Research ArticleNEUROSCIENCE

Multiple sclerosis iPS-derived oligodendroglia conserve their properties to functionally interact with axons and glia in vivo

See allHide authors and affiliations

Science Advances  04 Dec 2020:
Vol. 6, no. 49, eabc6983
DOI: 10.1126/sciadv.abc6983
  • Fig. 1 MS hiOLs do not exhibit aberrant survival or proliferation in the developing Shi/Shi:Rag2−/− brain.

    (A and C) Immunodetection of the human nuclei marker STEM101 (red) combined with OLIG2 (green) and the proliferation marker Ki67 (white) shows that a moderate proportion of MS-hiOLs sustains proliferation (empty arrowheads in the insets) following transplantation in their host developing brain, with no significant difference in the rate of proliferation between MS- and control-hiOLs over time. (B and D) Immunodetection of the apoptotic marker Caspase3 (green) indicates that MS-hiOLs survive as well as control-hiOLs 8 wpg. Two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison or Mann-Whitney t tests were used for the statistical analysis (n = 3 to 4 mice per group). Error bars represent SEMs. H, Hoechst dye. Scale bars, 100 μm.

  • Fig. 2 Differentiation of MS-hiOLs into mature oligodendrocytes is timely regulated in the corpus callosum of the developing Shi/Shi:Rag2−/− brain.

    (A) Combined immunodetection of human nuclei marker STEM101 (red) with CC1 (green) and SOX10 (white) for control (top) and MS-hiOLs (bottom) at 8, 12, and 16 wpg. (B and C) Quantification of SOX10+/STEM+ cells (B) and CC1+ SOX10+ over STEM+ cells (C). While the percentage of human oligodendroglial cells increased only slightly with time, the percentage of mature oligodendrocytes was significantly time regulated for both MS- and control-hiOLs. Two-way ANOVA followed by Tukey’s multiple comparison tests were used for the statistical analysis of these experiments (n = 3 to 4 mice per group). Error bars represent SEMs. *P < 0.05 and ****P < 0.0001. Scale bar, 100 μm.

  • Fig. 3 MS-hiOL–derived progeny extensively myelinates the dysmyelinated Shi/Shi:Rag2−/− corpus callosum.

    (A) Combined detection of human nuclei (STEM101) and human cytoplasm (STEM 121) (red) with MBP (green) in the Shi/Shi Rag2−/− corpus callosum at 8, 12, and 16 wpg. General views of horizontal sections at the level of the corpus callosum showing the progressive increase of donor-derived myelin for control- (top) and MS- (bottom) hiOLs. (B) Evaluation of the MBP+ area over STEM+ cells. (C and D) Quantification of the percentage of (C) MBP+ cells and (D) MBP+ ensheathed cells. (E) Evaluation of the average sheath length (μm) per MBP+ cells. No obvious difference was observed between MS and control-hiOLs. Two-way ANOVA followed by Tukey’s multiple comparison tests were used for the statistical analysis of these experiments (n = 6 to 14 mice per group). Error bars represent SEMs. *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bar, 200 μm. See also figs. S3 and S5.

  • Fig. 4 MS-hiOLs produce compact myelin in the dysmyelinated Shi/Shi:Rag2 −/− corpus callosum.

    (A to F) Ultrastructure of myelin in sagittal sections of the core of the corpus callosum 16 wpg with control-hiOLs (A to C) and MS-hiOLs (D to F). (A and D) General views illustrating the presence of some electron dense myelin, which could be donor derived. (B, C, E, and F) Higher magnifications of control (B and C) and MS (E and F) grafted corpus callosum validate that host axons are surrounded by thick and compact donor derived myelin. Insets in (C) and (F) are enlargements of myelin and show the presence of the major dense line. No difference in compaction and structure is observed between the MS and control myelin. (G) Quantification of g-ratio revealed no significant difference between myelin thickness of axons myelinated by control- and MS-hiOLs. Mann-Whitney t tests were used for the statistical analysis of this experiment (n = 4 mice per group). Error bars represent SEMs. Scale bars, (A and D) 5 μm , (B and E) 2 μm, and (C and F) 500 nm [with 200 and 100 nm, respectively in (C) and (F) insets].

  • Fig. 5 MS-hiOLs improve transcallosal conduction of the dysfunctional Shi/Shi:Rag2−/− axons to the same extent than control-hiOLs.

