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Generation and isolation of oligodendrocyte progenitor cells from human pluripotent stem cells Panagiotis Douvaras & Valentina Fossati The New York Stem Cell Foundation Research Institute, New York, New York, USA. Correspondence should be addressed to V.F. ([email protected]).

© 2015 Nature America, Inc. All rights reserved.

Published online 2 July 2015; doi:10.1038/nprot.2015.075

In the CNS, oligodendrocytes act as the myelinating cells. Oligodendrocytes have been identified to be key players in several neurodegenerative disorders. This protocol describes a robust, fast and reproducible differentiation protocol to generate human oligodendrocytes from pluripotent stem cells (PSCs) using a chemically defined, growth factor–rich medium. Within 8 d, PSCs differentiate into paired box 6–positive (PAX6+) neural stem cells, which give rise to OLIG2+ progenitors by day 12. Oligodendrocyte lineage transcription factor 2–positive (OLIG2+) cells begin to express the transcription factor NKX2.2 around day 18, followed by SRY-box 10 (SOX10) around day 40. Oligodendrocyte progenitor cells (OPCs) that are positive for the cell surface antigen recognized by the O4 antibody (O4+) appear around day 50 and reach, on average, 43% of the cell population after 75 d of differentiation. O4+ OPCs can be isolated by cell sorting for myelination studies, or they can be terminally differentiated to myelin basic protein–positive (MBP+) oligodendrocytes. This protocol also describes an alternative strategy for markedly reducing the length and the costs of the differentiation and generating ~30% O4+ cells after only 55 d of culture.

INTRODUCTION Oligodendrocytes are CNS cells present in vertebrates. They enable a fast and energy-efficient transmission of electrical signals, and they ultimately allow complex motor, sensory and cognitive functions to occur simultaneously. Oligodendrocytes produce a laminated, lipid-rich myelin sheath that wraps the neuronal axons and creates defined segments of electrical insulation to maximize the speed of action potential conduction. Myelin is also important for axonal integrity and survival, and it has been shown that even small changes affecting oligodendrocyte metabolism can lead to neurodegeneration1. The myelination process is particularly important in humans, as human brains have a higher content of myelinated neurons (white matter) and myelination continues after birth and throughout life2. This suggests that oligodendrocytes are not merely providing inert insulation, but rather that myelination is a dynamic process that affects cognitive function and even behavior. Many aspects of human oligodendrocyte biology and human myelination are still largely unknown, and studies have been hampered by the limited access to primary cells from biopsies or autopsies. PSCs, encompassing embryonic stem cells (ESCs) and induced PSCs (iPSCs) have been used within the past two decades as an alternative, useful source from which any desired cell type can be generated. This has been achieved by recapitulating in vitro the fundamental steps of embryonic development3. Notably, most of the crucial pathways of lineage commitment are highly conserved between mice and humans, and therefore insights gained from mouse developmental biology have been successfully applied to produce numerous cell types, including oligodendrocytes, from human PSCs4. Development of protocols for oligodendrocyte differentiation Most of the current protocols for oligodendrocyte differentiation are based on knowledge derived from mouse spinal cord development. Although the developmental origin of oligodendrocytes has long been debated, there is a general consensus that, in mice, oligodendrocyte precursors in the spinal cord arise within the pMN domain, where motor neurons are also formed. During mouse

embryonic development, retinoic acid (RA) and sonic hedgehog (SHH) pathways are crucial in defining the pMN domain. This finding has been exploited in in vitro cultures to convert neuroepithelial cells into progenitors of the pMN domain, expressing the transcription factor OLIG2 (ref. 5). RA at 100 nM to 10 µM concentration has been used in all methods to differentiate human ESCs from oligodendrocytes6–8. SHH was first used later, by the Zhang laboratory, which demonstrated that SHH is necessary for the induction of OLIG2+NKX2.2+ progenitors and for their transition to OPCs, expressing SOX10, PDGFR-α and ultimately O4 (ref. 8). It is noteworthy that this study demonstrated that the appearance of OPCs in vitro is substantially delayed in the human model compared with the mouse model, as there is a protracted period of 10 weeks between the appearance of OLIG2+ progenitors and the appearance of PDGFR-α+ OPCs in the human model9. During the past few years, several groups have devised improved protocols to differentiate hPSCs from OPCs to improve disease modeling and to potentially develop clinical applications10–12. Specifically, the Deng laboratory generated an OLIG2-GFP knock-in hESC line and developed a protocol based on the direct visualization of the reporter gene13. Stacpoole et al.11 demonstrated that low oxygen tension is a suitable culture condition for oligodendrocyte differentiation, and they identified the pathways involved in forebrain oligodendrocyte specification. Numasawa-Kuroiwa et al.10 have used patient-derived oligodendrocytes to model a genetic dysmyelinating disorder, Pelizaeus-Merzbacher disease, in vitro in order to better understand the pathogenic mechanisms linked to the missense mutation in the proteolipid protein 1 (PLP1) gene. Goldman and colleagues pioneered the in vivo myelination studies of human OPCs, first by showing that PDGFR-α+ cells isolated from fetal brains are able to engraft and myelinate the shiverer mouse14, and then by demonstrating that transplantation of hiPSC-derived PDGFR-α+ OPCs can rescue this hypomyelinated mouse12. However, these protocols are still largely limited in application by the lengthy culture times required, as they require from 80 to over 200 d of differentiation to obtain OPCs expressing the O4 antigen9–12. Furthermore, nature protocols | VOL.10 NO.8 | 2015 | 1143

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Figure 1 | Timeline of oligodendrocyte differentiation. (a,b) Timelines show the differences in following the original protocol (Step 32A, a) and the fast protocol (Step 32B, b). Adapted with permission from ref. 16. Colored triangles represent the recommended time points to evaluate the expression of stage-specific markers through immunofluorescence. SB, SB431542; LDN, LDN193189; SAG, smoothened agonist; T3, thriiodothytonine; RA, all-trans retinoic acid; PDGF, platelet-derived growth factor; HGF, hepatocyte growth factor; IGF-I, insulin-like growth factor-1; NT3, neurotrophin 3; AA, ascorbic acid.

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Applications of our protocol The substantial reduction in differentiation time compared with other published protocols9,11,12 makes this protocol preferable for research aimed at understanding human oligodendrocyte biology and the process of myelination. Patient-specific OPCs can be generated for the study of demyelinating or dysmyelinating disorders, such as multiple sclerosis, adrenoleukodystrophy, vanishing white matter disease, Pelizaeus-Merzbacher disease and all leukodystrophies. Furthermore, a crucial role for oligodendrocytes is emerging in many neurodegenerative and neurological disorders, including amyotrophic lateral sclerosis, Huntington’s disease, Alzheimer’s disease and schizophrenia17–20. Human OPCs can be used to develop in vitro myelination assays, to screen for myelinating compounds, and ultimately to become a source for autologous cell replacement therapies. Experimental design The timeline of our oligodendrocyte differentiation protocol is shown in Figure 1. At all stages of differentiation, cells are cultured in 5% CO2 incubators; however, the hypoxic cell culture system can be an efficient alternative condition, as demonstrated

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the reported efficiencies of O4+ cells obtained range from 4 to 47% (refs. 7,9,11,12). It has also been found that efficiencies of O4+ cell production fluctuate depending on the cell line used, owing to variability in their differentiation capacities15, and thus multiple PSC lines need to be initially tested to fully optimize conditions. Building on earlier studies, we developed a robust differentiation protocol that generates 28–80% O4+ OPCs within 75 d (ref. 16). When they were purified, the O4+ cells were able to engraft, and they showed comparable myelination potential to fetal human OPCs in short-term (16 weeks) transplantation studies into the shiverer mouse model16. We FACS-purify OPCs at the stage when they express O4 rather than PDGFR-α, as this eliminates contaminant cells and minimizes the tumorigenic potential. This will be important if the protocol is adapted for future applications as cell replacement therapies. In this context, sorting OPCs using PDGFR-α+ as a marker is a valid alternative strategy, as proven by the long-term transplantation studies performed by the Goldman laboratory12. We validated the protocol using >9 hPSC lines, with very consistent results seen for each line. In this protocol, we also provide an alternative, faster strategy for obtaining ~30% O4+ OPCs after just 55 d of differentiation, resulting in a substantial cost reduction (Fig. 1).

