This work was supported by a fellowship for advanced researchers from the Swiss National Science Foundation and through grants from NYSTEM (C026446; C026447) and the Tri-institutional stem cell initiative (Starr Foundation).

1. Preparation of Culture Media, Coated Dishes and Maintenance of hPSCs
1.1 Media preparation
Note: Filter all media for sterilization and store at 4 &#176;C in the dark for up to 2 weeks. Reagent names, company and catalog numbers are listed in the Materials&#160;Table.
<ol>
	<li>DMEM/10%FBS: Combine 885 ml DMEM, 100 ml FBS, 10 ml Pen/Strep and 5 ml L-Glutamine.</li>
	<li>HES-medium: Combine 800 ml DMEM/F12, 200 ml KSR, 5 ml L-Glutamine, 5 ml Pen/Strep, 10 ml MEM minimum essential amino acids solution, 1 ml &#946;-Mercaptoethanol. Add 10 ng/ml FGF-2 after filtering the medium. 
	CAUTION: &#946;-Mercaptoethanol is toxic, avoid inhalation, ingestion and skin contact.</li>
	<li>KSR-differentiation medium: Combine 820 ml Knockout DMEM, 150 ml KSR, 10 ml L-Glutamine, 10 ml Pen/Strep, 10 ml MEM minimum essential amino acids solution and 1 ml &#946;-Mercaptoethanol.</li>
	<li>N2-differentiation medium: Dissolve 12 g DMEM/F12 powder in 980 ml dH2O, add 1.55 g Glucose, 2 g Sodium Bicarbonate and 100 mg APO human transferrin. Mix 2 ml dH2O with 25 mg human insulin and 40 &#956;l 1 N NaOH, add the dissolved solution to the medium. Add 100 mM putrescine dihydrochloride, 0.5 mM selenite, 1 mM progesterone and bring the volume up to 1 L with dH2O.</li>
</ol>
1.2 Coating of culture dishes
<ol>
	<li>Matrigel coating: Thaw 1 ml frozen matrigel aliquot by pipetting 19 ml DMEM/F12 over the aliquot until it has dissolved. Remove clumps by passing it through a 40 &#956;m cell strainer and plate 8 ml/10 cm dish. Incubate the dishes for 1 hr at RT. Aspirate the matrigel immediately before plating the cells. 
	Note: Work quickly with matrigel because it can clump at temperatures above 4 &#176;C.</li>
	<li>PO/Lam/FN coating: Add 10 ml of 1x PBS containing 15 &#956;g/ml Poly-L Ornithin hydrobromide to a 10 cm dish. Incubate the dish over night at 37 &#176;C. Wash plates with 1x PBS once and add 10 ml/10 cm dish of 1x PBS containing 2 &#956;g/ml mouse Laminin-I and 2 &#956;g/ml fibronectin. Incubate the dishes over night at 37 &#176;C. Before plating the cells, completely remove the solution and let the plates dry thoroughly at RT for 15-20 min by standing them up onto the tissue culture hood wall without the lid on.&#160; 
	Note: The dishes are completely dry and ready for cell plating when one can see crystal structures on the surface (visible by eye). The plates can be kept at RT for a few hours in this dried state. PO/Lam/FN dishes have to be prepared 2 days ahead of time. In emergency cases Lam/FN can be incubated for 2-4 hr&#160;only. However, this may risk suboptimal differentiation/survival results.</li>
</ol>
1.3 Maintenance of hPSCs
Note: hPSCs are maintained on 0.1% gelatin and mitotically inactivated mouse embryonic fibroblasts (MEFs) in HES-medium supplemented with 10 ng/ml FGF-2 as described previously 10,12. The cells should be split every 6-8 days.
<ol>
	<li>Coat a 10 cm dish with 8 ml 0.1% gelatin (in 1x PBS without Magnesium or Calcium) at RT for 5 min.</li>
	<li>Thaw frozen MEFs quickly in a 37 &#176;C water bath. Add 1 million MEFs to 10 ml DMEM/10% FBS.</li>
