Human axial progenitors generate trunk neural crest cells in vitro
- The University of Sheffield, United Kingdom
- High Performance Computing and Networking Institute, National Research Council of Italy, Italy
- Technische Universität Dresden, Germany
- University of Sheffield, United Kingdom
- University of Cambridge, United Kingdom
- Max Delbrück Center for Molecular Medicine, Germany
- The Francis Crick Institute, United Kingdom
- University of Edinburgh, United Kingdom
Figures
Figure 1 with 1 supplement
Transcriptome analysis of in vitro-derived human axial progenitors.
(A) Immunofluorescence analysis of expression of indicated markers in day 3 hPSC-derived axial progenitors. Magnified regions corresponding to the insets are also shown. Scale bar = 100 µm. (B) …
Figure 1—figure supplement 1
RNASeq analysis of in vitro-derived axial progenitors.
(A) Dendrogram showing the clustering of the individual axial progenitor (NMP) and hESC sample replicates based on RNAseq expression data. (B) Principal Component Analysis (PCA) plot indicates a …
Figure 2 with 1 supplement
Human axial progenitor cultures exhibit a neural crest/border signature.
(A) Log-fold induction of representative neural crest/neural plate border and BMP-associated transcripts in axial progenitors compared to hESCs. (B) Immunofluorescence analysis of expression of …
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Figure 2—source data 1
Raw data for Figure 2.
- https://doi.org/10.7554/eLife.35786.007
Figure 2—figure supplement 1
Dissecting the expression of neural crest/border markers in cultures of in vitro-derived axial progenitors.
(A) qPCR expression analysis of indicated NC/border and pluripotency markers in axial progenitors vs hPSCs. Error bars = S.E.M. (n = 3). (B) Quantification of cells marked by different combinations …
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Figure 2—figure supplement 1—source data 1
Raw data for Figure 2—figure supplement 1.
- https://doi.org/10.7554/eLife.35786.006
Figure 3 with 3 supplements
In vitro-derived axial progenitors generate trunk neural crest efficiently.
(A) Diagram depicting the culture conditions employed to direct trunk NC, posterior neurectoderm (PNE) and paraxial mesoderm (PXM) differentiation from hPSC-derived axial progenitors. (B) …
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Figure 3—source data 1
Raw data for Figure 3.
- https://doi.org/10.7554/eLife.35786.015
Figure 3—figure supplement 1
Dynamics of trunk neural crest differentiation from axial progenitors.
(A) Immunofluorescence analysis of HOXC9 and ETS1 expression in cranial NC cells generated from hPSCs as described in (Hackland et al., 2017). Scale bar = 100 µm. (B) Immunofluorescence analysis of …
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Figure 3—figure supplement 1—source data 1
Raw data for Figure 3—figure supplement 1.
- https://doi.org/10.7554/eLife.35786.010
Figure 3—figure supplement 2
Characterisation of hPSC- derived axial progenitor differentiation products.
(A) Representative section of a chick embryo grafted with ZsGREEN+ human axial progenitor-derived trunk NC cells. The arrowhead indicates a migratory stream of donor human trunk NC cells emerging …
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Figure 3—figure supplement 2—source data 1
Raw data for Figure 3—figure supplement 2.
- https://doi.org/10.7554/eLife.35786.012
Figure 3—figure supplement 3
Quantification of pluripotency marker expression in hPSC-derived axial progenitors.
Quantification of cells marked by different combinations of NANOG(A) or OTX2 (B) and T-VENUS expression in undifferentiated hPSCs and axial progenitors. The data in the graph were obtained after …
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Figure 3—figure supplement 3—source data 1
Raw data for Figure 3—figure supplement 3.
- https://doi.org/10.7554/eLife.35786.014
Figure 4 with 1 supplement
Transcriptome analysis of in vitro derived neural crest cells corresponding to distinct axial levels.
(A) Diagrams showing the culture conditions employed for generating NC cells of distinct axial identities using hPSCs. Asterisks indicate the timepoints used for sample harvesting and transcriptome …
Figure 4—figure supplement 1
Microarray analysis of hPSC-derived neural crest cells of distinct axial identities.
(A) Heatmap showing Pearson’s signal correlation between indicated samples. (B) Venn diagram showing the overlap between transcription factors (TFs) enriched (≥2 fold relative to undifferentiated …
Figure 5 with 1 supplement
Axial progenitor-derived trunk neural crest is an optimal source of sympathoadrenal cells.
(A) Diagram depicting the culture conditions employed to direct axial progenitors (‘NMPs’) toward trunk NC and subsequently sympathoadrenal progenitors (SAP) and sympathetic neurons. (B) FACS …
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Figure 5—source data 1
Raw data for Figure 5.
- https://doi.org/10.7554/eLife.35786.020
Figure 5—figure supplement 1
Characterisation of axial progenitor-derived sympathoadrenal progenitors and sympathetic neurons.
(A) Immunofluorescence analysis of PHOX2B-GFP and ASCL1 expression in d12 SAP cells derived from axial progenitors as shown in Figure 5A. Scale bar = 100 µm. (B) Immunofluorescence analysis of DBH, …
Figure 6
A-P patterning of in vitro derived human NC cells.
Diagrammatic model summarising our findings on the in vitro generation of NC subtypes of distinct A-P identity from hPSCs. Examples of unique genes that were found to mark each NC population …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Additional information |
|---|---|---|---|
| cell line (Homo Sapiens) | T-VENUS | 68 | Parental hES cell line = H9 |
| cell line (Homo Sapiens) | SOX10-GFP | 15 | Parental hES cell line = H9 |
| cell line (Homo Sapiens) | PHOX2B-GFP | 18 | Parental hES cell line = H9 |
| cell line (Homo Sapiens) | MSGN1-VENUS | Unpublished | Not previously described, parental line = NCRM1 iPSCs (source = NIH) |
| cell line (Homo Sapiens) | Sox2-GFP | 44 | Parental hES cell line = Shef4 |
| cell line (Homo Sapiens) | MIFF1 | 102 | iPSC line from healthy donor |
| cell line (Homo Sapiens) | SFCi55-ZsGr | 64 | iPSC line from healthy donor containing a constitutive fluorescent ZsGreen reporte |
| cell line (Homo Sapiens) | MasterShef7 | 44 | Wild type hES cell line |
Additional files
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Supplementary file 1
Significantly up- and downregulated transcripts, GO enrichment and TF signatures from RNAseq analysis
- https://doi.org/10.7554/eLife.35786.022
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Supplementary file 2
List of genes upregulated in different NC populations and GO enrichment analysis
- https://doi.org/10.7554/eLife.35786.023
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Supplementary file 3
List of transcription factors shared between different NC populations
- https://doi.org/10.7554/eLife.35786.024
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Supplementary file 4
List of all genes up- and down-regulated in indicated NC populations and their progenitors.
- https://doi.org/10.7554/eLife.35786.025
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Supplementary file 5
List of primers
- https://doi.org/10.7554/eLife.35786.026
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Transparent reporting form
- https://doi.org/10.7554/eLife.35786.027
