Modeling hereditary diffuse leukoencephalopathy with axonal spheroids using microglia-sufficient brain organoids

Abstract

Hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS) is a rare, fatal, adult-onset neurodegenerative disease that is most often caused by mutations affecting the colony stimulating factor-1 receptor (CSF-1R). To understand how CSF-1R-mutation affects human microglia - the specialized brain-resident macrophages of the central nervous system - and the downstream consequences for neuronal cells, we used a macrophage and forebrain organoid co-culture system based on induced pluripotent stem cells generated from two patients with HDLS, with CSF-1R gene-corrected isogenic organoids as controls. Macrophages derived from iPSC (iMacs) of patients exhibited a metabolic shift toward the glycolytic pathway and reduced CSF-1 sensitivity, which was associated with higher levels of IL-1β production and an activated inflammatory phenotype. Bulk RNA sequencing revealed that iMacs adopt a reactive state that leads to impaired regulation of neuronal cell populations in organoid cultures, thereby identifying microglial dysregulation and specifically IL-1β production as key contributors to the degenerative neuro-environment in HDLS.

Keywords: CSF1R; HDLS; human; immunology; inflammation; macrophage; microglia; organoid.

Figure 1.. Generation and characterization of iPSC lines from patients with HDLS.

(A) Schematic representation of the generation of patient-derived induced pluripotent stem cells (iPSC) from dermal fibroblasts, and the subsequent generation of isogenic controls via gene editing with CRISPR/Cas9. (B) Sequencing of the genomic CSF-1R locus in donor-derived iPSC displaying mutant allele or genetically edited allele in isogenic control cell lines. (C) Phase-contrast microscopy of mature reprogrammed iPSC colonies displaying typical hESC morphology. Images were acquired with a standard microscope (Nikon) with a 10× objective. Scale bar represents 250 µM. (D) Immunofluorescence imaging of Mut HD1 iPSC colonies for the pluripotency markers SOX2, OCT4, TRA-1-60, and SSEA-4. Scale bar represents 150 and 100 µM for left and right panels, respectively. (E) Cytogenetic analysis of IsoHD1 (left) and IsoHD2 (right) iPSC clone showing a normal karyotype.

Figure 1—figure supplement 1.. Donor-derived human dermal fibroblast cells and induced pluripotent stem cells (iPSC) and off-target events (OTE) analysis.

(A) Human dermal fibroblast outgrowth from skin biopsies after 3 weeks of culture. Images were acquired with a standard microscope (Nikon) with 10× (left panel) and 20× (right panel) objective, respectively. Scale bar represents 200 µM. (B) Phase-contrast microscopy (20×) of emerging immature iPSC clones after reprogramming. Scale bar represents 200 µM. (C) Cytogenetic analysis of Mut HD1 (left) and Mut HD2 (right) iPSC clone showing a normal karyotype. (D) Sequencing of genomic CSF-1R locus displaying OTE analysis for IsoHD1 and IsoHD2 iPSC cell lines.

Figure 2.. Generation and characterization of patient iPSC-derived macrophages.

(A) Schematic representation of the derivation of primitive macrophages from donor-derived iPSC. (B) iMacrophage (iMac) yields across all cell line variants. (C) Flow cytometry analysis of iPSC and iMac expression of macrophage surface markers CD45 and CD14. (D) Morphology of iMacs visualized by Giemsa staining (left panel), and confocal imaging (middle panel); and internalization of PE-conjugated beads visualized by confocal imaging. Scale bar represents 30 µM. (E) Uptake of pHrodo-labeled beads by iMacs in the presence and absence of cytochalasin D (Cyto D). (F) Cell viability of iMacs used to determine CSF-1 sensitivity via an MTT assay, after incubating with varying concentrations of CSF-1 for 7 days. Data are presented as mean ± SEM. Statistical significance was assigned as: p < 0.05 was considered significant: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 3.. Mutant and isogenic control iMacs have distinct gene expression profiles.

(A, D) Principal component analysis (PCA), (B, E) volcano plots, and (C, F) GO analysis, of mutant and isogenic iMacs derived from HD1 and HD2 differentially expressed genes (DEGs), respectively. Representative curve of extracellular acidification rate (ECAR) (G, O) and their respective quantified data (H–J, P–R) are shown. Representative curve of oxygen consumption rate (OCR) (K, S) and their respective quantified data (L–N, T–V) are shown. Real-time assessment of bioenergetic profile between mutant and isogenic HD1 iMacs, measured using extracellular flux assay and a mitochondrial stress test. The following glycolytic parameters were calculated based on ECAR: (B) glycolysis, (C) glycolytic capacity, and (D) glycolytic reserve. Data shown as means + SEM, n = 9. The following parameters were calculated based on OCR: (F) basal, (G) ATP production, and (H) maximal respiration. 2-DG = 2-deoxy-glucose, FCCP = carbonyl cyanide 4-trifluoromethoxy-phenylhydrazone (n = 3). Data are presented as mean ± SEM. Statistical significance was assigned as: p < 0.05 was considered significant: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4.. Cytokine profiling of iMacs saw upregulated levels of IL-1β when probed with apoptotic neuronal cells.