	<li>Aspirate the gelatin and plate the cells. Incubate at 37 &#176;C for at least 6 hr.</li>
	<li>Aspirate the DMEM/10% FBS from the prepared MEF plate, wash plate with 1x PBS once and add 10 ml HES-medium supplemented with 10 &#956;M Y-27632 dihydrochloride. Allow the medium to warm up at 37 &#176;C for 20 min.&#160; 
	Note: For manual splitting of hPSCs a laminar flow hood with an embedded microscope is used. However, the cells can be passaged using appropriate alternative methods.</li>
	<li>Under a laminar flow hood with an embedded microscope, detach separate colonies using a cell lifter and let them float.</li>
	<li>Use a 1 ml syringe to aspirate the floating colonies and dispatch them onto the fresh, warm MEF plate. Transfer approximately one fourth of the cells to the new dish. Incubate at 37 &#176;C.</li>
	<li>Feed cells daily with fresh HES medium.</li>
</ol>
2. Plating of hPSCs for Differentiation
Note: hPSCs should be split or plated for differentiation when the colonies are large, but still have sharp edges with as little as possible differentiating cells at their borders (see Figure 1B). When the cells are maintained using manual passaging the colonies should be large enough to easily be seen by eye. To get the right feel for this time point one can maintain a separate hPSC dish for two weeks without passaging and watch the cells reach and pass the ideal time point for passaging/differentiating them.
<ol>
	<li>Prepare 10 cm dishes with matrigel 1 hr before starting the differentiation.&#160;Successful matrigel coating can be verified at 4x magnification.&#160;</li>
	<li>When the hPSCs are ready to be split, aspirate the medium and add 4 ml of 0.05% trypsin-EDTA to the cells.</li>
	<li>Shake the dish horizontally for 2 min vigorously until one can see the MEFs as single cells lifting off the plate under the microscope. The hPSC colonies remain attached as colonies.</li>
	<li>Immediately and thoroughly, aspirate the trypsin and let the plate stand at RT for 2-4 min.</li>
	<li>Add 2 ml of HES-medium to the plate and using a P1000 pipette, detach the cells. 
	Note: If the cells cannot be lifted off the surface easily, the plate can be incubated empty at RT for another 2-3 min.</li>
	<li>Transfer the cells to 8 ml HES-medium supplemented with 10 &#956;M Y-27632 dihydrochloride.</li>
	<li>Aspirate the matrigel from a previously prepared 10 cm dish. Plate the cells at a 1:1 or 1:2 ratio (for example: The cells from one 10 cm dish are split into two 10 cm dishes) onto the matrigel plate. Incubate at 37 &#176;C over night. 
	Note: This plating should result in approximately 100,000 cells/cm2.</li>
</ol>
3. Induction of Neural Differentiation
Note: The differentiation can be initiated (day 0) when the cells are 90-100% confluent (see Figure 1C), usually the following day. If the accurate confluency is not reached yet, the cells can be fed daily with HES-medium until they are ready for differentiation. Alternatively, the initial number of cells plated can be increased.
<ol>
	<li>On day 0 to 3, feed the cells daily with 10 ml/10 cm dish KSR-differentiation medium containing 0.1 &#956;M LDN193189 and 10 &#956;M SB431542.</li>
	<li>On day 4 and 5 feed the cells with 75% KSR-differentiation medium and 25% N2-differentiation medium both containing LDN193189 and SB431542.</li>
	<li>On day 6 and 7 feed the cells with 50% KSR-differentiation medium and 50% N2-differentiation medium both containing LDN193189 and SB431542.</li>
