This work was supported by a fellowship for melanoma researchers from the Joanna M. Nicolay Foundation and by the National Institutes of Health under Ruth L. Kirschstein National Research Service Award F31. This work was further supported through grants from NYSTEM and the Tri-institutional stem cell initiative (Starr Foundation).

NOTE: The melanocyte protocol outlined here was first demonstrated by Mica et al.
1. Preparation of Culture Medium, Coated Dishes and Maintenance of hPSCs
<ol>
	<li>Medium Preparation 
	Note: Store all medium at 4 &#176;C in the dark for up to 2 weeks. Filter all medium for sterilization.
	<ol>
		<li>Prepare DMEM/10% FBS. Mix 885 ml DMEM, 100 ml FBS, 10 ml Pen/Strep and 5 ml L-Glutamine. Filter for sterilization.</li>
		<li>Prepare hESC-medium. Mix 800 ml DMEM/F12, 200 ml KSR, 5 ml L-Glutamine, 10 ml MEM minimum essential amino acids solution, 1 ml &#946;-Mercaptoethanol, and 5 ml Pen/Strep. After filtering add 10 ng/ml FGF-2.</li>
		<li>Prepare KSR-differentiation medium: Mix 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. Filter for sterilization.</li>
		<li>Prepare 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 &#181;l 1 N NaOH; once dissolved, add the mixture to the medium. Add 100 &#181;l putrescine dihydrochloride, 60 &#181;l selenite, 100 &#181;l progesterone. Bring the final volume to 1 l with dH2O before filtering.</li>
		<li>Prepare Full melanocyte medium. Combine 50% Neurobasal medium, 30% Low glucose DMEM, and 20% MCDB201. To this add: 0.8% ITS+, 250 nM L-glutamine, 100 &#181;M Ascorbic Acid (L-AA), 50 ng/ml Cholera toxin, 50 ng/ml SCF, 0.05 &#181;M Dexamethasone, 100 nM EDN3, 4 ng/ml FGF2. Sterile filter then add remaining reagents: 2% B27 Supplement, 25 ng/ml BMP4, 3 &#181;M CHIR99021, 500 &#181;M cAMP.</li>
	</ol>
	</li>
	<li>Coating of Culture Dishes
	<ol>
		<li>Carry out coating using gelatinous protein such as Matrigel. Upon opening, aliquot and freeze Matrigel into 1 ml parts to avoid repetitive freeze thaw cycles. Thaw and re-suspend a 1 ml frozen aliquot with 19 ml DMEM/F12. Plate 5 ml onto a 10 cm dish. Incubate the dishes for 1 hr at RT. Aspirate the gelatinous protein immediately before plating the cells and wash with DMEM/F12.</li>
		<li>Carry out coating using Poly-L ornithin hydrobromide/ mouse Laminin-I/ fibronectin (PO/Lam/FN). Coat dish with PBS containing 15 &#181;g/ml Poly-L ornithin hydrobromide. Incubate O/N at 37 &#176;C in a humidified incubator. Aspirate the solution and wash the plates with PBS three times before coating with PBS containing 1 &#181;g/ml mouse Laminin-I and 2 &#181;g/ml fibronectin. Incubate the dishes O/N at 37 &#176;C in a humidified incubator.
		<ol>
			<li>Prior to plating cells, aspirate the solution and let the plates dry thoroughly without the lid in the tissue culture hood. Allow the plates to dry for approximately 10-15 min. 
			Note: The dishes are dry and ready for cell plating when crystal structures appear on the surface by eye. The plates can be kept with PBS containing LAM/FN in the incubator for two weeks, as long as the liquid does not evaporate. The plates can be kept dried at RT for a few hr.&#160;</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Maintenance of hPSCs 
	Note: hPSCs are maintained on 0.1% gelatin and mitotically inactivated mouse embryonic fibroblasts (MEFs) in hESC-medium. The cells should be split every 6-8 days.
	<ol>
		<li>Coat a 10 cm dish with 0.1% gelatin in PBS at RT for 5 min.</li>
		<li>Thaw frozen MEFs quickly in a 37 &#176;C water bath.</li>
		<li>Aspirate the gelatin and plate the cells. Plate MEFs at a density of ~50,000 cells/cm2 in DMEM/10% FBS. Incubate MEFs at 37 &#176;C O/N prior to adding hPSCs.</li>
		<li>Aspirate the DMEM/10% FBS from the prepared MEF plate, wash plate with PBS and add 10 ml hESC-medium with hPSCs. Passage the hPSCs at a ratio of 1:5/10 depending on density prior to passaging. 
		Note: If the hPSCs are being thawed or passaged as single cells, supplement with 10 &#181;M Y-27632 dihydrochloride (ROCKi) until the first passage.</li>
		<li>Feed cells daily with fresh 10 ml hESC-medium.</li>
		<li>Prior to starting a differentiation remove any pluripotent colonies that appear to contain differentiated cells, irregular borders, or transparent centers 28. Mechanically dislodge and aspirate the irregular colonies with a pipette under a laminar flow hood with a dissecting microscope.</li>
	</ol>
	</li>
</ol>
2. Plating of hPSCs for Differentiation
Note: Differentiation conditions are described for 10 cm dishes.
<ol>
	<li>Prepare 10 cm Matrigel dishes before starting the differentiation as described in Step 1.3.1.</li>
	<li>When plating hPSCs for a differentiation start with a plate of hPSCs at a density ready for passage (~80% confluent) aspirate the hESC-medium, wash with PBS and add 3 ml of 0.05% trypsin-EDTA to the cells.</li>
