SOX10 Single Transcription Factor-Based Fast and Efficient Generation of Oligodendrocytes from Human Pluripotent Stem Cells

•SOX10 is sufficient to generate myelinating human OLs from hPSCs in only 22 days•SOX10-induced OLs resemble primary human OLs at the transcriptome level•The methodology allows efficient generation of OLs from MS and ALS patients•OL-neuron co-cultures respond to myelinating drugs in a high-throughput setting Scarce access to primary samples and lack of efficient protocols to generate oligodendrocytes (OLs) from human pluripotent stem cells (hPSCs) are hampering our understanding of OL biology and the development of novel therapies. Here, we demonstrate that overexpression of the transcription factor SOX10 is sufficient to generate surface antigen O4-positive (O4+) and myelin basic protein-positive OLs from hPSCs in only 22 days, including from patients with multiple sclerosis or amyotrophic lateral sclerosis. The SOX10-induced O4+ population resembles primary human OLs at the transcriptome level and can myelinate neurons in vivo. Using in vitro OL-neuron co-cultures, myelination of neurons by OLs can also be demonstrated, which can be adapted to a high-throughput screening format to test the response of pro-myelinating drugs. In conclusion, we provide an approach to generate OLs in a very rapid and efficient manner, which can be used for disease modeling, drug discovery efforts, and potentially for therapeutic OL transplantation. Scarce access to primary samples and lack of efficient protocols to generate oligodendrocytes (OLs) from human pluripotent stem cells (hPSCs) are hampering our understanding of OL biology and the development of novel therapies. Here, we demonstrate that overexpression of the transcription factor SOX10 is sufficient to generate surface antigen O4-positive (O4+) and myelin basic protein-positive OLs from hPSCs in only 22 days, including from patients with multiple sclerosis or amyotrophic lateral sclerosis. The SOX10-induced O4+ population resembles primary human OLs at the transcriptome level and can myelinate neurons in vivo. Using in vitro OL-neuron co-cultures, myelination of neurons by OLs can also be demonstrated, which can be adapted to a high-throughput screening format to test the response of pro-myelinating drugs. In conclusion, we provide an approach to generate OLs in a very rapid and efficient manner, which can be used for disease modeling, drug discovery efforts, and potentially for therapeutic OL transplantation. Oligodendrocytes (OLs) are the central nervous system (CNS) glial cells responsible for axonal myelination. However, the complete roles of OLs are still only partially understood and vary depending on the CNS region wherein they reside (Marques et al., 2016Marques S. Zeisel A. Codeluppi S. van Bruggen D. Mendanha Falcão A. Xiao L. Li H. Häring M. Hochgerner H. Romanov R.A. et al.Oligodendrocyte heterogeneity in the mouse juvenile and adult central nervous system.Science. 2016; 352: 1326-1329Crossref PubMed Scopus (536) Google Scholar). Myelination in the CNS is essential for proper signal conduction along neuronal axons and for maintaining brain homeostasis. Defects in myelin production and/or maintenance are the predominant pathological feature of several diseases, including leukodystrophies and multiple sclerosis (MS) (Franklin et al., 2012Franklin R.J. ffrench-Constant C. Edgar J.M. Smith K.J. Neuroprotection and repair in multiple sclerosis.Nat. Rev. Neurol. 2012; 8: 624-634Crossref PubMed Scopus (207) Google Scholar). In addition to myelination, OLs have a role in trophic and metabolic support of neurons, fueling oxidative phosphorylation in the mitochondria of axons. This trophic support is disrupted in amyotrophic lateral sclerosis (ALS), and defects in OL function contribute to ALS onset and progression (Lee et al., 2012bLee Y. Morrison B.M. Li Y. Lengacher S. Farah M.H. Hoffman P.N. Liu Y. Tsingalia A. Jin L. Zhang P.W. et al.Oligodendroglia metabolically support axons and contribute to neurodegeneration.Nature. 