Two parallel neural ectoderm progenitors contribute to the developing brain

Loh, Kyle M. · Nature

36 min read Original article ↗

Data availability

Bulk-population RNAseq, scRNAseq and OmniATAC-sequencing datasets generated as part of this study are available at the NCBI Gene Expression Omnibus (accession nos. GSE286146 (OmniATAC-sequencing), GSE286147 (bulk RNAseq) and GSE286148 (scRNAseq)). These datasets are grouped under SuperSeries accession no. GSE286214. An interactive web browser to explore scRNAseq data of the hPS cell-derived cell types generated in this study is available at https://anglohlabs.shinyapps.io/dundes_jokhai_lohlab/. Source data are provided with this paper.

Code availability

Computational scripts used in this study to analyze sequencing datasets are available via GitHub at https://github.com/lohlaboratory/ane-pne.

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Acknowledgements

We thank J. Li, B. Conklin, T. Anbarchian, R. Nusse, F. Suchy, H. Nakauchi, A. Elefanty, Ed. Stanley and E. Ng for sharing reagents. We also thank S. Vijayakumar, B. Woodruff, C. Anselmi and A. Voskoboynik for contributing to experiments, and W. Talbot, A. Zorn, K. Campbell, R. Krumlauf, P. Beachy, C. Shatz, J. Day, A. Pollen, T. Nowakowski, L. T. Ang and N. Uchida for advice. Infrastructure support was provided by V. Park, L. Ou, K. Lee, C. Carswell-Crumpton, P. Lovelace, L. Dunkin-Hubby and the Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stem Cell FACS Core Facility, Veterinary Service Center, Cell Sciences Imaging Facility and Diabetes Research Center.

Funding

This work was supported by the US National Institutes of Health (grant nos. DP5OD024558 and P30DK116074 to K.M.L., DP2GM146258 and R00GM121852 to D.E.W., R01DK115728 to K.C.G., R01DE027538 to M.E.B., T32GM119995 to C.E.D., T32GM007365 and T32GM007790 to R.T.J., T32GM141828 to A.R. and F31DE031154 to H.A.U.), the US National Science Foundation (grant no. IOS1656628 to C.J.L.); the California Institute of Regenerative Medicine (grant nos. TB1-01195 to R.E.A.S.-S. and EDUC2-12677 to A.S.P.); the Spinal Muscular Atrophy Foundation (K.M.L.); the Stanford Maternal and Child Health Research Institute (K.M.L.); the Stanford Beckman Center (K.M.L.); the Stanford Ludwig Center (K.M.L.); the Siebel Stem Cell Institute (K.M.L.); the Stinehart-Reed Seed Grant (K.M.L.); Anonymous, Fickel and Gilbert families (K.M.L.); the Gatsby Charitable Foundation (M.M.); the Howard Hughes Medical Institute (M.M. and K.C.G.); the Ford Foundation, Stanford Graduate and Stanford DARE Fellowships (C.E.D.); the Stanford Medical Scholars Research Program (R.T.J.), the Yale Saybrook College and Stacey Leondis Fellowships (C.X.); and the Stanford MCHRI and Dean’s Postdoctoral Fellowships (Y.Q.). K.M.L. was supported as a Packard Foundation Fellow, Pew Scholar, Baxter Foundation Faculty Scholar, Human Frontier Science Program Young Investigator (RGY0069/2019) and Anthony DiGenova Endowed Faculty Scholar. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Author notes

  1. Yoon Seok Kim

    Present address: Institute of Bioengineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

  2. Yi Miao

    Present address: Division of Life Science, SIAT-HKUST Joint Laboratory for Brain Science, The Hong Kong University of Science and Technology, Hong Kong, China

  3. These authors contributed equally: Rayyan T. Jokhai, Carolyn E. Dundes.

Authors and Affiliations

  1. Department of Developmental Biology, Stanford University, Stanford, CA, USA

    Rayyan T. Jokhai, Carolyn E. Dundes, Hadia S. Ahsan, Rachel S. Kang, Rachel E. A. Salomon-Shulman, Arjun Rajan, Liam J. Stanton, Christine Xu, Stephanie Do, Alicia Wong, Yimiao Qu, Andrew S. Petkovic & Kyle M. Loh

