. 2024 Sep 10;19(9):1336-1350.

doi: 10.1016/j.stemcr.2024.07.004. Epub 2024 Aug 15.

Individual variation in the emergence of anterior-to-posterior neural fates from human pluripotent stem cells

Suel-Kee Kim¹, Seungmae Seo², Genevieve Stein-O'Brien², Amritha Jaishankar², Kazuya Ogawa², Nicola Micali¹, Victor Luria³, Amir Karger⁴, Yanhong Wang², Hyojin Kim⁵, Thomas M Hyde⁶, Joel E Kleinman⁷, Ty Voss⁸, Elana J Fertig⁹, Joo-Heon Shin², Roland Bürli¹⁰, Alan J Cross¹⁰, Nicholas J Brandon¹⁰, Daniel R Weinberger¹¹, Joshua G Chenoweth², Daniel J Hoeppner², Nenad Sestan¹², Carlo Colantuoni¹³, Ronald D McKay¹⁴

Affiliations

¹ Lieber Institute for Brain Development, 855 North Wolfe Street, Baltimore, MD 21205, USA; Department of Neuroscience, Yale School of Medicine, New Haven, CT 06510, USA.
² Lieber Institute for Brain Development, 855 North Wolfe Street, Baltimore, MD 21205, USA.
³ Department of Neuroscience, Yale School of Medicine, New Haven, CT 06510, USA; Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA; Division of Genetics and Genomics, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
⁴ IT-Research Computing, Harvard Medical School, Boston, MA 02115, USA.
⁵ Department of Neuroscience, Yale School of Medicine, New Haven, CT 06510, USA.
⁶ Lieber Institute for Brain Development, 855 North Wolfe Street, Baltimore, MD 21205, USA; Departments of Neurology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA; Departments of Psychiatry, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.
⁷ Lieber Institute for Brain Development, 855 North Wolfe Street, Baltimore, MD 21205, USA; Departments of Neurology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.
⁸ Division of Preclinical Innovation, Nation Center for Advancing Translational Science, NIH, Bethesda, MD 20892, USA.
⁹ Departments of Oncology, Biomedical Engineering, and Applied Mathematics and Statistics, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.
¹⁰ Astra-Zeneca Neuroscience iMED., 141 Portland Street, Cambridge, MA 01239, USA.
¹¹ Lieber Institute for Brain Development, 855 North Wolfe Street, Baltimore, MD 21205, USA; Departments of Neurology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA; Departments of Psychiatry, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA; Departments of Neuroscience, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA; McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.
¹² Department of Neuroscience, Yale School of Medicine, New Haven, CT 06510, USA; Departments of Genetics, Psychiatry, and Comparative Medicine, Kavli Institute for Neuroscience, Program in Cellular Neuroscience, Neurodegeneration and Repair, Child Study Center, Yale School of Medicine, New Haven, CT 06510, USA. Electronic address: nenad.sestan@yale.edu.
¹³ Lieber Institute for Brain Development, 855 North Wolfe Street, Baltimore, MD 21205, USA; Departments of Neurology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA; Departments of Neuroscience, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA; Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, USA. Electronic address: ccolantu@jhmi.edu.
¹⁴ Lieber Institute for Brain Development, 855 North Wolfe Street, Baltimore, MD 21205, USA; Departments of Cell Biology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA. Electronic address: ronaldmckay@mac.com.

PMID: 39151428
PMCID: PMC11411333
DOI: 10.1016/j.stemcr.2024.07.004

Individual variation in the emergence of anterior-to-posterior neural fates from human pluripotent stem cells

Suel-Kee Kim et al. Stem Cell Reports. 2024.

. 2024 Sep 10;19(9):1336-1350.

doi: 10.1016/j.stemcr.2024.07.004. Epub 2024 Aug 15.