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by Stacpoole et al.11. In the first stage, ‘Preparation of hPSC cultures for oligodendrocyte differentiation’ (Steps 1–20), PSCs are seeded as single cells at low density (10,000 cells per cm2) in adherent cultures. This setup does not require significant PSC expansion, as only one well (80% confluent) of a six-well plate contains enough cells to differentiate and isolate at least 2 × 106 oligodendrocytes. The signaling pathways that we manipulate to generate oligodendrocytes were selected on the basis of knowledge gained from studies of rodent spinal cord embryonic development8. In vitro RA and SHH signaling mimic the pMN environment, inducing differentiation of the PSCs to OLIG2 progenitor cells (Steps 21–23). In our cultures, SHH signaling is activated through the small-molecule smoothened agonist (SAG) instead of the human recombinant SHH protein16. We found that combining RA at a low concentration (100 nM) and SAG with SB431542 and LDN193189, two small molecules used to inhibit transforming growth factor (TGF)-β and bone morphogenetic protein (BMP) signaling, respectively, generated the highest yield of OLIG2+ progenitors. This is the key difference between our protocol and that of Wang et al12. SB431542 and LDN193189 have been shown to accelerate the production of PAX6+ neural stem cells from PSCs21 (Fig. 2). Furthermore, the addition of SB431542 to a SHH agonist upregulates OLIG2 in motor neurogenesis22 and BMP4 inhibits spinal cord oligodendrogenesis 23. Owing to the synergistic action of those compounds, >70% of the cells express OLIG2 at day 12 (ref. 16). For the first 8 d of culture, we use a customized mTeSR1 medium (defined as mTeSR custom), which has the same composition as mTeSR1 medium minus five factors that sustain pluripotency16. However, we have found that mTeSR custom medium can be replaced by the more readily available DMEM/F12 with the addition of 25 µg/ml insulin. The transition from PSCs to OLIG2+ progenitors is associated with massive proliferation, causing the cultures to become overconfluent and resulting in the cells forming 3D structures by day 12 (Fig. 2b). At this time point, the adherent cultures are dissociated to form spheres in suspension (Step 24). OLIG2- cells do not form aggregates, and thus this process enriches for the OLIG2+ population and OLIG2- cells are eliminated gradually during medium changes16. In this protocol, we describe two alternative methods for cell detachment: one using mechanical dissociation (Step 24A), and the second using enzymatic digestion (Step 24B). The first method offers the advantage of minimal cell death; however, it produces aggregates of variable size, and thus suitable spheres need to be selected through a manual picking process. Good spheres are defined as those having a round

Figure 2 | Fundamental steps of hPSC a b c differentiation to oligodendrocytes. (a) PAX6+ neural stem cells at day 8 of differentiation (PAX6, green; nuclei are stained with DAPI, blue). (b) Typical morphology of the culture at day 12, depicting 3D structures. (c) Expression of OLIG2 and NKX2.2 at day 12, as shown 500 µm 500 µm 200 µm PAX6 DAPI Phase OLIG2 NKX2.2 DAPI through immunofluorescence analysis (OLIG2, green; NKX2.2, red; nuclei are stained d e f with DAPI, blue). (d) Sphere selection at * Step 30. Arrows indicate the good spheres, which are round-shaped, golden or brown in color, with a darker core and with a diameter * between 300 and 800 µm. The exclamation mark (!) indicates a pair of joined spheres that 200 µm 200 µm 1 mm Phase NKX2.2 SOX10 OLIG2 Live O4 can be broken into single spheres by gentle pipetting. Aggregates that should be avoided are small and transparent (arrowheads) or g h i very large and irregular in shape, usually derived by mechanical digestion (asterisks,*). (e) Immunofluorescence staining of progenitor cells at day 56, coexpressing NKX2.2 (green), SOX10 (red) and OLIG2 (blue). (f) O4 (green) live staining showing the highly ramified morphology 500 µm 200 µm 100 µm Phase MBP DAPI MAP2 MBP GFAP of the cells. Reproduced in part from Douvaras et al.16 with permission. (g) MBP+ (red) oligodendrocytes at the end of the differentiation (nuclei are stained with DAPI, blue). The solid box is a magnification of the cells in the dashed box, to highlight the morphology. (h) Morphology of MBP+ (red) oligodendrocytes at higher magnification. MAP2+ (green) and GFAP+ (blue) cells are also present in the culture. (i) Purified O4+ cells 24 h after sorting still retain the typical ramified morphology. Scale bars, 500 µm (a,b,g); 200 µm (c,e,f,i); 1 mm (d); 100 µm (h).

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shape, golden or brown color, a darker core and with a diameter of 300–800 µm (Fig. 2d). Detaching the cells using enzymatic digestion predominantly produces spheres that are appropriate for further culture. Therefore, manual picking of spheres (Step 30) is not required, and the detachment steps can be adapted for automation and used in high-throughput studies. However, enzymatic digestion increases cell death, resulting in a lower number of spheres. Spheres obtained using either method are then plated into poly-l-ornithine/laminin-coated dishes (Step 31). We have found that plating spheres at a density of two spheres per cm2 provides optimal condiFast day 53 tions for migration and spread of the cells, a enabling coverage of the whole well without detachment by the end of the protocol. This also leads to the highest efficiency of O4+ cell production. The final stage of differentiation to OPCs and oligodendrocytes is initiated by exposing the cells to the key factors Figure 3 | Live imaging and quantification of O4+ OPCs. (a–f) Live O4 (green) imaging at day 53 (a,d), day 63 (b,e) and day 73 (c,f) of differentiation following the fast protocol. (a–c) Representative fields at low magnification showing an increase in the number of O4+ cells with time. (d–f) Higher-magnification images of a–c, respectively, to highlight the morphology of the cells. (g) Representative examples of O4+ cell frequencies calculated by flow cytometry at different time points using the fast protocol (days 55, 63 and 75) and the original protocol. Scale bars, 500 µm (a–c) and 200 µm (d,e,f).

known to drive oligodendrocyte differentiation or to promote oligodendrocyte survival, such as platelet-derived growth factor (PDGF), neurotrophin 3 (NT3), triiodo-l-thyronine (T3), insulinlike growth factor 1 (IGF-1) and hepatocyte growth factor (HGF)24. Our differentiation protocol is unique in that it does not require basic fibroblast growth factor (FGF) or epidermal growth factor (EGF), as required by the protocols described in Nistor et al.6, Hu et al.9 and Wang et al.12. Although neurons and astrocytes are the first cells to migrate out of the spheres, from Fast day 63

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day 50 highly ramified cells characterized by the expression of the sulfated glycolipid antigen recognized by the O4 antibody appear and by day 75 their frequency increases to up to 70% of the total population16 (Figs. 2f and 3). In this protocol, we have included an alternative strategy (defined as the ‘fast protocol’) for the differentiation of OPCs to oligodendrocytes that minimizes the costs of reagents and the duration of the differentiation. The mitogens PDGF, NT3, IGF-1 and HGF can be withdrawn from the medium as early as at day 30, when the selected spheres are seeded (Step 32B). This results in the appearance of O4+ cells at day 55. However, the proportion of O4+ cells seen is lower than that seen at day 75 of the original protocol (Step 32A). The cultures can be continued to increase the frequency of O4+ cells to levels comparable to the original protocol (Fig. 3). In both options, the withdrawal of the mitogens drives the terminal differentiation of OPCs to oligodendrocytes expressing myelin basic protein (MBP), although MBP+ cells do not align with axon fibers under these culture conditions (Fig. 2g,h). For

myelination studies, O4+ OPCs can be purified through FACS and transplanted in vivo. O4+ cells can also be cryopreserved immediately after sorting and thawed 24–48 h before transplantation. Limitations This protocol has been tested with several iPSC and ESC lines, including primary progressive multiple sclerosis patient–derived and healthy control lines, and O4+ cells were obtained at efficiencies ranging from 28% to 80%. However, owing to the variability among PSC lines, it is possible to observe lower efficiencies with certain refractory lines. We strongly recommend evaluating the differentiation at different stages (e.g., OLIG2+ progenitors, SOX10+ early OPCs) with RT-PCR analysis or immunofluorescence (Fig. 2c,e). The culture conditions described here are not optimized for culturing purified O4+ cells after sorting. To our knowledge, suitable conditions to expand purified human oligodendrocytes in the absence of supporting neurons and astrocytes have not yet been established.