  2. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA

    Rayyan T. Jokhai, Carolyn E. Dundes, Hadia S. Ahsan, Rachel S. Kang, Rachel E. A. Salomon-Shulman, Arjun Rajan, Yoon Seok Kim, Liam J. Stanton, Christine Xu, Stephanie Do, Alicia Wong, Yimiao Qu, Andrew S. Petkovic, Michelle Monje & Kyle M. Loh

  3. Department of Neurology and Neurological Sciences, Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA

    Yoon Seok Kim & Michelle Monje

  4. Hopkins Marine Station, Department of Biology, Stanford University, Pacific Grove, CA, USA

    Brennan D. McDonald, José Miguel Andrade López & Christopher J. Lowe

  5. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA

    Hugo A. Urrutia & Marianne E. Bronner

  6. Department of Obstetrics, Gynecology and Reproductive Science, Center for Reproductive Sciences, Eli and Edythe Broad Center for Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA, USA

    Hannah Greenfeld & Daniel E. Wagner

  7. Department of Molecular and Cellular Physiology, Department of Structural Biology, Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA

    Yi Miao & K. Christopher Garcia

Authors

  1. Rayyan T. Jokhai
  2. Carolyn E. Dundes
  3. Hadia S. Ahsan
  4. Rachel S. Kang
  5. Rachel E. A. Salomon-Shulman
  6. Arjun Rajan
  7. Yoon Seok Kim
  8. Liam J. Stanton
  9. Christine Xu
  10. Stephanie Do
  11. Brennan D. McDonald
  12. José Miguel Andrade López
  13. Hugo A. Urrutia
  14. Hannah Greenfeld
  15. Alicia Wong
  16. Yimiao Qu
  17. Andrew S. Petkovic
  18. Yi Miao
  19. K. Christopher Garcia
  20. Michelle Monje
  21. Daniel E. Wagner
  22. Marianne E. Bronner
  23. Christopher J. Lowe
  24. Kyle M. Loh

Contributions

R.T.J. and C.E.D. contributed equally as co-first authors. H.S.A., R.S.K. and R.E.A.S.-S. contributed equally as co-second authors. C.E.D. performed lineage tracing. C.E.D., R.T.J., R.E.A.S.-S., H.S.A., L.J.S., C.X., S.D., R.S.K., A.W., Y.Q., A.S.P. and K.M.L. differentiated and characterized hPS cells. C.E.D., A.R., R.T.J. and R.S.K. performed genomics analyses. Y.S.K. performed electrophysiological studies, supervised by M.M. Y.M. and K.C.G. provided synthetic WNT agonists. H.A.U. and M.E.B. stained chicken embryos. H.G. and D.E.W. stained zebrafish embryos. B.D.M., J.M.A.L. and C.J.L. stained acorn worm embryos. M.E.B., D.E.W. and C.J.L. contributed to evolutionary discussions. C.E.D., R.T.J. and K.M.L. designed the study. K.M.L. supervised the study.

Corresponding authors

Correspondence to Rayyan T. Jokhai or Kyle M. Loh.

Ethics declarations

Competing interests

Stanford University has filed patent applications related to neural differentiation, with K.M.L., R.E.A.S.-S., C.E.D., H.A. and R.T.J. listed as inventors. The other authors declare no competing interests.

Peer review

Peer review information

Nature Neuroscience thanks the anonymous reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Definitive ectoderm and neural ectoderm marker expression.

