Directed differentiation of human pluripotent cells to neural crest stem ...

protocol

Directed differentiation of human pluripotent cells to neural crest stem cells Laura Menendez1, Michael J Kulik1, Austin T Page2, Sarah S Park3, James D Lauderdale2, Michael L Cunningham3,4 & Stephen Dalton1 1Department of

Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA. 2Department of Cellular Biology, Paul D. Coverdell Center for Biomedical and Health Sciences, University of Georgia, Athens, Georgia, USA. 3Department of Pediatrics, University of Washington, Seattle, Washington, USA. 4Seattle Children’s Research Institute, Seattle, Washington, USA. Correspondence should be addressed to S.D. ([email protected]).

© 2013 Nature America, Inc. All rights reserved.

Published online 3 January 2013; doi:10.1038/nprot.2012.156

Multipotent neural crest stem cells (NCSCs) have the potential to generate a wide range of cell types including melanocytes; peripheral neurons; and smooth muscle, bone, cartilage and fat cells. This protocol describes in detail how to perform a highly efficient, lineage-specific differentiation of human pluripotent cells to a NCSC fate. The approach uses chemically defined media under feeder-free conditions, and it uses two small-molecule compounds to achieve efficient conversion of human pluripotent cells to NCSCs in ~15 d. After completion of this protocol, NCSCs can be used for numerous applications, including the generation of sufficient cell numbers to perform drug screens, for the development of cell therapeutics on an industrial scale and to provide a robust model for human disease. This protocol can be also be applied to patient-derived induced pluripotent stem cells and thus used to further the knowledge of human disease associated with neural crest development, for example, Treacher-Collins Syndrome.

INTRODUCTION For human pluripotent cells to have utility in cell therapy, disease modeling and drug discovery, desired cell types must be produced in an efficient manner, essentially free of contaminating lineages. In the case of neural progenitor cells (NPCs) and neural derivatives, this has been achieved by applying our understanding of lineage development in the embryo to tissue culture, enabling cranial neurons to be generated with high efficiency1. This general strategy has markedly enhanced the potential utility of pluripotent cells in approaches directed toward curing human neurological disease. In this protocol, we describe efficient lineage-specific differentiation of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) to a neural crest stem cell (NCSC) fate. This technology can be applied to the modeling of neural crest–related human diseases (neurocristopathies), and we illustrate how to do this using cells derived from patients with Treacher-Collins Syndrome (TCS). This protocol was proven to work in Menendez et al.2. NCSCs in embryonic development Neural crest cells are a transient population arising from the neural plate border at the time of neural tube closure. After delamination from the roof plate, NCSCs migrate to different regions of the embryo, depending on where they lie along the anterior-posterior (A-P) axis. During the migration process, NCSCs retain a characteristic phenotype, but upon reaching their target tissue they differentiate into a wide range of cell types depending on their origin. Cranial-derived NCSCs differentiate into sensory neurons, Schwann cells, melanocytes and cells that make up the craniofacial structures such as bone and cartilage. The more medial or vagal NCSC derivatives contribute to Schwann cells and melanocytes but also to enteric nerves, such as those associated with the gut, and to smooth muscle cells that contribute to the heart valves. The most posterior population of (trunk) NCSCs generate chromaffin cells, melanocytes, Schwann cells and a wide range of neurons that contribute to the sensory and autonomic nervous systems3–5.

Overall, NCSCs contribute to the craniofacial skeleton, the cornea, teeth, thyroid and thymus, septae of the heart, the adrenal gland, pigment cells in the skin, broadly distributed sensory nerves, autonomic nerves and Schwann cells. The broad developmental potential of NCSCs makes them of great interest to developmental biologists who often refer to them as being the fourth embryonic germ layer, in addition to the mesoderm, ectoderm and endoderm. These cells are not only of major developmental significance, but they are also related to a wide range of human diseases, which makes them of great clinical relevance (Table 1). Differentiation of human pluripotent cells to NCSCs Our strategy for developing a method for lineage-specific differentiation of human pluripotent cells to NCSCs was guided by our understanding of neural crest biology in the early vertebrate embryo (for detailed reviews see refs. 4,6) and from studies in which human pluripotent stem cells have been specified toward neuroectoderm fates1,7. In the latter case, simultaneous inhibition of transforming growth factor (TGF)-β- and bone morphogenetic protein (BMP)dependent Smad signaling directs cells toward a culture that predominantly consists of Pax6 + NPCs, with only small amounts of neural crest–like cells. As NCSCs and NPCs are derived from common ectoderm progenitor cells, we reasoned that dual Smad inhibition could also be required for NCSC specification, in combination with other factors. Clues that help in the identification of additional factors required for efficient NCSC differentiation came from studies showing that canonical Wnt signaling is required for neural crest induction at the neural plate border8,9. Consequently, we were able to show that Smad inhibition combined with activation of the Wnt pathway allows for a single-step, highly efficient method for the generation of NCSCs from hESCs and hiPSCs2. This is performed in chemically defined medium using smallmolecule inhibitors of TGF-β signaling and glycogen synthase kinase 3 (Gsk3) activity, both of which are available commercially. This method is a major improvement over previously developed

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protocol Table 1 | Neural crest–associated diseases. Disease DiGeorge syndrome Treacher-Collins syndrome

CHARGE syndrome


Waardenburg syndrome

Description Defects in heart, palate, thymus, facial features; learning disabilities Hypoplasia of facial bones, defects in ear development and eye function, cleft palate Defects in CNS, heart, hearing; growth and/or developmental retardation; genital and urinary tract defect Hearing loss; eye and hair pigment abnormality

Hirschsprung’s disease

Intestinal aganglionosis

Congenital heart disease

Outflow tract defects are common

Familial dysautonomia

Problems with formation and function of parasympathetic and sympathetic neurons

Pediatric and adult cancer

Examples include neuroblastoma and melanoma

Piebaldism

Pigment defects as a result of problems with melanocyte formation

Axenfeld-Rieger syndrome

Glaucoma; malformations of eye, teeth and skeleton

Goldenhar syndrome

Malformation of palate, ear, nose, lip and jaw

methods for several reasons. First, other methods often use coculture on feeder layers to generate neural crest. Second, by using other methods, generation of 10% neural crest cells in cultures is quite typical, and the cells require FACS sorting to obtain enriched populations7,10,11. Our protocol requires no feeder layers and achieves high enrichment (typically >90%) in a single-step method, all performed in chemically defined medium using small-molecule compounds to promote differentiation. NCSCs produced under the conditions described here can be maintained as a stable, self-renewing population for over 30 passages. NCSCs can be clonally amplified, frozen and thawed without loss of potency or self-renewal potential. Moreover, they can be differentiated into a wide range of differentiated cell types including smooth muscle cells, adipocytes, chondrocytes, osteocytes and peripheral neurons (Fig. 1).

