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A human-specific progenitor sub-domain extends neurogenesis and increases motor neuron production

Nature Neuroscience volume 27, pages 1945–1953 (2024)Cite this article

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Abstract

Neurogenesis lasts ~10 times longer in developing humans compared to mice, resulting in a >1,000-fold increase in the number of neurons in the CNS. To identify molecular and cellular mechanisms contributing to this difference, we studied human and mouse motor neurogenesis using a stem cell differentiation system that recapitulates species-specific scales of development. Comparison of human and mouse single-cell gene expression data identified human-specific progenitors characterized by coexpression of NKX2-2 and OLIG2 that give rise to spinal motor neurons. Unlike classical OLIG2+ motor neuron progenitors that give rise to two motor neurons each, OLIG2+/NKX2-2+ ventral motor neuron progenitors remain cycling longer, yielding ~5 times more motor neurons that are biased toward later-born, FOXP1-expressing subtypes. Knockout of NKX2-2 converts ventral motor neuron progenitors into classical motor neuron progenitors. Such new progenitors may contribute to the increased production of human motor neurons required for the generation of larger, more complex nervous systems.

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Fig. 1: Identification of species-specific progenitor cell states from temporal scRNA-seq of in vitro mouse and human motor neurogenesis.
Fig. 2: Human-specific, NKX2-2 and OLIG2 coexpressing mid-stage progenitors give rise to MNs.
Fig. 3: vpMNs undergo delayed and protracted motor neurogenesis and increase MN production, especially that of late-born, LMC-like MNs.
Fig. 4: NKX2-2 is necessary for the delayed neurogenic timeline of vpMNs and their increased MN production.

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Data availability

scRNA-seq datasets produced in this study are available in the GEO under GSE270069. Cell lines (ES and iPS), along with plasmids and viral constructs, will be made freely available to investigators at academic institutions for noncommercial research upon request. Public datasets for mouse in vivo single-cell RNA-seq data are available under accession E-MTAB-7320. Human in vivo single-cell RNA-seq data are available in the GEO under accessions GSE171890 and GSE219122. The TcoF database is accessible via https://tools.sschmeier.com/tcof/home/.

Code availability

Code used in this study is available at https://github.com/wichterle-lab/.

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Acknowledgements

We thank G. Yeo and D. Gifford for help with and access to their shared computing server, E. Bush and M. Finlayson of the Columbia University Sulzberger Genome Center for running the 10x single-cell library preparation and sequencing, and M. Kissner and the CSCI Stem Cell Facility for assistance with flow cytometry. We thank C. Marchetto for kindly sharing Rhesus macaque iPS cells and culture conditions. We thank S. DiIorio for designing and testing sequencing primers for barcode analysis. We are grateful to C. Mason, F. Polleux and M. Closser and members of the Wichterle, Chaolin Zhang and Edmund Au labs for discussion and feedback. H.W. holds an endowed chair from Jerry and Emily Spiegel. We acknowledge grants from the NIH (R01NS116141 and R01NS089676 to H.W.; K99MH130892 to S.J.) and Project ALS (to H.W.) and a seed grant from the Columbia Stem Cell Initiative (to S.J.).

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Authors and Affiliations

  1. Departments of Pathology and Cell Biology, Neuroscience, and Neurology, Center for Motor Neuron Biology and Disease, Columbia Stem Cell Initiative, Columbia University Irving Medical Center, New York, NY, USA

    Sumin Jang, Elias Gumnit & Hynek Wichterle

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Contributions

Conceptualization: S.J. and H.W. Experiments: S.J. Data analysis: S.J. Human NKX2-2-CreERT2 iPS cell line generation: E.G. Writing—original draft: S.J. and H.W. Writing—review and editing: S.J. and H.W.

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Correspondence to Sumin Jang or Hynek Wichterle.

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Extended data

Extended Data Fig. 1 Identification of human-specific progenitors in motor neurogenesis.