2012; 487: 443-448Crossref PubMed Scopus (1055) Google Scholar). Lack of insight in human OL biology is in large part a consequence of the limited access to human OLs and difficulties in maintaining these cells in vitro. Therefore, having access to human OLs would represent a major step forward in studies aimed at understanding mechanisms that are deregulated in diseases with OL involvement. Moreover, this would allow to test and study if and how candidate drugs affect the process of OL myelination and remyelination (Mei et al., 2014Mei F. Fancy S.P. Shen Y.A. Niu J. Zhao C. Presley B. Miao E. Lee S. Mayoral S.R. Redmond S.A. et al.Micropillar arrays as a high-throughput screening platform for therapeutics in multiple sclerosis.Nat. Med. 2014; 20: 954-960Crossref PubMed Scopus (365) Google Scholar, Lariosa-Willingham et al., 2016Lariosa-Willingham K.D. Rosler E.S. Tung J.S. Dugas J.C. Collins T.L. Leonoudakis D. Development of a central nervous system axonal myelination assay for high throughput screening.BMC Neurosci. 2016; 17: 16Crossref PubMed Scopus (21) Google Scholar), and/or the trophic support provided by OLs. With the isolation of human embryonic stem cells (hESCs) and the development of human induced pluripotent stem cell (hiPSC) technology, a number of protocols have been developed to generate OLs from hPSCs (Nistor et al., 2005Nistor G.I. Totoiu M.O. Haque N. Carpenter M.K. Keirstead H.S. Human embryonic stem cells differentiate into oligodendrocytes in high purity and myelinate after spinal cord transplantation.Glia. 2005; 49: 385-396Crossref PubMed Scopus (488) Google Scholar, Hu et al., 2009Hu B.Y. Du Z.W. Zhang S.C. Differentiation of human oligodendrocytes from pluripotent stem cells.Nat. Protoc. 2009; 4: 1614-1622Crossref PubMed Scopus (195) Google Scholar, Wang et al., 2013Wang S. Bates J. Li X. Schanz S. Chandler-Militello D. Levine C. Maherali N. Studer L. Hochedlinger K. Windrem M. Goldman S.A. Human iPSC-derived oligodendrocyte progenitor cells can myelinate and rescue a mouse model of congenital hypomyelination.Cell Stem Cell. 2013; 12: 252-264Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, Douvaras et al., 2014Douvaras P. Wang J. Zimmer M. Hanchuk S. O'Bara M.A. Sadiq S. Sim F.J. Goldman J. Fossati V. Efficient generation of myelinating oligodendrocytes from primary progressive multiple sclerosis patients by induced pluripotent stem cells.Stem Cell Reports. 2014; 3: 250-259Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), recapitulating in vitro the molecular signals and events that occur during in vivo OL development, leading to myelinating OLs (Wang et al., 2013Wang S. Bates J. Li X. Schanz S. Chandler-Militello D. Levine C. Maherali N. Studer L. Hochedlinger K. Windrem M. Goldman S.A. Human iPSC-derived oligodendrocyte progenitor cells can myelinate and rescue a mouse model of congenital hypomyelination.Cell Stem Cell. 2013; 12: 252-264Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, Douvaras et al., 2014Douvaras P. Wang J. Zimmer M. Hanchuk S. O'Bara M.A. Sadiq S. Sim F.J. Goldman J. Fossati V. Efficient generation of myelinating oligodendrocytes from primary progressive multiple sclerosis patients by induced pluripotent stem cells.Stem Cell Reports. 2014; 3: 250-259Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Despite recent optimizations (Douvaras and Fossati, 2015Douvaras P. Fossati V. Generation and isolation of oligodendrocyte progenitor cells from human pluripotent stem cells.Nat. Protoc. 2015; 10: 1143-1154Crossref PubMed Scopus (131) Google Scholar), these protocols remain inefficient and variable in terms of OL yield and, importantly, require very long differentiation times (>100 days to generate myelin basic protein (MBP)-positive OLs). These issues have precluded the use of patient-specific iPSC-derived OLs to elucidate human OL biology and disease, and use such cells as platform for drug screening. Here, we describe that, by the overexpression of the single transcription factor (TF) SOX10 in hPSC-derived neural precursors (NPCs), it is possible to generate surface antigen O4 (O4)-positive and MBP+ OLs within only ∼20 days from the PSC stage. The transcriptome of hPSC-derived O4+ cells resembles that of primary intermediate OLs. Similar OL production in terms of efficiency and time course was obtained from patients