	<li>On day 8 and 9 feed the cells with 25% KSR-differentiation medium and 75% N2-differentiation medium both containing LDN193189 and SB431542.</li>
	<li>On day 9 and 10 prepare PO/Lam/FN dishes as indicated in 1.2.2 for replating of the cells on day 11.</li>
	<li>On day 10 feed cells with N2-differentiation medium containing LDN193189 and SB431542.</li>
</ol>
4. Replating in Droplets for NC Specification
<ol>
	<li>On day 11 aspirate Lam/FN from the previously prepared plates and let them dry completely at RT for 20-30 min, as explained in 1.2.2.</li>
	<li>Remove medium from the differentiating cells, wash the cells once with 1x PBS and add 8 ml accutase/10 cm dish. Incubate for 20 min at 37 &#176;C.</li>
	<li>Using a cell lifter, detach the cells and resuspend them in accutase with a 5 ml pipette. Transfer the suspension to a 15 ml tube.</li>
	<li>Add 5 ml of 1x PBS and spin the cells for 5 min at 114 x g.</li>
	<li>Resuspend the cells in 10 ml 1x PBS. To ensure a single cell suspension, filter them through a 40 &#956;m cell strainer and count the cells using a hemocytometer or equivalent technique.</li>
	<li>Spin the cells down and resuspend them in N2-differentiation medium containing 200 &#956;M Ascorbic Acid (AA), 20 ng/ml BDNF, 100 ng/ml FGF8, 20 ng/ml SHH, 10 &#956;M Y-27632 dihydrochloride at the concentration of 100,000-150,000 cells/10 &#956;l.</li>
	<li>Using a repeat-pipettor, plate 10 &#956;l droplets close to each other without them touching onto the dried PO/Lam/FN 15 cm dishes. 
	Note: One 10 cm dish of differentiated cells normally results in approximately 2-3&#160;times 15 cm dishes or approximately 100 x 10 &#956;l droplets/15 cm dish.</li>
	<li>Let the droplets stand at RT for 20-30 min, then very carefully (not to disturb the droplets) add 30 ml of N2/AA/BDNF/FGF8/SHH/Y and incubate at 37 &#176;C.</li>
	<li>On day 12, carefully feed the cells with 20 ml/15 cm dish N2/AA/BDNF/FGF8/SHH.</li>
	<li>From day 14-17 feed the cells every second or third day with N2/AA/BDNF/FGF8.</li>
	<li>On day 16 and 17 prepare PO/Lam/FN plates to replate NC cells after FACS sorting.</li>
</ol>
5. Fluorescence Activated Cell Sorting (FACS) of NC cells
Note: The preparation of the cells for FACS requires approximately 2 hr.
<ol>
	<li>On day 18 remove medium, wash the cells once with 1x PBS in the dish and add 12 ml accutase. Incubate the cells for 20 min at 37 &#176;C.</li>
	<li>Using a cell lifter, detach the cells from the surface and let them float. Pipette the cells into suspension using a 5 ml pipette, transfer them to a 50 ml tube and add 30 ml of 1x PBS. Spin the cells for 5 min at 114 x g.</li>
	<li>Using a P1000 pipette, resuspend the cells in 1 ml of 2% FBS/HBSS (the HBSS contains 15 mM HEPES). Add 19 ml of 2% FBS/HBSS and filter the cells through a 40 &#956;m cell strainer to ensure a single cell suspension. Incubate the cells on ice for 15 min for antibody blocking and then count them using a hemocytometer.</li>
	<li>Spin the cells down and resuspend them at the concentration of 10 million cells/ml in 2% FBS/HBSS.</li>
	<li>Set aside 1 million cells each for the unstained, single-stained (HNK-1 only and p75 only) and secondary antibody stained only (APC only and 488 only) FACS controls.</li>
