Name: efficient differentiation of postganglionic sympathetic neurons using human pluripotent stem cells under feeder-free and chemically defined culture conditions
Uploaded: 2020-05-24T13:30:00
Description: Watch this Scientific Journal Video about Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

We would like to thank Heidi Ulrichs for critical reading and editing of the manuscript.

NOTE: The H9 PHOX2B:GFP reporter line was provided by Oh et al.19. Some qPCR primers used in this paper were obtained from OriGene Technologies, while a few sequences are obtained from Frith et al.20,30.
1. Set-up for dish coating, media preparation, and hPSC maintenance
<ol>
	<li>Dish coating
	<ol>
		<li>Vitronectin (VTN) coating
		<ol>
			<li>Place vials of VTN in a 37 &#176;C water bath unt...

<ol>
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J., 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+axial+progenitors+generate+trunk+neural+crest+cells+in+vitro.">Human axial progenitors generate trunk neural crest cells in vitro.</a> Elife. 7, (2018).</li><li>Kirino, K., Nakahata, T., Taguchi, T., Saito, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+derivation+of+sympathetic+neurons+from+human+pluripotent+stem+cells+with+a+defined+condition.">Efficient derivation of sympathetic neurons from human pluripotent stem cells with a defined condition.</a> Scientific Reports. 8 (1), 12865 (2018).</li><li>Wu, H. F., Zeltner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+Methods+to+Differentiate+Sympathetic+Neurons+from+Human+Pluripotent+Stem+Cells.">Overview of Methods to Differentiate Sympathetic Neurons from Human Pluripotent Stem Cells.</a> Current Protocols Stem Cell Biology. 50 (1), 92 (2019).</li><li>Zeltner, N., 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=Capturing+the+biology+of+disease+severity+in+a+PSC-based+model+of+familial+dysautonomia.">Capturing the biology of disease severity in a PSC-based model of familial dysautonomia.</a> Nature Medicine. 22 (12), 1421-1427 (2016).</li><li>Saito-Diaz, K., Wu, H. 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We recently published two reviews, one discussing the use of hPSC-derived symNs for disease modeling31 as well as an in-depth comparison of available differentiation protocols22. Thus, here we focus on troubleshooting the current protocol to help the interested researcher succeed in making symNs. During the entire differentiation process, in order to gain consistent data as well as healthy differentiated cells, contamination at all stages should be carefully controlled. In ...