(A, B) Levels of transcription of pro- and anti-inflammatory cytokine genes (IFNg, TNFa, IL-1b, IL-6, IL-18, IL-10, IL-12, and TGFb) quantified by qPCR (+uv depicts UV-treated, −uv depicts non-UV treated). n = 3 per cell line. Data are presented as mean ± SEM.

Figure 4—figure supplement 1.. Characterization of apoptotic SH-SY5Y neuronal cells.

(A) CFSE-labeled SH-SY5Y cells were either UV-treated (red) or untreated (blue) then after 24 hr were incubated with Annexin V/PE to determine the induction of apoptosis. Annexin V labeling was measured by flow cytometry (top panel) and visualized by fluorescence microscopy (bottom panel). (B) Percentage of CD45⁺ cells in iMac cultures from HD1 (top row) and HD2 (bottom row) that took up apoptotic SH-SY5Y cells, with cytochalasin D used as a negative control. Data are presented as mean ± SEM. Scale bar represents 30 µM.

Figure 5.. iMacs differentiate into microglia-like cells in co-culture with forebrain organoids.

(A) Schematic diagram of co-culture between day 31 iMacs and forebrain organoid. (B) Forebrain organoids depicted before, during, and after co-culture with iMacs. Day 31 isogenic forebrain organoids derived from the respective donor iPSC cell line were co-cultured with the same isogenic- and mutant-cell-line-derived day 31 iMacs. 30,000 iMacs were co-cultured with each respective organoid derived from HD1 and HD2. Figure shows matured organoids before and after the addition of iMacs during co-culture. (C) Three-dimensional visualization of co-cultured brain organoids after 23 days. Neurons were labeled for NESTIN and NPCs for SOX2. Microglia-like cells (IBA-1+) were visualized on the surface of organoids. (D) Flow cytometry analysis of neurons, NPCs, and macrophage populations within forebrain organoids after 23 days of co-culture. (E, F) Donor-derived iMacs in forebrain co-cultures undergo distinct changes in gene expression as observed in principal component analysis (PCA) (E, H), volcano plots (F, I), and GO analysis (G, J) in both HD1 and HD2 variants. Scale bar represents 200 µM.

Figure 5—figure supplement 1.. Phenotypic characterization of organoids and iMicro.

(A) Progression of forebrain organoid development across 31 days. Cells were initially seeded singly before the formation of embryoid bodies (EB) was observed after 2–3 days. Healthy EBs grew and matured, as seen by the formation of distinct neural epithelium in the organoids between days 12 and 31. Images were acquired with a standard microscope (Nikon) with a 10× objective. Scale bar represents 200 µM. (B) Immunofluorescence imaging of neuroepithelial rosette (red, SOX2+) in day 13 organoid. Developmental cues further supported the formation and expansion of the organoid to develop neuroepithelial tubes or rosettes, which contain predominantly neural stem cells (NPCs) that mimic the developing embryonic human cerebral cortex. (C) Immunocytochemical analysis of microglia-like cells (Iba1) after 23 days of co-culture with organoids. Scale bars represent 50 µM for low magnification and 10 µM for high magnification. (D) Three-dimensional visualization of non-co-cultured brain organoids (day 23). Neurons were labeled for NESTIN (green, cytoplasm) while the NPCs were labeled for SOX2 (red, cytoplasm). Microglia-like cells (IBA1+, white) were not identified, indicating the lack of formation of innately developing microglia. Scale bar represents 200 µM.

Figure 6.. Co-culture results in impaired regulation of neurons within organoids.

Size of (A) HD1 and (B) HD2 organoids measured before and after co-culture with mutant and isogenic iMacs in both HD1 and HD2 variants. The effects of co-culture on neuronal populations in (C) HD1 and (D) HD2 organoids after 23 days. Transcriptomic analysis of co-cultured (E) neurons and (F) NPCs from HD2 organoids after 23 days. Data are presented as mean ± SEM. Statistical significance was assigned as: p < 0.05 was considered significant: *p < 0.05; **p < 0.01.