with MS or familial ALS (fALS) compared with healthy donors. Finally, grafting into homozygous shiverer (Shi−/–) mouse brain slices and co-culture with hPSC-derived neurons confirmed the myelination capability of SOX10-induced OLs in in vivo and in vitro contexts. All hPSC-derived OL-neuron co-cultures were also adapted to high-throughput screening (HTS) formats allowing demonstration of enhanced myelin production by different compounds. To define which TFs could promote efficient OL differentiation from hPSCs, we selected 16 TFs known to function in OL specification and/or maturation: ASCL1, AXIN2, MYRF, MYT1, OLIG1, OLIG2, NKX2-2, NKX6-1, NKX6-2, SOX2, SOX8, SOX9, SOX10, ST18, ZEB2, and ZNF536 (Cahoy et al., 2008Cahoy J.D. Emery B. Kaushal A. Foo L.C. Zamanian J.L. Christopherson K.S. Xing Y. Lubischer J.L. Krieg P.A. Krupenko S.A. et al.A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function (2008).J. Neurosci. 2008; 28: 264-278Crossref PubMed Scopus (2238) Google Scholar, Pozniak et al., 2010Pozniak C.D. Langseth A.J. Dijkgraaf G.J. Choe Y. Werb Z. Pleasure S.J. Sox10 directs neural stem cells toward the oligodendrocyte lineage by decreasing suppressor of fused expression.Proc. Natl. Acad. Sci. USA. 2010; 107: 21795-21800Crossref PubMed Scopus (70) Google Scholar, Weng et al., 2012Weng Q. Chen Y. Wang H. Xu X. Yang B. He Q. Shou W. Chen Y. Higashi Y. van den Berghe V. et al.Dual-mode modulation of Smad signaling by Smad-interacting protein Sip1 is required for myelination in the central nervous system.Neuron. 2012; 73: 713-728Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, Najm et al., 2013Najm F.J. Lager A.M. Zaremba A. Wyatt K. Caprariello A.V. Factor D.C. Karl R.T. Maeda T. Miller R.H. Tesar P.J. Transcription factor-mediated reprogramming of fibroblasts to expandable, myelinogenic oligodendrocyte progenitor cells.Nat. Biotechnol. 2013; 31: 426-433Crossref PubMed Scopus (215) Google Scholar, Yang et al., 2013Yang N. Zuchero J.B. Ahlenius H. Marro S. Ng Y.H. Vierbuchen T. Hawkins J.S. Geissler R. Barres B.A. Wernig M. Generation of oligodendroglial cells by direct lineage conversion.Nat. Biotechnol. 2013; 31: 434-439Crossref PubMed Scopus (246) Google Scholar). The coding regions of these genes were individually cloned in the FUW lentiviral doxycycline-inducible expression vector. As reported (Carey et al., 2009Carey B.W. Markoulaki S. Hanna J. Saha K. Gao Q. Mitalipova M. Jaenisch R. Reprogramming of murine and human somatic cells using a single polycistronic vector.Proc. Natl. Acad. Sci. USA. 2009; 106: 157-162Crossref PubMed Scopus (402) Google Scholar), we demonstrated efficient overexpression of each TF in an inducible manner in our experimental settings (Figures S1A and S1B). An initial screen of the 16 selected TFs was performed to identify TFs that induced early, intermediate, and late OL fate. NPCs were generated from hPSCs by dual SMAD inhibition in the presence of retinoic acid (RA) and Sonic hedgehog (SHH) agonist (Chambers et al., 2009Chambers S.M. Fasano C.A. Papapetrou E.P. Tomishima M. Sadelain M. Studer L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling.Nat. Biotechnol. 2009; 27: 275-280Crossref PubMed Scopus (2431) Google Scholar). More than 95% of the day 12 PSC progeny stained positive for NPC markers (SOX2 and NESTIN) and most of them (81.8% ± 2.7%) expressed the ventral progenitor marker HOXB4 (Figure S1C). NPCs were further expanded using basic fibroblast growth factor (bFGF), and then transduced with each of the 16 individual TFs and cultured in OL differentiation medium (Figure 1A). To identify the TFs that enhanced OL lineage differentiation, we performed qRT-PCRs for early (OLIG2 and CSPG4), intermediate (ASCL1 and SOX10), and late (GALC and PLP1) OL lineage markers 7 days after transduction (Figure 1B). Overexpression of ASCL1, NKX6-2, or MYT1 induced a significant increase in endogenous (e) transcripts for OLIG2e, CSPG4, and ASCL1e, while expression of the more mature OL genes, GALC and PLP1, was significantly induced by overexpression of SOX8, SOX9, or SOX10. We also performed immunostaining for A2B5, a marker for intermediate oligodendrocyte precursor cells (OPCs) (Figures 