	<li>Add 5 &#956;l of HNK-1 and 5 &#956;l of p75 primary antibody per 10 million cells in 1 ml suspension to the proper samples and incubate for 20 min on ice.</li>
	<li>Wash cells in 10 ml 1x PBS (centrifuge 5 min at 114 x g) and resuspend the cells in 1 ml 2% FBS/HBSS. Add 2 &#956;l of APC and 1 &#956;l of 488 secondary antibody per 10 million cells in 1 ml suspension to the proper samples and incubate for 20 min on ice in the dark. 
	Note: APC and 488 are the secondary antibodies used here for the HNK-1 and p75 staining, respectively.</li>
	<li>Wash the cells in 10 ml 1x PBS twice, resuspend 10 million cells in 500 &#956;l of 2% FBS/HBSS containing DAPI (at 0.5 ng/&#956;l) or alternative appropriate live cell stain&#160;to exclude dead cells from the FACS analysis.</li>
	<li>Transfer the samples to appropriate FACS tubes.</li>
	<li>Prepare FACS tubes with 0.5 ml 2% FBS/HBSS for cell collection. 
	Note: The cells and collection tubes are kept on ice in the dark during the sorting time.</li>
	<li>Using a cell sorting machine with lasers that can detect DAPI, APC and 488 sort DAPI-/HNK-1+/P75+ double positive cells. 
	Note: Preparation time and handling of the cells leads to a small percentage of cell death, DAPI stains dead cells only and thus allows for these cells to be excluded from the sorted population. Any alternative live/dead stain works equally well for this purpose. DAPI itself does not cause cytotoxicity in the live cell population.</li>
</ol>
6. Replating of Sorted Cells, NC Maintenance and Expansion
Note: FACS sorted cells should be handled with special care to ensure optimal survival. Keep them on ice until replating them. Do not vortex or pipette them harshly. The cells can be resuspended by flicking the tube.
<ol>
	<li>After FACS sorting, count the NC cells, then spin them down and resuspend in N2-differentiation medium supplemented with 10 ng/ml FGF2, 20 ng/ml EGF and 10 &#956;M Y-27632 dihydrochloride in the volume appropriate to replate them at 30,000 cells/10 &#956;l droplets.</li>
	<li>Thoroughly dry previously prepared PO/Lam/FN plates and plate approximately 50-70 droplets per 10 cm dish. Let the dishes stand at RT for 20-30&#160;min.</li>
	<li>Very carefully add 20 ml/10 cm dish of N2/FGF2/EGF/Y-27632 without disturbing the droplets. Incubate the cells at 37 &#176;C.</li>
	<li>Feed the cells every 2-3 days with N2/FGF2/EGF. 
	Note: NC cells are expanded or passaged approximately every 4-5 days or when they start piling up within the droplets.</li>
	<li>To passage the NC cells, remove the medium, wash the cells once with 1x PBS and add 8 ml accutase/10 cm dish. Incubate for 20 min at 37 &#176;C.</li>
	<li>Pipette the cells off the plate using a 5 ml pipette and transfer them to a 15 ml tube. Add 6 ml 1x PBS.</li>
	<li>Spin the cells for 5 min at 114 x g.</li>
	<li>Resuspend the cells in N2-differentiation medium supplemented with 10 ng/ml FGF2, 20 ng/ml EGF and 10 &#956;M Y-27632 dihydrochloride in order to have 20,000 cells/10 &#956;l droplet.</li>
	<li>Replate the cells in 10 &#956;l droplets onto dried PO/Lam/FN plates. Let the dishes stand at RT for 20-30 min.</li>
	<li>Carefully add 20 ml/10 cm dish of N2/FGF2/EGF/Y-27632. Incubate at 37 &#176;C. 
	Note: NC cells can be maintained or expanded for up to 2 weeks as a relatively homogeneous progenitor population. Longer culture may lead them to differentiate into various progeny cell types.</li>
</ol>

