Name: feeder-free derivation of neural crest progenitor cells from human pluripotent stem cells
Uploaded: 2014-05-22T14:45:00
Description: Watch this Scientific Journal Video about Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells at JoVE.com

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/St...

<ol>
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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">
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	</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>
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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 ...

Research

JoVE Journal

Neuroscience

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

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). ...

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

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

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 ...

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 ...

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 ...

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 ...

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 ...