MATERIALS REAGENTS Human PSCs • The following hPSC lines have been differentiated to OPCs: RUES1 is a US National Institutes of Health (NIH)-approved hESC line generated at Rockefeller University. MS100101MR-01, MS100301MR-01, MS100601MR-01 and MS100801MR-01 are iPSC lines derived from primary progressive multiple sclerosis patients (male, 56 years old; female, 62 years old; male, 61 years old; female, 50 years old, respectively). MS100901MR-01, MS101001MR-01, PD002901MR-01 and PD009601MR-01 are iPSC lines derived from healthy subjects (male, 66 years old; male, 28 years old; male, 52 years old; male, 66 years old). All iPSC lines were derived at the New York Stem Cell Foundation Research Institute, and they can be requested through the following NYSCF Repository: http://www.nyscf.org/repository ! CAUTION iPSC and ESC research should always be conducted in accordance with all relevant governmental and institutional guidelines and regulations. • mTeSR1 (StemCell Technologies, cat. no. 05850) • mTeSR Custom (StemCell Technologies, customized) • DMEM: Nutrient Mixture F-12, DMEM/F12 (Life Technologies, cat. no. 11320082) • Distilled water (dH2O; Life Technologies, cat. no. 15230-204) • Dulbecco’s PBS (DPBS), 1× (Life Technologies, cat. no. 14190-250) • Accutase (Life Technologies, cat. no. A11105-01) • Penicillin-streptomycin (Life Technologies, cat. no. 15070063) • Y27632 (Rho-associated protein kinase (ROCK) inhibitor, Stemgent, cat. no. 04-0012) • GlutaMAX-I (100×; Life Technologies, cat. no. 35050079) • MEM non-essential amino acids (NEAA) solution (100×; Life Technologies, cat. no. 11140-050) • 2-Mercaptoethanol, 1,000× (Life Technologies, cat. no. 21985023) ! CAUTION 2-Mercaptoethanol is toxic if ingested, inhaled or absorbed through the skin or mucous membranes. • N2 supplement (Life Technologies, cat. no. 17502-048) • B27 Supplement without VitA (Life Technologies, cat. no. 12587-010) • DMSO (Sigma-Aldrich, cat. no. D2650) • All-trans retinoic acid (RA; Sigma-Aldrich, cat. no. R2625) • SB431542 (Stemgent, cat. no. 04-0010) • LDN-193189 (Stemgent, cat. no. 04-0074) • Smoothened agonist, (SAG; EMD Millipore, cat. no. 566660) • Recombinant human PDGF-AA, CF (R&D Systems, cat. no. 221-AA-050) • Recombinant human IGF-I, CF (R&D Systems, cat. no. 291-G1-200) • Recombinant human HGF (R&D Systems, cat. no. 294-HG-025) • Neurotrophin 3 (NT3; EMD Millipore, cat. no. GF031) • Insulin solution, human (Sigma-Aldrich, cat. no. 19278) • Biotin (Sigma-Aldrich, cat. no. 4639) 1146 | VOL.10 NO.8 | 2015 | nature protocols

• N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (cAMP analog; Sigma-Aldrich, cat. no. D0260) • 3,3,5-Triiodo-l-thyronine (T3; Sigma-Aldrich, cat. no. T2877) • l-Ascorbic acid (AA; Sigma-Aldrich cat. no. A4403) • HEPES (Sigma-Aldrich, cat. no. H4034) • Matrigel (BD Biosciences, cat. no. 354277) • Poly-l-ornithine hydrobromide (Sigma-Aldrich, cat. no. 3655) • Natural mouse laminin (Life Technologies, cat. no. 23017015) • ProFreeze-CDM (2×; Lonza, cat. no. 12-769E) • Paraformaldehyde solution, 4% (wt/vol) in PBS (Santa Cruz Biotechnology, cat. no. sc-281692) ! CAUTION Work in a fume hood. • Anti-O4 supernatant from hybridoma (Dr. James Goldman’s laboratory) • Anti-O4 clone 81 (EMD Millipore, cat. no. MAB345) • Pax-6 polyclonal antibody (Covance, cat. no. 1DB-001-0000751403) • Anti-Olig2 (EMD Millipore, cat. no. AB9610) • Mouse Nkx2.2 concentrate (DSHB, cat. no. 74.5A5) • hSox10 goat polyclonal (R&D Systems, cat. no. AF2864) • Anti-myelin basic protein, aa 82–87 (EMD Millipore, cat. no. MAB386) • Anti-chicken MAP2 (Abcam, cat. no. ab5392) • Polyclonal rabbit anti-glial fibrilliary acidic protein (GFAP) (Dako, cat. no. Z033401) • Alexa Fluor 647 goat anti-mouse IgM (µ-chain; Life Technologies, cat. no. A-21238) • Alexa Fluor 488 goat anti-mouse IgM (µ-chain; Life Technologies, cat. no. A-21042) • Alexa Fluor 488 goat anti-tabbit IgG (Life Technologies, cat. no. A-11034) • Alexa Fluor 488 donkey anti-rabbit IgG (Life Technologies, cat. no. A-21206) • Alexa Fluor 568 goat anti-mouse IgG (Life Technologies, cat. no. A-11004) • Alexa Fluor 488 donkey anti-mouse IgG (Life Technologies, cat. no. A-21202) • Alexa Fluor 555 donkey anti-goat IgG (Life Technologies, cat. no. A-21432) • Alexa Fluor 647 donkey anti-rabbit IgG (Life Technologies, cat. no. A-31573) • Alexa Fluor 568 goat anti-rat IgG (Life Technologies, cat. no. A-11077) • Alexa Fluor 647 donkey anti-rabbit IgG (Life Technologies, cat. no. A-31573) • Alexa Fluor 647 goat anti-chicken IgG (Life Technologies, cat. no. A-21449) • DAPI, FluoroPure (Life Technologies, cat. no. D21490) • Normal goat serum (Jackson ImmunoResearch Laboratories, cat. no. 005-000-121) • Sodium hydroxide solution (NaOH; Sigma-Aldrich, cat. no. 71436) • Hydrochloric acid solution (Fisher Scientific, cat. no. SA48-1) • BSA solution (Sigma-Aldrich, cat. no. A8412) EQUIPMENT • CO2 cell culture incubator, HERAcell 150i (Thermo Fisher Scientific) • Purifier biological safety cabinet (Labconco) • Inverted microscope Olympus CKX31 • Fluorescence microscope (Olympus IX71) equipped with Olympus DP30BW camera