	<li>Vigorously shake the dish horizontally for 2 min while visualizing under the microscope until the MEFs lift off as single cells. The hPSC colonies should remain attached as colonies.</li>
	<li>Aspirate the trypsin after the MEFs have lifted but before the hPSCs colonies detach.</li>
	<li>Add 10 ml of hESC-medium containing 10 &#181;M Y-27632 dihydrochloride to the plate and detach the cells by pipetting up and down over the colonies.</li>
	<li>Aspirate the Matrigel solution from the 10 cm dish prepared in step 2.1 and wash with DMEM/F12 to remove clumps. Plate the hPSCs at a 1:2 ratio onto the Matrigel plate. Add hESC-medium containing 10 &#181;M Y-27632 dihydrochloride up to 10ml. Incubate at 37 &#176;C O/N. 
	Note: This plating should result in approximately 100,000 cells/cm2.</li>
</ol>
3. Induction of Neural Differentiation
Note: The differentiation should be initiated (day 0) when the hPSCs are 80% confluent. The cells can be fed daily with hESC-medium containing 10 &#181;M Y-27632 dihydrochloride until beginning the differentiation.
<ol>
	<li>On day 0 and day 1, feed the cells with 10 ml of KSR-differentiation medium containing 100 nM LDN193189 and 10 &#181;M SB431542.</li>
	<li>On day 2, feed the cells with 10 ml of KSR-differentiation medium containing 100 nM LDN193189, 10 &#181;M SB431542 and 3 &#181;M CHIR99021.</li>
	<li>On day 3, feed the cells with 10 ml of KSR-differentiation medium containing 10 &#181;M SB431542 and 3 &#181;M CHIR99021.</li>
	<li>On days 4 and 5 feed the cells with 15 ml of 75% KSR-differentiation medium and 25% N2-differentiation medium containing 3 &#181;M CHIR99021. 
	Note: Do not be alarmed to see a large amount of cell death in terms of floating cells. This is normal and to be expected.</li>
	<li>On days 6 and 7 feed the cells with 15 ml of 50% KSR-differentiation medium and 50% N2-differentiation medium both containing 3 &#181;M CHIR99021, 25 ng/ml BMP4, and 100 nM EDN3.</li>
	<li>On days 8 and 9 feed the cells with 20 ml of 25% KSR-differentiation medium and 75% N2-differentiation medium both containing 3 &#181;M CHIR99021, 25 ng/ml BMP4, and 100 nM EDN3.</li>
	<li>On days 9 and 10 prepare PO/Lam/FN dishes as indicated in 1.2.2 for re-plating of the cells on day 11.</li>
	<li>On day 10 feed cells with 20 ml of N2-differentiation medium containing 3 &#181;M CHIR99021, 25 ng/ml BMP4, and 100 nM EDN3.</li>
</ol>
4. Replating in Droplets for NC Specification
<ol>
	<li>On day 11 aspirate Lam/FN from the prepared PO/LAM/FN plates and dry completely.</li>
	<li>Remove medium from the Day 11 cells, wash with PBS and add 4 ml cell detachment solution such as Accutase per 10 cm dish. Incubate for 25 min at 37 &#176;C.</li>
	<li>Add 5 ml of Full Melanocyte medium to the dish and resuspend the cells by manually pipetting up and down with a 10 ml pipette until all the cells have lifted off the plate. Transfer the suspension to a 15 ml tube.</li>
	<li>Spin the cells down for 5 min at 200 x g and resuspend the cells at 2 x 106 cells per ml in Full Melanocyte medium. Count the cells using the trypan blue exclusion on a hemocytometer or equivalent technique.</li>
	<li>Plate 10 &#181;l droplets close to each other (without them touching) onto the dried PO/Lam/FN 10 cm dishes. If the plate has been sufficiently dried, the droplets should have well defined edges and not run. 
	Note: This creates a high density local environment for the cells, which is important when replating the cells, while maintaining room within the dish for expansion.</li>
	<li>Allow the droplets to stand at RT for 10-20 min to allow cells to adhere, then slowly (not to disturb the attached cells) add 10 ml of Full Melanocyte medium. Move dish to the incubator.</li>
</ol>
5. Expanding Melanocyte Progenitors
<ol>
	<li>Continue feeding with Full Melanocyte medium every 2 to 3 days. 
	Note: Pigmentation should start to be visible within clusters of cells by the end of the first week and become very clear by the second week. View the plate over a white background such as a sheet of paper to discern these early small, dark clusters. Cells will become progressively more pigmented over time, until the entire plate is uniformly pigmented (See Figure 2B).</li>
	<li>Passage cells once a week at a ratio of ~1:6 (plating as droplets is no longer necessary at this stage). Use Accutase to dissociate cells and wash twice with plain Neurobasal medium before re-plating in Full Melanocyte medium. Maintain cells on PO/LAM/FN plates. 
	Note: We have found that the Full Melanocyte medium is best suited for maintaining and expanding hESC and iPSC-derived melanocytes. However, cells can be briefly cultured in commercially available M254 medium but the simplified media increases the risk of cells dying and lifting off the plate.</li>
</ol>