    (A) Scheme illustrating that intracallosal stimulation and recording are performed in the ipsi- and contralateral hemisphere, respectively. (B) N1 latency was measured following stimulation in different groups of Shi/Shi:Rag2−/−: intact or grafted with control or MS-hiOLs and WT mice at 16 wpg. MS-hiOL–derived myelin significantly restored transcallosal conduction latency in Shi/Shi:Rag2−/− mice to the same extent than control-derived myelin (P = 0.01) and close to that of WT levels. One-way ANOVA with Dunnett’s multiple comparison test for each group against the group of intact Shi/Shi:Rag2−/− was used. Error bars represent SEMs. *P < 0.05. (C) Representative response profiles for each group. Scales in Y axis is equal to 10 μV and in the X axis is 0.4 ms.

  • Fig. 6 Grafted MS-hiOLs show typical cell stage–specific electrophysiological properties.

    (A) Schematic representation of the concomitant Biocytin loading and recording of single RFP+ hiOL derivative in an acute coronal brain slice prepared from mice engrafted with hiOLs (control or MS) and analyzed at 12 to 14 wpg. (B and C) Currents elicited by voltage steps from −100 to +60 mV in a control-oligodendrocyte progenitor (B, left) and a MS-oligodendrocyte (C, left). Note that the presence of an inward Na+ current obtained after leak subtraction in the oligodendrocyte progenitor, but not in the oligodendrocyte (insets). The steady-state I-V curve of the oligodendrocyte progenitor displays an outward rectification (B, right) while the curve of the oligodendrocyte has a linear shape (C, right). (D) Mean amplitudes of Na+ currents measured at −20 mV in control and MS iPSCs-derived oligodendrocyte progenitors (n = 8 and n = 9, respectively, for four mice per condition; P = 0.743, Mann-Whitney U test). (E). Mean amplitudes of steady-state currents measured at +20 mV in control and patient differentiated iPSC-derived oligodendrocytes (n = 10 and n = 6 for 3 and four mice, respectively; P = 0.6058, Mann-Whitney U test). (F) A control iPSC-derived oligodendrocyte progenitor loaded with biocytin and expressing OLIG2, STEM101/121, and lacking CC1 (top) and an MS iPSC–derived oligodendrocyte loaded with biocytin and expressing SOX10, CC1, and STEM101/121 (bottom). Scale bar, 20 μm.

  • Fig. 7 Grafted hiOL derivatives are functionally connected to murine cells.

    (A) Z-stack identifying a target and connected cell. One single grafted human RFP+ cell (per acute slice) was loaded with biocytin by a patch pipette and allowed to rest for 30 min. The white arrowheads and insets in (A) illustrate biocytin diffusion up to the donut-shaped tip of the human oligodendrocyte processes. Another biocytin-labeled cell (empty yellow arrowhead) was revealed at different morphological level indicating diffusion to a neighboring cell and communication between the two cells via gap junctions. (B and C) Split images of (A) showing the target (B) and connected (C) cell separately at different levels. Immunolabeling for the combined detection of the human markers STEM101/121 (red), OLIG2 (blue), and CC1 (white) indicated that the target cell is of human origin (STEM+) and strongly positive for OLIG2 and CC1, a mature oligodendrocyte, and that the connected cell is of murine origin (STEM-) and weakly positive for OLIG2 and CC1, most likely an immature oligodendrocyte. Scale bars, 30 μm. See also fig. S7.

Supplementary Materials

  • Supplementary Materials

    Multiple sclerosis iPS-derived oligodendroglia conserve their properties to functionally interact with axons and glia in vivo

    Sabah Mozafari, Laura Starost, Blandine Manot-Saillet, Beatriz Garcia-Diaz, Yu Kang T. Xu, Delphine Roussel, Marion J. F. Levy, Linda Ottoboni, Kee-Pyo Kim, Hans R. Schöler, Timothy E. Kennedy, Jack P. Antel, Gianvito Martino, Maria Cecilia Angulo, Tanja Kuhlmann, Anne Baron-Van Evercooren

    Download Supplement

    This PDF file includes:

    • Figs. S1 to S8
    • Tables S1 and S2

    Files in this Data Supplement:

Stay Connected to Science Advances

Navigate This Article