Day 0 (Step 1)

Collection of scalp tissue

Week 1 (Steps 2–7)

TCS fibroblast culture


TCS hiPSC colony culture


TCS hiPSC singlecell culture


TCS NCSCs

Weeks 16–19 (Step 44)

TCS NCSCderived cells

Figure 1 | Schematic timetable of TCS hiPSC induction and differentiation to NCSCs.

Applications in human disease modeling Neurocristopathies are associated with a wide range of conditions including a spectrum of craniofacial defects, intestinal aganglionosis, pediatric tumors, melanoma and pediatric heart defects (Table 1). Although linkage studies have identified important genetic components associated with these diseases, a detailed mechanistic understanding is often lacking. Differentiating patient-derived induced pluripotent stem cells toward a neural crest fate therefore offers a potentially important approach to tackle this problem. Our strategy focuses on the inherited craniofacial defect TCS. Experimental design This protocol describes an efficient method for the generation of NCSCs from human pluripotent stem cells (hESCs and hiPSCs). NCSCs are capable of long-term self-renewal and retain multi potency, making them a good model for studying a wide range of neurocristopathies and for advancing our general understanding of NCSC biology. To emphasize the potential of NCSCs derived from pluripotent cells, we describe how to reprogram TCS patient cells to hiPSCs, followed by differentiation into NCSCs (Fig. 1). Although this protocol describes how to reprogram TCS patient cells, this general approach should be applicable to a wide range of neurocristopathies in addition to TCS. Alternatively, it can be used with hESCs or hiPSCs from alternative sources as the starting material, in which case the procedure should be started at Steps 37 and 22, respectively.

MATERIALS REAGENTS • TCS patient cells (containing a mutation in exon 21 of the TCOF1 gene, consistent with a diagnosis of TCS 1), hESCs or hiPSCs ! CAUTION Appropriate informed consent should be obtained from donors who contribute cells for research ! CAUTION Experiments involving the use of hESCs and hiPSCs should be performed in accordance with institutional and governmental guidelines and regulations. 204 | VOL.8 NO.1 | 2013 | nature protocols

• EmbyoMax primary mouse embryo fibroblast, neo resistant (pMEFs; Millipore, cat. no. PMEF-N) • CytoTune-iPS cell reprogramming kit (Invitrogen, cat. no. A13780) • Collagenase, type IV (Invitrogen, cat. no. 17104) • StemPro hESC serum- and feeder-free medium (SFM) (Invitrogen, cat. no. A1000701)


protocol • DMEM/F12 (Invitrogen, cat. no. 15090) • Probumin BSA (Millipore, cat. no. 82-100-5) ! CAUTION Each lot of Probumin needs to be tested for hESC culture. • Penicillin-streptomycin (Cellgro, cat. no. 30-002-CI) • l-Alanyl-l-glutamine (Cellgro, cat. no. 25-015-CI) • MEM non-essential amino acids (Cellgro, cat. no. 25-025-CI) • 2-Mercaptoethanol, 1,000× (Invitrogen, cat. no. 21985-023) ! CAUTION 2-mercaptoethanol is toxic on inhalation, on contact with skin and harmful if swallowed. • Trace elements A 1,000× (Cellgro, cat. no. 99-182-CI) • Trace elements B 1,000× (Cellgro, cat. no. 99-175CI) • Trace elements C 1,000× (Cellgro, cat. no. 99-176-CI) • ( + )-Sodium l-ascorbate (Sigma, cat. no. A4034) • Bovine transferrin (Holo form; Invitrogen, cat. no. 11107-018) • Recombinant human basic fibroblast growth factor (Fgf2; Invitrogen, cat. no. PHG0023) • Recombinant human activin A (R&D, cat. no. 338-AC) • LONGR3 IGF-I human (Sigma, cat. no. 85580C) • Heregulin β-1 (Peprotech, cat. no. 100-03) • Accutase (Innovative Cell Technologies, cat. no. AT104) • Geltrex reduced growth factor basement membrane matrix (Gibco, cat. no. A10480-02) • FBS, embryonic stem cell qualified (Atlanta Biologicals, cat. no. S10250) • Dulbecco’s PBS without calcium and magnesium (DPBS; Cellgro cat. no. 21-031) • Trypsin-EDTA, 0.05% (wt/vol) (Cellgro, cat. no. 25-052) • DMSO (Sigma-Aldrich, cat. no. D2650) • N2 plus media supplement (R&D, cat. no. AR003) • Waymouth’s medium (Invitrogen, cat. no. 11220035) • Knockout serum replacement (KSR, Invitrogen, cat. no. 10828) • SB431542 (Tocris Bioscience, cat. no. 1614) • GSK3 inhibitor IX (BIO, EMD Millipore, cat. no. 361550) • HNK1 antibody (Sigma, cat. no. C6680) • Nerve growth factor receptor (p75) antibody (Advanced Targeting Systems, cat. no. AB-N07) • Sox2 antibody (R&D, cat. no. MAB2018) • Oct4 antibody (Santa Cruz, cat. no. sc-8628) • Nanog antibody (Genetex, cat. no. GTX100863) • AP2 antibody (DSHB, cat. no. 3B5) • TRA-1-60 antibody (Invitrogen, cat. no. 41-1000) • Alexa Fluor 633 goat anti-mouse IgG1 (Invitrogen, cat. no. A21126) • Alexa Fluor 488 goat anti-mouse IgM (Invitrogen, cat. no. A21042) • Recombinant human brain-derived neurotrophic factor (BDNF; R&D, cat. no. 248-BD); Step 44B • Recombinant human β-nerve growth factor (NGF; R&D, cat. no. 256-GF); Step 44B • Recombinant human glial cell line-derived neurotrophic factor (GDNF; R&D, cat. no. 212-GD); Step 44B • Recombinant human neurotrophin-3 (NT3; R&D, cat. no. 267-N3); Step 44B • N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (cAMP; Sigma, cat. no. D0260); Step 44B • StemPro osteogenesis differentiation kit (Invitrogen, cat. no. A1007201); Step 44A • StemPro adipogenesis differentiation kit (Invitrogen, cat. no. A1007001); Step 44A • StemPro chondrogenesis differentiation kit (Invitrogen, cat. no. A1007101); Step 44A • Alizarin red S (Sigma, cat. no. A5533); Step 44A • Alcian blue 8GX (Sigma, cat. no. A5268); Step 44A • Oil Red O (Sigma, cat. no. O0625); Step 44A • Poly-l-ornithinine (Sigma, cat. no. P3655); Step 44 • Laminin (Sigma, cat. no. L2020); Step 44B • Vybrant DiO cell-labeling solution (Invitrogen, cat. no. V-22887); Step 44C • Molecular biology agarose (BioRad, cat. no. 161-3102); Step 44C • Larval zebrafish (Danio rerio, WIC strain; Zebrafish International Research Center (ZIRC)); Step 44C ! CAUTION Experiments involving the use of animals should be performed in accordance with all relevant institutional and governmental guidelines and regulations. • Sylgard (World Precision Instruments, cat. no. KwikGard); Step 44C • Protease from Streptomyces griseus (Pronase, Sigma cat. no. P5147); Step 44C

