Chimera production in iPSCs and iINTPSCs.

<p>Chimera production in iPSCs and iINTPSCs.</p

A Modified EpiSC Culture Condition Containing a GSK3 Inhibitor Can Support Germline-Competent Pluripotency in Mice

<div><p>Embryonic stem cells (ESCs) can contribute to the tissues of chimeric animals, including the germline. By contrast, epiblast stem cells (EpiSCs) barely contribute to chimeras. These two types of cells are established and maintained under different culture conditions. Here, we show that a modified EpiSC culture condition containing the GSK3 inhibitor CHIR99021 can support a germline-competent pluripotent state that is intermediate between ESCs and EpiSCs. When ESCs were cultured under a modified condition containing bFGF, Activin A, and CHIR99021, they converted to <u>int</u>ermediate <u>p</u>luripotent <u>s</u>tem <u>c</u>ell<u>s</u> (INTPSCs). These INTPSCs were able to form teratomas <i>in vivo</i> and contribute to chimeras by blastocyst injection. We also induced formation of INTPSCs (iINTPSCs) from mouse embryonic fibroblasts by exogenous expression of four reprogramming factors, Oct3/4, Sox2, Klf4, and c-Myc, under the INTPSC culture condition. These iINTPSCs contributed efficiently to chimeras, including the germline, by blastocyst injection. The INTPSCs exhibited several characteristic properties of both ESCs and EpiSCs. Our results suggest that the modified EpiSC culture condition can support growth of cells that meet the most stringent criteria for pluripotency, and that germline-competent pluripotency may depend on the activation state of Wnt signaling.</p></div

Establishment of Trophoblast Stem Cells under Defined Culture Conditions in Mice

<div><p>The inner cell mass (ICM) and trophoblast cell lineages duet early embryonic development in mammals. After implantation, the ICM forms the embryo proper as well as some extraembryonic tissues, whereas the trophoectoderm (TE) exclusively forms the fetal portion of the placenta and the trophoblast giant cells. Although embryonic stem (ES) cells can be derived from ICM in cultures of mouse blastocysts in the presence of LIF and/or combinations of small-molecule chemical compounds, and the undifferentiated pluripotent state can be stably maintained without use of serum and feeder cells, defined culture conditions for derivation and maintenance of undifferentiated trophoblast stem (TS) cells have not been established. Here, we report that addition of FGF2, activin A, XAV939, and Y27632 are necessary and sufficient for derivation of TS cells from both of E3.5 blastocysts and E6.5 early postimplantation extraembryonic ectoderm. Moreover, the undifferentiated TS cell state can be stably maintained in chemically defined culture conditions. Cells derived in this manner expressed TS cell marker genes, including <i>Eomes</i>, <i>Elf5</i>, <i>Cdx2, Klf5, Cdh1, Esrrb, Sox2</i>, and <i>Tcfap2c</i>; differentiated into all trophoblast subtypes (trophoblast giant cells, spongiotrophoblast, and labyrinthine trophoblast) <i>in vitro</i>; and exclusively contributed to trophoblast lineages in chimeric animals. This delineation of minimal requirements for derivation and self-renewal provides a defined platform for precise description and dissection of the molecular state of TS cells.</p></div

Differentiation capacity of TS cells <i>in vivo</i>.

<p>(a) Placenta-specific contribution of TS cells in an E14.5 embryo. (b) Merged image of DAPI staining (gray) and EGFP fluorescence (green) of a placental section at E14.5. de, decidua; gi, giant cells; sp, spongiotrophoblast cells; la, labyrinth; ch, chorion. Scale bar, 200 µm.</p

Derivation of TS cells in CDM/FAXY.

