1. Developmental Biology

A systematic review and embryological perspective of pluripotent stem cell-derived autonomic postganglionic neuron differentiation for human disease modeling

  1. Thomas A Bos  Is a corresponding author
  2. Elizaveta Polyakova
  3. Janine Maria van Gils
  4. Antoine AF de Vries
  5. Marie-José Goumans
  6. Christian Freund
  7. Marco C DeRuiter
  8. Monique RM Jongbloed  Is a corresponding author
  1. Department of Anatomy and Embryology, Leiden University Medical Centre, Netherlands
  2. Department of Cardiology, Leiden University Medical Centre, Netherlands
  3. Department of Cell and Chemical Biology, Leiden University Medical Centre, Netherlands
  4. Leiden hiPSC Centre, Leiden University Medical Centre, Netherlands
  5. Centre for Congenital Heart Disease Amsterdam-Leiden (CAHAL), Netherlands
https://doi.org/10.7554/eLife.103728
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Cite this article
  1. Thomas A Bos
  2. Elizaveta Polyakova
  3. Janine Maria van Gils
  4. Antoine AF de Vries
  5. Marie-José Goumans
  6. Christian Freund
  7. Marco C DeRuiter
  8. Monique RM Jongbloed
(2025)
A systematic review and embryological perspective of pluripotent stem cell-derived autonomic postganglionic neuron differentiation for human disease modeling
eLife 14:e103728.
https://doi.org/10.7554/eLife.103728
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7 figures, 2 tables and 8 additional files

Figures

Figure 1
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Study selection.

MEDLINE, Medical Literature Analysis and Retrieval System Online; PMC, PubMed Central; PRISMA, Preferred Reporting Items for Systematic reviews and Meta Analyses; PSCs, pluripotent stem cells.

Figure 2 with 1 supplement
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Quality assessment.

Quality assessment results per criterion. Criteria topics are indicated to the left of the criteria. See Figure 2—figure supplement 1 for results per article, and Supplementary file 2 for detailed criteria. iPSCs, Induced pluripotent stem cells; n/a, not applicable; PSCs, pluripotent stem cells.

Figure 2—figure supplement 1
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Quality assessment results per article.

Quality assessment results per article, grouped by protocol and neuron type, and listed chronologically. The earliest article per protocol is printed in bold. Reference numbers are indicated following the article authors and year. The quality criteria in Figure 2 have been indicated diagonally above each column. Topic categories have been indicated below the table. Please see Supplementary file 2 for detailed criteria. * Methodological articles were assessed using the same criteria as other articles. iPSCs, induced pluripotent stem cells; n/a, not applicable; PSCs, pluripotent stem cells.

Figure 3
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Neural crest cell induction.

Timings and signaling cues used during the first phase of differentiation until neural crest induction per unique protocol. Duration of this phase per protocol is indicated by the horizontal black bars. Categories of similar approaches are indicated to the right of the figure. Molecules targeting similar pathways have been grouped by color. Colors also match the signaling cues in Figures 4—6. * Selection step yields optimal cell purity, but this is not required. † Gomez et al., 2019 identified two optimal CHIR concentrations for neural crest induction, 3 µM and 10 µM. 10 µM was used for sympathetic neuron differentiation. BMP, bone morphogenetic protein; CD49d, Integrin subunit α4; CHIR, CHIR99021; DMH, dorsomorphin; eGFP, enhanced green fluorescent protein; FGF, fibroblast growth factor; GD2, disialoganglioside; HNK1, human natural killer-1; LDN, LDN193189; m3i, Modified three inhibitor approach (CHIR99021, DAPT, and PD173074); NGF, nerve growth factor; NGFR, nerve growth factor receptor; PHOX2B, paired-like homeobox 2b; PMP, purmorphamine; RA, retinoic acid; SB, SB431542; SDIA, stromal cell-derived inducing activity; SHH, Sonic hedgehog; TGFβ, transforming growth factor beta.

Figure 4 with 1 supplement
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In vivo neural crest induction signaling requirements.