a) Single-cell RNA-sequencing of E6.75 mouse embryos; analysis performed on a previously-published resource77. Embryonic cells are shown, with extraembryonic cells excluded (Elf5 and Rhox5 log2 normalized expression = 0). Otx2 is expressed in definitive ectoderm and pluripotent cells, consistent with previous studies4,32. Oct4 (Pou5f1) is broadly expressed across all cell-types, consistent with past studies110,111. b) Immunostaining of hPS cells before, or after, differentiation into definitive ectoderm for 24 hours. NANOG and SOX2 immunostaining was performed with H7 hPS cells. OTX2 immunostaining was performed with H1 hPS cells. Scale bar = 100 μm. Representative images from 2 biological replicates in 1 experiment. c) Flow cytometry of H1 hPS cells differentiated into definitive ectoderm for 24 hours. The percentage of SOX2 + NANOG- definitive ectoderm cells is shown. Each dot represents 1 independent differentiation experiment, with 3 experiments shown. d) Flow cytometry of HES3 MIXL1-GFP hPS cells differentiated into definitive ectoderm (day 1), anterior neural ectoderm (day 2), posterior neural ectoderm (day 2), border ectoderm (day 2), surface ectoderm (day 1), or mid primitive streak30 (day 1, as a positive control to induce MIXL1 expression). Negligible numbers of MIXL1+ primitive streak cells emerged after ectoderm differentiation, reaffirming the suppression of spurious primitive streak differentiation. Representative data from 2 biological replicates in 1 experiment. e) In situ staining of E7.5 mouse embryo, showing overlap of Sox2 and Gbx2 expression in pNE. Scale bar = 25 μm. Representative images from 40 embryos from 10 experiments. f) Single-cell RNA-sequencing of E7.5 mouse embryos; analysis performed on a previously-published resource77. Neural ectoderm cells (identified by log2 normalized expression of Sox2 > 0.5, and Nanog, Tfap2a, Rhox5, Foxj1, and Elf5 = 0) are shown. g) Intracellular flow cytometry for SOX2 protein expression in H1 hPS cells, prior to or after differentiation into anterior neural ectoderm or posterior neural ectoderm for 2 days. The percentage of SOX2+ cells, and median fluorescence intensity (MFI) of SOX2 expression, are both shown. Representative data from 2 biological replicates in 1 experiment. h) Immunostaining of H1 hPS cells differentiated into definitive ectoderm for 24 hours, followed by differentiation into aNE or pNE for 24 hours. Scale bar = 100 μm. Representative images from 2 biological replicates in 1 experiment.

Source data

Extended Data Fig. 2 Posterior neural ectoderm induction.

a) Western Blot to detect the expression of GBX2 protein in H1 hPS cell-derived day-2 aNE or day-2 pNE. VINCULIN protein serves as a loading control. Images from 2 biological replicates in 1 experiment. b) β-CATENIN immunostaining of H1 hPS cells that were differentiated into various cell-types. First, hPS cells were differentiated into definitive ectoderm for 24 hours, and then treated with posterior neural ectoderm-inducing media for 24 hours (A-83-01 [1 μM] + LDN193189 [100 nM] + TTNPB [50 nM] + FGF2 [20 ng/mL] + XAV939 [1 μM] + Thiazovivin [1 μM]), or as a positive control to induce nuclear β-CATENIN, modified posterior neural ectoderm media lacking WNT inhibitor and including a WNT agonist for 24 hours (A-83-01 [1 μM] + LDN193189 [100 nM] + TTNPB [50 nM] + FGF2 [20 ng/mL] + CHIR99021 [2 μM] + Thiazovivin [1 μM]). Alternatively, as a positive control to induce nuclear β-CATENIN, hPS cells were differentiated into mid primitive streak, with WNT agonist-containing media (Activin A [30 ng/mL] + BMP4 [40 ng/mL] + CHIR99021 [6 μM] + FGF2 [20 ng/mL]) for 24 hours. Nuclear β-CATENIN was absent from hPSC-derived posterior neural ectoderm, and either weakly or strongly positive in the two WNT agonist-treated cell populations. Scale bar = 100 μm. Representative images from 2 biological replicates in 2 experiments.

Source data

Extended Data Fig. 3 Differentiation of hPSCs into anterior neural, posterior neural, border or surface ectoderm.

a) Summary of the hPSC differentiation strategy described in this study. b) Top: In situ hybridization of H1 hPSCs differentiated into definitive ectoderm for 24 hours, followed by differentiation into aNE or pNE for 24 hours. Scale: 100 μm. Bottom: Immunostaining of H1 hPSCs differentiated into definitive ectoderm for 24 hours, followed by differentiation into border or surface ectoderm for 24 hours. Scale: 100 μm. Percentages of cells expressing a given marker are shown. Representative images from 2 biological replicates in 2 experiments.