1C and 1D). In the absence of TF overexpression, we detected 8.02% ± 2.46% A2B5+ cells, consistent with the fact that differentiation was induced for only 7 days. By contrast, and in line with the qRT-PCR data, 32.05% ± 4.04% and 47.63% ± 5.33% A2B5+ cells were identified following overexpression of ASCL1 and NKX6-2, respectively. We also transduced the NPCs with a vector containing the MCS5-SOX10 enhancer region, which is an efficient and specific reporter for human OL lineage cells (Pol et al., 2013Pol S.U. Lang J.K. O'Bara M.A. Cimato T.R. McCallion A.S. Sim F.J. Sox10-MCS5 enhancer dynamically tracks human oligodendrocyte progenitor fate.Exp. Neurol. 2013; 247: 694-702Crossref PubMed Scopus (13) Google Scholar). Following co-transduction of NPCs with the individual TFs combined with the MCS5-SOX10 vector, expression of GFP (activity of the reporter) was evaluated by fluorescence-activated cell sorting (FACS) 7 days later. As the MCS5-SOX10 vector also contained a constitutive mCherry cassette, the fraction of GFP+ cells within the mCherry+ population was quantified. A 5- to 6-fold increase in eGFP+/mCherry+ cells was identified in NPCs transduced with either SOX8, SOX9 or SOX10, in line with the increased expression of mature OL markers (Figures 1E and 1F). Thus, overexpression of ASCL1, NKX6-2, and MYT1 induced early-intermediate OL lineage transcripts and proteins, while overexpression of SOX8, SOX9, and SOX10 activated the MCS5-SOX10 enhancer-based reporter and induced expression of late OL genes (GALC and PLP1). The effect of these six TFs was then further analyzed. We used anti-O4 antibody staining (Sommer and Schachner, 1981Sommer I. Schachner M. Monoclonal antibodies (O1 to O4) to oligodendrocyte cell surfaces: an immunocytological study in the central nervous system.Dev. Biol. 1981; 83: 311-327Crossref PubMed Scopus (962) Google Scholar) to further define which of the six TFs caused differentiation to mid- and late-stage OL lineage cells. Day 12 NPCs, without bFGF expansion, were transduced with the six TFs individually to enable assessment of the shortest period required to generate OLs from hPSCs, and to avoid possible lineage skewing due to bFGF-based NPC expansion (Furusho et al., 2015Furusho M. Roulois A.J. Franklin R.J. Bansal R. Fibroblast growth factor signaling in oligodendrocyte-lineage cells facilitates recovery of chronically demyelinated lesions but is redundant in acute lesions.Glia. 2015; 63: 1714-1728Crossref PubMed Scopus (36) Google Scholar). OL differentiation was assessed on day 10 after transduction (22 days from undifferentiated hPSCs) (Figure 2A). Less than 1% of NPCs transduced with an eGFP control vector were O4+. Transduction of NPCs with ASCL1, NKX6-2, or MYT1 did not increase the fraction of O4+ cells, consistent with the finding that these TFs induced immature/intermediate OPC lineage. However, 50.02% ± 3.21%, 37.35% ± 4.51%, and 54.05% ± 2.52% of NPCs transduced with SOX8, SOX9 or SOX10 were O4+, respectively (Figure 2B). We next tested if combined overexpression of SOX10 with any of the other five TFs would further enhance the proportion of O4+ cells. However, no further increase in O4+ cells was seen with any TF combination over SOX10 alone (Figure 2D). This was confirmed by studies testing OPC/OL marker transcripts in cells transduced with SOX8, SOX9, or SOX10 alone, or SOX10 in combination with the other five TFs (Figure 2E). SOX10e, GALC, CNP, PLP1, and MBP expression was induced 5- to >100-fold following transduction with SOX10 alone. Transduction with either SOX8 or SOX9 induced similar, albeit somewhat lower levels of these transcripts. Combinations of SOX10 with any of the other TFs did not further enhance marker expression. We next FACS sorted O4+ and O4– subpopulations 10 days after transduction with SOX10. The O4+ fraction was highly enriched for cells expressing OPC/OL marker transcripts in comparison with the O4– fraction (Figure 2F), confirming that expression of O4 is specific for intermediate and late OL lineage cells. Thus, overexpression of the TF SOX10 alone is sufficient to induce differentiation of NPCs toward the OL lineage. Subsequent studies were designed to further