(A) General signaling requirements for distinct populations at the neural plate border. Left, a dorsal schematic view of the embryo and signaling cues (indicated in colored text) present near the neural plate border during early gastrulation are shown. Right, the temporal sequence of signaling cues required for distinct populations near the neural plate border in amniotes between gastrulation and neurulation is shown (based on Thawani and Groves, 2020). (B) Neuromesodermal progenitors arise in the tailbud during axial elongation under conditions of high Wnt and FGF signaling activation. Wnt and FGF concentrations form a rostrocaudal gradient, with highest concentrations in the tailbud. Right, neuromesodermal progenitors possibly contribute to posterior neural crest cell populations. BMP, bone morphogenetic protein; BMP-i, BMP signaling inhibition; FGF, fibroblast growth factor; SOX2, SRY-box transcription factor 2; TBXT, T-box transcription factor T; Wnt-i, Wnt signaling inhibition.

Figure 4—figure supplement 1
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Rostrocaudal boundaries of vagal and trunk neural crest.

Lateral view of an embryo around the time of neural crest delamination. The rostrocaudal boundaries of vagal neural crest, which gives rise to parasympathetic neurons (Figure 6), are indicated by the light purple line adjacent to the embryo. The darker purple line indicates the rostrocaudal boundaries of trunk neural crest, which gives rise to sympathetic neurons (Figure 5). In later developmental stages, as axial elongation progresses, (sacral) neural crest continues to be formed caudally from trunk neural crest.

Figure 5 with 1 supplement
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Sympathetic neurogenesis.

(A) Timings and signaling cues used from neural crest induction until the end of sympathetic neuron differentiation. Duration of this phase per protocol is indicated by the horizontal black bars. Total durations of this phase exceeding the width of the graph are indicated to the right of the graph. Molecules targeting similar pathways have been grouped by color. (B) Transverse cross section of the trunk of an embryo during neural crest migration. Signaling requirements for ventral neural crest migration and sympathetic specification are indicated by bold text. Signaling cues targeted by the protocols in (A) are indicated with colored text matching those in the figure legend. (C) Schematic view of the signaling requirements for sympathetic precursor proliferation and target innervation. The discontinuous axon and blood vessel represent the large distance from the sympathetic ganglia to their peripheral targets. * Aphidicolin selection yields optimal cell purity. However, this is not required. AA, ascorbic acid; BDNF, brain-derived neurotrophic factor; BMP, bone morphogenetic protein; CHIR, CHIR99021; CXCL12, C-X-C motif chemokine ligand 12; dbcAMP, dibutyryl cyclic adenosine monophosphate; EGF, epidermal growth factor; eGFP, enhanced green fluorescent protein; FGF, fibroblast growth factor; FSK, forskolin; GDNF, glial cell line derived neurotrophic factor; IGF, insulin-like growth factor; MYCN, MYCN proto-oncogene; NGF, nerve growth factor; NRG1, neuregulin 1; NRTN, neurturin; NTF3, neurotrophin 3; PHOX2B, paired-like homeobox 2b; PMP, purmorphamine; RA, retinoic acid; SEMA3A, semaphorin 3A; SB, SB431542; SDIA, stromal cell-derived-inducing activity; SHH, Sonic hedgehog; TGFβ, transforming growth factor beta.

Figure 5—figure supplement 1
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Total overview of all sympathetic protocols.

Overview of all unique sympathetic protocols. Protocol durations are indicated by the horizontal black bars. Total protocol durations exceeding the width of the graph are indicated to the right of the graph. Molecules targeting similar pathways have been grouped by color. AA, ascorbic acid; BDNF, brain-derived neurotrophic factor; BMP, bone morphogenetic protein; CD49d, integrin subunit α4; CHIR, CHIR99021; dbcAMP, dibutyryl cyclic adenosine monophosphate; DMH, dorsomorphin; EGF, epidermal growth factor; eGFP, enhanced green fluorescent protein; FGF, fibroblast growth factor; FSK, forskolin; GD2, disialoganglioside; GDNF, glial cell line-derived neurotrophic factor; HNK1, human natural killer-1; IGF1, insulin-like growth factor 1; LDN, LDN193189; m3i, modified three inhibitor approach (CHIR99021, DAPT, and PD173074); MYCN, MYCN proto-oncogene; NGF, nerve growth factor; NGFR, nerve growth factor receptor; NTF3, neurotrophin 3; PHOX2B, paired-like homeobox 2B; PMP, purmorphamine; RA, retinoic acid; SB, SB431542; SDIA, stromal cell-derived inducing activity; SHH, Sonic hedgehog; TGFβ, transforming growth factor.