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protocol • Sterile hood Cell logic (Labconco) equipped with a stereomicroscope (NIKON SMZ1500) • Centrifuge, Eppendorf 5810R • Freezer, −80 °C REVCO Ultima II and −20 °C (Isotemp, Fisher Scientific) • Refrigerator, 4 °C (NORLAKE Scientific) • Liquid nitrogen cell storage tank • Flow cytometer cell sorter BD FACSAria IIu ROU and multicolor analyzer with five-laser system (488, 640, 405, 355 and 532 nm) • Water bath, 37 °C (Isotemp Fisher Scientific) • Ice machine, Follett Symphony series • Hemocytometer (Fisher Scientific, cat no. 0267110) • Pipettes (Pipetman, Gilson) • Electronic pipettors (Accujet pro, BRAND) • Culture plates, six-well, tissue culture treated (Fisher Scientific, cat. no. 07-200-83) • Corning Costar ultra-low-attachment plates, six-well (Sigma-Aldrich, cat. no. 3471) • Falcon 15-ml tubes (Fisher Scientific, cat. no. 14-959-49D) • 5-ml polystyrene round-bottom tube with cell-strainer cap (BD Falcon, cat. no. 352235) • 5-ml polypropylene round-bottom tube (BD Falcon, cat. no. 352063) • Safe-Lock tubes, 1.5 ml (Eppendorf, cat. no. 022363204) • Cryogenic vials (cryovials; Thermo Scientific, cat. no. 5000-0012) • Mr. Frosty freezing container (Thermo Scientific, cat. no. 5100-0001) • Pipette tips (Denville Scientific, 10 µl, cat. no. P1096-FR; 20 µl, cat. no. P1121; 200 µl, cat. no. P1122; and 1,000 µl, cat. no. P1126) • Serological pipettes (Fisherbrand, 5 ml, cat. no. 13-678-11D; 10 ml, cat. no. 13-678-11E; and 25 ml, cat. no. 13-678-11) • Nalgene rapid-flow sterile disposable filter units (Thermo Scientific, 250 ml, cat. no. 168-0045 and 500 ml, cat. no. 166-0045) • Corning cell lifter (Sigma-Aldrich, cat. no. CLS3008-100EA) • Syringe filter unit, 0.22 µm (EMD Millipore, cat. no. SLGP033RS) • BD Luer-Lok disposable syringes without needles (Fisher Scientific, cat. no. 301604) REAGENT SETUP Y27632 (ROCK inhibitor)  Reconstitute 2 mg of powder in 625 µl of DMSO for a 10 mM stock solution. Make aliquots of the desired volume and store them at −80 °C for up to 6 months. All Trans RA  Reconstitute 500 mg of powder in 16.7 ml of DMSO for a 100 mM stock solution. Store the stock at −80 °C for up to 6 months. Prepare working aliquots by diluting the stock solution in DMSO 1,000 times, to obtain a final concentration of 100 µM. Store the working aliquots at −20 °C for up to 3 months. RA is light sensitive; keep the aliquots away from direct light exposure. SB431542  Reconstitute 5 mg of SB431542 in 650 µl of DMSO to make a 20 mM solution. Make aliquots of the desired volume, and store them, protected from light, at −80 °C for up to 6 months. LDN-193189  Reconstitute 2 mg of LDN-193189 in 450 µl of DMSO to make a 10 mM stock solution. Prepare working aliquots by diluting the stock solution in DMSO 40 times, to obtain a final concentration of 250 µM. Make aliquots of the desired volume and store them, protected from light, at −80 °C for up to 6 months. SAG  Reconstitute SAG in 1.67 ml of DMSO to make a 1 mM stock solution. Store it at −20 °C for up to 6 months. Recombinant human PDGF-AA  Reconstitute 10 µg of PDGF-AA in 100 µl of 4 mM HCl to make a 100 µg/ml stock. Make aliquots of the desired volume and store them at −80 °C for up to 6 months. Recombinant human IGF-I  Reconstitute 200 µg in 1 ml of DPBS to make a 200 µg/ml stock. Make aliquots of the desired volume and store them at −80 °C for up to 6 months. Recombinant human HGF  Reconstitute 25 µg in 2.5 ml of DPBS with 0.1% (wt/vol) BSA to make a 10 µg/ml stock. Make aliquots of the desired volume and store them at −80 °C for up to 6 months. NT3  Reconstitute 10 µg of NT3 in 100 µl of dH2O to make a 100 µg/ml stock. Make aliquots of the desired volume and store them at −80 °C for up to 6 months. Biotin  Reconstitute 100 mg of powder in 10 ml of NaOH 1 N to make a 10 mg/ml stock. Make 25-µl aliquots and store them at −80 °C. Dilute 25 µl in 250 µl of DMEM/F12 to make working aliquots of 1 mg/ml. Keep the working aliquots at 4 °C for up to 1 week. cAMP  Dissolve 5 mg in 1.017 ml of dH2O to make a 10-mM stock solution. Make aliquots of the desired volume and store them at −80 °C for up to 6 months.

T3  Weight 1.2 mg of T3 and dissolve it in 2 ml of 1 N NaOH to make a 600 µg/ml stock solution. Make aliquots of the desired volume and store them at −80 °C for up to 6 months. l-ascorbic acid (AA)  Reconstitute 100 mg of powder in 10 ml of dH2O to make a 10 mg/ml solution. AA can be stored at −80 °C for up to 6 months. HEPES  Weigh 2.382 g of HEPES and dissolve it in 10 ml of dH2O; filter it using a 10-ml syringe and a 0.22-µm syringe filter unit. Store it at 4 °C for up to 6 months. Matrigel  Thaw the bottle overnight at 4 °C. Prechill p1000 pipette tips at −20 °C for at least 10 min. Place the bottle of thawed Matrigel on ice and make 250-µl aliquots in 1.5-ml Eppendorf tubes. Store the aliquots at −80 °C for up to 6 months. To prepare working solution, thaw the stock aliquots on ice. Place a 50-ml conical tube on ice and add 25 ml of cold DMEM/F12. With a prechilled p1000 tip, transfer the stock aliquot to the 50-ml conical tube and mix well. The working solution can be stored at 4 °C for up to 2 weeks. Poly-l-ornithine hydrobromide  Reconstitute 50 mg of powder with 50 ml of dH2O to make a 1 mg/ml concentration. Make aliquots of the desired volume and store them at −20 °C for up to 1 year. DAPI  Reconstitute 10 mg of DAPI in 2 ml of dH2O to obtain a final concentration of 5 mg/ml. Make aliquots of the desired volume and store them at 4 °C for up to 6 months. DAPI is light sensitive; keep the aliquots protected from light. Freezing medium  To make the complete 2× freezing medium, add 15% (vol/vol) DMSO to the desired volume of ProFreeze-CDM NAO medium. Prepare complete 2× freezing medium only for a single use, and keep it on ice before adding it to the cell solution. mTeSR1  Prepare mTeSR1 medium by adding penicillin-streptomycin and mTeSR1 supplement to mTeSR basal medium to give a final concentration of 1× for all. Make 40-ml aliquots and keep them at −20 °C for up to 6 months. Thawed aliquots can be kept at 4 °C for 2 weeks. mTeSR custom  Prepare mTeSR custom medium by adding mTeSR custom supplement and penicillin-streptomycin to mTeSR basal medium to obtain a final concentration of 1× for all. Make 40-ml aliquots and keep them at −20 °C for up to 6 months. Thawed aliquots can be kept at 4 °C for 2 weeks. Basal medium  Basal medium is used to make N2 medium, N2B27 medium, PDGF medium and glial medium. Prepare it by adding NEAA, GlutaMAX, 2-mercaptoethanol and penicillin-streptomycin to DMEM/F12 to obtain final concentrations of 1× each. Sterilize the solution with a disposable filter unit. Basal medium can be stored at 4 °C for 1 month. Neural induction medium  Neural induction medium comprises mTeSR custom medium containing 10 µM SB431542, 250 nM LDN193189 and 100 nM RA added freshly on the day of use. mTeSR custom medium can be replaced by basal medium with 25 µg/ml insulin. N2 medium  Make up N2 medium by adding N2 supplement to the basal medium to obtain a final concentration of 1×. Keep it at 4 °C for 2 weeks, protected from light. Add 100 nM RA and 1 µM SAG freshly on the day of use. N2B27 medium  Prepare N2B27 medium by adding N2 and B27 supplements to the basal medium to obtain a final concentration of 1× of each. Add 25 µg/ml insulin. Keep the medium at 4 °C for 2 weeks, protected from light. Add 100 nM RA and 1 µM SAG freshly on the day of use. PDGF medium  Prepare PDGF medium as shown and keep it at 4 °C for 2 weeks, protected from light. Reagent

Final concentration

Basal medium N2 supplement (100×)

1×

B27 supplement (50×)

1×

PDGFaa

10 ng/ml

IGF-1

10 ng/ml

HGF

5 ng/ml

NT3

10 ng/ml

T3

60 ng/ml

Biotin

100 ng/ml

cAMP

1 µM

Insulin

25 µg/ml nature protocols | VOL.10 NO.8 | 2015 | 1147

protocol Glial medium  Prepare glial medium as shown and keep it at 4 °C for 2 weeks, protected from light. Reagent

Final concentration

Basal medium N2 supplement (100×)

1×

B27 supplement (50×)

1×

© 2015 Nature America, Inc. All rights reserved.