characterize the SOX10-induced cells. We next tested if SOX10-induced progeny expresses, in addition to O4, other typical OL markers. Immunostaining on day 10 SOX10-induced cells (22 days from hPSC stage; without prior O4+ enrichment), demonstrated that day 12 NPCs stained positive for OLIG2 but not O4 (Figure 3A), while SOX10-transduced cells 10 days after induction were negative for OLIG2 (not shown). Approximately 50%–60% of day 22 NPC progeny stained positive for O4 (in line with the FACS data; Figures 3B, 3C, and 3E) and O1 (Figure S2B), and that 97.15% ± 8.19% SOX10+ cells co-expressed O4. In addition, 21.48% ± 2.09% SOX10+ cells also stained positive for MBP (Figures 3D, 3F, and 3G), with 71.67% ± 2.43% of these cells co-expressing MOG (Figure 3H). PLP expression was found as well within the SOX10-induced cells (20.35% ± 3.19%), and remained expressed in most (94.60% ± 2.57%) of the O4+-purified cells (Figure 3J). These myelin protein-expressing cells displayed a more mature OL morphology, with extended membrane sheaths and highly branched processes (Figures 3C–3J). Aside from O4+ cells, we also found rare TUJI+ neurons (<5%; Figure 3I), but no GFAP+ astrocytes in the culture. We also assessed if Schwann cells (SCs) were present in the culture, by staining for the SC-specific peripheral myelin protein 22 (PMP22; Figures S2C and S2D). SOX10-induced progeny did not contain PMP22+ cells, indicating that only OLs and not peripheral SCs were generated. To further prove that generation of O4+/MBP+ cells was due to SOX10 overexpression, we co-stained SOX10-induced progeny with SOX10 and MBP. All MBP+ cells co-expressed SOX10, demonstrating that SOX10 expression was required for the generation of MBP+ OLs (Figure 3K). Furthermore, no O4 or MBP expression was observed in eGFP-transduced NPCs after 10 days of induction (Figures S2E and S2F). The yield of O4+ cells on day 22 was approximately 240% of the day 12 NPCs, and 24,000% of the day 0 PSCs. This high yield, also from NPCs, reflects the presence of proliferative OPCs in the culture that give rise not only to mature OLs but also to other OPCs. In fact, Ki67+ cells were present throughout differentiation (Figure 3L), with 24.22% ± 1.20% of Ki67+ cells present in the whole culture on day 10 after SOX10 induction. However, all MBP+ cells were Ki67− (Figure 3L), and hence post-mitotic, consistent with the acquisition of a mature OL phenotype. To assess if SOX10-induced OLs resembled primary OLs, we performed RNA-sequencing (RNA-seq) on purified O4+ cells derived from four different hPSC lines: the hESC H9 and the hiPSC ChiPSC6b, Sigma-iPSC0028, and BJ1 (healthy donor-derived) lines. The transcriptome of the O4+ cells was combined with published transcriptome data from different brain cells (GalC+ OLs, CD90+ neurons, CD45+ myeloid cells, HepaCAM+ astrocytes, BSL-1+ endothelial cells, and the whole cortex) (Zhang et al., 2016Zhang Y. Sloan S.A. Clarke L.E. Caneda C. Plaza C.A. Blumenthal P.D. Vogel H. Steinberg G.K. Edwards M.S. Li G. et al.Purification and characterization of progenitor and mature human astrocytes reveals transcriptional and functional differences with mouse.Neuron. 2016; 89: 37-53Abstract Full Text Full Text PDF PubMed Scopus (1129) Google Scholar, Abiraman et al., 2015Abiraman K. Pol S.U. O'Bara M.A. Chen G.D. Khaku Z.M. Wang J. Thorn D. Vedia B.H. Ekwegbalu E.C. Li J.X. et al.Anti-muscarinic adjunct therapy accelerates functional human oligodendrocyte repair.J. Neurosci. 2015; 35: 3676-3688Crossref PubMed Scopus (50) Google Scholar). Principal-component analysis and unsupervised hierarchical clustering on the entire transcriptome identified a very distinct cluster encompassing different brain-derived OL samples, as well as the SOX10-induced O4+ cells, separated from neurons, astrocytes and other non-ectodermal-derived cells (Figures 4A and 4B ). To further characterize the maturity of the generated O4+ OLs, we also included samples consisting of immature, intermediate, and mature OLs isolated from human fetal tissues (Abiraman et al., 2015Abiraman K. Pol S.U. O'Bara M.A. Chen G.D. Khaku Z.M. Wang J. Thorn D. Vedia B.H. Ekwegbalu E.C. Li J.X. et al.Anti-muscarinic adjunct therapy accelerates functional human oligodendrocyte repair.J. Neurosci. 