Figure 6 with 1 supplement
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Parasympathetic neurogenesis.

(A) Timings and signaling cues used from neural crest induction until the end of parasympathetic neuron differentiation. Duration of this phase per protocol is indicated by the horizontal black bars. Total duration of this phase per protocol is indicated to the right of the graph. Molecules targeting similar pathways have been grouped by color. (B) Migration of vagal neural crest-derived Schwann cell precursors along a cranial nerve. (C) Schematic view of the signaling requirements for parasympathetic precursor proliferation and target innervation. Signaling requirements are indicated by bold text. Signaling cues targeted by the protocols in (A) are indicated with colored text matching those in the figure legend. * Cytosine arabinoside selection yields optimal cell purity. However, this is not required. AA, ascorbic acid; AraC, cytosine arabinoside; BDNF, brain-derived neurotrophic factor; BMP, bone morphogenetic protein; CHIR, CHIR99021; CNTF, ciliary neurotrophic factor; dbcAMP, dibutyryl cyclic adenosine monophosphate; FGF, fibroblast growth factor; FSK, forskolin; GDNF, glial cell line-derived neurotrophic factor; NGF, nerve growth factor; NRG1, neuregulin 1; NRTN, neurturin; NTF3, neurotrophin 3; RA, retinoic acid; SHH, Sonic hedgehog; TGFβ, transforming growth factor beta.

Figure 6—figure supplement 1
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Total overview of all parasympathetic protocols.

Overview of all unique parasympathetic protocols. Protocol durations are indicated by the horizontal black bars. Total protocol durations are indicated to the right of the graph. Molecules targeting similar pathways have been grouped by color. AA, ascorbic acid; AraC, cytosine arabinoside; BDNF, brain-derived neurotrophic factor; BMP, bone morphogenetic protein; CHIR, CHIR99021; CNTF, ciliary neurotrophic factor; dbcAMP, dibutyryl cyclic adenosine monophosphate; DMH, dorsomorphin; FGF, fibroblast growth factor; FSK, forskolin; GDNF, glial cell line-derived neurotrophic factor; LDN, LDN193189; NGF, nerve growth factor; NRG1, neuregulin 1; NTF3, neurotrophin 3; RA, retinoic acid; SB, SB431542; SHH, Sonic hedgehog; TGFβ, transforming growth factor.

Figure 7
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Sympathetic neuron definitions and differentiation efficiency.

(A) All sympathetic neuron markers used in ≥3 articles, stratified by technique. (B) All combinations of sympathetic neuron markers used in ≥6 articles. Markers featured in multiple combinations are marked by colored text in (A) and (B). (C) Scatter plot of protocol purity and time of quantification per article. The graph shows only the latest timepoint per article %TH+ (or %GATA3 + or %DBH+, if %TH+ was not determined) was measured. Shapes indicate the markers used for quantification and protocol applied per article is indicated by color. #1 Gomez et al., 2019, #2 Cheng et al., 2024, #3 Frith et al., 2018, #4 Oh et al., 2016, #5 Wu et al., 2022b, #6 Zeltner et al., 2016, #7 Carr-Wilkinson et al., 2018, #8 Kirino et al., 2018, #9 Fan et al., 2024a, #10 Li et al., 2023, #11 Winbo et al., 2020. Sample sizes per article can be found in Source data 1. ASCL1, achaete-scute family bHLH transcription factor 1; DBH, dopamine beta-hydroxylase; GATA3, GATA binding protein 3; RT-qPCR, quantitative reverse transcriptase polymerase chain reaction; PHOX2B, paired-like homeobox 2B; PRPH, peripherin; SEM, standard error of the mean; TH, tyrosine hydroxylase; TUBB3, tubulin beta 3 class III.

