Differentiation and maturation of oligodendrocytes in human three-dimensional neural cultures

Abstract

Investigating human oligodendrogenesis and the interaction of oligodendrocytes with neurons and astrocytes would accelerate our understanding of the mechanisms underlying white matter disorders. However, this is challenging because of the limited accessibility of functional human brain tissue. Here, we developed a new differentiation method of human induced pluripotent stem cells to generate three-dimensional brain organoids that contain oligodendrocytes as well as neurons and astrocytes, called human oligodendrocyte spheroids. We found that oligodendrocyte lineage cells derived in human oligodendrocyte spheroids transitioned through developmental stages similar to primary human oligodendrocytes and that the migration of oligodendrocyte lineage cells and their susceptibility to lysolecithin exposure could be captured by live imaging. Moreover, their morphology changed as they matured over time in vitro and started myelinating neurons. We anticipate that this method can be used to study oligodendrocyte development, myelination, and interactions with other major cell types in the CNS.

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Fig. 1: Characterization of hOLS derived from hiPS cell lines.
Fig. 2: Transcriptional comparison of hOLS oligodendrocyte lineage cells to primary tissue cells.
Fig. 3: Developmental trajectory and expression of disease-related genes in hOLS.
Fig. 4: Oligodendrocyte maturation in hOLS.
Fig. 5: Oligodendrocyte–neuron interaction and myelination in hOLS.

Data availability

Gene expression data have been deposited in the Gene Expression Omnibus under accession number GSE115011. The data that support the findings of this study are available on reasonable request from the corresponding author.

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Acknowledgements

This paper is dedicated to the memory of our wonderful colleague and mentor Ben A. Barres. We thank B.A. Barres, B. Zuchero, and members of the Pasca laboratory for scientific input, J. Perrino (Stanford Cell Sciences Imaging Facility) for support with electron microscopy, as well as F. Sim (University of Buffalo) for providing the Sox10-MCS5::eGFP plasmid. This work was supported by the US National Institutes of Health BRAINS Award (MH107800), the MQ Fellow Award, the NYSCF Robertson Stem Cell Investigator Award, the Stanford Wu Tsai Neurosciences Institute’s Brain Rejuvenation Project and the Human Brain Organogenesis Project, the Kwan Research Fund and the California Institute of Regenerative Medicine, the Child Health Research Institute Pilot Award, and the NARSAD Independent Investigator Award from the Brain and Behavior Research Foundation (to S.P.P.); the National Science Foundation Graduate Research Fellowship and the Bio-X Stanford Interdisciplinary Graduate Fellowship (to R.M.M.); Stanford Medicine’s Dean’s Fellowship (to Y.M.); and NIMH T32GM007365, F30MH106261, and Bio-X Predoctoral Fellowship (to or supporting S.A.S.).

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Contributions

R.M.M. performed the differentiation experiments. Q.L. carried out single-cell library preparations and S.A.S. analyzed single-cell data. O.R. and J.R.H. conducted and analyzed electrophysiological experiments. R.M.M., Y.M., and R.J.L. carried out all other experiments and data analyses. R.M.M. and S.P.P. conceived the project, designed experiments, and wrote the manuscript with input from all authors. S.P.P. supervised the work.

Corresponding author

Correspondence to Sergiu P. Pașca.

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Stanford University has filed a provisional patent application that covers the generation of myelinating oligospheroids for studying human development and disease (US patent application number 15/953,197).

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Supplementary Figure 1 Characterization of oligodendrocytes, astrocytes, and neurons in hOLS.