<ol>
	<li>Lee, G., Studer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell+technology+for+the+study+of+human+disease.">Induced pluripotent stem cell technology for the study of human disease.</a> Nat Methods. 7, 25-27 (2010).</li><li>Lee, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+pathogenesis+and+treatment+of+familial+dysautonomia+using+patient-specific+iPSCs.">Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs.</a> Nature. 461, 402-406 (2009).</li><li>Ebert, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells+from+a+spinal+muscular+atrophy+patient.">Induced pluripotent stem cells from a spinal muscular atrophy patient.</a> Nature. 457, 277-280 (2009).</li><li>Lee, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+screening+using+familial+dysautonomia+induced+pluripotent+stem+cells+identifies+compounds+that+rescue+IKBKAP+expression.">Large-scale screening using familial dysautonomia induced pluripotent stem cells identifies compounds that rescue IKBKAP expression.</a> Nat Biotechnol. 30, 1244-1248 (2012).</li><li>Zimmer, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+developmental+toxicants+and+signaling+pathways+in+a+functional+test+based+on+the+migration+of+human+neural+crest+cells.">Evaluation of developmental toxicants and signaling pathways in a functional test based on the migration of human neural crest cells.</a> Environ Health Perspect. 120, 1116-1122 (2012).</li><li>Dupin, E., Sommer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+progenitors+and+stem+cells:+from+early+development+to+adulthood.">Neural crest progenitors and stem cells: from early development to adulthood.</a> Dev Biol. 366, 83-95 (2012).</li><li>McKeown, S. J., Stamp, L., Hao, M. M., Young, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hirschsprung+disease:+a+developmental+disorder+of+the+enteric+nervous+system.">Hirschsprung disease: a developmental disorder of the enteric nervous system.</a> Wiley Interdiscip Rev Dev Biol. 2, 113-129 (2013).</li><li>Slaugenhaupt, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+expression+of+a+splicing+mutation+in+the+IKBKAP+gene+causes+familial+dysautonomia.">Tissue-specific expression of a splicing mutation in the IKBKAP gene causes familial dysautonomia.</a> Am J Hum Genet. 68, 598-605 (2001).</li><li>Cheung, N. K., Dyer, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroblastoma:+developmental+biology,+cancer+genomics+and+immunotherapy.">Neuroblastoma: developmental biology, cancer genomics and immunotherapy.</a> Nat Rev Cancer. 13, 397-411 (2013).</li><li>Lee, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+directed+differentiation+of+neural+crest+stem+cells+derived+from+human+embryonic+stem+cells.">Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells.</a> Nat Biotechnol. 25, 1468-1475 (2007).</li><li>Lee, G., Chambers, S. M., Tomishima, M. J., Studer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+neural+crest+cells+from+human+pluripotent+stem+cells.">Derivation of neural crest cells from human pluripotent stem cells.</a> Nat Protoc. 5, 688-701 (2010).</li><li>Chambers, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+neural+conversion+of+human+ES+and+iPS+cells+by+dual+inhibition+of+SMAD+signaling.">Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling.</a> Nat Biotechnol. 27, 275-280 (2009).</li><li>Itoh, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducible+establishment+of+hemopoietic+supportive+stromal+cell+lines+from+murine+bone+marrow.">Reproducible establishment of hemopoietic supportive stromal cell lines from murine bone marrow.</a> Exp Hematol. 17, 145-153 (1989).</li><li>Barberi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+subtype+specification+of+fertilization+and+nuclear+transfer+embryonic+stem+cells+and+application+in+parkinsonian+mice.">Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice.</a> Nat Biotechnol. 21, 1200-1207 (2003).</li><li>Elkabetz, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+ES+cell-derived+neural+rosettes+reveal+a+functionally+distinct+early+neural+stem+cell+stage.">Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage.</a> Genes Dev. 22, 152-165 (2008).</li><li>Mica, Y., Lee, G., Chambers, S. M., Tomishima, M. J., Studer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+neural+crest+induction,+melanocyte+specification,+and+disease-related+pigmentation+defects+in+hESCs+and+patient-specific+iPSCs.">Modeling neural crest induction, melanocyte specification, and disease-related pigmentation defects in hESCs and patient-specific iPSCs.</a> Cell Rep. 3, 1140-1152 (2013).</li><li>Molofsky, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bmi-1+dependence+distinguishes+neural+stem+cell+self-renewal+from+progenitor+proliferation.">Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation.</a> Nature. 425, 962-967 (2003).</li></ol>

