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

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The authors have nothing to disclose.

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 &amp;mu;l/million cells in 100 &amp;mu;l volume</td></tr><tr><td>Anti-CD49D antibody (isotype)</td><td>BioLegend</td><td>400125</td><td>Host: Mouse IgG1; 5 &amp;mu;l/million cells in 100 &amp;mu;l volume</td></tr><tr><td>DAPI dye</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>Abcam</td><td>ab50839</td><td>Host: Mouse; 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>Santa Cruz Biotechnology</td><td>sc-365692</td><td>Host: Mouse IgG1; 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&amp;amp;D Systems</td><td>248-BD</td><td>Stock concentration: 10 &amp;mu;g/mL</td></tr><tr><td>BMP4</td><td>R&amp;amp;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&amp;amp;5424R</td><td></td></tr><tr><td>CHIR99021</td><td>R&amp;amp;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 &amp;mu;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&amp;amp;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 &amp;mu;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&amp;amp;D Systems</td><td>233-FB/CF</td><td>Stock concentration: 10 &amp;mu;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&amp;amp;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&amp;amp;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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Research

JoVE Journal

Developmental Biology

14.7K Views.  University of Georgia. Dit protocol maakt de afleiding van sympathische neuronen uit menselijke pluripotente stamcellen mogelijk. Deze cellen zullen een wetenschapper in staat stellen om menselijke aandoeningen te bestuderen die het sympathische zenuwstelsel beïnvloeden. Deze techniek maakt de afleiding van sympathische neuronen in verdeelde feeder-vrije vergaderomstandigheden op een hoge schaalbare efficiëntie.Het resulteert in elektrisch actieve neuronen in 20 dagen. Deze techniek kan worden toegepast op...

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

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycERTAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3&#8217;UTRs by dual reporter plasmid transfection and dual luciferase assay.

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

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

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

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

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

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

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Biology

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) pathophysiology in vitro. Multi-electrode array (MEA) recordings are a means to record electrical field potentials from large populations of neurons and analyze network activity over time. It was previously demonstrated that the presence of hiPSC-A that are differentiated using techniques to promote a spinal cord astrocyte phenotype improved maturation and electrophysiological activity of regionally specific spinal cord hiPSC-motor neurons (MN) when compared to those cultured without hiPSC-A or in the presence of rodent astrocytes. Described here is a method to co-culture spinal cord hiPSC-A with hiPSC-MN and record electrophysiological activity using MEA recordings. While the differentiation protocols described here are particular to astrocytes and neurons that are regionally specific to the spinal cord, the co-culturing platform can be applied to astrocytes and neurons differentiated with techniques specific to other fates, including cortical hiPSC-A and hiPSC-N. These protocols aim to provide an electrophysiological assay to inform about glia-neuron interactions and provide a platform for testing drugs with therapeutic potential in ALS.

We describe a method for differentiating spinal cord human induced pluripotent-derived astrocytes and neurons and their co-culture for electrophysiological recording.

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) ...

Derivation of Enteric Neurons from Human Pluripotent Stem Cells

Differentiation of Enteric Nervous System Lineages from Human Pluripotent Stem Cells

The human enteric nervous system, ENS, is a large network of glial and neuronal cell types with remarkable neurotransmitter diversity. The ENS controls bowel motility, enzyme secretion, and nutrient absorption and interacts with the immune system and the gut microbiome. Consequently, developmental and acquired defects of the ENS are responsible for many human diseases and may contribute to symptoms of Parkinson's disease. Limitations in animal model systems and access to primary tissue pose significant experimental challenges in studies of the human ENS. Here, a detailed protocol is presented for effective in vitro derivation of the ENS lineages from human pluripotent stem cells, hPSC, using defined culture conditions. Our protocol begins with directed differentiation of hPSCs to enteric neural crest cells within 15 days and yields diverse subtypes of functional enteric neurons within 30 days. This platform provides a scalable resource for developmental studies, disease modeling, drug discovery, and regenerative applications.

Author Spotlight: Robust Culture of Human Enteric Neurons from Human Pluripotent Stem Cells

Deriving enteric nervous system (ENS) lineages from human pluripotent stem cells (hPSC) provides a scalable source of cells to study ENS development and disease, and to use in regenerative medicine. Here, a detailed in vitro protocol to derive enteric neurons from hPSCs using chemically defined culture conditions is presented.

The human enteric nervous system, ENS, is a large network of glial and neuronal cell types with remarkable neurotransmitter diversity. The ENS controls ...

Generating Postganglionic Sympathetic Neurons from Human Pluripotent Stem Cells

Source: Wu, H. F., et al. <a href="https://app.jove.com/v/60843">Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions.</a> J. Vis. Exp. (2020).
This video demonstrates an in vitro method to generate postganglionic sympathetic neurons from human pluripotent stem cells (hPSCs). hPSCs are differentiated into neural crest cells (NCCs) on a basement membrane matrix support using a differentiation medium. The NCCs are then dissociated and formed into spheroids using another differentiation medium. Finally, the spheroids are transformed into postganglionic sympathetic neurons using a sympathetic neuron medium.

1. Set-up for dish coating, media preparation, and hPSC maintenance

	Dish coating
	
		Vitronectin (VTN) coating
		
			Place vials of VTN in a 37 &#176;C ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Advanced Neuronal Models

Differentiating Non-Transgenic and Transgenic Human Pluripotent Stem Cells into Neural Progenitor Cells

Source: Cvetkovic, C., et al. <a href="https://app.jove.com/v/58034">Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells.</a> J. Vis. Exp. (2018).
This video demonstrates the generation of neural progenitor cells from non-transgenic and transgenic human pluripotent stem cells (hPSCs). hPSCs are differentiated into neural progenitor cells using a neuronal differentiation medium. While differentiation is induced in non-transgenic cells using small molecules, the transgenic hPSCs are differentiated by adding an antibiotic that induces the promoter for a neuronal differentiation-specific transcription factor.

Ethic approvals were obtained when performing the experiments.
1. Cell Culture and Reagent Preparation

	Prepare coated plates for cell culture.
	
		...

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

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