• Instant ocean sea salt (Aquatic EcoSystems, cat. no. IO200); Step 44C • Sodium chloride (NaCl; Sigma Aldrich, cat. no. S7653); Step 44C • Potassium chloride (KCl; Sigma Aldrich, cat. no. P9333); Step 44C • Calcium chloride (CaCl2, Sigma Aldrich, cat. no. C1016); Step 44C • HEPES (Cellgro, cat. no. 25-060-Cl); Step 44C EQUIPMENT • Laminar flow hood • CO2 incubator • Microscope (i.e., Leica DMIL) • Dissecting microscope (i.e., Leica MZ16 F) • Cell culture centrifuge (i.e., AccuSpin 1R, Fisher Scientific; or similar) • Flow cytometer (i.e., CyAn ADP or MoFlo, Beckman Coulter; or similar) • Liquid nitrogen tank • Manual microsyringe pump (i.e., World Precision Instruments) • Micromanipulator (Marzhauser Wetzlar, cat. no. 00-42-107-0000) • Flaming/Brown micropipette puller (i.e., Model P-97) • Microinjection mold (Adaptive Science Tools, cat. no. PT-1) • Glass hemocytometer • Tissue culture dishes (60 mm; BD, cat. no. 35004) • Tissue culture six-well plates (six-well; BD, cat. no. 353046) • Tissue culture 12-well plates (12-well; BD, cat. no. 353043) • Petri dishes (Fisher Scientific, cat. no. 087579B) • Pyrex glass Petri dishes (VWR, cat. no. 77777-034) • Lab-Tek chamber slides (Thermo Scientific. cat. no. 177437) • Tissue culture flasks (T75, BD, cat. no. 353136; T-25, BD, cat. no. 353808) • Stericup-GP vacuum filtration system, 0.22 µm, 500 ml (Millipore, cat. no. SCGPU05RE) • Conical bottom centrifuge tubes, 15 ml (Corning, cat. no. 430790) • Conical bottom centrifuge tubes, 50 ml (Corning, cat. no. 430828) • Glass Pasteur pipettes (Corning, cat. no. 136784A) • Glass capillaries (World Precision Instruments, cat. no. TW100-4) • Plastic pipettes • Polystyrene round-bottom test tube for flow cytometry applications (BD, cat. no. 352235) • Cryogenic tubes (NUNC, cat. no. 377267) • StemPro EZPassage disposable stem cell passaging tool (Invitrogen, cat. no. 23181) • Freezing container (i.e., CoolCell, BioCision) • Geltrex-coated plates • MEF-coated plates • Microinjection mold (Adaptive Science Tools, cat. no. PT-1) • Sylgard-coated Petri dish REAGENT SETUP Complete Waymouth’s medium (500 ml) Combine 440 ml of Waymouth’s medium with 50 ml of heat-inactivated FBS and 10 ml of penicillin-streptomycin. Filter-sterilize the medium before use. Store the medium for up to 2 weeks at 4 °C. hESC maintenance medium (500 ml) Combine 432 ml of DMEM/F12, 50 ml of Probumin (20% (vol/vol) stock solution), 5 ml of penicillinstreptomycin, 5 ml of l-alanyl-l-glutamine, 5 ml of MEM non-essential amino acids, 0.5 ml of trace elements A, 0.5 ml of trace elements B, 0.5 ml of trace elements C, 0.9 ml of 2-mercaptoethanol, transferrin (10 µg ml − 1), ( + )-sodium l-ascorbate (50 µg ml − 1), Heregulin β-1 (10 ng ml − 1), activin A (10 ng ml − 1), LONGR3 IGF-I (200 ng ml − 1), and Fgf2 (8 ng ml − 1) or Stempro (Invitrogen) supplemented with Fgf2 (8 ng ml − 1). Filter-sterilize the medium before use. Store the medium for up to 1 week at 4 °C.  CRITICAL If the medium is not going to be used within 1 week, add growth factors separately to smaller aliquots of medium. Neural crest differentiation medium (50 ml) Use the hESC maintenance medium without activin A and add GSK3 inhibitor IX (BIO) (2–4 µM) and SB431542 (20 µM). Store the medium for up to 1 week at 4 °C.  CRITICAL The use of commercially available stem cell medium, such as StemPro or mTesR, is not recommended for neural crest differentiation, as the presence of activin A and/or TGF-β does not support efficient NCSC differentiation. The use of serum-rich medium or KSR medium is also not recommended. FBS medium for pMEFs and fibroblasts, 10% (vol/vol) (500 ml) Combine 440 ml of DMEM/F12, 50 ml of FBS, 5 ml of penicillin-streptomycin, 5 ml of l-alanyl-l-glutamine and 0.9 ml of 2-mercaptoethanol. Filter-sterilize the medium before use. Store the medium for up to 2 weeks at 4 °C. KSR medium, 20% (vol/vol) (500 ml) Combine 390 ml of DMEM/F12, 100 ml of KSR, 5 ml of penicillin-streptomycin, 5 ml of l-alanyl-l-glutamine, nature protocols | VOL.8 NO.1 | 2013 | 205


