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. 2018 Sep;15(9):700-706.
doi: 10.1038/s41592-018-0081-4. Epub 2018 Jul 25.

Induction of Myelinating Oligodendrocytes in Human Cortical Spheroids

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Free PMC article

Induction of Myelinating Oligodendrocytes in Human Cortical Spheroids

Mayur Madhavan et al. Nat Methods. .
Free PMC article

Abstract

Cerebral organoids provide an accessible system for investigations of cellular composition, interactions, and organization but have lacked oligodendrocytes, the myelinating glia of the central nervous system. Here we reproducibly generated oligodendrocytes and myelin in 'oligocortical spheroids' derived from human pluripotent stem cells. Molecular features consistent with those of maturing oligodendrocytes and early myelin appeared by week 20 in culture, with further maturation and myelin compaction evident by week 30. Promyelinating drugs enhanced the rate and extent of oligodendrocyte generation and myelination, and spheroids generated from human subjects with a genetic myelin disorder recapitulated human disease phenotypes. Oligocortical spheroids provide a versatile platform for studies of myelination of the developing central nervous system and offer new opportunities for disease modeling and therapeutic development.

Conflict of interest statement

Competing Interests

P.J.T. and R.H.M. are consultants for Convelo Therapeutics, which has licensed patents from Case Western Reserve University. P.J.T., R.H.M., and Case Western Reserve University hold equity in Convelo Therapeutics. D.C.F. became an employee of Convelo Therapeutics subsequent to completion of these studies. P.J.T. is a consultant and on the Scientific Advisory Board of Cell Line Genetics. P.J.T. is Chair of the Scientific Advisory Board (volunteer position) for the Pelizaeus-Merzbacher Disease Foundation. All other authors have no competing interests.