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The authors have no conflicting interests to disclose.

For the successful differentiation of melanocytes from hPSCs the following suggestions should be taken into consideration. First and foremost, it is essential to work under sterile culture conditions at all times. Additionally, it is important to start with pluripotent, fully undifferentiated hPSCs; if the starting population contains differentiated cells the yield will invariably drop as the contaminants cannot be directed towards melanocytes and may even further disrupt the properly differentiating cells.
<p class=...

Human pluripotent stem cells (hPSCs) provide a platform to mimic normal differentiation in a scalable fashion for disease modeling, drug screening, and cell replacement therapies 1-6. Of particular interest, hPSCs open up avenues for studying difficult to isolate or rare/transient cell types where patient samples are scarce. Furthermore, induced pluripotent stem cells (iPSCs) enable researchers to study development and disease modeling in a patient specific manner to unravel unique mechanisms 1,2,7-11. The previously published protocol for differentiation of melanocytes from hPSCs requires up to 6 weeks of differentiations and involves culturing cells with conditioned medium from L-Wnt3a cells 12. The protocol first presented by Mica et al. and described here produces pigmented cells in three weeks and removes the ambiguity and inconsistencies associated with conditioned medium.
Melanocytes are derived from the neural crest, a migratory population of cells unique to vertebrates. The neural crest is defined during gastrulation and represents a population of cells at the edge of the neural plate, bordering between the neural and non-neural ectoderm. During neurulation, the nervous tissue evolves from a neural plate to form neural folds, which converge at the dorsal midline resulting in the neural tube 13,14.
The neural crest cells emerge from the roof plate of the neural tube, opposite the notochord, and undergo an epithelial to mesenchymal transition before migrating away to give rise to a diverse population of differentiated cells. The fates of the crest cells are defined in part by the anatomic location of the roof plate along the body axis of the embryo. Neural crest cell derivatives include lineages characteristic of both mesoderm (smooth muscle cells, osteoblasts, adipocytes, chondrocytes) and ectoderm cells (melanocytes, Schwann cells, neurons) 14. Neural crest stem cells upregulate the transcription factor SOX10 and can be isolated by fluorescence-activated cell sorting with antibodies to p75 and HNK1.
The neural crest cells fated to become melanocytes pass through a melanoblast stage and upregulate KIT and MITF (microphthalmia-associated transcription factor) 6,21 MITF is a master regulator of melanocyte development and is a transcription factor responsible for controlling much of melanocyte development 22-24. Human melanoblasts migrate to the basal layer of the epidermis where they reside either in the hair bulge or surrounded by keratinocytes in the epidermis (forming pigmentation units) to serve as precursors to the mature, pigmented melanocytes. The differentiation and maturation of melanoblasts into pigmented melanocytes occurs concomitant with colonization of the hair bulb and expression of the melanin production pathway (TYRP1, TYR, OCA2 and PMEL) 25,26.
Isolating human melanocytes and melanoblasts from patients is expensive, difficult and limiting in quantity. This protocol enables researchers to differentiate hPSCs (induced or embryonic) into melanocytes or melanocyte precursors in a well defined, rapid, reproducible, scalable, and inexpensive method without cell sorting. The protocol was used previously to identify disease-specific defects when differentiating iPSCs from patients with pigmentation disorders .