The authors have no conflicting interests to disclose.

For the successful differentiation of R-NC cells from hESC/hiPSCs the following considerations should be made. It is critical to work under sterile culture conditions at all times. In particular, it is important to test hPSC cultures for mycoplasma contamination regularly, since this contamination will hinder successful differentiation, but cannot readily be detected visually in hPSC cultures. The R-NC differentiation should be initiated at 90-100% cell density; lower cell density affects cell survival and efficiency of ...

Human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) have shown immense potential, in particular for the investigation and future treatment of human diseases for which neither good animal models nor primary tissues are available. Application examples for the hESC/hiPSC technology are the following: Cells of particular interest can be generated from hESC/hiPSCs for regenerative medicine at unlimited quantity1. Cells can be produced from patients carrying a specific disease and used to establish in vitro disease models2,3. Such disease models can then be employed for large-scale drug screening in the quest for new drug compounds4 as well as testing of existing drugs for efficacy and toxicity5. In vitro disease models can lead to the identification of novel disease mechanisms. For all applications of the hESC/iPSC technology it is important to work with specific, well-defined cell types affected in the disease of interest. Thus, the availability of solid and reproducible in vitro differentiation protocols is crucial for all applications of the hESC/hiPSC technology. Protocols are desirable that show minimal variability, time expense, effort, difficulty and cost as well as maximal reproducibility among hESC/hiPSC lines and different researchers.
Neural crest (NC) cells emerge during vertebrate neurulation between the epidermis and the neural epithelium. They proliferate and migrate extensively throughout the developing embryo and give rise to an impressive diversity of progeny cell types, including bone/cartilage, the craniofacial skeleton, sensory nerves, Schwann cells, melanocytes, smooth muscle cells, enteric neurons, autonomic neurons, chromaffin cells, cardiac septum cells, teeth and adrenal/thyroid glandular cells6. Thus, NC cells are an attractive cell type for the stem cell field and important for the modeling of a variety of diseases, such as Hirschsprung's disease7, Familial Dysautonomia8 as well as cancers such as neuroblastoma9. Furthermore, they offer the possibility to study aspects of human embryonic development in vitro.
The currently available and widely applied in vitro differentiation protocol for the derivation of NC cells from hESCs10,11 requires up to 35 days of differentiation and it involves neural induction on stromal feeder cells such as MS5 cells and is thus performed under poorly defined conditions. While it can be up-scaled to generate large quantities of NC cells, for example required for high-throughput drug screening4, this is labor and cost intensive. Furthermore, it involves manual passaging of neural rosettes, which can be difficult to reproduce and thus is subject to overall variability, in particular when it is applied to a large variety of hESC or hiPSC lines. Here, the stepwise derivation of NC cells in an 18-day protocol that is free of feeder cells is shown. This method is shorter and more defined than the currently used protocol. Furthermore, it is very robust in generating NC cells among different hiPSC lines. Importantly, it is shown that the NC cells yielded by both protocols emerge at the border of neural rosettes (hereafter termed rosette-NC or R-NC). The cells derived using either of the two protocols look morphologically identical, they express the same NC markers and cluster together in microarray analysis. NC cells derived using the new protocol (R-NC) are functional, similar to NC cells derived using the old protocol (MS5-R-NC) such that they can migrate and further differentiate into neurons. Therefore, the cells can be used concurrently with the MS5-R-NC cells. The R-NC cell protocol for the derivation of NC cells from hESC/iPSC will be useful for all applications of the hESC/iPSC technology involving the NC lineage.

