Journal: Cellular and molecular bioengineering

Article Title: Mouse Pluripotent Stem Cell Differentiation Under Physiological Oxygen Reduces Residual Teratomas.

doi: 10.1007/s12195-021-00687-8

Figure Lengend Snippet: FIGURE 2. Expression of pluripotency markers decrease substantially with time during differentiation and extended culture under reduced oxygen. Cells were differentiated for 10 days followed by up to 80 days of extended culture (90 days total). (a) Flow cytometric quantification of the fraction and number of Oct4-GFP1 for differentiating mESCs and miPSCs at 142, 36, and 7 mmHg pO2gas (n = 3). (b) Representative en face bright field and Oct4-GFP images of cell aggregates after extended culture at 142 or 7 mmHg pO2gas. (c) Relative expression of Oct4 and Nanog mRNA with time in mESCs, miPSCs, and HIF-1a2/2 mESCs at 142, 36, and 7 mmHg pO2gas (n = 3) measured with qPCR.

Article Snippet: Cells were incubated overnight at 4 C with primary antibodies against Oct4 (sc-9081; Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:100, Nanog (IHC-00205; Bethyl Laboratories, Montogomery, TX, USA) diluted 1:100, SSEA-1 (MC-480; DSHB) diluted 1:10, Foxa2 (sc-6554; Santa Cruz Biotechnology) diluted 1:200, Nestin diluted 1:50, cardiac troponin T (cTnT; MS-295-P1; NeoMarkers, Fremont, CA, USA) diluted 1:50, or Nkx2.5 (sc-8697; Santa Cruz Biotechnology) in 3% DS, 0.1% Triton X-100 solution, washed once with PBS, and incubated with secondary donkey AlexaFluor 488 (green) or 594 (red) antibodies (Invitrogen) against the appropriate species diluted 1:250 in 3% DS, 0.1% Triton X-100 solution for 2 h in the dark.

Techniques: Expressing

Journal: Development (Cambridge, England)

Article Title: Oct4 is required for lineage priming in the developing inner cell mass of the mouse blastocyst

doi: 10.1242/dev.096875

Figure Lengend Snippet: Consequences of deleting Oct4 in the developing oocyte and zygote. (A) Breeding scheme for the generation of female mice in which Oct4 is deleted during oocyte maturation by recombination of LoxP sites in one Oct4 allele mediated by Cre recombinase driven by the ZP3 promoter. These females are termed ODE for ‘ Oct4 deletion in the egg’. (B) The number of offspring produced from ODE females following mating with F1 males, as compared with a control female from the same breeding programme but carrying a wild-type Oct4 allele. (C) Scheme to illustrate genotypes of embryos generated by mating ODE females with males heterozygous for Oct4 . (D-F) Example confocal images, maximum projection of Oct4 +/- heterozygote (Het, top) and Oct4 maternal-zygotic mutant embryos (Null, bottom) at E3.5 stained for Oct4 (white) and (D) Nanog (green), (E) Gata6 (red) or (F) Sox17 (blue). All embryos contained 34-58 cells. (G-I) Mean Nanog (G), Gata6 (H) and Sox17 (I) levels in trophectoderm (TE) or ICM cells obtained by QIF analysis from Oct4 +/- (Het, red bars) and Oct4 maternal-zygotic mutant embryos (KO, grey bars). Single-cell fluorescence levels were quantified for seven Oct4 +/- and five Oct4 maternal-zygotic mutant embryos. Error bars indicate s.e. The raw data are presented in supplementary material Fig. S1.

Article Snippet: The following TaqMan probes were used: Pou5f1, Mm03053917_g1; Nanog, Mm02019550_s1; Pdgfrα, Mm00440701_m1; Fgf4, Mm00438916_g1; Fgfr2, Mm01269930_m1.