Figures

Figure 1 |
Figure 1 |. Generation of oligodendrocytes in human cortical spheroids.
a, Schematic of spheroid generation. The protocols to generate neurocortical spheroids (NCS) and oligocortical spheroids (OCS) were the same until week 8, after which time neurocortical spheroids were grown in basal media, while oligocortical spheroids were treated with PDGF-AA/IGF-1 from day 50–60 and T3 from day 60–70. Differentiation of oligodendrocytes was assessed at week 14 (dotted box), and these data are presented in Fig. 1. Colors in the schematic simulate neurons (magenta), astrocytes (red) and OPCs/Oligodendrocytes (green). Maturation of oligodendrocytes was evaluated at weeks 20 and 30, and these data are presented in Fig. 2. b, Representative fluorescence images of week 14, H7 spheroids generated with the neurocortical protocol. These spheroids generate neurons (Neurofilament:magenta) and astrocytes (GFAP:red), but no oligodendrocytes (PLP1:green). Similar results were obtained from 3 independent batches of spheroids generated from 4 separate lines. Scale bar, 50um. c, Representative fluorescence images of week 14, H7 spheroids generated with the oligocortical protocol. These spheroids generate neurons (Neurofilament:magenta), astrocytes (GFAP:red), and oligodendrocytes (PLP1:green). Scale bar, 50um. Higher magnification of oligodendrocyte morphology is shown in inset. Representative fluorescence images of H9, CWRU191, and RUES1 spheroids generated with the oligocortical protocol. Similar results were obtained from 3 independent batches of spheroids generated from 4 separate lines. Scale bar, 50um. d, Quantification of MYRF, a nuclear marker of the oligodendrocyte lineage, in week 14 spheroids generated with the neurocortical or oligocortical protocols as well as with either PDGF-AA and IGF-1 or T3 only. MYRF-positive cells were counted from four planes from four or five individual spheroids (n=4, PDGF/IGF or T3 treatments, n=5, NCS and OCS) for each treatment condition from lines H7, H9 and CWRU191 cell line and averaged (white boxes). Error bars are standard deviation. Three spheroids (n=3) from the same batch were used for the externally validated line RUES1. Representative fluorescence images of MYRF expression in additional lines and a schematic of MYRF quantification are shown in Supplementary Figure 2b–c. e, RNAseq comparing neuron, astrocyte, and oligodendrocyte gene expression in neurocortical and oligocortical spheroids. The heat maps consist of the 100 most cell specific transcripts for each cell type. Oligodendrocyte as well astrocyte specific genes are upregulated in oligocortical spheroids compared to neurocortical spheroids. f, Neuron, astrocyte, and oligodendrocyte-specific gene expression from the RNAseq shown in e. Box plots display the first and third quartiles, split by the mean; whiskers extend to include the maximum and minimum values. Oligocortical spheroids show a statistically significant change in astrocyte and oligodendrocyte specific gene expression compared to neurocortical spheroids. RNAseq experiment was performed using 5 spheroids from each condition, Paired non-parametric Wilcoxon matched pairs signed-rank test was used to determine statistical significance.
Figure 2 |
Figure 2 |. Maturation of oligodendrocytes oligocortical spheroids.
a, Schematic depicting the generation of oligocortical spheroids. Colors in the schematic simulate neurons (magenta), astrocytes (red) and OPCs/Oligodendrocytes (green). Maturation of oligodendrocytes was evaluated at weeks 20 and 30 (dotted box), and these data are presented in Fig. 2. b, Representative fluorescence image of week 20, H7 oligocortical spheroids show robust generation of the oligodendrocyte lineage (MYRF:magenta) as well as early born CTIP2-positive (yellow) neurons and SATB2-positive (cyan) late born neurons. Scale bar, 50um. Representative fluorescence images of week 20 MYRF expression in additional lines are shown in Supplementary Fig. 3a. Similar results were obtained from 2 independent batches of spheroids. c, Representative fluorescence image of week 20, H7 oligocortical spheroids demonstrate linear process formation in maturing oligodendrocytes (PLP1:green). Scale bar, 50um for c and d. Representative fluorescence images of week 20 PLP1 expression in additional lines are shown in Supplementary figure 3. Similar results were obtained from 2 independent batches of spheroids. d, Representative fluorescence image of week 20, H7 oligocortical spheroids immunostained for MBP (red), a marker of mature myelin, shows punctate MBP expression indicative of an early stage of maturation. Similar results were obtained from 2 independent batches of spheroids. e, Representative EM of week 20, H7 oligocortical spheroids showing a cluster of neurons that are being myelinated by oligodendrocytes. Scale bar, 1um. EM results were obtained from 3 spheroids from a single batch of spheroids. f, Representative EM of week 20, H7 oligocortical spheroids shows an axon encircled by multiple layers of loosely compacted myelin. Scale bar, 1um. Representative EMs of myelin in additional lines are shown in Supplementary Fig. 3b. EM results were obtained from 3 spheroids from a single batch of spheroids. g, Representative EM of week 20, H7 oligocortical spheroids shows more extensive wrapping of loosely compacted myelin encircling an axon. Scale bar, 1um. EM results were obtained from 3 spheroids from a single batch of spheroids. h, Representative fluorescence image of week 30, H9 oligocortical spheroids shows cortical lamination and separation of CTIP2-positive (yellow) deep layers from SATB2-positive (cyan) superficial layers. MYRF-positive (magenta) oligodendrocytes are interspersed within the cortical layers. Similar results were obtained from 4 spheroids from a single batch of oligocortical spheroids. Scale bar, 50um. i-j, Representative images of week 30, H9 oligocortical spheroids show oligodendrocyte processes (PLP1:magenta) tracking (arrows) neuron axons (neurofilament:yellow). Higher Magnification images of inset in figure i is shown in j. Similar results were obtained from 4 spheroids from a single batch of oligocortical spheroids. Scale bar, 50um. k, Electron micrograph of week 30 H9 oligocortical spheroids showing compact myelin around axons. EM results were obtained from 3 spheroids from a single batch of spheroids Scale bar, 1um. l, 3D reconstruction from block face EM sections taken along the length of an axon.
