 13 weeks (D and E), and only modest GFAP⁺ astrocytic maturation at 18 weeks (F). G-H, The mature astrocytic morphologies of mHTT-expressing and control astrocytes differed, in that mHTT astrocytes typically failed to manifest the degree of radial symmetry of their control-derived counterparts. I, The proportion of GFAP-expressing cells among all donor cells was consistently lower in mHTT hGPC-engrafted than control-engrafted mice. J-K, Sholl analysis of cells traced in NeuroLucida in 3D and shown flattened (O-P), revealed that normal donor astrocytes exhibited greater fiber complexity (J), more primary processes (K), yet shorter average and maximal fiber lengths (L-M), than mHTT-expressing astroglia. N-P, Fan-in radial analysis of volume occupancy (Dang et al., 2014) revealed that mHTT astrocytes had significantly more regions unoccupied by glial processes than did control astrocytes (N; illustrations in O and P), indicating their discontiguous domain structure. Means ± SEM. *p<0.05; **p<0.01; ***p<0.001 by: I, 2-way ANOVA with Bonferroni’s post-hoc tests; J, comparison of non-linear regressions, p<0.0001; K-N, unpaired t-tests comparing per-mouse average values across all cells scored (n=4 control, 7 mHTT mice). Scale: A-F, 50 μm; G-H, O-P, 25 μm.

Figure 7. HD hESC-derived CD44+ astroglia exhibit mHTT-dependent changes in gene expression

A, Principal component analysis performed as in Figure 1 but using CD44-sorted astroglia and their precursors validates the segregated expression signatures of HD-derived and normal cells. B, The Venn diagram highlights the intersection of lists of differentially expressed genes (DEGs) (green, down-regulated; red, up-regulated; FDR 5%), obtained by comparing astroglia derived from 3 HD patients against pooled control cells, using the same cell lines and analytic pipeline as in Figure 1. The list of DEGs shared by the 3 HD patients was filtered by those genes differentially expressed by patient HD20 (GENEA20) relative to its sibling donor CTR19 (GENEA19). C, The heatmap based on log2-transformed TPM values calculated from raw counts of the 114 DEGs highlighted in B, shows clustering by disease status. D, Network representation of functional annotations (Gene Ontology: Cellular Component, FDR-corrected p<0.1) for the 114 intersection DEGs highlighted in B. Genes are designated as round nodes (green, down-regulated; red, up-regulated); rounded rectangular nodes represent annotation terms. Nodes are sized by degree and grouped as interconnected modules (M1 though M4) identified by community detection. For each colored module, three of the top significant annotations are listed and labeled in the network. E, Expression heatmap of 14 conserved DEGs identified in M1 (yellow in D), annotations related to post-synaptic and receptor complex components. F, Heatmap of 9 conserved DEGs identified in M2 (grey in D), annotated to perinuclear and early endosome components. G, Heatmap of 11 conserved DEGs identified in M3 (blue in D), annotations related to plasma membrane, cell-cell junction, and desmosomal components. H, Heatmap of 8 DEGs identified in M4 (orange in D); annotations related to extracellular matrix components.