Extended Data Fig. 4 Lineage tracing of neural ectoderm cells within mouse embryos.

a) In situ staining of E9.5 Gbx2-CreER; ROSA26-tdTomato mouse embryo that was not exposed to 4OHT. This negative control revealed minimal Gbx2-CreER-driven recombination in the absence of 4OHT. Scale bar = 500 μm. Image of 1 embryo from 1 experiment. b) E7.0 Gbx2-CreER; ROSA26-LoxP-STOP-LoxP-tdTomato mouse embryos were exposed to 1 mg 4OHT (intraperitoneally delivered into pregnant females) to label Gbx2+ pNE with tdTomato. Subsequently, in situ staining was performed on E8.25 embryos to visualize pNE-derived progeny in relation to the expression of Wnt1 (which marks the midbrain/hindbrain boundary) and Gbx2 (which marks the hindbrain). Scale bar = 100 μm. Representative images from 18 embryos from 4 experiments. c) E7.5 Sox2-CreER; Confetti mouse embryos were exposed to 0.5 mg 4OHT (intraperitoneally delivered into pregnant females) to label single E7.5 Sox2+ neural ectoderm progenitors with one of three detectable fluorescent colors (red, cyan, or yellow). Subsequently, E9.5 embryos were immunostained to visualize neural ectoderm-derived cell clusters and where they resided in relation to the forebrain, midbrain, and hindbrain; photos of three independent embryos are shown (left). The forebrain/midbrain domain was molecularly determined by Otx2 immunostaining (left). Quantification of the locations of all neural ectoderm-derived cell clusters analyzed in this study, totaling 494 cell clusters from 16 embryos from 7 experiments (right). Scale bar = 100 μm. d) Detailed analysis of the spatial position of a E7.5 Sox2+ neural ectoderm-derived cell cluster relative to the midbrain/hindbrain boundary. E7.5 Sox2-CreER; Confetti mouse embryos were exposed to 0.5 mg 4OHT (intraperitoneally delivered into pregnant females) to label single E7.5 Sox2+ neural ectoderm progenitors with one of three detectable fluorescent colors (red, cyan, or yellow). Subsequently, E9.5 embryos were immunostained to visualize neural ectoderm-derived cell clusters and where they resided in relation to the forebrain, midbrain, and hindbrain. The forebrain/midbrain domain was molecularly determined by Otx2 immunostaining. This is a higher magnification image of an embryo shown in Extended Data Fig. 4c, which was taken from 1 experiment. Asterisks indicate autofluorescence from red blood cells (RBCs). Scale bar = 100 μm.

Extended Data Fig. 5 Differentiation of hPSCs into forebrain or midbrain progenitors.

a) qPCR of H1 hPSCs differentiated into definitive ectoderm for 24 hours, followed by anterior neural ectoderm for 24 hours, and then subsequently treated with the following WNT pathway modulators for 48 hours: WNT inhibitor (XAV939, 1 μM), control media (no WNT modulator, depicted as “-”), RSPO2 (25 nM), RSPO2 + WNT3A (increasing concentrations of 10, 100 or 1000 ng/mL), RSPO2 + NGS WNT-Fc (increasing concentrations of 0.03, 0.3 or 3 nM), or CHIR99021 (increasing concentrations of 0.75, 1.5 or 3 μM). For WNT3A or NGS WNT-Fc treatment conditions, RSPO2 was added to increase cell-surface levels of FRIZZLED receptors, thereby enhancing cellular responsiveness to these WNT pathway agonists112. NGS-WNT-Fc refers to an Fc-tagged synthetic WNT pathway agonist that acts by heterodimerizing FZD and LRP6 receptors112. Heatmap depicts row Z-score, calculated across the 12 treatment conditions from the mean normalized expression of 2 biological replicates from 1 experiment. b) In situ staining of an E8.5 Tcf/Lef:H2B:Venus reporter mouse embryo82 for Foxg1 (forebrain marker) and Venus (indicating WNT transcriptional response). This disclosed a posteriorizing gradient of WNT signaling, with minimal WNT signaling in Foxg1+ forebrain, but high WNT signaling in the midbrain. Representative image from 15 embryos from 2 experiments.