protocol 0.9 ml of 2-mercaptoethanol and Fgf2 (4 ng ml − 1). Filter-sterilize the medium before use. Store the medium for up to 2 weeks at 4 °C. Peripheral neuron medium (50 ml) (see PROCEDURE Step 44B) Combine DMEM/F12, 0.5 ml of N2 supplement, 0.5 ml of penicillin-streptomycin, BDNF (10 ng ml − 1), NGF (10 ng ml − 1), GDNF (10 ng ml − 1), NT3 (10 ng ml − 1), ascorbic acid (200 ìm) and cAMP (0.5 mM). Store the medium for up to 1 week at 4 °C. Cryoprotective medium (20 ml) For TCS fibroblasts, combine 18 ml of FBS and 2 ml of DMSO. For hiPSCs and hESCs, combine 18 ml of hESC maintenance medium and 2 ml of DMSO. Store the medium for up to 1 month at 4 °C. Geltrex Thaw Geltrex on ice or overnight at 4°C. Dilute it to a 1:1 dilution with DMEM/F12. Store the diluted Geltrex in 1-ml aliquots at − 20 °C.  CRITICAL Geltrex must be kept cold at all times in order to avoid gelatinization. Collagenase Mix collagenase powder (400 units) with DMEM/F12 to a final concentration of 400 units per ml. Filter-sterilize the solution before use. Collagenase solution can be kept at 4 °C for a week, or it can be stored at − 20 °C for up to 2 months. Ringer’s solution (see PROCEDURE Step 44C) Combine NaCl (final concentration 116 mM), KCl (final concentration 2.9 mM), CaCl2 (final concentration 1.8 mM) and HEPES (final concentration 5 mM) in water. Filter-sterilize the solution before use. Store the solution indefinitely at room temperature (20°–25 °C). Water, 8× (see PROCEDURE Step 44C) Dissolve 0.48 g of instant ocean sea salt in 1 liter of dH2O. Store it indefinitely at room temperature. EQUIPMENT SETUP Geltrex-coated plate Thaw a 1-ml aliquot and dilute it to a final dilution of 1:200 in DMEM/F12 before plating. Add 2 ml of the final dilution to a 60-mm tissue culture plate. Let it stand for at least 30 min at 37 °C.  CRITICAL Diluted Geltrex can be frozen again as long as it is kept cold during the process.  CRITICAL Coated plates can be stored at 37 °C in a 5% CO2 incubator for up to 5 d as long as the plates do not dry out. For longer storage at 37 °C, extra DMEM/F12 can be added after the 30-min solidification step. MEF-coated plate Thaw one tube of mitotically inactivated ( + mitomycin C) pMEFs quickly at 37 °C. Transfer the pMEFs to a tube with 10 ml of 10% (vol/vol) FBS medium and centrifuge it for 4 min at 200g at room temperature.

Aspirate the supernatant and resuspend the cells in 10% (vol/vol) FBS medium. Count the cells using a hemocytometer and plate them at a density of ~21,000 cells per cm2 in 10% (vol/vol) FBS medium. The plates should be prepared at least 4 h before use and can be stored at 37 °C in a 5% CO2 incubator for up to 5 d. Poly-l-ornithine/laminin plate Add Poly-l-ornithine (final concentration 100 µg ml − 1) to a tissue culture plate or Lab-Tek chamber slide and incubate it at 4 °C overnight or for 2 h at 37 °C. Rinse the plate or slide three times with sterile dH2O. Add laminin (final concentration 1 µg ml − 1) to a tissue culture plate or slide and incubate it at 4 °C overnight or for 2 h at 37 °C. Rinse the plate or slide three times with sterile dH2O and once with DPBS. Store the plate at 4 °C for up to 3 weeks. Rinse the plate with DMEM/F12 before use. Agarose-coated dish Mix agarose in DPBS at a concentration of 5 mg ml − 1 and heat it until the agarose dissolves completely. Filter-sterilize the agarose before use. Use a small (2–3 ml) amount to coat a 10-cm Petri dish. Swirl the plate to coat it evenly, and then remove excess liquid agarose. Let it cool down to room temperature before use.  CRITICAL Use coated plates immediately to prevent agarose from drying out. Sylgard transplant dish Fill the bottom half of a 100 × 15 mm Petri dish with Sylgard (~30 ml, Kwik-Gard start-up kit). Place the microinjection mold, supported on four small plastic supports, carefully on the Sylgard. Avoid the formation of bubbles. Allow the transplant dish to cure at 37 °C for 2 d, or until the Sylgard solidifies. By means of a scalpel, lift the microinjection mold from the dish. The transplant dish can be reused indefinitely; clean the dish with ethanol between uses and store it at room temperature. Sylgard-coated Petri dish By using the Kwik-Gard kit, coat the bottom of several Petri dishes with Sylgard. Place the dishes at 37 °C for 2 d or until the Sylgard solidifies. The Sylgard coating prevents dechorionated host embryos from sticking to the plastic and the dishes can be reused indefinitely. Zebrafish transplant apparatus Pull a glass capillary (without internal filament) on a Flaming/Brown micropipette puller (model P-97) to create a long, fine tapered tip. Break the tip to the desired diameter (typically two to three times the cell diameter) with a pair of fine-tipped forceps. Place the micropipette in a micropipette holder connected via Teflon tubing to a manual microsyringe pump filled with mineral oil. Secure the micropipette holder to the micromanipulator.

