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, 22 (3), 484-491

Differentiation and Maturation of Oligodendrocytes in Human Three-Dimensional Neural Cultures


Differentiation and Maturation of Oligodendrocytes in Human Three-Dimensional Neural Cultures

Rebecca M Marton et al. Nat Neurosci.


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.


FIGURE 1.. Characterization of hOLS derived from hiPS cell lines.
a, Schematic for generating hOLS. Human iPS cells are enzymatically dissociated and aggregated in microwells to form spheroids. DM, dorsomorphin; SB, SB-431542; SAG, smoothened agonist; HGF, hepatocyte growth factor; AA, ascorbic acid to the end of the description of 1a. b–d, Relative gene expression (normalized to GAPDH) as determined by qPCR at day 100 of in vitro culture in hCS and hOLS of (b) OLIG2 (two-tailed Mann-Whitney test, ****P< 0.0001), (c) NKX2–2 (two-tailed Mann-Whitney test, ****P<0.0001) and (d) MBP (two-tailed t-test, t = 2.97, df=15, ***P=0.009). In b–d, 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. Lines are shown in different colors; each point represents 2–4 hOLS pooled from one differentiation experiment. e–h, Day 54 immunostaining and quantification of OLIG2 and NKX2–2 double positive cells (e, f) (two-tailed t-test, t=0.55, df=7, P= 0.59), and of PDGFRα (g, h) (two-tailed t-test, t=1.68, df=7, P= 0.13) out of Hoechst in dissociated hOLS at day 54 and day 110 (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). i–l, Immunostaining of (i) O4, (j) O1, (k) MBP, (l) MBP and O4 in cryosections. Immunostainings were repeated on hOLS from 6 independent inductions for O4, 3 independent inductions for O1, 5 independent inductions for MBP, and 3 independent inductions for MBP and O4 together with similar results. m, n, Immunostaining (m) and quantification (n) of MBP+ cells over time in whole hOLS cryosection (day 50–60: n=12 hOLS from 5 hiPS cell lines; day 100–110: n=17 hOLS from 5 hiPS cell lines; day 150–160: n=9 hOLS from 4 hiPS cell lines, each point represents one hOLS; see Supplementary Table 1; Kruskal-Wallis test, P< 0.0001 with Dunn’s multiple comparison test day 50–60 versus day 100–110, ***P=0.0005 and day 50–60 versus day 100–110, ****P<0.0001). o, Immunostaining of MBP, GFAP and NF-H in hOLS cryosections. Immunostainings were repeated on hOLS from 3 independent inductions with similar results. Data are mean ± s.e.m. Scale bars, 10 μm (k, o), 20 μm (i, j, l), and 50 μm (e, g), and 100 μm (m).
FIGURE 2.. Transcriptional comparison of hOLS oligodendrocyte-lineage cells to primary tissue cells.
a, Schematic showing the isolation of O4+ cells from hOLS. b, tSNE clustering single cell RNA-seq data from hOLS (n = 295 cells), primary human brain tissue and hCS (n= 1473 cells total; colored by cell type). c, Gene expression of oligodendrocyte-lineage related SOX10 in single cells. d, O4+ hOLS-derived single cells. e, Oligodendrocyte cluster from tSNE map in (b) with three distinct k-means subclusters of hOLS. f, Mean expression of oligodendrocyte lineage-specific genes in hOLS as well as primary OPCs and mature oligodendrocytes isolated from adult human brain tissue (log2 data normalized across rows). g, Single cell gene expression of subcluster-specific markers in the oligodendrocyte-lineage cluster. h, O4+ single cells derived from hOLS indicated by hiPS cell line.
FIGURE 3.. Developmental trajectory and expression of disease-related genes in hOLS.
a, Monocle pseudotime analysis of all oligodendrocyte-lineage cells colored by tissue of origin. Most mature time points are shown on right. b, Hierarchical clustered heat map depicting gene modules whose expression patterns covary across pseudotime (z-scores normalized by row). c, Expression of oligodendrocyte lineage markers across pseudotime (colored by tissue of origin; log2 data normalized by gene). d, Singe cell gene expression pattern of disease-implicated genes in the oligodendrocyte lineage cluster from (Fig. 2b).
FIGURE 4.. Oligodendrocyte maturation in hOLS.
a, Example of time lapse imaging of a migrating Sox10-MCS5::eGFP+ cell in hOLS. b, Percentage of migrating Sox10-MCS5::eGFP+ cells over differentiation time in vitro. (day 65–85: n=8 hOLS from 3 hiPS cell lines; day 115: n=5 hOLS from 3 hiPS cell lines; day 150: n=6 hOLS from 3 hiPS cell lines; day 170–180: n=6 hOLS from 3 hiPS cell lines; day >200: n=8 hOLS from 4 hiPS cell lines; see Supplementary Table 1). Lines are shown in different colors; Kruskal-Wallis test, P=0.003). Data are mean ± s.e.m. asterisk indicates the soma of a migrating cell c, Slice recordings in hOLS showing membrane voltage responses in bipolar and multipolar cells following intracellular current injections. d, Example of an I-V curve showing an outward rectifying current in a bipolar cell but not in a multipolar cell. For c and d, recordings were repeated in 12 bipolar cells and 13 multipolar cells from 3 independent inductions with similar results. e, Quantification of the rectification index in bipolar and multipolar Sox10-MCS5::eGFP+ cells (n=13 multipolar cells, n=12 bipolar cells, two-tailed Mann-Whitney test, ***P= 0.0009). 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 percentile of the population. f, Voltage clamp recording from a representative bipolar Sox10-MCS5::eGFP+ cell showing a reduction in holding current variance in response to the blockers of glutamate receptors APV (40 μM) and NBQX (20 μM). Recordings were repeated in 5 cells from 2 independent inductions with similar results.
FIGURE 5.. Oligodendrocyte-neuron interaction and myelination in hOLS.
a, b, Immunostaining of hOLS cryosections with MBP and NF-H (n=9 hOLS from 3 hiPS cell lines). c, Example images of interactions between MBP+ cells and NF-H+ processes in cryosections at day 150–158 of in vitro culture imaged by confocal microscopy. The first and seconds panels of each row are max projections, the third panel of each row is an individual z-section, and the right most panels are cross sections. d, Quantification of MBP+ cells that interact with NF-H+ neurons out of total MBP+ cells. e, f, Transmission electron microscopy images of hOLS at day 103 of differentiation showing myelin in line 2242–1. Examples from hOLS derived from other hiPS cell lines are shown in Supplementary Fig. 4. Electron microscopy was performed on hOLS from 3 independent inductions with similar results. g, Example images of Sox10-MCS5::eGFP+ cells in untreated and lysolecithin-treated hOLS after 13 hours of live imaging. h, Percentage of Sox10-MCS5::eGFP+ cells that disappear during the 13-hour live imaging experiment following lysolecithin treatment (n=8 untreated hOLS from 3 hiPS cell lines, n=7 lysolecithin-treated hOLS from 3 hiPS cell lines, two-tailed Mann-Whitney test, **P=0.001). Data are mean ± s.e.m. Scale bars, 200 μm (g) 50 μm (c left panel) 10 μm (a, b, c middle panel) 200 nm (e), 50 nm (f).

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