HEPES

10 mM

T3

60 ng/ml

Biotin

100 ng/ml

cAMP

1 µM

Insulin

25 µg/ml

Ascorbic acid

20 µg/ml

EQUIPMENT SETUP Matrigel-coated plates  Prechill the six-well plates to be coated and a 5-ml serological pipette at −20 °C for at least 10 min. Take the working solution of Matrigel from the 4 °C refrigerator and keep it on ice. Place the chilled plates on ice, and with the chilled pipette add 1 ml of Matrigel per well of a sixwell plate. Swirl the plate to cover the entire well with the Matrigel solution. Incubate the plate at 37 °C for at least 2 h. Aspirate the solution just before adding the cells to the wells. Matrigel-coated plates, sealed with Parafilm, can be stored at 4 °C for up to 2 weeks. Poly-l-ornithine– and laminin-coated plates  Add 1 ml of 50 µg/ml poly-l-ornithine in dH2O per well of a six-well plate. Swirl the plate to cover the entire well and incubate the plate at 37 °C overnight. Poly-l-ornithine–coated plates can be stored at 37 °C for up to 4 d. The following day, aspirate the poly-l-ornithine solution and leave the plate to air-dry for 5 min. Add 1 ml of 20 µg/ml natural mouse laminin in DMEM/F12 per well of a six-well plate, swirl the plate to cover the entire well and incubate it at 37 °C for 4 h. Poly-l-ornithine– and laminin-coated plates can be stored at 37 °C for up to 2 d. Aspirate the laminin and leave the plate to dry for 5 min. Wash it two times with DMEM/F12.

PROCEDURE Preparation of hPSC cultures for oligodendrocyte differentiation ● TIMING 4–5 d 1| Partially thaw a frozen cryovial containing hPSCs in a 1-ml volume by holding the vial in a 37 °C water bath for 1–2 min. 2| Slowly add 1 ml of mTeSR1 medium to the partially thawed cryovial, and transfer the 2 ml of cells from the cryovial to a 15-ml conical tube. 3| Add mTeSR1 medium to the 15-ml conical tube to a total volume of 10 ml. 4| Collect the cells by centrifugation at 200g for 4 min at 20–23 °C (room temperature, RT). 5| Remove the supernatant and resuspend the cell pellet in 2 ml of mTeSR1 medium containing 10 µM ROCK Inhibitor (Y27632).  CRITICAL STEP Y27632 should be freshly added and kept in the culture for the first 24 h, as this greatly helps the survival of the single-cell suspension of hPSCs. 6| Transfer the cells to a Matrigel-coated well of a six-well plate. 7| Incubate the six-well plate for 24 h in a 37 °C incubator at 5% CO2. 8| Remove the medium and add 2 ml of fresh mTeSR1 medium. 9| Incubate the cells in a 37 °C incubator, with 5% CO2, and replace mTeSR1 medium every day. 10| When the cells reach 70–90% confluency, remove the medium and add 1 ml of prewarmed Accutase.  CRITICAL STEP The growth rate of different cell lines may vary, and it also depends on the quality and number of the frozen cells. 11| Incubate the plate in a 37 °C incubator for 5 min. 12| Dilute the Accutase solution by adding 2 ml of DMEM/F12 medium. 13| Gently pipette the mixture 2–5 times with the p1000 pipette to fully dissociate the hPSC colonies to single cells. 14| Transfer the cells in a 15-ml conical tube, and dilute the solution further by adding 5 ml of DMEM/F12. 15| Centrifuge the cells at 200g for 4 min at RT. 1148 | VOL.10 NO.8 | 2015 | nature protocols

protocol 16| Resuspend the cell pellet in 1 ml of mTeSR1 containing 10 µM Y27632, and count the cells with a hemocytometer. 17| Plate 8 × 104 to 1 × 105 cells per well on a Matrigel-coated six-well plate that already contains 1.5 ml of mTeSR1 medium supplemented with 10 µM Y27632 per well.  CRITICAL STEP This density of plated hPSCs is optimized to give a confluent well by day 8 and multilayered structures at day 12 of differentiation. 18| Incubate the cells for 24 h in a 37 °C incubator at 5% CO2. 19| Remove the old medium and add 2 ml of fresh mTeSR1 medium in each well.

© 2015 Nature America, Inc. All rights reserved.

20| Incubate the cells for 1–2 d, until hPSC colonies reach a diameter of 100–250 µm. Differentiation to OLIG2+ progenitors ● TIMING 12 d 21| Once colonies are ~100–250 µm in diameter, aspirate the old medium and induce differentiation by adding 2 ml of neural induction medium to each well. This is day 0.  CRITICAL STEP Adding RA at a low concentration (100 nM) from day 0 of the differentiation greatly improves the yield of OLIG2+ progenitors at day 12. 22| Incubate the plate in a 37 °C, 5% CO2 incubator and perform media changes every day for 8 d.  CRITICAL STEP Add fresh RA, SB431542 and LDN193189 to the medium every day. 23| On day 8, switch to N2 medium and incubate it for 4 d, by changing the medium daily.  CRITICAL STEP By day 8, cells should be confluent and PAX6 expression should be at its peak, as shown in Figure 2a. By day 12, overconfluent cells should be piling up and 3D structures should be clearly visible, as shown in Figure 2b. This is an important checkpoint before proceeding with the differentiation.  CRITICAL STEP Add fresh RA and SAG to the medium every day.  CRITICAL STEP The medium will become yellowish owing to the rapid expansion of the cells and their increasing numbers. This is why it is important to feed the cells every day to provide the required nutrients. ? TROUBLESHOOTING Cell detachment and formation of OLIG2-enriched aggregates ● TIMING 18 d 24| On day 12, detach adherent cells. Follow Step 24A to obtain the highest number of spheres and ultimately of O4+ cells. Follow Step 24B to obtain more uniform spheres, although this step will result in a lower number of spheres.  CRITICAL STEP Sphere formation enriches for the OLIG2+ progenitors. Only the OLIG2+ cells aggregate into spheres, whereas the OLIG2- cells remain as single cells. (A) Aggregation of OLIG2+ cells after mechanical dissociation (i) Remove the old medium and add 1 ml of fresh N2B27 medium per well of a six-well plate. (ii) Use a cell lifter and place it perpendicular to the bottom of the well. (iii) Press the cell lifter against the bottom of the plate and create a cut to the cell layer. Create at least 20 such lines parallel to each other, to cover the whole well. (iv) Turn the well 90° and repeat Step 24A(ii,iii). (v) Turn the well 45° and repeat Step 24A(ii,iv).  CRITICAL STEP It is important to break the monolayer of cells into small clumps so that nutrients will reach all the cells within the aggregate. (vi) Use the same cell lifter to detach the remaining adherent cells by scraping the whole well.  CRITICAL STEP Check under the microscope to ensure that there are no cells left attached.  PAUSE POINT Cultures can be frozen at this stage (Box 1). (vii) With a p1000 pipette, gently pipette the clumps of cells 3–5 times, and transfer the contents of one well into two wells of an ultra-low-attachment six-well plate. (viii) Add N2B27 medium to a total of 3 ml in each well and incubate the plate for 2 d in an incubator at 37 °C, 5% CO2.  CRITICAL STEP During the first 48 h before medium change, the culture will look turbid owing to the single cells that did not aggregate and died. However, small aggregates should be already visible. The debris and dead cells will be eliminated with the medium changes in the following days. (ix) Transfer the medium containing the cell aggregates to a 15-ml conical tube and wait for 3–5 min for the aggregates to sink to the bottom of the tube. nature protocols | VOL.10 NO.8 | 2015 | 1149