Source data

Extended Data Fig. 6 Lineage commitment of hPSC-derived anterior and posterior neural ectoderm.

a) scRNAseq of H7 hPSC-derived day-4 hindbrain progenitors, and day-7 dorsal hindbrain or ventral hindbrain progenitors. Colors denote differentiation conditions. An asterisk (*) indicates that the legend is identical to that shown in Fig. 4c, and is reproduced here to aid the interpretation of other images shown in this subpanel. Data from 1 biological replicate per cell-type. b) H1 hPSCs were differentiated into definitive ectoderm for 24 hours, and then subsequently differentiated into either anterior neural ectoderm (aNE) or posterior neural ectoderm (pNE) within an additional 24 hours. hPSC-derived aNE and pNE cells were then dissociated and then replated in either forebrain-, midbrain-, or hindbrain-inducing signals for 48 hours, and then immunostaining was performed. Scale bar = 50 μm. Representative images from 4 biological replicates from 4 experiments.

Extended Data Fig. 7 Accessible chromatin landscapes and lineage commitment of hPSC-derived anterior vs. posterior neural ectoderm.

a) H1 hPSCs were differentiated into either anterior or posterior neural ectoderm for 2 days. They were then respectively challenged with either forebrain-, midbrain-, or hindbrain-inducing signals for 4 days. Immunostaining was performed on day 6 of differentiation. Scale = 100 μm. Representative images from 2 biological replicates from 1 experiment. b) H1 hPSCs were differentiated into either aNE or pNE within 2 days; pNE was fluorescently labeled, and then aNE (uncolored) and pNE (dye-labeled) were mixed. Cocultures were treated with either forebrain-, midbrain-, or hindbrain-inducing signals for 4 days, prior to immunostaining (that is, immunostaining was performed on day 6 of hPSC differentiation). Quantification indicates the percentage of pNE cells (that is, dye-labeled) that express a given marker protein. aNE: day-2 anterior neural ectoderm. pNE: day-2 posterior neural ectoderm. Representative images from 4 biological replicates from 2 experiments. c) OmniATACseq was performed on day 2 H7 hPSC-derived aNE vs. pNE. A Z-score heatmap is shown, depicting all differentially accessible regions between these two cell-types (false discovery rate [FDR] < 0.05). Each row indicates an individual genomic element. Data from 3 biological replicates from 1 experiment. d) OmniATACseq was performed on day 2 H7 hPSC-derived aNE vs. pNE that were challenged with forebrain-inducing signals for 2 additional days (1 day of XAV939 [1 μM] + LDN193189 [100 nM] + FGF2 [20 ng/mL], followed by 1 day of XAV939 (1 μM) and LDN193189 [100 nM]). A Z-score heatmap is shown, depicting all differentially accessible regions between these two cell-types (FDR < 0.05). Each row indicates an individual genomic element. Data from 3 biological replicates from 1 experiment. e) OmniATACseq and RNAseq of hPSC-derived aNE and pNE that were treated with either forebrain- or hindbrain-inducing signals for 48 hours. Data from 3 biological replicates from 1 experiment. f) OmniATACseq was performed on day 2 H7 hPSC-derived aNE vs. pNE that were challenged with hindbrain-inducing signals (CHIR99021 [750 nM] + FGF2 [20 ng/mL]) for 2 additional days. A Z-score heatmap is shown, depicting all differentially accessible regions between these two cell-types (FDR < 0.05). Each row indicates an individual genomic element. Data from 3 biological replicates from 1 experiment. g) Single-cell RNA-sequencing of H7 hPSCs differentiated into day-1 definitive ectoderm, day-2 anterior neural ectoderm, day-2 posterior neural ectoderm, day-2 border ectoderm, or day-2 surface ectoderm. Colors denote differentiation conditions. Fzd5, Lhx5, and Fezf1 have previously been associated with anterior neural identity in vivo at later developmental stages113,114,115, whereas Meis2 has been previously associated with posterior neural identity in vivo116. Data from 1 biological replicate per cell-type.

Source data

Extended Data Fig. 8 Differentiation of hPSCs into dorsal forebrain and ventral forebrain progenitors within 7 days.

scRNAseq of H7 hPSC-derived day-4 hindbrain progenitors, and day-7 dorsal hindbrain or ventral hindbrain progenitors. Colors denote differentiation conditions. An asterisk (*) indicates that the legend is identical to that shown in Fig. 4c, and is reproduced here to aid the interpretation of other images shown in this subpanel. Data from 1 biological replicate per cell-type.