PROCEDURE Collection of fibroblasts ● TIMING 1 week 1| Slice scalp tissue from a TCS patient into pieces of 3–5 mm in diameter and culture it at 37 °C in a 5% CO2 incubator in a 12-well plate (one or two pieces per well) with 2 ml of complete Waymouth’s medium warmed at 37 °C. 2| Monitor the cells daily. No medium changes are necessary. Once the cells are confluent (5–10 d), wash the cells twice with DPBS, aspirate the DPBS and add 1 ml of 0.05% (wt/vol) trypsin-EDTA. 3| Incubate the cells at 37 °C for 4–5 min; when the cells have detached, add 1 ml of complete Waymouth’s medium warmed at 37 °C. 4| Transfer the cells to a 15-ml tube and centrifuge them for 4 min at 200g at room temperature. 5| Aspirate the supernatant and plate the cells in a T75 flask complete Waymouth’s medium warmed at 37 °C. 6| Replace complete Waymouth’s medium the next day. 7| Monitor the cells until they are confluent, and then wash and trypsinize them as described in Steps 2–4, but by using 5 ml of 0.05% (wt/vol) trypsin-EDTA instead of 1 ml. If you are not cryopreserving the cells, aspirate the supernatant and resuspend the cells in 10% (vol/vol) FBS medium.  PAUSE POINT The cells can be stored under liquid nitrogen indefinitely. To cryopreserve the cells, aspirate the supernatant and resuspend the pellet in 1 ml of fibroblast cryoprotective medium. Place the cells in a freezing container (such as a CoolCell) 206 | VOL.8 NO.1 | 2013 | nature protocols

protocol


Figure 2 | Generation of hiPSCs from TCS patients. (a) TCS fibroblasts seeded before viral transduction. (b,c) TCS hiPSC colonies grown after reprogramming (b) can be stained with Tra-1-60 antibody (c) (Steps 1–13). (d) After several passages, colonies can be adapted to single-cell culture conditions. (e–i) TCS hiPSCs should express pluripotent markers Nanog (e,f), Sox2 (g,i) and Oct4 (h,i) (Steps 14–36). See Table 2 for antibody concentrations. Scale bars, 100 µM.

and leave them in a − 80 °C freezer. After 4 h, the cells can be transferred to a liquid nitrogen tank. To recover fibroblasts, partially thaw the vial in a 37 °C water bath and transfer the contents into a T25 flask containing complete Waymouth’s medium. Incubate the cells overnight to ensure adequate cell adhesion. The next day, remove residual cryoprotective medium by replacing the medium with 10% (vol/vol) FBS medium.

a

b

d

e

Nanog

f

h

Oct4

i

g

Sox2

c

Tra-1-60

Nanog/DAPI

Sox2/Oct4/DAPI

hiPSC generation from fibroblasts using the CytoTune-iPS kit ● TIMING 6 weeks 8| Seed TCS fibroblasts on a six-well plate in 10% (vol/vol) FBS medium and incubate at 37 °C in a 5% CO2 incubator for 2 d (Fig. 2a). 9| Two days later, aspirate the medium, mix CytoTune Sendai virus with fresh 10% (vol/vol) FBS medium and add it to TCS fibroblasts (as recommended in the CytoTune-iPS reprogramming kit). 10| Aspirate the medium every other day and add 10% (vol/vol) FBS medium warmed at 37 °C. 11| Six days after viral transduction, passage the transduced fibroblasts with trypsin-EDTA as described in Steps 2–4. Aspirate the supernatant and plate the cells on mitotically inactivated MEF-coated plates in 20% (vol/vol) KSR medium. 12| Replace the 20% (vol/vol) KSR medium every day. 13| Monitor the cells daily. The colonies should start to become visible ~2 weeks after passage. To check for reprogrammed colonies, probe live cultures with Tra-1-60 antibody. Aspirate the medium and add Tra-1-60 antibody in 20% (vol/vol) KSR medium for 60 min at 37 °C (Table 2). Remove the antibody solution, wash the cells three times with 20% (vol/vol) KSR medium and add secondary antibody; incubate for 60 min at 37 °C. Remove the secondary solution and wash the cells three times with 20% (vol/vol) KSR medium. Tra-1-60 + cells can be visualized with a fluorescent microscope (Fig. 2b,c). 14| Manually pass uniform, tightly packed Tra-1-60 + colonies with a diameter between 200 and 500 µM by using a sharp tool (i.e., a 21G needle) to grid an individual colony and cut it into small pieces. 15| Use a pipette tip to lift the pieces from the plate. 16| Transfer the pieces from an individual colony to a single well of a 12-well MEF-coated plate in 20% (vol/vol) KSR medium. Incubate the cells. 17| Repeat Steps 14–16 in order to transfer multiple Tra-1-60 + colonies to separate wells. 18| Replace 20% (vol/vol) KSR medium every day. nature protocols | VOL.8 NO.1 | 2013 | 207

protocol 19| Continue expanding individual clones by manually passaging all the colonies from a well when they reach 80% confluency, as described in Steps 14–18. Rather than plating in a 12-well plate, transfer the colonies first to six-well MEF-coated plates and then to 60-mm MEF-coated plates at 37 °C in a 5% CO2 incubator.


20| Confirm that the cells are hiPSCs. We recommend doing this by karyotyping hiPSCs by fluorescence in situ hybridization (FISH) and G-banding analysis12. In addition, to be confident of their pluripotency potential, perform teratoma assays or in vitro differentiation to all three germ layers (endoderm, ectoderm and mesoderm), followed by rigorous marker analysis13. 21| Maintain TCS hiPSCs colonies by manually passaging colonies at a 1:3 (vol/vol) ratio in 60-mm MEF-coated plates as described in Steps 14–16 every 4–7 d until they have been passaged two or three times. Cells can then be adapted to collagenase passaging (as described below), maintained indefinitely by manually passing colonies, or frozen for long-term storage.  CRITICAL STEP hiPSC aliquots should be frozen and stored under liquid nitrogen at several different passages. ? TROUBLESHOOTING  PAUSE POINT For long-term storage, when cells are ready to be passaged, manually pick all colonies, centrifuge the cells for 4 min at 200g at room temperature and resuspend the pellet in 3 ml of cryoprotective medium. Freeze 1-ml aliquots of cells as detailed in the PAUSE POINT after Step 7.

Table 2 | Antibodies used in this study.

Name

ml per 106 cells

Company

Cat. no.

CD13-APC

10

EBioscience

17-0138-73

CD73-PE

20

BD

550257 17-0441-82

CD44-APC

0.33

EBIOS

p75

0.2

Advance targeting ABN07 systems

HNK1

0.2

Sigma

C6608

Name

Dilution for staining

Company

Cat. no.

p75

1:100

Advance Targeting ABN07 Systems

HNK1

1:300

Sigma

C6608

β-Tubulin III

1:500

Sigma

T8660

AP2

1:50

DSHB

3B5

Pax6

1:200

DSHB

PAX6

Sox2

1:200

R&D

MAB2018

Peripherin

1:200

Chemicon

AB1530

OCT3/4

1:200

Santa Cruz

SC-8628

Nanog

1:200

Genetex

GTX10086

Tra-1-60 1:100 Invitrogen 41-1000 Adaption of hiPSCs to StemPro medium via collagenase passaging ● TIMING 2 weeks 22| When the colonies reach ~80% confluency, aspirate the medium and add collagenase (2 ml to a 60-mm plate).