<p>(a) E3.5 blastocyst with the GOF18-EGFP (<i>Oct3/4</i>-EGFP) reporter transgene. Scale bar, 50 µm. (b) Outgrowths arising at day 5 in CDM/LIF/2i (left) and CDM/FAXY (FGF2, 12.5 ng/ml) (right) on fibronectin. Scale bar, 100 µm. (c) Morphology of TS cell colonies at day 1 (left), day 2 (center), and day 3 (right) after passage. Scale bar, 100 µm. (d) Derived TS cells from E3.5 blastocyst (center) and E6.5 ExE (right). Conventional TS cells from an E3.5 blastocyst (left) are shown as a control. Scale bar, 100 µm. (e) Immunofluorescence staining of TS cells on a fibronectin-coated plastic-bottom dish. Scale bar, 100 µm. (f) Proliferation of conventional TS cells (red) and E3.5 blastocyst–derived TS cells in CDM/FAXY (blue). (g) Proliferation of ES cells in CDM/LIF/2i (red) and E3.5 blastocyst–derived TS cells in CDM/FAXY (blue). (h) Comparison of expression levels of ES cell–specific genes, commonly expressed genes, and TS cell–specific genes among ES cells (red), E3.5 blastocyst–derived TS cells (blue), and E6.5 ExE–derived TS cells (cyan). (i) Comparison of expression levels of ectodermal genes, mesodermal genes, and endodermal genes among ES cells (red), E3.5 blastocyst–derived TS cells (blue), and E6.5 ExE–derived TS cells (cyan).</p

Differentiation capacity of TS cells <i>in vitro</i>.

<p>(a) Morphological changes of TS cells upon removal of FGF2 (-F, second from the left), activin A (-A, second from the right), or XAV939 (-X, right). FAXY represents undifferentiated TS cells as a control (left). Scale bar, 100 µm. (b) Changes in expression levels of TS cell marker genes upon removal of FGF2 (-F). (c) Changes in expression levels of TS cell marker genes upon removal of activin A (-A). (d) Changes in expression levels of TS cell marker genes upon removal of XAV939 (-X). (e) Changes in expression levels of differentiated trophoblast lineage marker genes upon removal of FGF2 (-F). (f) Changes in expression levels of differentiated trophoblast lineage marker genes upon removal of activin A (-A). (g) Changes in expression levels of differentiated trophoblast lineage marker genes upon removal of XAV939 (-X).</p

Establishment of TS cell lines in various conditions.

a<p>, EpiSC-like;</p>b<p>, partially differentiated TSCs;</p>c<p>, ESCs.</p><p>Establishment of TS cell lines in various conditions.</p

Gene-expression analyses of TS cells.

<p>(a) Dose-dependent effect of FGF2 on expression levels of TS cell marker genes and differentiated trophoblast lineage marker genes at 12.5 ng/ml (cyan), 25 ng/ml (green), and 50 ng/ml (magenta). (b) Comparison of expression levels of FGF ligand family genes between ES cells (red) and E3.5 blastocyst–derived TS cells (blue). (c) Comparison of expression levels of FGF receptor genes between ES cells (red) and E3.5 blastocyst–derived TS cells (blue). (d) Comparison of global gene expression analysis between conventional and new TS cells. (e) Highlighted expression levels of trophoblast stem-cell marker genes and differentiated trophoblast-subtype marker genes.</p

Conversion of ESCs into INTPSCs in a modified EpiSC culture condition containing CHIR99021.

<p>(A) Proliferation of ESCs in the presence of LIF/2i, F, FA, and FAC. (B) Proportions of Oct3/4-GFP–positive ESCs in the presence of LIF/2i, F, FA, and FAC. (C) Phase-contrast images of ESCs, ESC-INTPSCs, and EpiSCs. Scale bar, 50 µm. (D) Alkaline-phosphatase staining of ESCs and ESC-INTPSCs. Scale bar, 500 µm. (E and G) Immunostaining of ESCs and ESC-INTPSCs. Scale bar, 500 µm. (F) Percentage of OCT3/4-positive or -negative cells in GOF18 Oct3/4-GFP–positive or –negative ESCs, ESC-INTPSCs, and EpiSCs. (H) Immunostaining for H3K27me3 in E6.0 epiblasts, ESCs, and ESC-INTPSCs. Scale bar, 10 µm.</p

Requirement for Y27632.

<p>(a) Fluorescence-based detection of poly-caspase–positive cells by FAM-FLICA. Undifferentiated control (FAXY, left) and Y27632 removed (-Y, right). Scale bar, 100 µm. (b) Quantitation of poly-caspase–positive cells, expressed as a percentage (%) (c) Morphology of TS cell colonies on fibronectin (left) and Matrigel (right). Scale bar, 100 µm.</p