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 ...

This protocol allows the derivation of sympathetic neurons from human pluripotent stem cells. These cells will enable a scientist to study human disorders that affect the sympathetic nervous system. This technique allows the derivation of sympathetic neurons in divided feeder-free meeting conditions at a high scalable efficiency.It results in electrically active neurons in 20 days. This technique can be applied to diseases caused by a dysfunctional sympathetic nervous system, it also can be used for disease modeling, regenerative medicine, and drug screening. These cells could be used in vitro to innovate any tissue within a co-culture system, for example, for the study of cardiac tissue regulation.When the human pluripotent stem cell culture reaches 80 to 90%confluency, big colonies with smooth bright edges should be observed. For splitting, wash the culture with PBS before treating the cells with four milliliters of 0.25 molar EDTA at 37 degrees Celsius and 5%carbon dioxide. After two minutes, flush the plate with 10 milliliters of E8 medium and transfer the detached cell suspension to a 15 milliliter conical tube.Then transfer 500 microliters of cells into a 9.5 milliliter aliquot of fresh E8 medium. And plate the stem cells onto new vitronectin coated 100 millimeter dishes. One day before differentiation induction, coat the appropriate number of six well plates with two milliliters of basement membrane matrix per well.And store the plates at four degrees Celsius overnight. The next morning, warm the plates to room temperature and wash the ready to split human pluripotent stem cell culture two times with fresh PBS per wash. After the second wash, treat the cells with seven milliliters of 0.5 millimolar EDTA for 15 minutes in the cell culture incubator before aspirating the floating human pluripotent stem cells and transferring the harvested cells into a 50 milliliter tube.Add an equal volume of PBS to the tube to dilute the EDTA and collect the cells by centrifugation. Resuspend the pellet in one milliliter of day 01 differentiation medium before adding an appropriate volume of medium for accounting. Dilute the cells to a 1.25 times 10 to the fifth cells per square centimeter concentration into milliliters of differentiation medium per well.Aspirate all of the basement membrane matrix solution from each well of the previously prepared to six well plate. Then seed two milliliters of cells into each well and place the plate in the cell culture incubator. The next morning, feed the cells with three milliliters of day 01 differentiation medium per well.On day two of differentiation, feed the cells with three milliliters of day two to 10 differentiation medium per well, then feed the cells every other day until day 10. To aggregate the neural crest cells into spheroids, on day 10, wash the cells with PBS. And add two milliliters of dissociation medium to each well.After 20 minutes in the cell culture incubator, transfer the floating cells into a 50 milliliter conical tube and bring the volume of suspension in the tube to 50 milliliters with fresh PBS. Collect the cells by centrifugation. And resuspend the pellet in a sufficient volume of day 10 to 14 spheroid medium for counting.After counting, dilute the cells to a five times 10 to the fifth cells per 500 microliters concentration using day 10 to 14 spheroid medium. And plate 500 microliters of cells into each well of an ultra low attachment 24-well plate. Then return the cells to the cell culture incubator.To induce a minimal spheroid culture, on day 11 of culture, feed the neural crest spheroids with an additional 500 microliters of day 10 to 14 spheroid medium per well. On day 12, tilt the plate to accumulate the spheroids on one side of the plate. And carefully aspirate as much medium as possible from each well without aspirating spheroids.Then feed the spheroids with one milliliter of day 10 to 14 spheroid medium per well. To induce an expanded spheroid culture, on day 15, feed the spheroids with 1.5 milliliters of day 10 to 14 spheroid medium supplemented with 0.5 micromolar retinoic acid per well. Then return the spheroids to the cell culture incubator.For sympathetic neuron differentiation, on day 14 or 28, tilt the plate to accumulate the spheroids on one side of the plate and carefully aspirate as much medium as possible. Feed the cells with one milliliter of sympathetic neuron medium. To break up big spheroid aggregates into smaller spheroids, add no more than one milliliter of medium per well and pipette the spheroids five to 10 times before splitting.And remove the excess laminin fibronectin from previously prepared 24-well plates. Split the plate one milliliter of spheroids from each well on the 24-wells spheroid culture plate between four separate wells of the new coated 24-well plate. Add 250 microliters of fresh sympathetic neuron medium to each well.And place the plate in the cell culture incubator. The next morning, replace the medium in each well with one milliliter of sympathetic neuron medium supplemented with 0.125 micromolar retinoic acid and return the plate to the cell culture incubator. After day 35, feed the neurons by carefully replacing only half of the existing medium in each well with fresh medium.Before differentiation, the human pluripotent stem cell colonies should be round with shiny, smooth edges and little to no differentiation. The cells express the neural crest marker Sox10 from days four through 10. Neural crest cells emerge in dense darkened ridges that are visible from day six on.These ridges also express Sox10. At the neural crest cells stage, Sox10 correlates with the cell surface marker CD49D, which can be used to sort the neural crest cells. Positive sorted and unsorted populations produced neural crest spheroids in a similar fashion.While negative sorted cells do not aggregate properly, do not produce round, smooth healthy looking spheroids and die within three to four days. Furthermore, when neural crest spheroids are compared at day 14, the quantitative reverse transcriptase PCR for neural crest and sympathetic nerve progenitor markers, significant differences between the sorted and unsorted cells cannot be detected. On day 20 or 35, the respective minimal or extended protocols, neuroids can be observed growing in a radial pattern from the attached spheroids.Markers related to norepinephrine synthesis and transportation are expressed at this stage, as well as Hox6 through nine trunk neural crest genes. Electrophysiological recording also detects neural activity from day 20 on. Such activity can be enhanced or suppressed by drugs that target autoreceptors of sympathetic neurons adjusting the functionality of differentiated neurons.The human proponent themselves must be healthy starting on day zero of differentiation, otherwise, the experiment will likely fail. Pharmacological inhibitors or activators can be added to the neurons to understand their normal or pathological behavior. This method opens a new avenue for studying sympathetic biology and pathology since it is now possible to obtain biopsies of sympathetic neurons from humans easily.

efficient-differentiation-postganglionic-sympathetic-neurons-using

Research

JoVE Journal

Developmental Biology

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

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 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 ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...