23| Use the StemPro passaging tool to cut the colonies into uniform pieces. 24| Leave the cells in collagenase at 37 °C for 15–30 min or until the colony fragments begin to lift up from the plate. 25| Collect the colonies in a 15-ml tube and centrifuge the cells for 4 min at 200g at room temperature. 26| Aspirate the supernatant and resuspend the cell pellet in 4 ml of StemPro medium warmed at 37 °C. 27| Plate the cells at a 1:4 (vol/vol) ratio onto Geltrex-coated plates. Add extra StemPro medium (a total volume of 3 ml for a 60-mm plate). 28| Maintain the cells at 37 °C in a 5% CO2 incubator. 29| Replace the StemPro medium every day. Steps 22–28 can be repeated indefinitely to maintain TCS hiPSCs, as colonies or cells can be adapted to Accutase passage as described below. Passage of hiPSCs colonies with Accutase to adapt them to single-cell growth conditions ● TIMING 2 weeks 30| When colonies reach ~80% confluency, aspirate the medium and add Accutase (2 ml to a 60-mm plate). 31| Incubate the plate at room temperature or at 37 °C for 5–10 min, or until the cells detach from the plate.

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protocol

Hnk1/DAPI

FoxD3/DAPI

i

3

10

2

10

1

10

0


TC NCSC isotype

TC NCSC P3 d16 90

2

0

10

1

Sox2/Oct4/DAPI

700 600 500 400 300 200 100 0

PAX3

hi PS N C C p4

h

10

TC hiPSC

TC hiPSC isotype

10

2

10

3

10

4

p75

Relative transcript expression

AP2/DAPI

4

10

k g

10

250

ZIC1

200 150 100

6,000

TFAP2A

5,000 4,000 3,000 2,000

50

1,000

0

0

35 30 25 20 15 10 5 0

SOX9

45 40 35 30 25 20 15 10 5 0

SOX10

25

P75NTR

20 15 10 5 0

hi PS N C C p4

f

j

p75/DAPI

hi PS N C C p4

p75/DAPI

c

hi PS N C C p4

e

TC NCSC P2 d8

hi PS N C C p4

Hnk1/DAPI

b

hi P N SC C p4

d

TC NCSC P0 d3

Hnk1

a

Figure 3 | Differentiation of TCS hiPSCs to NCSCs. (a,b) Bright-field images of TCS NCSCs after day 3 (d3) and day 8 (d8) of differentiation (Steps 40–41). (c,d) Immunocytochemistry analysis shows that TCS hiPSCs are negative for p75 and low to moderate for Hnk1 expression before differentiation to NCSCs. (e–i) At day 16 of differentiation (Step 42), TCS NCSCs are positive for p75 (e), Hnk1 (f), AP2 (g) and FoxD3 (h) and negative for Sox2 and Oct4 (i). See Table 2 for antibody concentrations. Scale bars, 100 µM. (j) Flow cytometry analysis for TCS hiPSCs before and after neural crest differentiation. The percentage of p75 + Hnk1 + cells is shown in the graph in the circled areas. (k) Real-time PCR data showing the expression of PAX3, ZIC1, TFAP2A, SOX9, SOX10 and P75NTR in TCS NCSCs relative to TCS hiPSCs (Step 42). P0, passage 0; P2, passage 2; TC, Treacher-Collins (Syndrome).

32| Collect the cells in a 15-ml tube and centrifuge for 4 min at 200g at room temperature. 33| Aspirate the supernatant and resuspend the cells in 4–5 ml of StemPro medium. 34| Count the cells with a hemocytometer and replate them at a density of ~5–8 × 104 cells per cm2 or at a 1:4 to 1:5 (vol/vol) ratio on a Geltrex-coated plate. 35| Maintain the cells at 37 °C in a 5% CO2 incubator. 36| Replace the medium every day. hiPSCs can be maintained indefinitely in StemPro or in maintenance medium (Fig. 2d). Alternatively, proceed to the next step when cells are 90% confluent. TCS hiPSCs maintained in these conditions should be positive for pluripotent markers such as Sox2, Oct4 and Nanog (Table 2, Fig. 2e–i). ? TROUBLESHOOTING Differentiation of hESC and hiPSC to NCSC ● TIMING ~15 d 37| Passage 90% confluent hiPSCs or hESCs using Accutase as described in Steps 30–35, except that cells should be resuspended and seeded in maintenance medium at a density of ~9.2 × 104 cells per cm2 onto Geltrex-coated plates. 38| The next day, aspirate the medium and add neural crest medium. 39| Replace the neural crest medium every day. 40| After 3–4 d, the cultures should be very confluent (Fig. 3a). Pass the cells with Accutase as described in Steps 30–35, except that cells should be resuspended with neural crest medium. Seed the cells at a density of 9.2 × 104 cells per cm2 onto Geltrex-coated plates. 41| Incubate the cells, replace neural crest medium daily and pass NCSCs with Accutase every 4–5 d. Morphology changes should be noticeable after day 4, and neural crest morphology should be obvious around 7–10 d of growth in neural crest medium (Fig. 3b). ? TROUBLESHOOTING 42| After 15 d of growth in neural crest medium, confirm neural crest identity. This can be done by immunocytochemistry, flow cytometry and/or RT-PCR. If you are using immunocytochemistry, NCSCs should be positive for NCSC markers such as nature protocols | VOL.8 NO.1 | 2013 | 209

protocol a

MSC d3

b

c

MSC d24

WA09 MSC d10

4

10

WA09 MSC d15

98

3

10

d

99

Chondrocytes

e

Adipocytes

2

CD73

10

1

10

0

10

0

10

10

1

CD44

f

Osteocytes

g

β-Tubulin/peripherin

h

β-Tubulin

10

i

2

3

10

0

10

10

1

10

CD13

DiO/β-tubulin

2

3

10

j

β-Tubulin/DAPI

k

β-Tubulin

l

β-Tubulin

Right eye HB D P

A


V

MB Yolk sac

Figure 4 | Differentiation of hESC-derived NCSCs to MSCs and peripheral neurons (Step 44). (a,b) Bright-field images of MSCs differentiated from WA09 hESC-derived NCSC (days 3 and 24). (c) Flow cytometry analysis for MSCs showing CD73 + CD44 + and CD73 + CD13 + cells at day 10 and day 15, respectively. (d–f) MSCs can be further differentiated to chondrocytes, adipocytes and osteocytes as shown by Alcian blue staining, Oil Red O staining and Alizarin Red staining, respectively. (g) hESC-derived NCSCs are differentiated to peripheral neurons as shown by peripherin and β-tubulin III immunocytochemistry. (h–l) hiPSCs-derived NCSCs implanted in zebrafish embryos differentiate to neurons as shown by β-tubulin III/DiO immunocytochemistry. The box in k is enlarged in l. See Table 2 for antibody concentrations. A, anterior; P, posterior; D, dorsal; V, ventral. HB, hindbrain; MB, midbrain. Scale bars, 100 µM.