Extended Data Fig. 9 Differentiation of hPSCs into dorsal and ventral forebrain progenitors, and subsequently, dorsal and ventral hindbrain neurons.

a) scRNAseq of H7 hPSC-derived dorsal forebrain, ventral forebrain, dorsal hindbrain and ventral hindbrain populations on day 14 of differentiation. An asterisk (*) indicates images identical to those shown in Fig. 5d, which were reproduced here to aid the interpretation of other images shown in this subpanel. Data from 1 biological replicate per cell-type. b) scRNAseq of H7 hPSC-derived dorsal forebrain populations on day 14 of differentiation reveals expression of Cajal-Retzius neuron markers117. Data from 1 biological replicate per cell-type. c) scRNAseq of H7 hPSC-derived ventral forebrain populations on day 14 of differentiation reveals expression of cortical interneuron markers. Data from 1 biological replicate per cell-type. d) scRNAseq of H7 hPSC-derived ventral forebrain populations on day 14 of differentiation reveals expression of hypothalamus-associated markers. hPSC-derived groups I and II express PROX1 (ref. 118), LHX5 (ref. 119), and ADCYAP1 (ref. 120,121), which are expressed in vivo by various regions of the hypothalamus, including the preoptic area, paraventricular nucleus, and supraoptic nucleus. hPSC-derived group I is additionally defined by OTP, the archetypic transcription factor required for the development of paraventricular and supraoptic nuclei122. Instead, hPSC-derived group II is additionally defined by POU4F1 (BRN3A) and EBF1, and may correspond to an early Pou4f1+ Ebf1+ Pomc- hypothalamic lineage in vivo123. hPSC-derived group II also expresses ADCYAP1R1 (the ADYCAP1 receptor), which is expressed by multiple brain regions, including the preoptic area, paraventricular nucleus, and supraoptic nucleus124. Finally, hPSC-derived group III is demarcated by ISL1 and POMC, which are expressed within the arcuate nucleus of the hypothalamus64. Data from 1 biological replicate per cell-type. e) scRNAseq of H7 hPSC-derived ventral hindbrain populations on day 14 of differentiation reveals expression of multiple ventral hindbrain neuron subtype markers (left), whose identities were extrapolated from ventral spinal cord neuron subtype markers125,126. Cartoon of progenitor domains within the developing ventral hindbrain, based on embryological studies (right)125. The box reflects the neuron subtypes putatively produced in our in vitro differentiation system. Data from 1 biological replicate per cell-type. f) scRNAseq of H7 hPSC-derived dorsal hindbrain populations on day 14 of differentiation reveals expression of multiple dorsal hindbrain neuron subtype markers (left). Cartoon of progenitor domains within the developing ventral hindbrain, based on embryological studies (right). The box reflects the neuron subtypes putatively produced in our in vitro differentiation system. Data from 1 biological replicate per cell-type.

Extended Data Fig. 10 Functional assessment of hPSC-derived dorsal and ventral hindbrain neurons.

a) Day 28 H1 hPSC-derived ventral hindbrain neurons transduced with AAVdj-CAG-bReaChES-eYFP were stimulated with either prolonged (1 second), or pulses of (5 ms, 5 Hz), 560 nm light, and electrophysiological recording was performed to detect action potentials. Representative data from 4 cells from 1 experiment. b) Electrophysiological activity of H1 hPSC-derived dorsal hindbrain neurons on day 30 of differentiation, showing voltage-dependent K+ (left) or Na+ (right) channel currents elicited by depolarization of the holding potential from -90 mV to 50 mV in voltage-clamp mode. Representative data from 4 cells in 1 experiment. c) Current-clamp characterization of H1 hPSC-derived dorsal hindbrain neurons on day 30 of differentiation, showing action potentials elicited by injection of pulsed (500 pA, 5 ms, 10 Hz, left) or prolonged (50 pA, 1.5 seconds, right) currents in current-clamp mode. Representative data from 4 cells in 1 experiment. d) Live Ca2+ imaging of WTC11 AAVS1-CAG-GCaMP6f hPSCs differentiated into dorsal hindbrain neurons for 30 days, which exhibited spontaneous Ca2+ transients as assessed within individual cells (left) and across the culture (right). Scale bar = 50 μm. Representative data from 7 single cells in 1 experiment.

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Jokhai, R.T., Dundes, C.E., Ahsan, H.S. et al. Two parallel neural ectoderm progenitors contribute to the developing brain. Nat Neurosci (2026). https://doi.org/10.1038/s41593-026-02433-7

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