Tables

Table 1
Characteristics of the included articles.

Articles are grouped per protocol and neuron type, in chronological order. Rows in bold indicate the earliest article per protocol.

ReferenceJournal (ISO 4)Neuron typeArticle typeSource cells
Huang et al., 2016Sci RepSympatheticProtocol developmentPSCs
Cheng et al., 2024*Sci RepSympatheticProtocol applicationiPSCs
Zhang et al., 2016PLoS OneSympatheticProtocol development/applicationESCs
Oh et al., 2016Cell Stem CellSympatheticProtocol developmentPSCs
Zeltner et al., 2016Nat MedSympatheticProtocol development/applicationPSCs
Saito-Diaz et al., 2019Curr Protoc Stem Cell BiolSympatheticMethodologicalPSCs
Frith et al., 2018eLifeSympatheticProtocol developmentPSCs
Frith and Tsakiridis, 2019Curr Protoc Stem Cell BiolSympatheticMethodologicalPSCs
Saldana-Guerrero et al., 2024Nat CommunSympatheticProtocol applicationESCs
Kirino et al., 2018Sci RepSympatheticProtocol developmentPSCs
Amer-Sarsour et al., 2024Embo JSympatheticProtocol applicationiPSCs
Saleh et al., 2024Free Radic Biol MedSympatheticProtocol applicationiPSCs
Carr-Wilkinson et al., 2018Stem Cells IntSympatheticProtocol developmentESCs
Hackland et al., 2019Stem Cell ReportsSympatheticProtocol development/applicationPSCs
Gomez et al., 2019DevelopmentSympatheticProtocol development/applicationPSCs
Wu and Zeltner, 2020J Vis ExpSympatheticMethodologicalESCs
Wu et al., 2022aClin Auton ResSympatheticProtocol applicationPSCs
Wu et al., 2022bNat CommunSympatheticProtocol development/applicationPSCs
Wu et al., 2023Front NeurosciSympatheticProtocol applicationESCs
Wu et al., 2024aSTAR ProtocSympatheticMethodologicalESCs
Winbo et al., 2020Am J Physiol Heart Circ PhysiolSympatheticProtocol developmentiPSCs
Winbo et al., 2021Am J Physiol Heart Circ PhysiolSympatheticProtocol applicationiPSCs
Bernardin et al., 2022CellsSympatheticProtocol applicationiPSCs
Li et al., 2023Philos Trans R Soc Lond B Biol SciSympatheticProtocol applicationiPSCs
Van Haver et al., 2024iScienceSympatheticProtocol development/applicationPSCs
Fan et al., 2024aJ Mol NeurosciSympatheticProtocol developmentPSCs
Fan et al., 2024bCell Rep MedSympatheticProtocol applicationPSCs
Takayama et al., 2020Sci RepSympathetic or parasympatheticProtocol developmentPSCs
Takayama et al., 2023Int J Mol SciSympathetic or parasympatheticProtocol applicationPSCs
Akagi et al., 2024aFEBS Open BioParasympatheticProtocol applicationiPSCs
Akagi et al., 2024bMoleculesSympathetic or parasympatheticProtocol applicationiPSCs
Goldsteen et al., 2022Front PharmacolParasympatheticProtocol developmentESCs
Wu et al., 2024bCell Stem CellParasympatheticProtocol development/applicationPSCs

  1. ESCs, embryonic stem cells; iPSCs, induced pluripotent stem cells; ISO, International Organization for Standardization; PSCs, pluripotent stem cells (iPSCs or ESCs).

  2. *

    Cheng et al., 2024 applied three of the protocols included in this review, by Huang et al., 2016, Frith et al., 2018, and Kirino et al., 2018.

Table 2
Patch clamp recordings of hPSC-derived sympathetic neurons.

Electrophysiological characteristics of hPSC-derived sympathetic neurons determined by whole-cell patch clamp. Data from primary adult murine thoracic sympathetic neurons is included for reference. Tabulation is in chronological order. Data is reported as mean ± SEM or range, unless indicated otherwise. AP, action potential; hPSC, human pluripotent stem cell; NR, not reported; SEM, standard error of the mean.