protocol

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Box 1 | Freezing and thawing cells or aggregates ● TIMING 20–60 min For freezing aggregates on day 30 (Step 29), transfer the aggregates to a 15-ml conical tube and then continue to step 6. For freezing cells on day 55 or 75 (Step 32), first detach the cells with Accutase, as described in steps 1–5. 1. Aspirate the medium and add 1 ml of prewarmed Accutase per well of a six-well plate. 2. Incubate the plate at 37 °C for 25–35 min, until most of the cells that have migrated out of the spheres are visibly floating. 3. Dilute the Accutase solution by adding 2 ml of DMEM/F12. 4. Gently pipette the mixture 2–3 times with a p1000 pipette to disperse as many cells as possible.  CRITICAL STEP Do not pipette extensively, as this will result in high cell death. 5. Transfer the diluted Accutase solution containing single cells and aggregates into a 15-ml conical tube 6. Add 5 ml of DMEM/F12 into the 15-ml conical tube containing the cells. 7. Centrifuge the mixture at 200g for 5 min at RT. 8. Resuspend the pelleted cells into 500 µl of their respective medium. 9. Transfer the cells into a cryovial on ice. 10. Gently add 500 µl of the freezing medium in each cryovial and pipette 1–2 times. 11. Store the cells overnight at −80 °C in a Mr. Frosty container that has a cooling rate very close to −1 °C/min. 12. The next day, transfer the cryovials into a liquid nitrogen tank for long-term storage.  PAUSE POINT Cells can be stored in liquid nitrogen indefinitely. 13. For thawing the cells, follow Steps 1–5 of the main PROCEDURE substituting mTeSR1 medium with the medium appropriate for the differentiation stage of the cells. When thawing the aggregates, let them recover for 48 h in ultra-low-attachment plates before proceeding with ‘Selection and plating of aggregates’ (Step 30).

(x) Aspirate two-thirds of the medium and replenish it with fresh N2B27 medium. (xi) R  eturn the aggregates to the same ultra-low-attachment plate and redistribute an approximately equal number of aggregates in each well with a p1000 pipette. (xii) Repeat Step 24A(ix–xi) every other day until day 20 of differentiation, at which point proceed to Step 25. (B) Aggregation of OLIG2+ cells after enzymatic digestion to single cells (i) Aspirate the medium and add 1 ml of prewarmed Accutase. (ii) Incubate the medium at 37 °C for 5 min. (iii) Dilute the Accutase solution by adding 2 ml of DMEM/F12 medium. (iv) With a p1000 pipette, pipette the mixture 2–5 times to dissociate the culture into a single-cell suspension. (v) Transfer the cells to a 15-ml conical tube and dilute the solution further by adding another 5 ml of DMEM/F12. (vi) Collect the cells by centrifugation at 200g for 4 min at RT. (vii) Resuspend the cell pellet in 1 ml of N2B27 medium and distribute the single-cell suspension into two wells of a six-well ultra-low-attachment plate. (viii) Add N2B27 medium to a total of 3 ml per well, and incubate the plate for 2 d in the incubator at 37 °C, 5% CO2. (ix) Transfer the medium containing the cell aggregates into a 15-ml conical tube and spin it for 2 min at 100g at RT. (x) Remove two-thirds of the medium and add 2 ml of fresh N2B27 medium. (xi) Return the aggregates to the same ultra-low-attachment plate and redistribute an approximately equal number of aggregates in each well with a pipette. (xii) Repeat Steps 24B(ix–xi) every other day until day 20 of differentiation, at which point proceed to Step 25. 25| On day 20, transfer the aggregates to a 15-ml conical tube and wait for 3 min for the aggregates to sink to the bottom of the tube. 26| Remove two-thirds of the medium and replenish it with PDGF medium. 27| Gently pipette five times up and down with a p1000 pipette.  CRITICAL STEP It is important to break apart the aggregates that stick to each other through gentle pipetting. 28| Return the aggregates to the same ultra-low-attachment plate and redistribute an approximately equal number of aggregates in each well with the p1000 pipette. 29| Repeat Steps 25–28 every other day until day 30 of differentiation.  PAUSE POINT Aggregates can be frozen at this stage (Box 1).

1150 | VOL.10 NO.8 | 2015 | nature protocols

protocol Selection and plating of aggregates ● TIMING 1–4 h 30| On day 30, use a microscope under sterile conditions, and with a p200 pipette pick the aggregates that are round, that have a diameter between 300 and 800 µm, and that appear golden or brown with a dark center (Fig. 2d).  CRITICAL STEP Aggregates created with mechanical dissociation (Step 24A) will vary greatly, whereas aggregates formed after the single-cell suspension (Step 24B) will be more uniform in size and appearance. Selecting the best aggregates at this point, especially when Step 24A has been followed, is important. Spheres that are completely transparent should be avoided altogether, as the cells produced by them do not differentiate to oligodendrocytes.  CRITICAL STEP If two or three good-looking spheres are sticking together, try to disaggregate them by gently pipetting with the p200 pipette. ? TROUBLESHOOTING

© 2015 Nature America, Inc. All rights reserved.

OPC differentiation in adherent cultures ● TIMING 15–60 d 31| Plate 20 spheres in a well of a six-well plate coated with poly-l-ornithine and laminin.  CRITICAL STEP Plating two spheres per cm2 ensures that cells will migrate out of the spheres and fill the whole well around day 60 without the need for passaging the cells. ? TROUBLESHOOTING 32| Plated spheres can be cultured in a medium containing mitogens (Step 32A) or in a medium without any mitogen (Step 32B). Step 32A was optimized to obtain the highest yield of O4+ cells, whereas Step 32B was developed to provide a shorter and cheaper version of the protocol, although the tradeoff is a possible reduction in efficiency. (A) Expansion of adherent cells in PDGF medium (‘original’ protocol) (i) Add 3 ml of PDGF medium per well to the poly-l-ornithine– and laminin-coated wells with the picked spheres, and then swirl the plate to disperse the aggregates. (ii) Incubate the plate in the incubator at 37 °C, 5% CO2. (iii) Every other day, carefully replenish two-thirds of the medium with fresh PDGF medium until day 75 of differentiation. The appearance of O4+ cells can be assessed by live O4 staining from day 55 onward (Box 2 and Fig. 3).  CRITICAL STEP Be extremely gentle during medium changes. Do not tilt the plate to aspirate the old medium, so that the cell aggregates are covered with liquid at all times. Gently aspirate the old medium with a p1000 pipette, by placing the tip close to the wall, without disturbing the cells. Add fresh medium very slowly by aiming at the wall of the well. Avoid rough and sudden movements even when you transfer the plate from the incubator, especially after day 40, because the cells will detach from the plate, typically in the form of a sheet. ? TROUBLESHOOTING  PAUSE POINT Cells can be frozen at day 75 (Box 1). (iv) At day 75, OPCs can be isolated through FACS using the O4 surface antigen (Box 3 and Fig. 2i). Alternatively, proceed to Step 32A(v) to continue the differentiation. (v) For terminal differentiation to MBP+ cells (Fig. 2g,h), from day 75, switch to glial medium. (vi) Change two-thirds of the glial medium every 2–3 d for 2 weeks.