p75, Hnk1, AP2 and FoxD3 (Fig. 3c–h), but they should be negative for hESCs markers (Sox2, Oct4, Nanog) (Fig. 3i) and neural progenitor markers (Pax6, Sox2). If you are performing flow cytometry, NCSCs should be p75 + and Hnk1 + (Fig. 3j). If you carry out RT-PCR, NCSCs should express genes such as PAX3, AP2, ZIC1, SOX9 and SOX10, among others (Fig. 3k)2.  CRITICAL STEP As both antibodies are raised in mice, make sure to use Alexa Fluor 488 IgM and Alexa Fluor 633 IgG1 secondary antibodies to detect Hnk1 and p75, respectively.  CRITICAL STEP Different NCSCs lines will have different levels of expression for these markers, and thus you should always use the correspondent hiPSCs as a negative control. 43| If desired, proceed to Step 44 to differentiate NCSCs. Alternatively, maintain NCSCs as described in Steps 37–41 for more than 15 passages. Periodically, flow cytometry should be used to confirm that cells are maintaining their NCSC identity. Differentiation of NCSCs ● TIMING 2–5 weeks 44| If desired, differentiate NCSCs to mesenchymal stem-like cells (MSCs; option A) and/or peripheral neurons (option B). Alternatively, NCSCs can be implanted in vivo after DiO staining (option C). (A) Differentiation to MSCs ● TIMING 5–10 d (plus 3–4 weeks of further differentiation time) (i) Treat NCSCs with Accutase as described in Steps 30–34, and then plate them at a density of 6.5 × 104 cells per cm2 in 10% (vol/vol) FBS medium onto a noncoated tissue culture dish. (ii) Change the medium every other day. (iii) Passage the cells every 4–5 d using trypsin-EDTA, as described in Steps 2–4, and plate them at a 1:4 (vol/vol) ratio. MSCs should become CD73 + , CD44 + and CD13 + by day 5 after plating (Fig. 4a–c). (iv) If desired, further differentiate these cells over 3–4 weeks to osteocytes, adipocytes and chondrocytes using StemPro osteogenesis, adipogenesis and chondrogenesis differentiation kits from Invitrogen (Fig. 4d–f). ? TROUBLESHOOTING (B) Neuronal differentiation ● TIMING 12–14 d (i) Treat NCSCs with Accutase as described in Steps 30–34, and then plate them at a density of 1 × 105 cells per cm2 in neural crest medium onto Geltrex- or Poly-l-ornithine/laminin–coated plates. (ii) After 24 h, replace it with peripheral neuron medium. (iii) Change the medium every 2 d for 12–14 d. Peripheral neurons should be β-tubulin III + and peripherin + (Fig. 4g). By using these conditions, the percentage of β-tubulin III cells should be between 70–85% (on the basis of counting at least 300 cells from three different fields of view in three different experiments11). (C) Implantation of NCSCs in vivo into zebrafish after DiO staining ● TIMING 4 d (i) Treat NCSCs with Accutase as described in Steps 30–34, and then resuspend the cells at a density of 1 × 106 cells per ml in DMEM/F12. (ii) Add 5 µl of DiO per million cells and incubate at 37 °C for 15 min. (iii) Add 5 ml of DMEM/F12 and centrifuge the mixture for 4 min at 200g at room temperature. 210 | VOL.8 NO.1 | 2013 | nature protocols


protocol (iv) Aspirate the supernatant, resuspend the cells in 5 ml of DMEM/F12 and centrifuge the mixture for 4 min at 200g at room temperature. (v) Aspirate the supernatant and plate the cells at a density of 0.5 to 1 × 105 cells per cm2 in neural crest medium onto an agarose-coated Petri dish to make NCSC spheres. (vi) Maintain the cells at 37 °C in a 5% CO2 incubator for 24–48 h. (vii) Collect the spheres in a 15-ml tube and centrifuge the tube for 1 min at 200g at room temperature. (viii) Aspirate the supernatant and wash the spheres twice with DMEM/F12 as described in Step 44C(iii − iv). (ix) Resuspend the spheres in DMEM/F12 before injections. (x) Collect wild-type zebrafish embryos in egg water and grow them to the 6-h shield stage at 28.5 °C (refs. 14–16).  CRITICAL STEP Alternatively, cells can be transplanted into chick embryos as detailed in ref. 2. Chick embryo experiments show that NCSCs can differentiate to peripheral neurons, as shown by double staining with β-tubulin III and peripherin2. (xi) Place the embryos in a glass Petri dish and dechorionate them by incubating in 0.5 mg ml − 1 Pronase for 10 min (ref.16). Rinse the embryos three times with 8× water. The chorions should be brittle, and they should fall off with agitation during rinsing. If they are not fully removed, forceps can be used to remove the remaining chorions. Care must be taken not to puncture the yolk or expose embryos to air, which will cause them to rupture. (xii) With a wide-tipped glass pipette, transfer dechorionated embryos to a Sylgard transplant mold covered with Ringer’s solution with penicillin-streptomycin solution. (xiii) Place the embryos and mold on the stage of a dissection microscope. (xiv) By using a manual microsyringe pump, aspirate the DiO-labeled NCSCs into a micropipette tip. (xv) Orient the embryo for transplant such that its midline and shield are visible and opposing the tip of the micropipette. This can be accomplished by using the micromanipulator to gently nudge the embryo with the micropipette. (xvi) By means of a micromanipulator, and under visual control, insert the micropipette into the appropriate position of the host embryo, for example, the neural crest fate map region14. (xvii) Slowly expel the NCSCs into host zebrafish embryo, taking care to minimally disturb the embryo. (xviii) Transfer the host embryos to Sylgard-coated Petri dishes in Ringer’s solution with penicillin-streptomycin solution. (xix) Grow the embryos overnight at 28.5 °C. (xx) Perform whole-mount embryo immunocytochemistry with β-tubulin III to confirm that NCSCs differentiate to neurons 24–36 h after implantation (Fig. 4h–l). ? TROUBLESHOOTING Troubleshooting advice can be found in Table 3. Table 3 | Troubleshooting table. Step