Authors

Affiliations

¹ Lieber Institute for Brain Development, 855 North Wolfe Street, Baltimore, MD 21205, USA; Department of Neuroscience, Yale School of Medicine, New Haven, CT 06510, USA.
² Lieber Institute for Brain Development, 855 North Wolfe Street, Baltimore, MD 21205, USA.
³ Department of Neuroscience, Yale School of Medicine, New Haven, CT 06510, USA; Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA; Division of Genetics and Genomics, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
⁴ IT-Research Computing, Harvard Medical School, Boston, MA 02115, USA.
⁵ Department of Neuroscience, Yale School of Medicine, New Haven, CT 06510, USA.
⁶ Lieber Institute for Brain Development, 855 North Wolfe Street, Baltimore, MD 21205, USA; Departments of Neurology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA; Departments of Psychiatry, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.
⁷ Lieber Institute for Brain Development, 855 North Wolfe Street, Baltimore, MD 21205, USA; Departments of Neurology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.
⁸ Division of Preclinical Innovation, Nation Center for Advancing Translational Science, NIH, Bethesda, MD 20892, USA.
⁹ Departments of Oncology, Biomedical Engineering, and Applied Mathematics and Statistics, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.
¹⁰ Astra-Zeneca Neuroscience iMED., 141 Portland Street, Cambridge, MA 01239, USA.
¹¹ Lieber Institute for Brain Development, 855 North Wolfe Street, Baltimore, MD 21205, USA; Departments of Neurology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA; Departments of Psychiatry, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA; Departments of Neuroscience, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA; McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.
¹² Department of Neuroscience, Yale School of Medicine, New Haven, CT 06510, USA; Departments of Genetics, Psychiatry, and Comparative Medicine, Kavli Institute for Neuroscience, Program in Cellular Neuroscience, Neurodegeneration and Repair, Child Study Center, Yale School of Medicine, New Haven, CT 06510, USA. Electronic address: nenad.sestan@yale.edu.
¹³ Lieber Institute for Brain Development, 855 North Wolfe Street, Baltimore, MD 21205, USA; Departments of Neurology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA; Departments of Neuroscience, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA; Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, USA. Electronic address: ccolantu@jhmi.edu.
¹⁴ Lieber Institute for Brain Development, 855 North Wolfe Street, Baltimore, MD 21205, USA; Departments of Cell Biology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA. Electronic address: ronaldmckay@mac.com.

PMID: 39151428
PMCID: PMC11411333
DOI: 10.1016/j.stemcr.2024.07.004

Abstract

Variability between human pluripotent stem cell (hPSC) lines remains a challenge and opportunity in biomedicine. In this study, hPSC lines from multiple donors were differentiated toward neuroectoderm and mesendoderm lineages. We revealed dynamic transcriptomic patterns that delineate the emergence of these lineages, which were conserved across lines, along with individual line-specific transcriptional signatures that were invariant throughout differentiation. These transcriptomic signatures predicted an antagonism between SOX21-driven forebrain fates and retinoic acid-induced hindbrain fates. Replicate lines and paired adult tissue demonstrated the stability of these line-specific transcriptomic traits. We show that this transcriptomic variation in lineage bias had both genetic and epigenetic origins, aligned with the anterior-to-posterior structure of early mammalian development, and was present across a large collection of hPSC lines. These findings contribute to developing systematic analyses of PSCs to define the origin and consequences of variation in the early events orchestrating individual human development.

Keywords: SOX21; acid signaling; anterior or posterior neural fates; cell line variation; early embryo; human population; pluripotent stem cells; retinoic.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

**Figure 1**
Cell line variation in the emergence of neural fate from pluripotency (A) Spatial expression on day 6 in SR condition in SA01 and i04 lines. (B) Variation in NANOG and SOX21 expression across PSC lines. (i) Representative images. Scale bar, 100 μm. (ii) Expression levels in each line across time (n = 5, independent experiments; ^∗, Comparison between SA01 and i04: p < 0.001; r and p value refer to the Pearson correlation coefficient between SOX21 levels in SR and NSB). (C) PCA showing differentiation trajectories. See also Figure S1.

**Figure 2**
Decomposing dynamic and cell line-specific transcriptomic modules (A) Hierarchical clustering of GWCoGAPS-I patterns and p values from ANOVA analysis of effects of line, day, and condition in each pattern. (B) (i) GWCoGAPS-I patterns representing loss of pluripotency (P7) or mesendoderm induction (P3). (ii) Projections of mouse gastrula data. (C) (i) NSB patterns delineating early and later stages of neuroectoderm differentiation. (ii) Projection of mouse gastrula data. (D) (i) H9-specific transcriptomic signature. (ii) Projection of embryoid body (EB) data from the same lines (Comparison between H9 and other lines, p = 3.0e−4). (iii) Projection of multiple hPSC line data. H9 samples are circled in green. See also Figure S2.

