Volume 38, Issue 1, Pages 100-115 (July 2016) Spatiotemporal Reconstruction of the Human Blastocyst by Single-Cell Gene- Expression Analysis Informs Induction of Naive Pluripotency  Jens Durruthy-Durruthy, Mark Wossidlo, Sunil Pai, Yusuke Takahashi, Gugene Kang, Larsson Omberg, Bertha Chen, Hiromitsu Nakauchi, Renee Reijo Pera, Vittorio Sebastiano  Developmental Cell  Volume 38, Issue 1, Pages 100-115 (July 2016) DOI: 10.1016/j.devcel.2016.06.014 Copyright © 2016 Terms and Conditions

Developmental Cell 2016 38, 100-115DOI: (10.1016/j.devcel.2016.06.014) Copyright © 2016 Terms and Conditions

Figure 1 Single-Cell Gene-Expression Analysis on Human Blastocysts (A) Overview of experimental strategy to reconstruct early- and late-stage human blastocysts by single-cell gene-expression analysis. (B and C) Principal component analysis (PCA) on gene-expression analysis of 88 single cells and 153 single cells collected from early-stage (B) and late-stage (C) blastocysts identified two and three subpopulations, respectively, that are color coded. Subpopulations are defined according to their gene-expression and cluster analysis (see Figures S1M and S1N). (D and E) Principal component (PC) projections of 84 analyzed genes, showing the contribution of each gene to the first two PCs. The first PC in early blastocysts is discriminating between TE and ICM (D), while PC1 and PC2 are discriminating all three subpopulations in late blastocysts (E). See also Figure S1 and Tables S1 and S2. Developmental Cell 2016 38, 100-115DOI: (10.1016/j.devcel.2016.06.014) Copyright © 2016 Terms and Conditions

Figure 2 3D Reconstruction of the Inner Cell Mass of Human Blastocysts (A) 3D in silico reconstruction of the ICM and trophectoderm (TE). From left to right: bright-field image of a human blastocyst with ICM and TE indicated. Scale bar, 40 μm. Model of ICM and TE, which resembles two sphere-like tissue structures. Single cells of the TE and ICM are dissociated and projected in the 3D space onto a unit sphere based on the first three PCs after PC analysis. Different colors symbolize heterogeneity between single cells of the same tissue. Scale bar, 30 μm. (B) PCA on single cells collected from late blastocysts. Cells that were defined as ICM (n = 129) that were subject to subsequent 3D PC analysis are highlighted in red. (C) First three PCs of late ICM cells projected in 3D space and projected onto a sphere in the 3D space. A 2D view of the sphere is shown. View from the XY axes (PC1 and PC2) with single cells projected onto the sphere. (D) Cells of the late ICM are defined as epiblast (EPI)-like and primitive endoderm (PE)-like origin and color coded accordingly. Grouping of single cells based on k means and specific expression of EPI and PE markers. View from the XY axes of color-coded cells projected onto the sphere. (E) Gene-expression analysis of representative epiblast marker for cells projected onto the first three PCs and projected onto a sphere. XY view facilitates locating each cell on the sphere. Cells in gray represent undetectable gene expression. Expression values for cells that were defined as EPI and PE, respectively, were combined and plotted as bar plots with +SEM shown. (F) Gene-expression analysis of PE marker GATA4. See (E) for details. See also Figures S2 and S3. Developmental Cell 2016 38, 100-115DOI: (10.1016/j.devcel.2016.06.014) Copyright © 2016 Terms and Conditions

Figure 3 Salt-and-Steak Model for NANOG and GATA4 Expression in Human Blastocysts (A) PCA on gene-expression analysis of single cells collected from early blastocysts. Cells that were defined as ICM (n = 67) that were subject to subsequent 3D PCA are highlighted in red. (B) Early ICM cells projected onto a sphere in the 3D space. (C) View from the XY (PC1/PC2) and XZ (PC1/PC3) axes with single cells projected onto the sphere. (D–F) 3D PCA performed with ICM cells of late blastocysts. See (A–C) for details. (G–J) GATA4+ and NANOG+ cells are expressed in a salt-and-steak-like fashion (see text) in the early ICM of human blastocysts. Cells in gray represent undetectable expression levels. (K) NANOG+ and GATA4+ cells presented in one sphere (3D PCA) of an early ICM. (L–O) GATA4+ and NANOG+ cells are separated in the late ICM of human blastocysts. Cells in gray represent undetectable expression levels. (P) NANOG+ and GATA4+ cells presented in one sphere (3D PCA) of a late ICM. (Q) Representative immunostaining of GATA4 (gray) and NANOG (red) in early and late human blastocysts validates 3D modeling using PCA. N = 16 early human blastocysts; scale bar, 40 μm. See also Figure S4 and Movies S1, S2, and S3. Developmental Cell 2016 38, 100-115DOI: (10.1016/j.devcel.2016.06.014) Copyright © 2016 Terms and Conditions