Problem

Solution

21

Low yield of hiPSC colonies

Check the troubleshooting recommendations in the CytoTune-iPS reprogramming kit

36

Low survival of hiPSCs as single cells

Maintain cells as colonies for more than one passage before switching to Accutase Seed cells at a higher density for the first passage Wait until the plate of colonies is 80 − 90% confluent and then passage with Accutase After passing with Accutase for the first time, wait until plate is at least 90% confluent to passage again Check Geltrex plates to make sure they are not dried

41

Low percentage of p75 + or HNK1 + cells

Try different BIO concentrations (0.5 − 4 mM) Check cells at a later passage

44A(iv)

Low efficiency of adipocytes, osteocytes and/or chondrocytes

Try differentiating from MSCs between day 5 and day 10 Change from FBS to differentiation medium when the plate is confluent

nature protocols | VOL.8 NO.1 | 2013 | 211

protocol ● TIMING Steps 1–36, generation of hiPSCs and further adaptation to single-cell culture: ~11 weeks Steps 37–43, differentiation of hESCs and hiPSCs to neural crest–like cells: ~15 d Step 44A, NCSC differentiation to MSCs: ~10 d (further differentiation of MSCs to osteocytes, adipocytes and chondrocytes takes 3–4 more weeks) Step 44B, NCSC differentiation to MSCs: 12–14 d Step 44C, in vivo implantation of NCSCs into zebrafish after staining: 4 d


ANTICIPATED RESULTS We have used this protocol to efficiently derive hiPSCs from TCS patient samples. These cells, or alternatively other hiPSCs and hESCs, can be consistently differentiated to NCSCs with reproducible results. Note that efficiencies and rates of differentiation can vary in different lines. Efficiencies ranging from 80 to 99% p75 + Hnk1 + cells can be generally expected. Differentiated NCSCs can be maintained for several passages; however, with time, a small percentage of cells could further differentiate into neuronal lineages. NCSCs can be differentiated efficiently after day 20 to neuronal and mesenchymal lineages. Previous reports suggest that differentiation to glial lineages might require further maturation (past day 60) of NCSCs7.

Acknowledgments This work was supported by a grant to S.D. from the National Institute for General Medical Sciences (GM085354) and a grant to J.D.L. from the Children’s Glaucoma Foundation. AUTHOR CONTRIBUTIONS L.M. performed all differentiation experiments, analyzed the data and contributed to writing the manuscript; M.J.K. generated hiPSCs from fibroblasts and performed quality control analysis; S.S.P. maintained patient fibroblasts; A.T.P. designed and performed zebrafish in vivo experiments; J.D.L. designed zebrafish in vivo experiments; M.L.C. supervised the isolation of patient fibroblasts and obtained patient consent; S.D. provided overall direction for the project, analysis of data and writing of the manuscript. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Published online at http://www.nature.com/doifinder/10.1038/nprot.2012.156. Reprints and permissions information is available online at http://www.nature. com/reprints/index.html. 1. 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). 2. Menendez, L., Yatskievych, T.A., Antin, P.B. & Dalton, S. Wnt signaling and a Smad pathway blockade direct the differentiation of human pluripotent stem cells to multipotent neural crest cells. Proc. Natl. Acad. Sci. USA 108, 19240–19245 (2011). 3. Shakhova, O. & L., Sommer Neural crest–derived stem cells. in StemBook (ed. the Stem Cell Research Community) doi:10.3824/stembook.1.51.1 (4 May 2010).

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4. Sauka-Spengler, T. & Bronner-Fraser, M. A gene regulatory network orchestrates neural crest formation. Nat. Rev. Mol. Cell Biol. 9, 557–568 (2008). 5. Le Douarin, N.M. & Dupin, E. Multipotentiality of the neural crest. Curr. Opin. Genet. Dev. 13, 529–536 (2003). 6. Betancur, P., Bronner-Fraser, M. & Sauka-Spengler, T. Assembling neural crest regulatory circuits into a gene regulatory network. Ann. Rev. Cell Dev. Biol. 26, 581–603 (2010). 7. Lee, G., Chambers, S.M., Tomishima, M.J. & Studer, L. Derivation of neural crest cells from human pluripotent stem cells. Nat. Protoc. 5, 688–701 (2010). 8. Patthey, C., Edlund, T. & Gunhaga, L. Wnt-regulated temporal control of BMP exposure directs the choice between neural plate border and epidermal fate. Development 136, 73–83 (2009). 9. Garcia-Castro, M.I., Marcelle, C. & Bronner-Fraser, M. Ectodermal Wnt function as a neural crest inducer. Science 297, 848–851 (2002). 10. Jiang, X. et al. Isolation and characterization of neural crest stem cells derived from in vitro–differentiated human embryonic stem cells. Stem Cells Dev. 18, 1059–1070 (2009). 11. Pomp, O. et al. PA6-induced human embryonic stem cell-derived neurospheres: a new source of human peripheral sensory neurons and neural crest cells. Brain Res. 1230, 50–60 (2008). 12. Heng, H.H., Windle, B. & Tsui, L.C. High-resolution FISH analysis. Curr. Protoc. Hum. Genet. 4.5.1–4.5.23 (2005). 13. Wesselschmidt, R.L. The teratoma assay: an in vivo assessment of pluripotency. Methods Mol. Biol. 767, 231–241 (2011). 14. Woo, K. & Fraser, S.E. Order and coherence in the fate map of the zebrafish nervous system. Development 121, 2595–2609 (1995). 15. Deschene, E.R. & Barresi, M.J. Tissue targeted embryonic chimeras: zebrafish gastrula cell transplantation. J. Vis. Exp. doi:10.3791/1422 (11 September 2009). 16. Westerfield, M. The Zebrafish Book: A Guide For the Laboratory Use of Zebrafish (Danio rerio) (University of Oregon Press, 2000).