Arrowheads mark extraembryonic endoderm (black) and trophoblast (red). Black bars indicate the plane of section. Embryos were collected at E6.5 and X-gal stained. Subpopulations of ESCs Cultured in 2i Demonstrate Extraembryonic Potential In Vivo (A and B) HV cells, expressing a constitutive LACZ marker, cultured in 2i (A) or 2i/LIF (B), were sorted via FACS into HV − (A: n = 18 B: n = 60) and HV + (A: n = 26 B: n = 55) PECAM-1 + cells and aggregated with wild-type F1 morulae. Error bars indicate mean ± SD of biological replicates. (G) Neural differentiation was quantified by counting elongated TUJ1-positive neurons (n = 5). (F) Endoderm differentiation was quantified by counting the number of GATA6-positive endodermal clusters (see also Figures S3A and S3C n = 3). (E) Trophoblast differentiation was quantified by counting CDX2-positive cells, which were negative for GATA6 and BRACHYURY (n = 3). (E and F) Quantification of in vitro differentiation of sorted HV − and HV + ESCs after differentiation in trophoblast stem cell conditions for 7 days (E), LIF withdrawal (F), or neural differentiation (G). High-magnification images are shown as insets. (D) Immunostaining displaying representative images of CDX2-positive cells in trophoblast differentiation. Red represents upregulation and green represents downregulation of expression. Two biological replicates are shown per sample. Genes are hierarchically clustered by average Euclidean distance. Data were normalized by subtracting the average log expression from all samples. Heat map shows differentially expressed genes identified by pairwise comparison of all sorted fractions. (C) Heat map of sorted HV − and HV + populations from serum/LIF and 2i/LIF culture, based on gene expression data from RNA-seq. (B) Flow cytometry of clones (represented by individual bars) after expansion from single HV − or HV + sorted cells from 2i or 2i/LIF. Black boxes indicate sorting gates for the upper and lower 25% of HV expression used to separate HV − and HV + populations. Gates set using unstained E14 ESCs (Figure S1B). (E) Confocal optical sections of immunostained transgenic HV embryos after flushing at E2.5 and 3 days in culture either in control or 2i medium.ĮSCs Cultured in 2i Are Heterogeneous (A) Flow cytometry of HV and ESC marker PECAM-1 in cells cultured in serum/LIF, 2i, and 2i/LIF. Red arrowheads indicate HV-expressing trophoblast cells, whereas white arrowheads indicate HV + PE cells. (D) HV embryos were flushed at E2.5 and cultured in 2i media for 3 days before live confocal microscopy imaging. Although in vivo PE is specified by E4.5, this segregation is delayed in vitro, and, hence, embryos were cultured until E5.5 to observe differentiated PE. Red arrowheads indicate trophoblast cells coexpressing HV and CDX2. (C) Confocal optical section of an E2.5 HV embryo cultured for 3 days immunostained for the trophoblast marker CDX2. Only HV-expressing transgenic embryos were scored. Day 0 = E2.5, day 1 = E3.5 (early blastocyst), day 2 = E4.5 (midblastocyst), and day 3 = E5.5 (late blastocyst, equivalent to E4.5 in vivo due to culture induced delay). (B) Scoring of the HV expression pattern in embryos collected at E2.5 and cultured in either control or 2i medium. E3.5 and E4.5 images show confocal optical sections. At E2.5, an extended focus image is shown. Representative images are shown for each stage. All rights reserved.Ĭulture in 2i Does Not Eliminate Endoderm Marker Expression in the Early Embryo (A) HV transgenic embryos were collected at E2.5 and cultured in vitro for different time periods before live confocal microscopy imaging. Thus, 2i and LIF support a totipotent state comparable to early embryonic cells that coexpress embryonic and extraembryonic determinants.Ĭopyright © 2013 The Authors. The cytokine LIF, necessary for ESC self-renewal, supported the expansion of this population but did not directly support Nanog-positive epiblast-like ESCs. Single Hex-positive ESCs coexpressed epiblast and extraembryonic genes and contributed to all lineages in chimeras. Similarly, 2i ESC cultures were heterogeneous and contained a Hex-positive fraction primed to differentiate into trophoblast and extraembryonic endoderm. However, we observed heterogeneous expression of the extraembryonic endoderm marker Hex in 2i-cultured embryos, suggesting that 2i blocked development prior to epiblast commitment. ESCs maintained with inhibitors of MEK and GSK3 (2i) are thought to represent an embryonically restricted ground state. Embryonic stem cells (ESCs) are derived from mammalian embryos during the transition from totipotency, when individual blastomeres can make all lineages, to pluripotency, when they are competent to make only embryonic lineages.
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