Figure 4 Identification of Progenitor Epiblast and Primitive Endoderm Cells (A) PCA on gene-expression analysis of single cells collected from early and late blastocysts. Cells that were defined as ICM and GATA4+ or NANOG+ are highlighted in red (n = 85). (B) PCA on GATA4+ and NANOG+ cells. Cells are color coded according to early- and late-stage blastocysts. Based on expression analysis of lineage-specific markers, arrows indicate epiblast- and PE-lineage specification path. Progenitor epiblast and PE cells encircled based on expression analysis. Vertical line indicates separation between PE (negative PC1) and epiblast (positive PC1) subpopulations. (C) Normalized and scaled gene-expression changes over time (along positive PC2) of PE markers during PE differentiation. Data points are plotted via third-order polynomial curve fitting. (D) Normalized and scaled gene-expression changes over time (along positive PC1) of epiblast markers and epiblast-inducing markers during epiblast differentiation. Data points are plotted via third-order polynomial curve fitting. (E) Normalized gene-expression analysis of precursor epiblast cells; see encircled cells in (B). Cells express NANOG and previously uncharacterized epiblast-inducing genes and low levels of POU5F1, while other epiblast-specific markers are not yet expressed. n = 5 with +SEM shown. (F) Gene-expression analysis of epiblast-inducing markers for cells projected onto the first two PCs. Cells are plotted along PC1 (X axis) and a pseudo vector (Y axis) to highlight lineage specification of epiblast cells. Curve presents gene-expression changes of single cells that are ranked along PC1. Expression values were scaled and curves fit with the LOWESS method (Prism 6). ∗High expression of the respective gene. See also Figure S5. Developmental Cell 2016 38, 100-115DOI: (10.1016/j.devcel.2016.06.014) Copyright © 2016 Terms and Conditions

Figure 5 Single-Cell Gene-Expression Analysis in NANOG−/GATA4− Cells of the Human Blastocyst (A) PCA on single cells collected from all blastocysts. Cells that were defined as ICM and GATA4− and NANOG− (n = 108) are highlighted in red. (B) PCA on GATA4−/NANOG− cells. Cells are color coded according to early or late blastocyst origin. Based on expression analysis of lineage-specific markers, arrows indicate epiblast- and PE-lineage specification path. The horizontal line indicates separation between PE (positive PC2) and epiblast (negative PC2) subpopulations. (C) Gene-expression analysis of CXCR4 (PE) and LIN28A (epiblast) markers for cells projected onto the first two PCs. (D) Gene-expression analysis of PE and epiblast markers in lineage 1 and 2, respectively, see (B), with +SEM shown. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. See also Figure S5. Developmental Cell 2016 38, 100-115DOI: (10.1016/j.devcel.2016.06.014) Copyright © 2016 Terms and Conditions

Figure 6 MCRS1, TET1, and THAP11 Induce Naive Pluripotency in Human Embryonic Stem Cells (A) Fluorescence activity of the GFP reporter line. No GFP activity in hESC conditions and without overexpression of specific genes. GFP+ cells observed in NANOG + KLF4 and MCRS1+TET1+THAP11 overexpressed cells. Scale bar, 150 μm. (B) Fluorescence-activated cell sorting analysis of reporter line 7 days after naive pluripotency induction. (C) Continuous overexpression of MCRS1, TET1, and THAP11 ensures propagation under naive culture conditions on feeders. Cells were passaged as single cells and formed dome-like GFP-positive colonies. Withdrawal of overexpressed genes resulted in differentiation of colonies and GFP-negative forming colonies. Scale bar, 100 μm. (D) Immunostaining for H3K9me3. Intensity and distribution analysis by ImageJ. Scale bar, 20 μm. See also Figure S6. Developmental Cell 2016 38, 100-115DOI: (10.1016/j.devcel.2016.06.014) Copyright © 2016 Terms and Conditions

Figure 7 Stable Overexpression of MCRS1, THAP11, and TET1 Induces Naive Pluripotency in hESCs (A) Experimental overview of construction of the hESC line that expresses MTTH in a doxycycline-dependent manner. Scale bar, 100 μm. (B) Assessing clonal passaging efficiency. Scale bar, 100 μm. (C) Alkaline phosphatase (AP) positive colonies after 6 days of single-cell plating. (D) Assessing proliferation rate in hESCs. (E) Quantification of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) staining in MTTH overexpressing and primed hESCs. (F) Representative immunostaining for 5mC and 5hmC in naive and primed hESCs. MTTH overexpressing cells demonstrate decreased signal intensity for 5mC- and 5hmC-IF. Scale bar, 150 μm. (G) Volcano plot of genes differentially expressed in MTTH overexpressing and primed hESCs after microarray analysis. (H) Heatmap of significantly differentially expressed genes between samples in this study and the Jaenisch group (Theunissen et al., 2014). (I) Interspecies chimera formation assay: Representative immunostainings for NUMA (nuclear mitotic apparatus, human-specific antibody), OCT4, and Cdx2 of mouse blastocysts injected with naive human MTTH-H9 overexpressing cells. A total of 16 derived mouse blastocysts were analyzed with primed and MTTH overexpression hESCs injected at the morulae stage. In 2 of 8 mouse blastocysts, MTTH overexpression hESCs contributed to the mouse ICM. Scale bar, 50 μm. See also Figure S7 for second interspecies chimera. (J) Magnification of the human cell in (I, lower panel) showing the metaphase chromosomes and the distinct NUMA signal for mitotic cells. Scale bar, 20 μm. See also Figure S7 and Movies S4 and S5. Developmental Cell 2016 38, 100-115DOI: (10.1016/j.devcel.2016.06.014) Copyright © 2016 Terms and Conditions