Postganglionic sympathetic neurons (symNs) belong to the autonomic nervous system (ANS) and have multiple important roles in responding and regulating homeostasis of the body independent of consciousness. For example, stress stimulates symNs and evokes the fight-or-flight response that leads to an increase in heart rate, blood pressure, and sweating. SymNs are affected in multiple human disorders due to genetics, toxicity/injury, or as companions to other diseases. An example of a genetic neuropathy is the childhood disorder Familial Dysautonomia (FD), where a severe dysregulation of symNs causes dysautonomic crisis, evident by sweating, blotching of the skin, vomiting attacks, hypertension, and anxiety1. An example of toxicity is chemotherapy treatment, which has been reported to have toxic side effects on autonomic neurons2. It is known that autonomic denervation and hyper-innervation can both lead to, or accompany, diseases such as Parkinson&#8217;s disease or hypertensive renal disease3,4. Thus, being able to conduct research and understand the mechanisms of symN biology and defects in the context of disease is beneficial for the search of novel and effective treatments.
Anatomy 
The peripheral nervous system branches into sensory and autonomic divisions. The afferent nerves of the sensory nervous system are responsible for sensation of pain and touch, whereas the ANS is responsible for relaying information from all organs to the brain. The ANS is divided into the enteric nervous system, innervating the gastrointestinal tract, the parasympathetic nervous system, which is important for relaxation, and the sympathetic nervous system (SNS), which is important for activation/regulation of organs. The SNS adapts a two-neuron system5. Preganglionic sympathetic neural axons in the spinal cord first project to the sympathetic ganglia, where postganglionic symN cell bodies are located. These neurons then send long projections to innervate the target tissues of every organ in the body. Signals transmitted by preganglionic neurons are cholinergic, whereas postganglionic symNs are adrenergic and thus express norepinephrine (NE) as their main neurotransmitter. There are few notable exceptions of postganglionic, sympathetic neurons that are cholinergic, including the ones innervating blood vessels. Adrenergic postganglionic neurons express the enzymes tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AAAD), dopamine &#946;-hydroxylase (DBH), and monoamine oxidase (MAO-A), all responsible for generating and metabolizing NE. Furthermore, they express the NE recycling transporters and/or receptors &#945;-adrenergic receptor (ADRA2), &#946;-adrenergic receptor (ADR2B), norepinephrine transporter (NET1), and vesicular monoamine transporter (VMAT1/2).
Development 
During embryonic development symNs are derived from the neural crest (NC), which emerges between the neural tube and overlaying ectoderm6, and can differentiate into multiple cell lineages, including melanocytes, osteoblasts, adipocytes, glia, enteric neurons, sensory neurons, and autonomic neurons7. Neural crest cells (NCCs) are highly migratory cells that take several routes through the embryo. At this early stage of NC development, the cells express the markers SNAIL1/2, FOXD3, and SOX108,9,10,11. The migration route together with the axial location they adopt determines the NC subtype into which they will develop. These NC subtypes can be distinguished by their specific HOX gene expression: Cranial NCCs do not express HOX genes, vagal NCCs express HOX 1&#8211;5, trunk NCCs express HOX 6&#8211;9, and sacral NCCs express HOX 10&#8211;1112. Among them, trunk NCCs are recognized as the main source of symNs. SymN precursors express the transcription factor MASH1/ASCL113, which promotes expression of PHOX2B14 and INSM115. The GATA family of transcription factors is expressed during late sympathetic development. GATA2 and GATA3 are expressed in the symNs, which in turn activates DBH16. The transcription factor HAND2 is also important for the expression and maintenance of DBH and TH17.
HPSCs (e.g., embryonic and induced pluripotent stem cells) are a powerful tool18 to recapitulate developmental paradigms and generate symNs that can then be employed for disease modeling of various human disorders. Thus, while generating symNs from hPSCs, it is crucial to follow developmental guidelines and assess expression of appropriate markers along the differentiation process.
Previous symN protocol 
Few research groups have previously reported the generation of symNs from hPSCs19,20,21. The direct comparison of these protocols to each other and ours was reviewed recently22. In 201623, we published a differentiation protocol for the generation of autonomic neurons with symN character (Figure 1A). This protocol used KSR-based medium, which was used in both the maintenance of undifferentiated hPSCs and cell differentiation. Furthermore, hPSCs were maintained on mouse embryonic fibroblasts (MEF feeder cells). We employed this protocol and PSCs from patients with FD to model the disorder23. In 2019, we described a more detailed version of this older protocol24. In summary, the neural fate was induced by dual SMAD inhibition25 to block TGF-&#946; and BMP signaling in the first 2 days. WNT activation using CHIR99021 promoted neural progenitors to become NC cells. On day 11, cells were sorted by FACS for CD49D+ or SOX10+ populations26,23, which yielded about 40% NC generation efficiency. Thus, sorting was needed to ensure the efficiency and purity for the next steps of differentiation. The NCCs were maintained and amplified as spheroids with the combined treatment of FGF2 and CHIR. After 4 days, the NC spheroids of maintenance were plated and given BDNF, GDNF, and NGF to finish the symN maturation. Although these symNs expressed strong symN markers such as ASCL1, TH, DBH, and PHOX2A, markers for more mature symNs, including expression of the nicotinic acetylcholine receptor (CHRNA3/CHRNB4) and vesicle transporter (VMAT1/2), were low even after 70 days of differentiation. HOX genes in this protocol were not formally tested, and mature neural properties, including electrophysiological activity of the cells, were not verified.
Here, we present an optimized protocol to generate symNs (Figure 1B). HPSCs are maintained in feeder-free conditions, on vitronectin (VTN)-coated dishes, using Essential 8 (E8) media27. The formula of the differentiation media has been modified at each stage, thereby increasing the percentage of the NC population28. The symN maturation can be done on CD49D+/SOX10+ sorted or unsorted bulk NCC populations. Both show high levels of symN marker expression by day 30. Moreover, the symNs generated with this protocol are responsive to electrophysiological recording and to treatments with symN activator and inhibitor compounds.

