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. 2018 Sep 13;9(1):3708.
doi: 10.1038/s41467-018-06102-7.

Rapid Functional Genetics of the Oligodendrocyte Lineage Using Pluripotent Stem Cells

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Free PMC article

Rapid Functional Genetics of the Oligodendrocyte Lineage Using Pluripotent Stem Cells

Angela M Lager et al. Nat Commun. .
Free PMC article

Abstract

Oligodendrocyte dysfunction underlies many neurological disorders, but rapid assessment of mutation-specific effects in these cells has been impractical. To enable functional genetics in oligodendrocytes, here we report a highly efficient method for generating oligodendrocytes and their progenitors from mouse embryonic and induced pluripotent stem cells, independent of mouse strain or mutational status. We demonstrate that this approach, when combined with genome engineering, provides a powerful platform for the expeditious study of genotype-phenotype relationships in oligodendrocytes.

Conflict of interest statement

P.J.T. is on the scientific advisory board of Cell Line Genetics. The remaining authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Reproducible generation of OPCs and oligodendrocytes from mESCs. a Graphical overview of the differentiation time course for generating OPCs and stage-specific oligodendrocytes from mESCs. b Quantification of immunocytochemistry for stage-specific markers demarcating the transition from pluripotency (Oct4) to neuroectoderm (Pax6) to ventral neural tube (Olig2) over 9 days. n = 4 independent biological replicates (mESC lines) with >25 colonies scored per cell line. Data are represented as means ± SEM. c Representative immunofluorescent images of starting mESCs (Oct4 and Nanog) and day 5 neuroectoderm (Pax6 and Sox1). Scale bar, 50 µm. d Quantification of OPC-defining transcription factors Olig2, Nkx2.2, and Sox10 at passages 1 and 3 of the differentiation protocol. n = 4 independent mESC lines; >114,500 cells scored per cell line. Data are represented as means ± SEM. ***P value <0.001; unpaired t test. e Immunofluorescent image of passage 1 cultures stained for Sox10, an OPC marker, and βIII-Tubulin, a marker of neurons. Scale bar, 50 µm. f Representative phase contrast image of mESC-derived OPCs exhibiting a canonical bipolar morphology. Scale bar, 50 µm. g Immunostained image of passage 3 mESC-derived OPCs co-expressing Olig2, Nkx2.2, and Sox10, canonical OPC transcription factors. Scale bar, 50 µm. h Cell surface immunostaining of the immature oligodendrocyte marker O4, after treatment with T3. Scale bar, 50 µm. i Representative images of differentiated OPCs immunostained for mature oligodendrocyte markers MBP and PLP1, 72 h post treatment with T3. Scale bar, 50 µm. j Representative images of OPC/DRG co-cultures stained for MBP and neurofilament (NF) at day 10. Scale bar, 50 µm. Unless otherwise noted, images presented in Fig. 1 are derived from the CBA/Ca mESC line, and are representative of results obtained with C57BL/6, PO, and 129P2/Ola lines, which are shown separately in Supplementary Fig. 2
Fig. 2
Fig. 2
Cellular profiling of spontaneous and purposely generated mutant oligodendrocyte alleles. a Diagram indicating that shiverer mice harbor a ~20-kilobase (kb) homozygous deletion encompassing exons 2–7 of the MBP gene. A Sanger sequencing trace shows the breakpoint of the shiverer deletion. b Diagram indicating the location of the two gRNAs designed to target MYRF. A Sanger sequencing trace shows the location of the homozygous deletion of exon 1. c Quantification of transcription factors Olig2, Nkx2.2, and Sox10 at passage 3 of the differentiation protocol. n = 3 shiverer cell lines; n = 3 replicate wells per cell line; >179,500 cells scored per well. n = 3 MYRF KO and wild-type (WT) mESC replicate wells per cell line; >700 cells scored per well. Data are represented as means ± SEM. d Fluorescent images of WT iPSC, shiverer, and MYRF KO OPCs expressing canonical OPC markers Olig2, Nkx2.2, and Sox10. Scale bar, 50 µm. e Cell surface immunostaining of the immature oligodendrocyte marker O4, after treatment with T3, of WT iPSC, shiverer, and MYRF KO OPCs. Scale bar, 50 µm. f Representative images of differentiated OPCs immunostained for mature oligodendrocyte markers MBP and PLP1, 72 h post treatment with T3 of WT iPSC, shiverer, and MYRF KO OPCs. Scale bar, 50 µm. g Representative images of OPC/DRG co-cultures stained for MBP and neurofilament (NF) at day 10 from WT iPSC, shiverer, and MYRF KO OPCs stained for PLP1 or MBP after being co-cultured for 10 days with NF+ embryonic rat DRGs. Scale bar, 50 µm
Fig. 3
Fig. 3
Molecular profiling of spontaneous and purposely generated mutant oligodendrocyte alleles. a Row normalized heatmap with genes (rows) sorted by time of maximum (TOM). The four independent WT mESC lines for each time point are shown compared to gene expression of shiverer and MYRF KO OPCs 72 h post T3 induction. b Boxplot of OPC-signature genes prior to T3 induction and after differentiation with T3 for 24, 48, and 72 h. c Boxplot of oligodendrocyte-signature genes prior to T3 induction and after differentiation with T3 for 24, 48, and 72 h. d Row normalized heatmap of the top 30 dysregulated oligodendrocyte-signature genes for shiverer 72 h post T3 induction. Gene expression for the four WT mESC strains for each time point are shown compared to gene expression of shiverer OPCs 72 h post T3 induction. Note the lack of MBP expression (red star). e Row normalized heatmap of the top 30 dysregulated oligodendrocyte-signature genes for MYRF KO OPCS at 72 h post T3 induction. Gene expression for the four WT mESC strains for each time point are shown compared to gene expression of MYRF KO OPCs 72 h post T3 induction. Note the lack of MYRF expression (red star)

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