Figure 3 |
Figure 3 |. Cortical patterning and organization in oligocortical spheroids.
a, Representative fluorescence image of week 8, H7 spheroids at the end of initial neurocortical patterning. These spheroids generate distinct populations of neural progenitors (SOX2:yellow and Nestin:blue) that organize into ventricular-like zones. These cells are also the only actively dividing cells as marked by Ki67 (magenta). Scale bar, 50um for a and b. b, Representative Fluorescence image of week 8, H7 spheroids show the presence of a TBR2-positive (blue) outer SVZ-like zone adjacent to the Sox2-positive (yellow) ventricular-like area. c, Representative fluorescence image of H7 spheroids generated with the oligocortical protocol up through PDGF-AA/IGF-1 treatment, then administered two doses of BrdU (magenta) during week 9 (day 58 and 60) to label dividing cells. BrdU-positive cells localize to SOX2-positive ventricular zones, identifying this as a primary germinal center. Scale bar, 50um for c-e. d-e, Representative fluorescence images of H7 spheroids generated with either the neurocortical (d) or oligocortical (e) protocol, treated with BrdU during week 9 (Day 58 and 60), and then maintained through week 14. Only oligocortical spheroids generate oligodendrocytes (MYRF:cyan), many of which are double positive for BrdU (arrows in high magnification inset).
Figure 4 |
Figure 4 |. Promyelinating drugs promote the generation of oligodendrocytes in oligocortical spheroids.
a-d, Representative fluorescence images of week 14, H7 spheroids treated with PDGF/IGF-1 (from day 50–60) and either a, DMSO, b, T3, c, clemastine, or d, ketoconazole (from day 60–70). Whereas DMSO produced few MYRF-positive cells, T3, clemastine, and ketoconazole produced robust MYRF signal. Four spheroids from the same batch were used for analysis. Scale bar, 50um for a-d. e, Quantification of MYRF from a-d. MYRF-positive cells were counted in four individual spheroids (n=4) per cell line (colored points) and averaged (white bars). Error bars are standard deviation and a two-tailed unpaired t-test with Welch’s correction was performed. f, Representative EM of week 14, H7 spheroids generated with T3 (i.e. the standard oligocortical protocol) demonstrates an absence of myelin. Scale bar, 500nm for f and g. g, Representative EM of week 14, H7 spheroids generated with ketoconazole in lieu of T3 demonstrates robust production of non-compact myelin encircling multiple neuronal axons.
Figure 5 |
Figure 5 |. Oligocortical spheroids recapitulate a human myelin disease phenotype.
a-b, Representative fluorescence images of week 14, CWRU198 oligocortical spheroids immunostained for a, PLP1:green or b, MYRF:red demonstrates abundant oligodendrocytes and robust PLP1 expression. Scale bar, 50um for a-l. Five spheroids from the same batch were used for analysis. c-d, Representative images of week 14, PLP1 deletion oligocortical spheroids immunostained for c, PLP1:green or d, MYRF:red demonstrates an expected lack of PLP1 despite abundant MYRF-positive oligodendrocytes. Four spheroids from the same batch were used for analysis. e-f, Representative images of week 14, PLP1 duplication oligocortical spheroids immunostained for e, PLP1:green or f, MYRF:red demonstrates robust PLP1 expression despite a decrease in the abundance of MYRF-positive oligodendrocytes. Four spheroids from the same batch were used for analysis. g-h, Representative images of week 14, PLP1 c.254T>G oligocortical spheroids immunostained for g, PLP1:green or h, MYRF:red demonstrates perinuclear retention of PLP1 and a decrease in MYRF-positive oligodendrocyte abundance. Four spheroids from the same batch were used for analysis. i-j, Representative images of week 14, PLP1 c.254T>G oligocortical spheroids treated with GSK2656157 and immunostained for i, PLP1:green or j, MYRF:red demonstrates mobilization of PLP1 into oligodendrocyte process and rescue of MYRF-positive oligodendrocyte abundance. Four spheroids from the same batch were used for analysis. k-l, Representative images of week 14, PLP1 CRISPR-corrected c.254TG>T oligocortical spheroids immunostained for k, PLP1:green or l, MYRF:red demonstrates rescue of both PLP1 perinuclear retention and oligodendrocyte abundance. Four spheroids from the same batch were used for analysis. m, %MYRF-positive oligodendrocytes per organoid in a-l. MYRF-positive cells were counted from five individual spheroids of control line CWRU 198 (n=5) and four individual spheroids (n=4) per cell line (colored points) and averaged (white boxes). Error bars are standard deviation and a two tailed unpaired t-test with Welch’s correction was performed. n, Representative EM of week 30, PLP1 CRISPR-corrected c.254G>T oligocortical spheroids demonstrates compact myelin encircling an axon. 3 spheroids from a single batch were used for EM analysis. Scale bar, 1um.

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