<table>
	<tbody>
		<tr>
			<td>100 mm cell culture dishes</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tissue culture tubes</td>
			<td>VWR/Corning</td>
			<td>89039-664</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue culture plates</td>
			<td>Falcon</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well ultra-low-attachment plates</td>
			<td>Corning</td>
			<td>07 200 601 and 07 200 602</td>
			<td></td>
		</tr>
		<tr>
			<td>5% CO2/20% O2 tissue culture incubator</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>Heracell VIOS 160i</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conical tissue culture tubes</td>
			<td>VWR/Corning</td>
			<td>89039-656</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plates</td>
			<td>Costar</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovation Cell Technologies</td>
			<td>AT104500</td>
			<td>Cell dissociation solution</td>
		</tr>
		<tr>
			<td>Anti-AP2a antibody</td>
			<td>Abcam</td>
			<td>ab108311</td>
			<td>Host: Rabbit; 1:400 dilution</td>
		</tr>
		<tr>
			<td>Anti-Ascl1 antibody</td>
			<td>BD Pharmingen</td>
			<td>556604</td>
			<td>Host: Mouse IgG1; 1:200 dilution</td>
		</tr>
		<tr>
			<td>Anti-CD49D antibody</td>
			<td>BioLegend</td>
			<td>304313</td>
			<td>Host: Mouse IgG1; 5 &#956;l/million cells in 100 &#956;l volume</td>
		</tr>
		<tr>
			<td>Anti-CD49D antibody (isotype)</td>
			<td>BioLegend</td>
			<td>400125</td>
			<td>Host: Mouse IgG1; 5 &#956;l/million cells in 100 &#956;l volume</td>
		</tr>
		<tr>
			<td>Anti-DAPI antibody</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Anti-DBH antibody</td>
			<td>Immunostar</td>
			<td>22806</td>
			<td>Host: Rabbit; 1:500 dilution</td>
		</tr>
		<tr>
			<td>Anti-GFP antibody</td>
			<td>Abcam</td>
			<td>ab13970</td>
			<td>Host: Chicken; 1:1000 dilution</td>
		</tr>
		<tr>
			<td>Anti-HOXC9 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-365692</td>
			<td>Host: Mouse IgG1; 1:100 dilution</td>
		</tr>
		<tr>
			<td>Anti-NET1 antibody</td>
			<td>Mab</td>
			<td>NET17-1</td>
			<td>Host: Mouse; 1:1000 dilution</td>
		</tr>
		<tr>
			<td>Anti-PRPH antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-377093/H0112</td>
			<td>Host: Mouse IgG2a; 1:200 dilution</td>
		</tr>
		<tr>
			<td>Anti-SOX10 antibody</td>
			<td>Abcam</td>
			<td>ab50839</td>
			<td>Host: Mouse; 1:100 dilution</td>
		</tr>
		<tr>
			<td>Anti-TH antibody</td>
			<td>Pel-Freez</td>
			<td>P40101- 150</td>
			<td>Host: Rabbit; 1:500 dilution</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma</td>
			<td>A8960-5G</td>
			<td>Stock concentration: 100 mM</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>12587-010</td>
			<td>Stock concentration: 50x</td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>R&#38;D Systems</td>
			<td>248-BD</td>
			<td>Stock concentration: 10 &#956;g/mL</td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>R&#38;D Systems</td>
			<td>314-BP</td>
			<td>Stock concentration: 6 mM</td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>Countess II</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counting chamber slides</td>
			<td>Invitrogen</td>
			<td>C10312</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>57021&#38;5424R</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>R&#38;D Systems</td>
			<td>4423</td>
			<td>Stock concentration: 6 mM</td>
		</tr>
		<tr>
			<td>Cryo-vial</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>375353</td>
			<td></td>
		</tr>
		<tr>
			<td>dbcAMP</td>
			<td>Sigma</td>
			<td>D0627</td>
			<td>Stock concentration: 100 mM</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>10829-018</td>
			<td>Stock concentration: 1x</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>11330-057</td>
			<td>Stock concentration: 1x</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>BP231-100</td>
			<td></td>
		</tr>
		<tr>
			<td>E6 medium</td>
			<td>gibco</td>
			<td>A15165-01</td>
			<td></td>
		</tr>
		<tr>
			<td>E8 medium</td>
			<td>gibco</td>
			<td>A15169-01</td>
			<td>Stock concentration: 1x</td>
		</tr>
		<tr>
			<td>E8 supplement</td>
			<td>gibco</td>
			<td>A15171-01</td>
			<td>Stock concentration: 50x</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>ED2SS</td>
