1. Overexpression of Trophoblast Stem Cell-Enriched MicroRNAs Promotes Trophoblast Fate in Embryonic Stem Cells 
Ursula Nosi, Fredrik Lanner, Tsu Huang, Brian Cox 
Cell Reports 
Volume 19, Issue 6, Pages (May 2017)
DOI: /j.celrep
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2. Cell Reports 2017 19, 1101-1109DOI: (10.1016/j.celrep.2017.04.040)
Copyright © 2017 The Author(s) Terms and Conditions

3. Figure 1 ESCs and TSCs Have Reciprocal miRNA and mRNA Networks
(A) Venn diagram of enriched TSC miRNAs and ESC mRNAs.
(B) Highly connected network of three TSC-enriched miRNAs targeting 54 ESC-enriched mRNAs. Known pluripotency genes have green outer ring, with conserved expression in humans in red.
(C) qRT-PCR TaqMan assay analysis of miR-15b, miR-322, or miR-467 g in ESCs and TSCs; sno142 and 202 were used as reference genes. SE is calculated between technical triplicates.
(D) Bright-field image of ESCs 5 days post-transfection with vectors constitutively expressing the three candidate miRNAs.
(E) Transfected cells can be passaged and maintain trophoblast-like morphology.
(F) Control culture of TSCs.
(G) Immunocytochemical analysis of transfected ESC colonies for the TSC markers Cdx2 and Gata3 and the ESC marker Pou5f1.
(H) Principle-component analysis of single-cell gene expression using Made4 (R library) revealed that iTCs cluster closely with blastocyst-derived TSCs and away from both ESCs and MEFs.
(I) Loadings plot of (H) highlighting genes driving sample clusters.
See also Figure S1.
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4. Figure 2 Activity of Induced Expression of TSC-Enriched miRNAs in ESCs
(A) Bright-field images of cell lines induced to express miR-15b, miR-322, or miR-467 g for 5 days in the presence of valproic acid (i–iii). Scale bar, 20 pixels. Bright-field image of induced cells after 9 days of induction culture (iv). Bright-field images of the same cell lines cultured in TSC media without the miRNA transgene-inducing agent (doxycycline) (i′–iii′). Non-induced cells cultured for 6 days in ESC media (iv′).
(B) qPCR analysis of miRNA predicted targets Sall1 and Sall4 and pluripotency genes Pou5f1 and Nanog. Shown are the average expression of three miR-467 g lines, two miR-15b lines, and two miR-322 lines. All lines show a downward trend beginning as early as 4 hr.
(C) Western blot of Sall4 and Oct4 protein expression in induced and non-induced cell lines compared to TSCs. Blot was cropped for clarity and white space.
(D) Quantification of western blot using ImageJ densitometry plug-in and actin as reference.
(E) qPCR assessment of mRNA expression of TSC marker genes Elf5 and Cdx2 in the same miRNA lines and biological replicates. Observed is a steady increase of these genes as early as 24 hr, throughout a 10-day induction period.
(F) Bright-field image of ESCs 3 days post-induction of miR-1 expression.
(G) Bright-field image of ESCs 6 days post induction of miR-1. Scale bar, 10 pixels.
(H) qPCR gene expression analysis comparing miR-15b, miR-1, and TSCs for TSC markers (Cdx2, Elf5, and Eomes), pluripotency markers (Pou5f1, Nanog, and Lin28), and cardiac lineage markers (Gata4 and Nkx2-5). qPCR data were normalized against Gapdh/Actb as reference and graphed as log2 normalized expression relative to ESCs. SE is calculated between two technical replicates of each biological replicate.
See also Figure S2.
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5. Figure 3
iTCs Are Stable and Propagate after Withdrawal of miRNA Induction
(A) A schematic of the experiment.
(B) Bright-field image of ESCs after 3-day miRNA expression and cultured until 9 days.
(C) Bright-field image of ESCs after 6-day miRNA expression and cultured for 9 days. Scale bar, 50 pixels.
(D) qPCR gene expression analysis of 3-day and 6-day induced ESC cell lines (NI is no induction of miR15b).
(E) Bright-field images of 6-day induced lines serially passaged without transgene induction.
(F) Gene expression of wild-type TSCs and passaged iTC lines corresponding to (E).
(G) qPCR of ESC lines induced for 6 days and serially passaged.
SE is calculated between technical replicates. See also Figure S3.
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6. Figure 4
Global Gene Expression Identifies a Significant Relationship between Induced and Embryo-Derived Trophoblast Cells
(A) A heatmap of Pearson correlation coefficients of microarray data for ESCs, TSCs, and iTCs (miRNA and Cdx2). A high degree of cross-correlation between miRNA induced trophoblast and embryo trophoblast is observed.
(B) Venn diagram of a three-way comparison of genes exhibiting increased expression relative to ESCs.
(C) A bi-graph network diagram of the relationship of enriched gene sets related to trophoblast development and function spanning Gene Ontology (blue), Mutant Phenotypes (green), and Anatomy Ontologies (orange) to either the co-expressed genes of miR-iTCs and TSCs or miR-iTC, Cdx2-iTCs, and TSCs (purple nodes).
See also Figure S4 and Tables S1 and S2.
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7. Figure 5
miRNA-iTCs Contribute to the Trophectoderm In Vivo and Have Physiological Properties of the Mural TE
(A) Bright-field (BF) images of representative E6.5 embryos are presented on the left and fluorescence (RFP) images of corresponding embryos on the right. mCherry localization within pockets of the Reichert’s membrane was observed. Control embryos were assessed under the same microscope settings. Scale bar, 5 pixels.
(B) Single optical sections of individual embryos cultured from E2.5 to E3.5, with green representing GFP-expressing iTCs, blue Hoechst-labeled nuclei, and merged channels. The approximate position of the inner cell mass is outlined by a white dashed line. Scale bar, 10 pixels.
(C) Fluorescent confocal microscopy of miR-15b day-6-induced cells, TSCs, and trophoblast giant cells (TGC) exposed to 1.75 μm fluorescently labeled microspheres (red). Scale bar, 10 pixels. Cell nuclei are labeled with Hoechst (blue).
See also Figure S5 and Table S3.
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