Human iPSC-derived myelinating organoids and globoid cells to study Krabbe disease

Abstract

Krabbe disease (Kd) is a lysosomal storage disorder (LSD) caused by the deficiency of the lysosomal galactosylceramidase (GALC) which cleaves the myelin enriched lipid galactosylceramide (GalCer). Accumulated GalCer is catabolized into the cytotoxic lipid psychosine that causes myelinating cells death and demyelination which recruits microglia/macrophages that fail to digest myelin debris and become globoid cells. Here, to understand the pathological mechanisms of Kd, we used induced pluripotent stem cells (iPSCs) from Kd patients to produce myelinating organoids and microglia. We show that Kd organoids have no obvious defects in neurogenesis, astrogenesis, and oligodendrogenesis but manifest early myelination defects. Specifically, Kd organoids showed shorter but a similar number of myelin internodes than Controls at the peak of myelination and a reduced number and shorter internodes at a later time point. Interestingly, myelin is affected in the absence of autophagy and mTOR pathway dysregulation, suggesting lack of lysosomal dysfunction which makes this organoid model a very valuable tool to study the early events that drive demyelination in Kd. Kd iPSC-derived microglia show a marginal rate of globoid cell formation under normal culture conditions that is drastically increased upon GalCer feeding. Under normal culture conditions, Kd microglia show a minor LAMP1 content decrease and a slight increase in the autophagy protein LC3B. Upon GalCer feeding, Kd cells show accumulation of autophagy proteins and strong LAMP1 reduction that at a later time point are reverted showing the compensatory capabilities of globoid cells. Altogether, this supports the value of our cultures as tools to study the mechanisms that drive globoid cell formation and the compensatory mechanism in play to overcome GalCer accumulation in Kd.

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.