<table><tbody><tr><td>Accutase</td><td>Innovative Cell Technologies</td><td>AT104</td><td></td></tr><tr><td>apo human transferrin</td><td>Sigma</td><td>T1147</td><td></td></tr><tr><td>Ascorbic Acid (L-AA)</td><td>Sigma</td><td>A4034</td><td>100 mM</td></tr><tr><td>B27 (B27 Supplement)</td><td>Invitrogen</td><td>17504044</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Gibco-Life Technologies</td><td>21985-023</td><td>10 mg/ml</td></tr><tr><td>BMP4</td><td>R&amp;amp;D Systems</td><td>314-bp</td><td></td></tr><tr><td>CHIR99021</td><td>Tocris-R&amp;amp;D Systems</td><td>4423</td><td>6 mM</td></tr><tr><td>Cholera toxin</td><td>Sigma</td><td>C8052</td><td>50 mg/ml</td></tr><tr><td>cAMP (cyclicAMP)</td><td>Sigma</td><td>D0627</td><td>100 mM</td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D2915-100MG</td><td>50 &amp;mu;M</td></tr><tr><td>DMEM - Dulbecco&#039;s Modified Eagle Medium</td><td>Gibco-Life Technologies</td><td>11985-092</td><td></td></tr><tr><td>DMEM/F12 - Dulbecco&#039;s Modified Eagle Medium: Nutrient Mixture F-12</td><td>Gibco-Life Technologies</td><td>1133--032</td><td></td></tr><tr><td>DMEM/F12 powder</td><td>Invitrogen</td><td>12500-096</td><td></td></tr><tr><td>EDN3 (Endothelin-3, human)</td><td>American Peptide Company</td><td>88-5-10B</td><td>100 &amp;mu;M</td></tr><tr><td>Fibronectin</td><td>BD Biosciences</td><td>356008</td><td>200 &amp;mu;g/ml</td></tr><tr><td>gelatin (PBS without Mg/Ca)</td><td>in house</td><td></td><td>0.1% in PBS</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G7021</td><td></td></tr><tr><td>Human insulin</td><td>Sigma</td><td>I2643</td><td></td></tr><tr><td>ITS+ Universal Culture Supplement Premix&amp;nbsp;</td><td>BD Biosciences</td><td>354352</td><td></td></tr><tr><td>KSR (Knockout Serum Replacement)</td><td>Gibco-Life Technologies</td><td>10828-028</td><td></td></tr><tr><td>Knockout DMEM</td><td>Gibco-Life Technologies</td><td>10829-018</td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco-Life Technologies</td><td>25030-081</td><td></td></tr><tr><td>LDN193189</td><td>Stemgent</td><td>04-0074</td><td>100 mM</td></tr><tr><td>Low glucose DMEM</td><td>Invitrogen</td><td>11885-084</td><td></td></tr><tr><td>Matrigel matrix</td><td>BD Biosciences</td><td>354234</td><td>Dissolve 1:20 in DMEM/F12</td></tr><tr><td>MCDB201 Medium</td><td>Sigma</td><td>M6770</td><td></td></tr><tr><td>MEM minimum essential amino acids solution</td><td>Gibco-Life Technologies</td><td>11140-080</td><td></td></tr><tr><td>Mouse embryonic fibroblasts (7 million cells/vial)</td><td>GlobalStem</td><td>GSC-8105M</td><td></td></tr><tr><td>Mouse Laminin-I</td><td>R&amp;amp;D Systems</td><td>3400-010-01</td><td>1 mg/ml</td></tr><tr><td>Neurobasal medium</td><td>Invitrogen</td><td>21103049</td><td></td></tr><tr><td>Penicilin/Streptomycin</td><td>Gibco-Life Technologies</td><td>15140-122</td><td>10,000 U/ml</td></tr><tr><td>Poly-L Omithin hydrobromide</td><td>Sigma</td><td>P3655</td><td>15 mg/ml&amp;nbsp;</td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P8783</td><td>0.032 g in 100 ml 100% ethanol</td></tr><tr><td>Putrescine dihydrochloride</td><td>Sigma</td><td>P5780</td><td></td></tr><tr><td>FGF2 (Recombinant human FGF basic)</td><td>R&amp;amp;D Systems</td><td>233-FB-001MG/CF</td><td>10 mg/ml</td></tr><tr><td>SB431542</td><td>Tocris-R&amp;amp;D Systems</td><td>1814</td><td>10 mM</td></tr><tr><td>Selenite</td><td>Sigma</td><td>S5261</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>SCF (Stem Cell Factor, recombinant Human)&amp;nbsp;</td><td>Peprotech Inc.</td><td>300-07</td><td>50 &amp;mu;g/ml</td></tr><tr><td>TYRP1 (G-17) Antibody</td><td>Santa Cruz</td><td>10443</td><td>1:200</td></tr><tr><td>TYRP2 Antibody</td><td>Abcam</td><td>74073</td><td>1:200</td></tr><tr><td>Trypsin-EDTA (0.05%)</td><td>Gibco-Life Technologies</td><td>25300-054</td><td></td></tr><tr><td>Y-27632 dihydrochloride</td><td>Tocris-R&amp;amp;D Systems</td><td>1254</td><td>10 mM</td></tr></tbody></table>