(a) Culture conditions for motor neuron differentiation. (b) UMAP of all high-quality single-cell RNA-seq profiles after removal of interneuron-lineage/fibroblast-like cells, colored based on replicate. (c) Timestamp distribution in human single-cell gene expression datasets across the two replicates, normalized by cell cluster (day 10 cells in replicate 1 were not timestamped and therefore inferred based on absence of timestamp expression; see Methods) (d) Timestamp distribution (day 4–6) in mouse single-cell gene expression datasets across the two replicates. (Day 7 was not collected for first replicate.) (e) UMAP of all high-quality single-cell RNA-seq profiles, clusters colored based on identity. (f) UMAP of combined human and mouse single-cell gene expression profiles following CCA-mediated integration, colored by normalized expression level of key marker genes. (g) Chi-square distance between human and mouse clusters. Smaller distances indicate greater similarity in constituent cells’ distribution across common clusters. (h) Classification of human (top) and mouse (bottom) cells according to human-based random-forest classifier shows that the vast majority of mouse cells classified as H0-2 (pMN), H6 (late-appearing NKX2-2+/OLIG2+), or H7-9 (MN), leaving the H3-5 (vpMN) category void of classified mouse cells. (i) Macaque iPSC cultures display NKX2-2 and OLIG2 co-expressing cells when ISL1+ MNs begin to appear, mimicking human cultures (scale bar = 50μm). (j) UMAP of scRNA-seq data from Carnegie Stage 12 human embryonic spinal cords30, colored based on cluster identity or NKX2-2/OLIG2 expression. (k) Left: Alignment of human in vitro and human CS12 embryonic scRNA-seq data shows that human-specific clusters map onto distinct clusters found in vivo. Right: Alignment of human in vitro and mouse E9.5-10.5 spinal cord shows that H4 cells show poor overlap with all embryonic mouse clusters, suggesting that H4-like cells are found in human (but not mouse) embryonic spinal cords. (l) Normalized expression levels of key marker genes in CS12 human (top) and E9.5-E10.5 mouse (bottom) clusters. (m) Alignment of human in vitro and human CS14/pcw5 (CS14) embryonic scRNA-seq data. (n) Normalized expression levels of key marker genes in CS14/pcw5 human clusters.

Extended Data Fig. 2 Characterization of NKX2-2-dependent lineage tracing.

(a) Left: varying lengths and concentrations of 4OHT pulse affect the efficiency of recombination-based RFP expression (mean ± SD; two-way ANOVA; n = 3 biological replicates; time \(p < 1\times {10}^{-4}\); concentration \(p=0.03\)). Right: proportions of NKX2-2-positive cells within RFP-positive populations on day 13, 48 hours post 4OHT removal (two-way ANOVA; n = 3 biological replicates). (b) The same panel of cells as shown in Fig. 2c, immunolabeled for OLIG2 and NKX2-2 and counter-stained with DAPI (scale bar = 50μm). (c) Day 12 human embryoid bodies differentiated with and without SAG (Smoothened agonist), and with 4OHT pulse between days 9-11 show that in the absence of SAG, NKX2-2 expression as well as Cre-dependent recombination is lost (scale bar = 50μm). (d) Representative flow cytometry gating strategy to remove debris and clumps of cells. (e) Day 16 human culture immunostained for pan-neuronal marker NEUN and ISL1 shows that the vast majority of neurons produced are motor neurons (scale bar = 50μm). (f) Flow cytometry analysis of day 16 human cultures shows that the vast majority of ISL1-positive cells are MNX1-positive and vice versa in both RFP+ and RFP− populations (representative differentiation, n = 1, sample pooled across 50+ EBs). (g) Human cultures following 4OHT treatment on days 9–11 show that many RFP+ cells retain progenitor identity (OLIG2+ or OLIG2+/NKX2-2+) on day 14; however, with DAPT treatment, virtually no cells express OLIG2 or NKX2-2 (scale bar = 50μm). (h) Majority of cells in human cultures express ISL1 in response to DAPT, with a small fraction (<1%) of cells expressing V2 interneuron marker CHX10 (scale bar = 50μm). (i) UMAP of all high-quality human scRNA-seq profiles, colored based on expression of V2 and V3 interneuron markers (CHX10 and SIM1), fibroblast marker (COL1A2), and timestamp identity.

Extended Data Fig. 3 vpMNs display delayed and protracted neurogenesis.

