Table 1.
Antibodies.
Fig 1.
Kd iPSCs do not show any morphological and proliferation defects.
(A) GALC activity measurements of Ctrol and Kd iPSCs. (B) Immunohistochemistry of Ctrol and Kd iPSCs for the pluripotency markers Nanog, TRA1-60 and OCT4. (C) Proliferation assessment by EdU incorporation assay of Ctrol and Kd iPSCs. Colors indicate replicates from individual iPSC lines. Values are expressed as the means ± SEMs. ***P < 0.001 by Student’s t test.
Fig 2.
Kd organoids show normal neurogenesis, astrogenesis and oligodendrogenesis.
(A) Graphical abstract of the protocol used to produce myelinating organoids. (B) Brightfield images of Ctrol and Kd organoids at D60 of culture. (C) Immunohistochemistry and quantifications of the number of Sox2+ neural stem cells in Ctrol and Kd organoids at D20. (D) Immunohistochemistry and quantifications of the number of Ki67+ proliferating progenitors in Ctrol and Kd organoids at D37. (E) Immunohistochemistry and quantifications of the number of NeuN+ pan-neuronal marker in Ctrol and Kd organoids at D37. (F) Immunohistochemistry and quantifications of the number of Islet1/2+ neuronal progenitors in Ctrol and Kd organoids at D37. (G) Immunohistochemistry and densitometric quantifications of GFAP+ astrocytes in Ctrol and Kd organoids at D37. (H) Immunohistochemistry and quantifications of the number of Olig2+/Nkx2.2+ OPCs in Ctrol and Kd organoids at D37 and D60. Values are expressed as the number of Olig2+/Nkx2.2+ per area (upper chart) or the number of Olig2+/Nkx2.2+ relative to the Olig2+ population (lower chart). Colors indicate replicates from individual iPSC lines. Values are expressed as the means ± SEMs.
Fig 3.
Kd organoids signs of demyelination and minimal mTOR pathway perturbation.
(A) Immunohistochemistry for MBP+/Olig2+ mature oligodendrocytes at 8 or 12 weeks of myelination. Immunohistochemistry at 12 weeks for MBP/NF myelinated axons and MBP/Nfasc/Caspr fully developed nodes of Ranvier. (B) Immunohistochemistry for MBP in Ctrol and Kd organoids at 12 and 20 weeks of myelination. Quantifications of the number and length of MBP+ myelin internodes in Ctrol and Kd organoids at 12 and 20 weeks of myelination. Each point in the internode’s length graph represents the average internode’s length per organoid. (C) Electron micrographs of Ctrol and Kd organoids at 12 weeks of myelination showing lysosomal enlargement, vacuolation, lipid droplets and myelin debris in Kd organoids. (D) Western blot of Ctrol and Kd organoid’s lysates at 8 and 12 weeks of myelination for the lysosomal marker LAMP1, the ribosomal protein S6 (S6) and the autophagy proteins p62 and LC3B. Colors indicate replicates from individual iPSC lines. Values are expressed as the means ± SEMs. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test or two-way ANOVA followed by Newman–Keuls multiple-comparisons post-test.
Fig 4.
Globoid cell formation is drastically increased in Kd microglia by GalCer feeding.
(A) Differentiated microglia (D50) from Ctrol and Kd iPSCs treated with vehicle (DMSO + HMCD) or 10 μM GalCer for 24 or 48 hrs. Phalloidins’ staining of actin to identify cell morphology. Quantification of the number of globoid shaped cells. Arrow indicates globoid cell in Kd cultures fed with DMSO. (B) Phalloidin staining for globoid multinucleated cells in Kd cultures exposed to GalCer for 24 or 48 hrs. (C) Western blot analysis of untreated Ctrol and Kd microglia for the lysosomal marker LAMP1, the ribosomal protein S6 (S6) and the autophagy proteins p62 and LC3B. (D) Western blot analysis of Ctrol (left) and Kd (right) microglia exposed to GalCer for 16 or 48 hrs. for the lysosomal marker LAMP1, the ribosomal protein S6 (S6) and the autophagy proteins p62 and LC3B. Colors indicate replicates from individual iPSC lines. Values are expressed as the means ± SEMs. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test.