Adult murine thoracic sympathetic neurons (McKinnon et al., 2019) (n=35)Oh et al., 2016 (n=9)Frith et al., 2018 (n=14)Winbo et al., 2020 (n=30)Takayama et al., 2023 (n=113)
Age (days)37–379 (postnatal)28>2048–76>42
Membrane capacitance (pF)89 ± 4.6
(n=34)		NR11 ± 0.685 ± 5.1NR
Current injection range (pA)0–2000–800–10 to 1000–300–100 to 300
Proportion neurons firing repetitive APs, %10056217336
Resting membrane potential (mV)−60 ± 1.1−46 ± 5.4−54 to –60−60 ± 1.9NR
AP amplitude (mV)54 ± 2.7NRNR93 ± 3.974 ± 4.3
(n=20)
AP duration, half-width (ms)4.6 ± 0.2NRNR2.8 ± 0.2NR

Additional files

Supplementary file 1

List of excluded articles.

A list of all articles that were excluded during full-text screening, including the reason of exclusion. PSCs, pluripotent stem cells.

https://cdn.elifesciences.org/articles/103728/elife-103728-supp1-v2.docx
Supplementary file 2

Quality assessment criteria and definitions.

Detailed criteria and definitions per judgement per criterion.

https://cdn.elifesciences.org/articles/103728/elife-103728-supp2-v2.docx
Supplementary file 3

Methodological details of included protocols.

Additional culture details per original protocol, relating to Figures 3, 5 and 6.

https://cdn.elifesciences.org/articles/103728/elife-103728-supp3-v2.docx
Supplementary file 4

Small molecules used in autonomic neuron protocols.

Modes of actions for all small molecules featured in this article.

https://cdn.elifesciences.org/articles/103728/elife-103728-supp4-v2.docx
Supplementary file 5

Molecular autonomic neuron markers.

Function and expression profiles of molecular autonomic neuron markers.

https://cdn.elifesciences.org/articles/103728/elife-103728-supp5-v2.docx
Supplementary file 6

Preferred Reporting Items for Systematic Reviews and Meta-Analyses 2020 Main Checklist.

Locations of all Preferred Reporting Items for Systematic Reviews and Meta-Analyses 2020 Main Checklist items.

https://cdn.elifesciences.org/articles/103728/elife-103728-supp6-v2.docx
MDAR checklist
https://cdn.elifesciences.org/articles/103728/elife-103728-mdarchecklist1-v2.docx
Source data 1

Dataset relating to Figure 1.

All title-abstract exclusion reasons per record. Please see Supplementary file 1 for exclusion reasons during full-text screening. n/a, not available. Dataset relating to Figure 7A and B. Definitions of sympathetic neurons per article. Only one of quantification, immunofluorescence or RT-qPCR data was collected per article. Quantification data was preferentially collected over immunofluorescence or RT-qPCR. Immunofluorescence data was collected preferentially over RT-qPCR. n/a, not available; RT-qPCR, quantitative reverse transcriptase polymerase chain reaction. Dataset relating to Figure 7C: sympathetic neuron efficiency quantification per article. Only one efficiency marker was collected per article. %TH+ was collected preferentially over the other markers. %TH+ PRPH+ was collected preferentially over %GATA3+ and %DBH+. DBH, dopamine beta-hydroxylase; GATA3, GATA binding protein 3; n/a, not available; NR, not reported; PRPH, peripherin; TH, tyrosine hydroxylase.

https://cdn.elifesciences.org/articles/103728/elife-103728-data1-v2.xlsx

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  1. Thomas A Bos
  2. Elizaveta Polyakova
  3. Janine Maria van Gils
  4. Antoine AF de Vries
  5. Marie-José Goumans
  6. Christian Freund
  7. Marco C DeRuiter
  8. Monique RM Jongbloed
(2025)
A systematic review and embryological perspective of pluripotent stem cell-derived autonomic postganglionic neuron differentiation for human disease modeling
eLife 14:e103728.
https://doi.org/10.7554/eLife.103728