<table border="0" cellpadding="0" cellspacing="0" style="width:897px;" width="897">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Regents</td>
			<td style="width:219px;">company</td>
			<td style="width:160px;">cataloge number</td>
			<td style="width:165px;">comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Gibco-Life Technologies</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM/F12</td>
			<td>Gibco-Life Technologies</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Knockout Serum Replacement</td>
			<td>Gibco-Life Technologies</td>
			<td>10828-028</td>
			<td>Lot should be tested</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine</td>
			<td>Gibco-Life Technologies</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicinlin/Streptomycin</td>
			<td>Gibco-Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM minimum essential amino acids solution&#160;</td>
			<td>Gibco-Life Technologies</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#946;-Mercaptoethanol&#160;</td>
			<td>Gibco-Life Technologies</td>
			<td>21985-023</td>
			<td>toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recombinant human FGF basic (FGF2)</td>
			<td>R&#38;D Systems</td>
			<td>233-FB-001MG/CF</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Knockout DMEM</td>
			<td>Gibco-Life Technologies</td>
			<td>10829-018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM/F12 powder&#160;</td>
			<td>Invitrogen</td>
			<td>12500-096</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Bicarbonate&#160;</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">APO human transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human insulin&#160;</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Putriscine dihydrochloride</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel matrix</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Poly-L Ornithin hydrobromide</td>
			<td>Sigma</td>
			<td>P3655</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse Laminin-I</td>
			<td>R&#38;D Systems</td>
			<td>3400-010-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin</td>
			<td>BD Biosciences</td>
			<td>356008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dispase in Hank&#39;s Balanced Salt Solution 5U/ml</td>
			<td>Stem Cell Technologies</td>
			<td>7013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA&#160;</td>
			<td>Gibco-Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y-27632 dihydrochloride</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LDN193189</td>
			<td>Stemgent</td>
			<td>04-0074</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SB431542</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1614</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ascorbic Acid&#160;</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BDNF</td>
			<td>R&#38;D Systems</td>
			<td>248-BD</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FGF8</td>
			<td>R&#38;D Systems</td>
			<td>423-F8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse recombinant sonic hedgehog (SHH)</td>
			<td>R&#38;D Systems</td>
			<td>464SH</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>Gibco-Life Technologies</td>
			<td>14170-112</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Gibco-Life Technologies</td>
			<td>1563-080</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human recombinant EGF</td>
			<td>R&#38;D Systems</td>
			<td>236EG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">gelatin (PBS without Mg/Ca)</td>
			<td>in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibodies:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti HNK-1/N-Cam (CD57) mIgM</td>
			<td>Sigma</td>
			<td>C6680-100TST</td>
			<td>lot should be tested</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-p75 mIgG1 (Nerve Growth factor receptor)</td>
			<td>Advanced Targeting Systems</td>
			<td>AB-N07</td>
			<td>lot should be tested</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">APC rat anti-mIgM</td>
			<td>BD Parmingen</td>
			<td>550676</td>
			<td>lot should be tested</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AlexaFluor 488 goat anti-mouse IgG1&#160;</td>
			<td>Invitrogen</td>
			<td>A21121</td>
			<td>lot should be tested</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti Oct4 mIgG2b (used at 1:200 dilution)</td>
			<td>Santa Cruz</td>
			<td>sc-5279</td>
			<td>lot should be tested</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Material/Equipment:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse embryonic fibroblasts (7 million cells/vial)</td>
			<td>GlobalStem</td>
			<td>GSC-6105M</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture dishes: 10cm and 15 cm plates, centrifuge tubes, FACS tubes,&#160; pipetts, pipet tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass hematocytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture incubator (CO2, humidity and temperature controlled)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture laminar flow hood with embedded microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture biosafety hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell sorting machine, i.e. MoFlo</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Inverted microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 ml TB syringe 27Gx1/2</td>
			<td>BD Biosciences</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell lifter Polyethylene</td>
			<td>Corning Incorporated</td>
			<td>3008</td>
			<td></td>
		</tr>
	</tbody>
</table>

feeder-free derivation of neural crest progenitor cells from human pluripotent stem cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

The two most important improvements of the R-NC protocol over the MS5-R-NC protocol&#160;11 are the feeder-free, defined differentiation conditions and the overall shortening of the time requirement. MS5 feeder cells&#160;13 are murine bone-marrow derived stromal cells that have been shown to support neural differentiation from hESCs&#160;14. HESCs cultured on MS5 feeder cells at low density form epithelial structures and neural rosettes&#160;15, at the periphery of which NC ce...

Watch this Scientific Journal Video about Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells at JoVE.com

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

feeder-free-derivation-neural-crest-progenitor-cells-from-human

Research

JoVE Journal

Neuroscience

14.1K Görüntüleme.  Sloan-Kettering Institute for Cancer Research. Insan pluripotent kök hücreleri (hPSC) türetilmiş nöral krest (NC) hücreler, insan gelişimi ve hastalık modelleme ve hücre replasman tedavileri için büyük bir potansiyele sahiptir. Burada, şu anda yaygın olarak kullanılan bir besleyici ücretsiz adaptasyon In vitro HPSCs NC hücrelerin derivasyon için farklılaşma protokolü sunulmuştur.

Video: Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...