(a) Top enriched pathways in Reactome analysis of genes upregulated in vpMN relative to pMN (q-value\(\,\le\) 0.001). (b) Differential gene expression analysis between in vivo vpMN (HE4) and pMN (HE1) clusters (Supplemental figure 1J) shows that, similar to in vitro, vpMNs display characteristics of higher Notch activity relative to pMNs (DEsingle). (c) Schematic of cumulative BrdU labeling assay to derive MN birthcurve for vpMN and pMN lineages. (d) Changes in the proportion of BrdU+, day 21 MNs for vpMN (RFP+), total and pMNIMPUTED lineages following cumulative BrdU labeling at progressively later timepoints (mean ± SD; n = 3 biological replicates). Despite all three curves starting at close to 100% (indicating that most vpMNs and pMNs are mitotic at day 9), the vpMN curve is right-shifted, indicating that RFP+ vpMNs remain mitotic for longer. (e) Flow cytometry plots for day 21 human cultures (only ISL1/2+ MNs shown) treated with BrdU starting at progressively later timepoints show slower decrease in BrdU+ MNs in RFP+ compared to RFP− populations. (f) Estimation of cell cycle length based on EdU pulse-labeling shows that RFP+ have similar cell cycle length compared to the total cells (mean ± SD; unpaired two-sided t-test; n = 3 biological replicates; \(p=0.07\)). (g) RFP+ and RFP- cells display similar proportions of cleaved Caspase-3−positive progenitors and motor neurons, indicating that cell death rates are similarly low for vpMN and pMN lineages (mean ± SD; unpaired two-sided t-test; n = 3 biological replicates; from left to right: \(p=0.36,\,0.29\)). (h) Proportion of RFP+ cells within newborn motor neuron population (as determined by BrdU labeling) increases over time, indicating that vpMNs undergo delayed neurogenesis (mean ± SD; n = 3 biological replicates). (i) Numbers of motor neuron progenitors and motor neurons at the onset of and tail-end of neurogenesis in both mouse and human, numbers for each replicate shown separately. (j) Day 18 human cultures immunostained for FOXP1, LHX3 and MNX1 show that FOXP1+ cells are LHX3− and MNX1+, indicating that they are LMC-like MNs (scale bar = 50 µm). (k) Day 18 human cultures immunostained for FOXP1, LHX1 and ISL1 show that LHX1 is expressed in a subset of FOXP1 cells but is not co-expressed with ISL1, suggesting that FOXP1+/LHX1+ cells are LMC-l-like MNs (scale bar = 50μm). (l) Human cultures show sequential appearance of MMC/HMC-, LMCm- and LMCl-like MNs (mean ± SD; chi-squared test; n = 3 biological replicates; \(p < 1\times {10}^{-5}\)). (m) Cumulative BrdU labeling, followed by immunolabeling for FOXP1 and ISL1/2 reveal that the birthcurve of FOXP1+ MNs is slightly late-shifted relative to the total birthcurve (mean ± SD; unpaired two-sided t-test; n = 3 biological replicates; ** from left to right: \(p=0.003,\,0.001\)). (n) Early (day 8) treatment with DAPT produces ~99% FOXP1− MNs (left), supporting the finding that early-born MNs are void of FOXP1-expressing subtypes. In contrast, adding DAPT on day 10 results in ~30% FOXP1+ MNs (right).

Extended Data Fig. 4 NKX2-2 knockout functionally reverts vpMNs to pMNs.

(a) Both WT and NKX2-2 KO cultures display broad OLIG2 expression and begin to show ISL1+ MNs at day 10 (scale bar = 50μm). (b) NKX2-2 KO cells lack NKX2-2 immuno-reactivity in both RFP+ and RFP− cells (scale bar = 50μm). (c) GFP-expressing WT cells can easily be distinguished from NKX2-2 KO cells in mosaic co-cultures based on flow cytometry. (d) Schematic for BrdU pulse-labeling. (e) Proportion of MNs born after day 17 (assessed at day 19) in WT and NKX2-2 KO MNs (RFP+ and total) show an even greater fold-change between WT and KO cells (~2.5-fold, mean ± SD; unpaired two-sided t-test; n = 3 biological replicates; *** \(p < 1\times {10}^{-4}\); n.s. \(p=0.27\); ** \(p < 0.01\)). (f) The proportion of GFP- NKX2-2 KO cells decreases over time in co-cultures, both in total and MN populations (mean ± SD; n = 3 biological replicates). (g) Relative numbers of progenitors and MNs in WT and NKX2-2 KO populations in mosaic co-cultures at the beginning (day 11) and end (day 19) of neurogenesis, normalized to number of WT progenitors on day 11 (mean ± SD; n = 5 biological replicates).

Extended Data Table 1 Number of RFP+ and total cells analyzed via flow cytometry
Full size table
Extended Data Table 2 Genes upregulated in vpMN (H3-5) compared to pMN (H1−2) based on Desingle
Full size table

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Jang, S., Gumnit, E. & Wichterle, H. A human-specific progenitor sub-domain extends neurogenesis and increases motor neuron production. Nat Neurosci 27, 1945–1953 (2024). https://doi.org/10.1038/s41593-024-01739-8

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