**Figure 3**
SOX21 mediates early forebrain fate (A) Spatial expression on D3 in NSB condition. (B) Expression of the top 100 genes in P9 and P3 on D2 SR (S2) and 24 h after BMP4 treatment on D2 (B3). (C) (i) Expression of mesendodermal regulators 24 h after BMP4 treatment on D2. Arrowheads indicate coexpression of TBXT and CDX2. Scale bar, 100 μm. (ii) Spatial expression. ^∗, Comparison between WT (n = 3, replicate cell lines) and SOX21-KO (n = 3): p < 0.05. (D) Expression of NMP genes 24 h after BMP4 D2T. See also Figure S3.

**Figure 4**
Cell line-specific transcriptomic signatures underlie variation in forebrain versus hindbrain fate bias (A) MDS plot of gene amplitudes showing correlation between GWCoGAPS-I patterns. ^∗, patterns with RA-responsive gene enrichment (P3, p = 9.7e−08; P6, p = 0.014; P14, p = 0.008). (B) MDS plot colored by correlation of each pattern’s gene weights with DEGs in SOX21-KO cells in SR condition. (C) Average expression of RA genes. (D) Distribution of gene weights of RA genes in cell line-specific patterns. (E) (i) Proportion of HOXB1^hi cells after RA treatment in NSB condition. ^∗, comparison between SA01 and i04 (p < 0.05). (ii) Correlation of HOXB1^hi cell proportions and RA gene enrichment in each cell line-specific pattern. n = 3, independent experiments. (F) Different production of hindbrain neurons in response to RA Comparison between SA01 and i04 (∗, p < 0.05; ^∗∗, p < 0.01). n = 3, independent experiments. (G) Proportion of SOX21^hi cells after RAR inhibitor treatment on D4 in NSB condition (^∗, p < 0.05; ^∗∗∗∗, p < 0.0001). n = 6, technical replicates. (H) Proportion of HOXB1^hi cells in SOX21-KO lines after RA treatment in NSB condition. Comparison between WT and SOX21-KO (^∗, p < 0.05). n = 3, replicate cell lines. See also Figure S4.

**Figure 5**
Genetic and epigenetic elements contribute to donor- and line-specific transcriptomic signatures (A) (i) Donor-specific patterns. (ii) Projection of 260 human brain data. Significance confirmed by permutation: 2,053, p = 3.8e−6; 2075, p = 1.7e−3; 2063, p = 3.1e−5. (B) Contribution of genes of different evolutionary eras to GWCoGAPS-II patterns. Ancient genes (era 1) show high gene amplitudes in conserved dynamic patterns (compared to era 5, Wilcoxon rank-sum test: p < 1e−16 for all 3 dynamic patterns). Primate-specific genes (era 5) show higher gene amplitudes in cell line-specific patterns (p < 1e−16 for all 4 line-specific patterns). (C) 2053-6 line-specific pattern and projection of brain data. (D) Correlation of HOXB1^hi cell proportions and RA gene enrichment in cell line-specific patterns (R² = 0.75 in 1 μM RA and R² = 0.87 in 10 μM RA). The proportion in line 2053-2 was correlated with 2053 donor-specific pattern. n = 3, independent experiments. (E) Projection of ChIP-seq data from lines 2053-2 and 2053-6 in SR into the 2053-6 line-specific pattern. (F) H3K9me3 ChIP-seq data at the *GBX2* locus. See also Figure S5.

**Figure 6**
Early developmental bias and expression of RA-responsive genes define hPSC variation in the human population (A) PCs of the NextGen RNA-seq data. Donors are ordered along the X axis by the average PC1 level of all replicates. Pearson’s R and p values indicate correlations of PC1 and PC2 with mean RA gene expression. Blue circles highlight lines from one donor with high variance in PCs and RA gene expression. Intraclass correlation coefficient (ICC) estimates proportion of transcriptomic variation across lines attributed to the donor of origin. (B) NextGen PC projection into mouse gastrula data. (C and D) NextGen PC projections into macaque gastrula data. (E) (i) PC3 of the mouse (embryonic day 7) embryo data. (ii) Projection into the NextGen PCs. (F) Average RA gene expression in NextGen PCs. (G) (i) PC1 represents higher gene expression in mESCs. (ii) Projection into NextGen PCs. (H) Projection of 6 hPSC line data into NextGen PCs. (I) A model illustrating how biased gene expression in hPSCs drives (arrows) anterior/neuroectodermal or posterior/mesendodermal differentiation. See also Figure S6.

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References

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Individual variation in the emergence of anterior-to-posterior neural fates from human pluripotent stem cells

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Individual variation in the emergence of anterior-to-posterior neural fates from human pluripotent stem cells

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Abstract

Conflict of interest statement

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