Techniques: Produced, Control, Generated, Mutagenesis, Staining, Fluorescence

Journal: Development (Cambridge, England)

Article Title: Oct4 is required for lineage priming in the developing inner cell mass of the mouse blastocyst

doi: 10.1242/dev.096875

Figure Lengend Snippet: Deletion of Oct4 at the morula/blastocyst stage to investigate specification of PrE. (A) Scheme for inducible deletion of Oct4 in IOD embryos isolated at the 8-cell stage and subsequent culture. (B) Confocal images of IOD embryos. The top row shows an untreated embryo with 36 cells following culture from the 8-cell stage (E2.5) for 24 hours; the bottom row is a 32-cell embryo following treatment with 4-OHT from the 8-cell stage for 24 hours, showing immunofluorescence for Oct4 (red) and DAPI staining (blue). (C) Single confocal section of IOD embryo treated from E2.5 with 4-OHT for 24 hours, then cultured for a further 24 hours without 4-OHT showing Oct4 (white), Nanog (green), Cdx2 (red) and DAPI (blue). (D) Confocal images of two untreated IOD embryos (top two panels) and two 4-OHT-treated embryos (bottom two panels) cultured from the 8-cell stage (E2.5) for 48 hours, showing Oct4 (white), Nanog (green), Sox17 (red) and DAPI (blue). Arrowhead indicates Sox17-positive cells. Mean embryo cell numbers are presented in . (E) Single-cell gene expression analysis on single ICM cells isolated from IOD embryos with or without 4-OHT treatment from the 8-cell stage (E2.5) for 24 hours. Gene expression values were normalised to Gapdh . Error bars indicate s.d.

Article Snippet: The following TaqMan probes were used: Pou5f1, Mm03053917_g1; Nanog, Mm02019550_s1; Pdgfrα, Mm00440701_m1; Fgf4, Mm00438916_g1; Fgfr2, Mm01269930_m1.

Techniques: Isolation, Immunofluorescence, Staining, Cell Culture, Gene Expression

Journal: Development (Cambridge, England)

Article Title: Oct4 is required for lineage priming in the developing inner cell mass of the mouse blastocyst

doi: 10.1242/dev.096875

Figure Lengend Snippet: Effect of addition of excess FGF2 or FGF4 to Oct4 -deleted IOD embryos. (A) Confocal images of two untreated IOD embryos (top two panels) and two 4-OHT-treated embryos (bottom two panels) cultured from the 8-cell stage (E2.5) for 48 hours, as indicated in , supplemented with 1 mg/ml FGF4 (or FGF2) from 24-48 hours showing Oct4 (white), Nanog (green), Sox17 (red) and DAPI (blue). (B) Average numbers of cells expressing Sox17, Nanog and total cell number. The number of embryos used for each data point is indicated beneath the x -axis. Error bars represent s.d. (C-E) Phase contrast images of whole blastocyst outgrowths from IOD embryos following 5 days in culture after (C) culture for 24 hours from the 8-cell stage with treatment, (D) 24 hours with 4-OHT, (E) 24 hours with 4-OHT and FGF2. Scale bar: 50 μm.

Article Snippet: The following TaqMan probes were used: Pou5f1, Mm03053917_g1; Nanog, Mm02019550_s1; Pdgfrα, Mm00440701_m1; Fgf4, Mm00438916_g1; Fgfr2, Mm01269930_m1.

Techniques: Cell Culture, Expressing

Journal: Development (Cambridge, England)

Article Title: Oct4 is required for lineage priming in the developing inner cell mass of the mouse blastocyst

doi: 10.1242/dev.096875

Figure Lengend Snippet: Model of Oct4 functions during preimplantation development. As shown in , levels of Nanog protein are higher when Oct4 is subject to maternal-zygotic deletion, implying that Oct4 plays a role in restricting Nanog to maintain flexibility for formation of PrE. It is known from previous work ( ; ) that Oct4 is required to suppress Cdx2 and trophectoderm differentiation in the mid-late ICM. Single-cell expression analysis suggests that sustained expression of Oct4 beyond the 8-cell stage is required to enable the segregation of molecular programmes for either epiblast or PrE. However, expression of Sox17 can be induced by addition of excess FGF2 or FGF4, and overt differentiation of PrE derivatives is possible in cells from which Oct4 was conditionally deleted from the morula stage following ESC complementation from the 8-cell stage.

Article Snippet: The following TaqMan probes were used: Pou5f1, Mm03053917_g1; Nanog, Mm02019550_s1; Pdgfrα, Mm00440701_m1; Fgf4, Mm00438916_g1; Fgfr2, Mm01269930_m1.

Techniques: Expressing