Figure 1.
Characterization of embryo-derived β-catΔ/Δ mESCs.
(A): Morphological appearance of β-catΔ/Δ mESCs is shown in the left panel. ALP staining of β-catΔ/Δ mESCs is shown in the right panel. Scale bars are 150 µm. (B): Electrophoretic analysis of the genotyping PCR for wild-type (NCH4.3), β-catfl/fl (fl/fl1 and fl/fl2), β-catfl/Δ (fl/Δ2 and fl/Δ3) and β-catΔ/Δ (Δ/Δ1, Δ/Δ2 and Δ/Δ6) mESCs. (C): Western blots of β-catenin and Gapdh in wild-type (NCH4.3 and R1), β-catfl/fl (fl/fl2), β-catfl/Δ (fl/Δ2 and fl/Δ3), β-catΔ/Δ (Δ/Δ2, Δ/Δ6 and Δ/Δ8) and β-catenin rescued β-catΔ/Δ mESCs (rsc). (D and E): Immunofluorescence staining for β-catenin (red), α-catenin (green), E-cadherin (red) and Oct3/4 (red), Nanog (green) of β-catfl/fl and β-catΔ/Δ mESC colonies as observed under confocal microscopy. Nuclei are stained for DAPI (blue). Scale bars in (D) and (E) are 20 µm.
Figure 2.
Differentiation potential of embryo-derived β-catΔ/Δ mESCs.
(A): Phase contrast pictures of EBs derived from β-catfl/fl and β-catΔ/Δ mESCs on day 7 of differentiation. Scale bars are 500 µm. (B): Immunofluorescence staining for Afp (green), α-SMA (red) and Tuj-1 (green) of β-catfl/fl, β-catΔ/Δ and β-catenin rescued β-catΔ/Δ mESCs on day 14 of differentiation. Nuclei are stained for DAPI (blue). Scale bars are 200 µm. (C): Expression levels of Sox17 and Brachyury(T) relative to Gapdh in β-catfl/fl (blue bar), β-catΔ/Δ (light blue bar) and β-catenin rescued β-catΔ/Δ (yellow bar) mESCs on day 6 of differentiation. (D): Fluorescence microscopic images of EGFP-β-catΔ/Δ mESC colonies. They constitutively expressed EGFP. Scale bars are 500 µm. (E): Fluorescent stereomicroscopic image of an embryo on E12.5 generated from blastocysts injected with EGFP-β-catΔ/Δ mESCs. The contribution of β-catΔ/Δ mESCs were barely detected anywhere in the whole body. Scale bars are 2 mm. (F): Chimeras generated by injection of β-catfl/fl and β-catfl/Δ mESCs into ICR host blastocysts. Offspring coat color demonstrates high contribution chimeras.
Figure 3.
Immunohistochemical examination of tumors generated from ESCs either β-catΔ/Δ or β-catfl/fl.
(A): The gross image of a tumor mass derived from β-catfl/fl mESC is shown on the left and that of a tumor derived from β-catΔ/Δ mESCs is shown on the right. β-catΔ/Δ tumor mass was characterized by extensive intra-tumoral hemorrhage (white arrow head). Scale bars are 5 mm. (B): Immunohistochemical staining for Cytoketratin 5/8, Desmin and Neuronal nuclear antigen (NeuN) in tumors derived from mESCs of β-catfl/fl, β-catΔ/Δ or res-β-catΔ/Δ. Tissue sections of β-catΔ/Δ tumors displayed high level staining only for the neuronal differentiation marker NeuN, while there was no detectable staining for Cytoketratin 5/8 or Desmin. Tissue sections of both β-catfl/fl and res-β-catΔ/Δ tumors displayed multiple differentiations as shown in three markers’ positive staining. The left bar graph shows percentages of Cytoketratin 5/8, Desmin and NeuN positive areas with standard deviation (n = 3) as the vertical axis and each tumor as the horizontal axis. Scale bars are 100 µm.
Figure 4.
Histological and immunohistochemical examination of β-catΔ/Δ tumors.
Careful examination of numerous tumor sections stained with hematoxylin and eosin (HE) from different β-catΔ/Δ mESC lines generated independently in separate animals revealed that each β-catΔ/Δ tumor contained three subtypes (seminoma, embryonal carcinoma, choriocarcinoma) of carcinomatous germ cell tumor components. The “seminoma” component is characterized by large uniform dispersed tumor cells displaying clear cytoplasm and distinct cell membrane. Histologic features of the “seminoma” were “fried-egg” appearance: central nucleus with nucleolus, clear cytoplasm and well-defined cell borders. The “embryonal carcinoma (EC)” displayed a papillary pattern, with cohesive clustered growth and marked cytologic atypia. The “choriocarcinoma” was identified by syncytiotrophoblastic giant cells with extensive hemorrhage. The syncytiotrophoblastic giant cells had huge, pleomorphic nuclei and abundant eosinophilic cytoplasm. The “seminoma” displayed very high levels of nuclear OCT4 and Sall4 immunoreactivity, but was negative for pan-cytokeratin. “EC” was strongly positive for pan-cytokeratin, Oct3/4 and Sall4. The syncytiotrophoblastic giant cells of the “choriocarcinoma” showed positive cytoplasmic staining for pan-cytokeratin, but negative nuclear immunostaining for Oct3/4 and Sall4. Scale bars are 100 µm.
Figure 5.
The histological tumorigenic properties of β-catΔ/Δ ESCs can be restored to wild-type ESCs.
β-catfl/fl teratomas consisted of well-differentiated mesodermal tissues (bone and cartilage), endodermal tissues (glandular epithelial structures) and ectodermal tissues (pigmented cells). β-catenin-rescued β-catΔ/Δ mESCs (res-β-catΔ/Δ mESCs) gave rise to multilineage differentiated teratoma possessing mesoderm (bone and cartilage), endoderm (glandular epithelial structures) and ectoderm (epidermal tissue). Scale bars are 100 µm.
Figure 6.
Quantitative PCR and western blot examination of wild-type ESCs derived teratomas, res-β-catΔ/Δ teratomas and β-catΔ/Δ tumors.
(A and B): Expression levels of self-renewal marker genes (oct3/4, nanog, lefty1) and the downstream genes of Wnt/β-catenin signaling (Ctnnb1, tcf3, T) and early differentiation markers (afp, neuroD1, T) relative to Gapdh in β-catfl/fl (blue bar), res-β-catΔ/Δ (yellow bar) and β-catΔ/Δ (light blue bar) mESC derived teratomas or tumors. (C) Western blots of Nanog, Oct3/4 and Gapdh for teratomas derived from β-catfl/fl (fl/fl1,fl/fl2), β-catfl/Δ, res-β-catΔ/Δ (rsc) and R1 mESCs, tumors derived from β-catΔ/Δ (Δ/Δ1, Δ/Δ2, Δ/Δ6) mESC. MEF was used as a control. Abbreviation: MEF, mouse embryonic fibroblast.