2015; 35: 3676-3688Crossref PubMed Scopus (50) Google Scholar). Comparison of OL-specific markers (Cahoy et al., 2008Cahoy J.D. Emery B. Kaushal A. Foo L.C. Zamanian J.L. Christopherson K.S. Xing Y. Lubischer J.L. Krieg P.A. Krupenko S.A. et al.A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function (2008).J. Neurosci. 2008; 28: 264-278Crossref PubMed Scopus (2238) Google Scholar, Nielsen et al., 2006Nielsen J.A. Maric D. Lau P. Barker J.L. Hudson L.D. Identification of a novel oligodendrocyte cell adhesion protein using gene expression profiling.J. Neurosci. 2006; 26: 9881-9891Crossref PubMed Scopus (62) Google Scholar) revealed that O4+ cells derived from H9 and Sigma-iPSC0028 lines co-clustered with intermediate OLs, while ChiPSC6b and BJ1 O4+ cells clustered more closely to immature OLs (Figure 4C). Next, we compared SOX10-PSC O4+ cells (average values of all PSC-derived cells) with different primary OLs using a sequence alignment map (Figures 4D and 4E). We identified 916 (13.48%) differentially expressed genes between mature OLs and PSC-O4+ cells (fold change >2 and false discovery rate < 0.05) (699 up- and 217 downregulated genes in PSC-O4+ cells) and 240 (3.53%) differentially expressed genes between intermediate OLs and PSC-O4+ cells (all upregulated in PSC-O4+ cells) (Table S1), confirming that O4+ cells were highly similar to mature/intermediate primary OLs (Figure 4E). Over 85% (47/53) of OL-specific genes (including MAG, MOG, SOX10, and OLIG2) were expressed at comparable levels in O4+ cells and primary OLs (Figure 4D). Lastly, we performed gene ontology (GO) analysis to identify classes of genes that were similarly expressed between O4+ hPSC-derived cells and primary OLs. When compared with primary intermediate OLs, a higher number of shared GO pathways were obtained, including those referred to CNS development as well as to OL development (Table S2). In addition, other terms were related to cytoskeleton organization and protein modifications, pathways associated to OLs (Nielsen et al., 2006Nielsen J.A. Maric D. Lau P. Barker J.L. Hudson L.D. Identification of a novel oligodendrocyte cell adhesion protein using gene expression profiling.J. Neurosci. 2006; 26: 9881-9891Crossref PubMed Scopus (62) Google Scholar). Overall, these results support the notion that SOX10-induced O4+ cells are highly comparable at the transcriptome level with primary OLs, especially intermediate OLs. To avoid effects of random integration of the SOX10 transgene resulting from lentiviral transduction, and also to avoid the use of this technology for OL generation, we created an hESC line wherein SOX10 was introduced in the safe harbor locus AAVS1, using recombinase-mediated cassette exchange in hPSC lines containing an FRT-flanked cassette, which contained a hygromycin-resistance/thymidine kinase selection cassette (Ordovás et al., 2015Ordovás L. Boon R. Pistoni M. Chen Y. Wolfs E. Guo W. Sambathkumar R. Bobis-Wozowicz S. Helsen N. Vanhove J. et al.Efficient recombinase-mediated cassette exchange in hPSCs to study the hepatocyte lineage reveals AAVS1 locus-mediated transgene inhibition.Stem Cell Reports. 2015; 5: 918-931Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). This created 100% homogeneous hESCs containing either an SOX10 or an SOX10-eGFP cassette under a doxycycline-inducible promoter (Figures S4A and S4B). Addition of doxycycline to hESCs induced the expression of SOX10 or SOX10-eGFP in >99% of cells (Figures S4C and S4D). Induction of SOX10 on day 0, without prior neural commitment, did not result in the generation of MBP+ OLs (not shown). When hPSCs were first fated to NPCs for 8 days, followed by addition of doxycycline and culture in OL differentiation medium, already 50% O4+ cells were found on day 4, and 89.3% ± 0.6% by day 7, which was sustained at later time points (Figure S4E). The emergence of O4+ cells was accompanied by progressively increased levels of OPC/OL markers, in both SOX10 and SOX10-eGFP transgenic lines (Figure S4F). Immunostaining further demonstrated the OL identity of the cells: MBP+ cells could be detected by day 7, and its