a, Relative gene expression (normalized to GAPDH) as determined by qPCR at day 25 of in vitro culture of markers for pluripotency and germ layers (OCT4, SOX2, BRACH, SOX17) (n = 4 RNA samples of 2 hOLS each from 4 hiPS cell lines; see Supplementary Table 1; Kruskal–Wallis test, P = 0.001). b, Relative gene expression (normalized to GAPDH) in hCS and hOLS as determined by qPCR at day 37 of in vitro culture hOLS of CNS markers (FOXG1, SIX3, NKX2.1, OTX2, LMX1B, RAX1, HOXB4, PAX6, and LHX2) (n = 5 samples of 2 hOLS each from 5 hiPS cell lines; see Supplementary Table 1; Kruskal–Wallis test, P = 0.0001). c, Day 51 immunostaining of hOLS cryosections for OLIG2 and NKX2-2. Immunostainings were repeated on hOLS from four independent inductions with similar results. d, Histogram of the spatial distribution of MBP+ cells in hOLS cryosections. The number of MBP+ cells at various proportions of the radius (distance from center to edge) is shown. e, Relative gene expression (normalized to GAPDH) as determined by qPCR at day 100 of in vitro culture in hCS and hOLS of RBFOX3 (NEUN) (two-tailed t-test, t = 1.23, d.f. = 15, P = 0.23) and GFAP (two-tailed Mann–Whitney test, **P = 0.001) (for hCS n = 8 and for hOLS n = 9 RNA samples from spheroids derived from 4 hiPS cell lines in 1–4 differentiation experiments; see Supplementary Table 1). fi, Immunostaining of MAP2 at day 54 (f) and GFAP at day 110 (h) and quantification of MAP2+ (g) (two-tailed t-test, t = 3.82, d.f. = 7, **P = 0.006) and GFAP+ (i) (two-tailed t-test, t = 4.615, d.f. = 7, **P = 0.002) at day 54 and day 110 in dissociated hOLS (n = 5 samples each consisting of 4–6 hOLS derived from 3 hiPS cell lines; hiPS cell lines shown in different colors; see Supplementary Table 1). j, Relative expression of VGLUT1 (SLC17A7) (two-tailed Mann–Whitney test, ***P <0.0003) and GAD1 (two-tailed t-test, t = 2.92, d.f. = 17, **P = 0.009), in hCS and hOLS (normalized to GAPDH), as determined by qPCR at day 100 of in vitro culture (for hCS n = 8 and for hOLS n = 11 RNA samples from spheroids derived from 7 hiPS cell lines in 1–3 differentiation experiments; see Supplementary Table 1). k, Day 100 immunostaining of hOLS cryosections for GABA and MAP2. Immunostainings were repeated on hOLS from six independent inductions with similar results. Data are mean ± s.e.m. Scale bars, 50 μm (c, f, h, k upper panel), and 10 μm (k lower panel).

Supplementary Figure 2 Single cell characterization of hOLS.

a, Source of cells in the cluster in Fig. 2b. b, Single cell gene expression pattern of the markers for astrocytes (SOX9), endothelial cells (FLT1), neurons (STMN2), and myeloid cells (CX3CR1). c, Unsupervised hierarchical clustering of all single cells, colored by source. d, Relative gene expression (normalized by GAPDH) as determined by qPCR of MKI67, TOP2A, PDGFRA, and MBP in pooled cDNA from 50 single cells from the proliferating cells, OPCs, NFOs, and myelinating oligodendrocytes clusters (Fig. 2e-g). e, Percentage of cells in the proliferating cells, OPCs, NFOs, and myelinating oligodendrocytes subclusters in two hiPS cell lines. f, Pearson correlation values between the log normalized gene expression data for each oligodendrocyte subcluster between two hiPS cell lines.

Supplementary Figure 3 Primary and hOLS-derived oligodendrocyte differential gene expression and pattern of expression of disease-related genes.

a, Genes that were enriched in oligodendrocyte lineage cells isolated from primary tissue versus hOLS. b, Expression of the housekeeping gene GAPDH across pseudotime (colored by tissue of origin; log2 data normalized by gene). c, Single cell gene expression pattern of disease-implicated genes ARSA, RNASEH2A, and GALC in hOLS, hCS, and primary samples.

Supplementary Figure 4 Electrophysiological characterization of hOLS and myelination.

a, Quantification of the capacitance in bipolar and multipolar Sox10-MCS5::eGFP+ cells (n = 12 bipolar cells, n = 13 multipolar cells, two-tailed Mann–Whitney test, ***P = 0.0006). b, Quantification of the input resistance in bipolar and multipolar Sox10-MCS5::eGFP+ cells (n = 12 bipolar cells, n = 13 multipolar cells, two-tailed Mann–Whitney test, *P = 0.01). For a and b, dots represent individual cells, box edges represent s.e.m., the middle horizontal lines within the box represent the mean, and whiskers represent the 10th and 90th percentiles of the population. c, (upper left) Voltage clamp recording of a Sox10-MCS5::eGFP+ cell showing lack of inward current generation following electrical stimulation indicated by the red dot. (upper right) Voltage clamp recording of Sox10-MCS5::eGFP+ cell showing holding current variance in response to TTX (1 μM) and after treatment with NBQX and APC (lower right). Recordings were repeated in five cells from two independent inductions with similar results. d, Example images of interactions between MBP+ cells and NF-H+ processes in cryosections at days 150–158 of in vitro culture imaged by confocal microscopy. The first and seconds panels of each row are maximum projections, the third panel of each row is an individual z-section, and the right most panels are cross-sections. Immunostainings were repeated on hOLS from three independent inductions with similar results. e,f, Transmission electron microscopy images of hOLS from 8858-3 (e) and 0524-1 (f) at days 150–170 of differentiation showing stages of myelination. Electron microscopy was repeated on hOLS from three independent inductions with similar results. Scale bars, 1 μm (e, f), 50 μm (d left panel), and 10 μm (d middle panel).

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Marton, R.M., Miura, Y., Sloan, S.A. et al. Differentiation and maturation of oligodendrocytes in human three-dimensional neural cultures. Nat Neurosci 22, 484–491 (2019). https://doi.org/10.1038/s41593-018-0316-9

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