Box 2 | Live O4 staining ● TIMING 1–2 h 1. In the culturing medium, add 5% (vol/vol) goat serum and primary O4 antibody in the appropriate concentration. 2. Incubate in the incubator at 37 °C, 5% CO2 for 45 min. 3. Remove the medium and wash by adding 1 ml of DMEM/F12. 4. Aspirate the washing medium.  PAUSE POINT At this point cells can be fixed by adding 4% (wt/vol) paraformaldehyde in PBS for 10 min at RT. Fixed cells can be stored at 4 °C for weeks. Immunofluorescence staining can be continued at any time by adding a goat anti-mouse IgM secondary antibody conjugated to a fluorochrome. 5. Add 1 ml of glial medium containing goat anti-mouse IgM secondary antibody conjugated with Alexa Fluor 488 (1:500 dilution). 6. Incubate the cells at 37 °C for 15 min. 7. Remove the medium and wash the cells by adding 1 ml of DMEM/F12. 8. Aspirate the washing medium and add 2 ml of glial medium 9. O4+ cells can be visualized under a fluorescence microscope.  CRITICAL STEP After this procedure, if cells were not fixed, they will still be alive and can be cultured further by following any of the protocols described herein. The complex O4–Alexa Fluor 488 is very stable and O4+ ‘fragments’ can be detected in the culture for at least 1 week after staining. nature protocols | VOL.10 NO.8 | 2015 | 1151

protocol

© 2015 Nature America, Inc. All rights reserved.

Box 3 | Preparation of cells for FACS ● TIMING 2.5 h The following procedure describes how to isolate O4+ cells by FACS from cells in the format of one well of a six-well plate, seeded with 20 aggregates at day 30 of differentiation. It can be linearly scaled up, depending on the number of wells you wish to use for O4 isolation.  CRITICAL STEP We would not advise processing and pulling together more than a full six-well plate at a time, as the large cell number combined with the manipulations described below will result in extensive cell death and debris that will interfere with the FACS results. 1. Aspirate the old medium and add 1 ml of prewarmed Accutase. 2. Incubate the mixture at 37 °C for 25–35 min, or longer, until most of the cells are visibly floating. 3. Dilute the Accutase solution by adding 2 ml of DMEM/F12. 4. Gently pipette 2–3 times with a p1000 pipette to disperse as many cells as possible.  CRITICAL STEP Do not pipette extensively, because this will result in high cell death.  CRITICAL STEP Most of the spheres will look intact after this step. It is mainly the cells that have migrated out of the sphere that are usually processed. 5. Transfer the total volume containing single cells and aggregates into a 15-ml conical tube 6. Add 5 ml of DMEM/F12 and centrifuge at 200g for 5 min at RT. 7. Aspirate the supernatant and resuspend the cells in 100 µl of DMEM/F12 containing 5% (vol/vol) goat serum and primary O4-specific antibody at the appropriate concentration 8. Incubate the cells for 45 min on ice. 9. Wash the cells by adding 1 ml of DMEM/F12. 10. Centrifuge the cells at 200g for 5 min at RT. 11. Aspirate the supernatant and resuspend the cells in 100 µl of DMEM/F12 containing goat anti-mouse IgM secondary antibody conjugated with a fluorochrome. 12. Incubate the cells on ice for 25 min. 13. Wash the cells by adding 1 ml of DMEM/F12. 14. Centrifuge the cells at 200g for 5 min at RT. 15. Aspirate the supernatant. 16. Repeat steps 13–15. 17. Resuspend the cells in 250 µl of glial medium. 18. Pass the solution through a FACS tube with a cell-strainer cap using a p1000 pipette to obtain a single-cell suspension. 19. With the p1000 pipette, add 250 µl of glial medium to wash the strainer. 20. Collect the flow-through and FACS sort with a 130-µm nozzle, at a sheath pressure of 25 p.s.i. Sort slowly, at 1,500–1,800 events per second.  CRITICAL STEP The large nozzle greatly improves cell survival after sorting.  CRITICAL STEP We recommend the use of a sample stained with the secondary antibody only as a negative control to set the gates in the FACS.

? TROUBLESHOOTING

(B) Fast expansion of adherent cells in glial medium (‘fast’ protocol) (i) Add 3 ml of glial medium to each well of the poly-l-ornithine– and laminin-coated wells with the picked spheres, and swirl the plate to disperse the aggregates. (ii) Incubate the plate in the incubator at 37 °C, 5% CO2. (iii) Every other day, carefully replenish two-thirds of the medium with fresh glial medium until day 55 of differentiation. (iv) At day 55, O4+ cells can be visualized by live O4 staining (Box 2; Figs. 2f and 3a–f) or isolated by FACS (Fig. 3g and Box 3).  CRITICAL STEP Cultures can be kept in glial medium until day 75 to increase the efficiency of O4+ cells (Fig. 3).  CRITICAL STEP As in Step 32A, be extremely gentle during medium changes. Do not tilt the plate to aspirate the old medium, so that the cell aggregates are covered with liquid at all times. Gently aspirate the old medium with a p1000 pipette, by placing the tip close to the wall, without disturbing the cells. Add fresh medium very slowly by aiming at the wall of the well. Avoid rough and sudden movements, even when you transfer the plate from the incubator, especially after day 40, because the cells will detach from the plate, typically in the form of a sheet. ? TROUBLESHOOTING  PAUSE POINT Cells can be frozen at day 55 (Box 1). ? TROUBLESHOOTING Troubleshooting advice can be found in Table 1. 1152 | VOL.10 NO.8 | 2015 | nature protocols

protocol

© 2015 Nature America, Inc. All rights reserved.

Table 1 | Troubleshooting table. Step

Problem

Solution

23

Well is not confluent

Check the plating number Check the quality of PSCs (e.g., spontaneous differentiation, mycoplasma) Check the concentration, activity, lot and expiration of reagents Adjust the plating density according to the growth rate of the cell line

30

Aggregates stick together

Increase the pipetting speed and repetition during medium changes at Step 27

31

Well is not confluent at day 60

Consider increasing the number of plated spheres to 25 spheres per well of a six-well plate

The well is overconfluent before day 60

Consider decreasing the number of plated spheres to 18 spheres per well of a six-well plate Avoid the transparent spheres during picking at day 20 Count the large aggregates (>1 mm) as two spheres If you are following original protocol, switch to glial medium at day 55 to delay cell growth

32A(iii)

Cell detachment

Select only the spheres that fulfill the criteria described in Step 30 Avoid aggregates that are completely transparent Be very gentle when handling the cultures

32B(iv)

Poor efficiency of O4+ cells

Make sure that the starting hPSC lines are free of spontaneous differentiation Select good spheres following the criteria at Step 30 Wait for a few more days to increase the number of oligodendrocytes

Box 3

Increased cell death or debris

Be gentle after the Accutase treatment Handle up to four wells of a six-well plate at a time Use multiple strainers to filter the cells, as one might get clogged

● TIMING Steps 1–9, expansion of hPSCs: 3 d Steps 10–20, plating of hPSCs and preparation for induction: 2 d Steps 21 and 22, neural induction: 8 d Step 23, induction to OLIG2+ cells: 4 d Steps 24–29, sphere formation and culture in suspension: 18 d Step 30, sphere selection: 1–4 h Steps 31 and 32, differentiation to OPCs and oligodendrocytes in adherent cultures: 15–60 d for the original protocol; 25 d for the fast protocol Box 1, freezing and thawing cells or aggregates 20–60 min Box 2, live O4 staining 1–2 h Box 3, preparation of cells for FACS 2.5 h ANTICIPATED RESULTS The protocol described here provides a reproducible way to differentiate 105 hPSCs into >2.5 × 105 O4+ oligodendrocytes within 75 d. As shown in Table 2, O4 efficiencies ranged from 28% to 80% with nine different PSC lines, and the average was >60% in four lines. Reported efficiencies are based on manual selection of the good spheres and plating at the aforementioned density of two spheres per cm2. The reproducibility is very high across different PSC lines that we and other groups have already tested (P. Tesar, M. Wernig and C. Desponts, unpublished data), but deviations from the stated percentages may be expected. These discrepancies can be attributed to several factors including the methods to generate iPSC lines (integration methods, viral transductions, Cre-resulting footprint and so on), the quality of the PSCs and/or the lot-to-lot variability for the reagents composing the medium. Cultures can be checked for the expression of appropriate markers by either quantitative RT-PCR or immunofluorescence at various stages of the protocol. When performing immunofluorescence analysis of the cultures at day 8 for PAX6 (Fig. 2a) and at day 12 for OLIG2 and NKX2.2 (Fig. 2c), the frequencies should be always >90% for the PAX6+ cells, 70% for OLIG2+ cells and 30% for the OLIG+NKX2.2+ cells. By days 40–50, SOX10 should be expressed and should colocalize with OLIG2