expression increased progressively by day 10 of induction (day 18 of the overall differentiation culture; Figure S4G). No contamination with neurons was observed. To demonstrate that SOX10-induced O4+ cells have functional characteristics of OLs, we tested if they were capable of myelinating neuronal axons. To address myelination in an in vivo context, purified O4+ cells were injected in brain slices from homozygous shiverer (shi/shi) mice (MBP deficient) and slices were analyzed 10 days later. Immunostaining demonstrated efficient engraftment and homogeneous spreading of the transplanted human hNA+ cells within the tissue, with 48.13% ± 4.15% of cells also expressing MBP (Figures 5A–5C ). Moreover, MBP+ OL projections wrapping NF200+ neuronal axons could be observed 10 days after injection (Figures 5D and 5E). We also assessed if myelination occurred in in vitro co-cultures. We generated cortical neurons from human iPSCs as described previously (Shi et al., 2012Shi Y. Kirwan P. Smith J. Robinson H.P. Livesey F.J. Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses.Nat. Neurosci. 2012; 15 (477–486, S1)Crossref PubMed Scopus (588) Google Scholar). Neuronal progenitors were replated and allowed to mature for 10–14 days. O4+ cells, isolated and purified on day 10 following SOX10 induction, were co-cultured with cortical neuronal progeny for an additional 20 days in OL myelination medium. Regions wherein TUJI+ axons were aligned with MBP+ OLs could already be seen a few days later (not shown). By day 20 of co-culture, O4+ cells extended MBP+ regions aligned with axons at multiple locations (Figures 5F–5K). Transverse sections of reconstructed confocal microscopy images demonstrated the presence of MBP+ extensions fully wrapping neuronal axons (Figures 5H and 5I). 3D reconstructions also demonstrated the presence of MBP+ sheaths surrounding TUJ1+ axonal prolongations (Figures 5J and 5K). At the ultrastructural level, cytoplasmic regions of OLs were frequently observed surrounding neuronal axons (Figures 5L and 5M) and were able to form multilayer compact myelin sheaths (Figure 5Q). Early myelination of neuronal axons was also observed (Figures 5O and 5P). These results indicate that SOX10-induced OLs matured into myelinating OLs that ensheathed and wrapped axons in both an in vivo context, as well as when co-cultured with hPSC-derived neurons in vitro. To determine the robustness of the protocol, and to demonstrate that OLs can also be generated from iPSCs of patients with neurodegenerative diseases wherein OLs have been shown to play a causal role, we compared the generation of OLs from hPSCs from healthy donors (hESC-H9 and hiPSC ChiPSC6b, Sigma-iPSC0028, and BJ1 lines), with iPSC lines from two primary progressive MS (PPMS) patients and from two patients with a familial form of ALS (fALS) caused by mutations in the genes superoxide dismutase (SOD1A4V) or C9ORF72. We found no substantial differences in the expression of intermediate and late OL marker transcripts among the eight cell lines analyzed at different time points (Figure 6A). In addition, no significant differences in the efficiency of generating O4+ cells were seen among the lines (50%–65% O4+ cells; Figure 6B). Finally, approximately 10% of MBP+ cells were present on day 22 in the SOX10-induced NPC progeny from all lines examined, with the exception of BJ1-derived cells, which contained only 6.34% ± 0.70% MBP+ cells (Figure 6C). In addition, MBP+ progeny from PSCs of healthy donors and from PPMS and fALS patients had similar morphology (Figure 6D). Thus, the SOX10-mediated differentiation protocol could generate intermediate and mature OLs with similar efficiencies also from PPMS and fALS iPSC lines only after 22 days of differentiation. This proves the robustness of the protocol irrespective of the iPSC lines used to generate O4+/MBP+ OLs. Currently, no good assays are available to identify and validate drugs that can enhance myelination. The existing platforms are based mostly on primary murine OLs cultured in the absence (Mei et al., 2014Mei F. Fancy S.P. Shen Y.A. Niu J. Zhao C. Presley B. Miao E. Lee S. Mayoral S.R. 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