feeder-free derivation of melanocytes from human pluripotent stem cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This protocol provides a method for deriving fully pigmented, mature melanocytes from hPSCs in an in vitro feeder-free, cost efficient, and reproducible manner. In contrast to the previously established Fang et al. protocol for hPSC-derived melanocytes, the outlined protocol does not require conditioned medium and decreases the time requirement. The Fang et al. protocol utilized conditioned medium from a WNT3A-producing murine cell line and took up to 6 weeks to...

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

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

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Research

JoVE Journal

Developmental Biology

10.2K Views.  Memorial Sloan-Kettering Cancer Center. This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Video: Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

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 ...

Neuroscience

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

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.

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 ...

Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously presented a differentiation protocol for the derivation of autonomic neurons with sympathetic character that has been applied to patients with autonomic neuropathy. However, the protocol was built on Knock Out Serum Replacement (KSR) and feeder-based culture conditions, and to ensure high differentiation efficiency, cell sorting was necessary. These factors cause high variability, high cost, and low reproducibility. Moreover, mature sympathetic properties, including electrical activity, have not been verified. Here, we present an optimized protocol where PSC culture and differentiation are performed in feeder-free and chemically defined culture conditions. Genetic markers identifying trunk neural crest are identified. Further differentiation into postganglionic sympathetic neurons is achieved after 20 days without the need for cell sorting. Electrophysiological recording further shows the functional neuron identity. Firing detected from our differentiated neurons can be enhanced by nicotine and suppressed by the adrenergic receptor antagonist propranolol. Intermediate sympathetic neural progenitors in this protocol can be maintained as neural spheroids for up to 2 weeks, which allows expansion of the cultures. In sum, our updated sympathetic neuron differentiation protocol shows high differentiation efficiency, better reproducibility, more flexibility, and better neural maturation compared to the previous version. This protocol will provide researchers with the cells necessary to study human disorders that affect the autonomic nervous system.

In this protocol, we describe a stable, highly efficient differentiation strategy for the generation of postganglionic sympathetic neurons from human pluripotent stem cells. This model will make neurons available for the use of studies of multiple autonomic disorders.

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously ...

Direct Reprogramming of Mouse Fibroblasts into Melanocytes

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, there is no effective long-term treatment for vitiligo. Therefore, it is imperative to develop a convenient and effective treatment for vitiligo. Regenerative medicine technology for direct reprogramming of skin cells into melanocytes seems to be a promising novel treatment of vitiligo. This involves the direct reprogramming of the patient's skin cells into functional melanocytes to help ameliorate the loss of melanocytes in patients with vitiligo. However, this method needs to be first tested on mice. Although direct reprogramming is widely used, there is no clear protocol for direct reprogramming into melanocytes. Moreover, the number of available transcription factors is overwhelming. 
Here, a concentrated lentivirus packaging system protocol is presented to produce transcription factors selected for reprogramming skin cells to melanocytes, including Sox10, Mitf, Pax3, Sox2, Sox9, and Snai2. Mouse embryonic fibroblasts (MEFs) were infected with the concentrated lentivirus for all these transcription factors for the direct reprogramming of the MEFs into induced melanocytes (iMels) in vitro. Furthermore, these transcription factors were screened, and the system was optimized for direct reprogramming to melanocytes. The expression of the characteristic markers of melanin in iMels at the gene or protein level was significantly increased. These results suggest that direct reprogramming of fibroblasts to melanocytes could be a successful new therapeutic strategy for vitiligo and confirm the mechanism of melanocyte development, which will provide the basis for further direct reprogramming of fibroblasts into melanocytes in vivo.

Here, we describe an optimized direct reprogramming system for melanocytes and a high-efficiency, concentrated virus packaging system that ensures smooth direct reprogramming.

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, ...

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout SR.

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

Biology

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...