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© 2015 Nature America, Inc. All rights reserved.

protocol and NKX2.2 (Fig. 2e). From days 50–75, live O4 staining can be performed to detect the appearance of O4+ cells and their expansion (Figs. 2f and 3a–f ). Finally, O4+ cells can either be isolated via FACS or further differentiated to MBP+ oligodendrocytes (Fig. 2g,h). Other cell types also exist in our day 75 cultures, although at lower percentages. We normally find GFAP+ cells in ~15% of the total cell population (13.81 ± 0.12, mean percentage ± s.e.m., n = 3) and ~20% βIII-tubulin+ cells (20.07 ± 1.01, mean% ± s.e.m., n = 3; Fig. 2h). After the final differentiation step, when cells are cultured in glial medium for 2 weeks, ~35% of the O4+ cells should also express MBP16. Aggregates at day 30 (Step 29) and cells at the end of the differentiation can be cryopreserved with a viability >70%. Aggregates’ viability is based on the number of thawed spheres that reattach onto poly-l-ornithine– and laminin-coated dishes after thawing. The sorted O4+ cells can be frozen, by following steps 7–9 in Box 1, immediately after sorting. The expected post-thaw viability of the sorted O4+ cells is 70–80%.

Table 2 | Percentages of O4+ OPCs after ~75 d of differentiation.

Acknowledgments We thank M. Zimmer for excellent assistance with cell sorting and J. Goldman (Columbia University) for providing the O4 antibody. We are thankful to D. Paul and B. Corneo for their insightful comments. This work was supported by a New York Stem Cell Foundation (NYSCF)-Helmsley Early Career Investigator Award, The NYCSF, and The Leona M. and Harry B. Helmsley Charitable Trust. P.D. is a NYSCF-Druckenmiller postdoctoral fellow.

10. Numasawa-Kuroiwa, Y. et al. Involvement of ER stress in dysmyelination of Pelizaeus-Merzbacher disease with PLP1 missense mutations shown by iPSC-derived oligodendrocytes. Stem Cell Rep. 2, 648–661 (2014). 11. Stacpoole, S.R. et al. High yields of oligodendrocyte lineage cells from human embryonic stem cells at physiological oxygen tensions for evaluation of translational biology. Stem Cell Rep. 1, 437–450 (2013). 12. Wang, S. et al. Human iPSC-derived oligodendrocyte progenitor cells can myelinate and rescue a mouse model of congenital hypomyelination. Cell Stem Cell 12, 252–264 (2013). 13. Liu, Y., Jiang, P. & Deng, W. OLIG gene targeting in human pluripotent stem cells for motor neuron and oligodendrocyte differentiation. Nat. Protoc. 6, 640–655 (2011). 14. Sim, F.J. et al. CD140a identifies a population of highly myelinogenic, migration-competent and efficiently engrafting human oligodendrocyte progenitor cells. Nat. Biotechnol. 29, 934–941 (2011). 15. Boulting, G.L. et al. A functionally characterized test set of human induced pluripotent stem cells. Nat. Biotechnol. 29, 279–286 (2011). 16. Douvaras, P. et al. Efficient generation of myelinating oligodendrocytes from primary progressive multiple sclerosis patients by induced pluripotent stem cells. Stem Cell Rep. 3, 250–259 (2014). 17. Kang, J. et al. Granulocyte colony-stimulating factor minimizes negative remodeling of decellularized small diameter vascular graft conduits but not medial degeneration. Ann. Vasc. Surg. 27, 487–496 (2013). 18. Fennema-Notestine, C. et al. In vivo evidence of cerebellar atrophy and cerebral white matter loss in Huntington disease. Neurology 63, 989–995 (2004). 19. Behrendt, G. et al. Dynamic changes in myelin aberrations and oligodendrocyte generation in chronic amyloidosis in mice and men. Glia 61, 273–286 (2013). 20. Bernstein, H.G., Steiner, J., Guest, P.C., Dobrowolny, H. & Bogerts, B. Glial cells as key players in schizophrenia pathology: recent insights and concepts of therapy. Schizophr. Res. 161, 4–18 (2015). 21. Chambers, S.M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275–280 (2009). 22. Patani, R. et al. Retinoid-independent motor neurogenesis from human embryonic stem cells reveals a medial columnar ground state. Nat. Commun. 2, 214 (2011). 23. Miller, R.H. et al. Patterning of spinal cord oligodendrocyte development by dorsally derived BMP4. J. Neurosci. Res. 76, 9–19 (2004). 24. Tomassy, G.S. & Fossati, V. How big is the myelinating orchestra? Cellular diversity within the oligodendrocyte lineage: facts and hypotheses. Front. Cell. Neurosci. 8, 201 (2014).

AUTHOR CONTRIBUTIONS P.D. performed the experiments. P.D. and V.F. designed the protocol, analyzed the data and wrote the manuscript. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Reprints and permissions information is available online at http://www.nature. com/reprints/index.html. 1. Kassmann, C.M. et al. Axonal loss and neuroinflammation caused by peroxisome-deficient oligodendrocytes. Nat. Genet. 39, 969–976 (2007). 2. Bartzokis, G. Brain myelination in prevalent neuropsychiatric developmental disorders: primary and comorbid addiction. Adolesc. Psychiatry 29, 55–96 (2005). 3. Irion, S., Nostro, M.C., Kattman, S.J. & Keller, G.M. Directed differentiation of pluripotent stem cells: from developmental biology to therapeutic applications. Cold Spring Harb. Symp. Quant. Biol. 73, 101–110 (2008). 4. Murry, C.E. & Keller, G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 132, 661–680 (2008). 5. Wichterle, H., Lieberam, I., Porter, J.A. & Jessell, T.M. Directed differentiation of embryonic stem cells into motor neurons. Cell 110, 385–397 (2002). 6. Nistor, 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 49, 385–396 (2005). 7. Izrael, M. et al. Human oligodendrocytes derived from embryonic stem cells: effect of noggin on phenotypic differentiation in vitro and on myelination in vivo. Mol. Cell. Neurosci. 34, 310–323 (2007). 8. Hu, B.Y., Du, Z.W., Li, X.J., Ayala, M. & Zhang, S.C. Human oligodendrocytes from embryonic stem cells: conserved SHH signaling networks and divergent FGF effects. Development 136, 1443–1452 (2009). 9. Hu, B.Y., Du, Z.W. & Zhang, S.C. Differentiation of human oligodendrocytes from pluripotent stem cells. Nat. Protoc. 4, 1614–1622 (2009).

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Cell line

N

O4+ (%)

Mean ± s.e.m. (% )

MS100101MR-01

4

40, 71, 72, 74

61.8 ± 7.6

MS100301MR-01

4

55, 61, 61, 68

61.3 ± 2.7

MS100601MR-01

1

48

MS100801MR-01

3

55, 60, 70

61.7 ± 4.2

MS100901MR-01

3

29, 37, 73

46.2 ± 13.7

MS101001MR-01

2

46, 47

46.4 ± 0.4

PD002901MR-01

4

43, 47, 58, 76

56.1 ± 7.2

PD009601MR-01

1

28

RUES1

5

36, 54, 68, 78, 80

62.9 ± 8.2

Cells were stained with O4 antibody and analyzed by flow cytometry. One hESC line (RUES1) and eight hiPSC lines were tested. Technical replicates were performed using different batches of each line, at different passages. N = number of technical repeats. Results are also expressed as mean percentages ± s.e.m.