			<td>Stock concentration: 0.5 M</td>
		</tr>
		<tr>
			<td>Electrophysiology plates (AXION cytoview MEA96)</td>
			<td>Axion BioSystems</td>
			<td>M768-tMEA-96W</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS machine</td>
			<td>Beckman Coulter</td>
			<td>CytoFLEX (for FACS)</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS machine</td>
			<td>Beckman Coulter</td>
			<td>MoFlo Astrios EQ (for sorting)</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tubes (blue filter cap)</td>
			<td>Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tubes (white cap)</td>
			<td>Falcon</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>PeproTech</td>
			<td>450</td>
			<td>Stock concentration: 10 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Geltrex</td>
			<td>Invitrogen</td>
			<td>A1413202</td>
			<td>Basement membrane matrix; Stock concentration: 100x</td>
		</tr>
		<tr>
			<td>hPSCs</td>
			<td>Thomson et al., (1998)</td>
			<td>WA09</td>
			<td></td>
		</tr>
		<tr>
			<td>hPSCs</td>
			<td>Oh et al. (2016)</td>
			<td>H9-PHOX2B::eGFP</td>
			<td></td>
		</tr>
		<tr>
			<td>Human fibronectin (FN)</td>
			<td>VWR/Corning</td>
			<td>47743-654</td>
			<td>Stock concentration: 1 mg/mL</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Thermo Fisher/Gibco</td>
			<td>25030-081</td>
			<td>Stock concentration: 200 mM</td>
		</tr>
		<tr>
			<td>LN tank</td>
			<td>Custom Biogenic Systems</td>
			<td>V-1500AB</td>
			<td></td>
		</tr>
		<tr>
			<td>MEA reader</td>
			<td>Axion BioSystems</td>
			<td>Maestro Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse laminin I (LM)</td>
			<td>R&#38;D Systems</td>
			<td>3400-010-01</td>
			<td>Stock concentration: 1 mg/mL</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>17502-048</td>
			<td>Stock concentration: 100x</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>gibco</td>
			<td>21103-049</td>
			<td>Stock concentration: 1x</td>
		</tr>
		<tr>
			<td>NGF</td>
			<td>PeproTech</td>
			<td>450-01</td>
			<td>Stock concentration: 25 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>14190-136</td>
			<td>Stock concentration: 1x</td>
		</tr>
		<tr>
			<td>Poly-L-ornithine hydrobromide (PO)</td>
			<td>Sigma</td>
			<td>P3655</td>
			<td>Stock concentration: 15 mg/mL</td>
		</tr>
		<tr>
			<td>Primocin (antibiotics)</td>
			<td>InvivoGen</td>
			<td>ANTPM1</td>
			<td>Stock concentration: 50 mg/mL</td>
		</tr>
		<tr>
			<td>qPCR machine</td>
			<td>Bio-Rad Laboratories</td>
			<td>C1000 Touch</td>
			<td></td>
		</tr>
		<tr>
			<td>qPCR plates</td>
			<td>Bio-Rad Laboratories</td>
			<td>HSP9601</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant FGF2</td>
			<td>R&#38;D Systems</td>
			<td>233-FB/CF</td>
			<td>Stock concentration: 10 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td>Stock concentration: 1 mM</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Tocris/R&#38;D Systems</td>
			<td>1614</td>
			<td>Stock concentration: 10 mM</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Corning</td>
			<td>MT-25-900-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitronectin (VTN)</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>A14700</td>
			<td>Stock concentration: 0.5 mg/mL</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>VWR/Corning</td>
			<td>706308</td>
			<td></td>
		</tr>
		<tr>
			<td>Y27632</td>
			<td>R&#38;D Systems</td>
			<td>1254</td>
			<td>Stock concentration: 10 mM</td>
		</tr>
	</tbody>
</table>

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 give instructions on how to generate symNs from hPSCs. The culture conditions demonstrated here were improved from an earlier published protocol23,24 (Figure 1A) to feeder-free and chemically defined conditions (Figure 1B). Two options are provided, one where symNs are made within 20 days, and another where the NCCs can be expanded for 2 weeks to generate more cells that can then be ...

Watch this Scientific Journal Video about Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

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

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 ...

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