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. 2018 Mar 25;2018:7906531.
doi: 10.1155/2018/7906531. eCollection 2018.

Comparison of Teratoma Formation Between Embryonic Stem Cells and Parthenogenetic Embryonic Stem Cells by Molecular Imaging

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Free PMC article

Comparison of Teratoma Formation Between Embryonic Stem Cells and Parthenogenetic Embryonic Stem Cells by Molecular Imaging

Hongyan Tao et al. Stem Cells Int. .
Free PMC article

Abstract

With their properties of self-renewal and differentiation, embryonic stem (ES) cells hold great promises for regenerative therapy. However, teratoma formation and ethical concerns of ES cells may restrict their potential clinical applications. Currently, parthenogenetic embryonic stem (pES) cells have attracted the interest of researchers for its self-renewing and pluripotent differentiation while eliciting less ethic concerns. In this study, we established a model with ES and pES cells both stably transfected with a double-fusion reporter gene containing renilla luciferase (Rluc) and red fluorescent protein (RFP) to analyze the mechanisms of teratoma formation. Transgenic Vegfr2-luc mouse, which expresses firefly luciferase (Fluc) under the promoter of vascular endothelial growth factor receptor 2 (Vegfr2-luc), was used to trace the growth of new blood vessel recruited by transplanted cells. Bioluminescence imaging (BLI) of Rluc/Fluc provides an effective tool in estimating the growth and angiogenesis of teratoma in vivo. We found that the tumorigenesis and angiogenesis capacity of ES cells were higher than those of pES cells, in which VEGF/VEGFR2 signal pathway plays an important role. In conclusion, pES cells have the decreased potential of teratoma formation but meanwhile have similar differentiating capacity compared with ES cells. These data demonstrate that pES cells provide an alternative source for ES cells with the risk reduction of teratoma formation and without ethical controversy.

Figures

Figure 1
Figure 1
Generation of pES cells and ES cells labeled with double-fusion reporter genes. (a) Schema of lentiviral construct showing EF1α promoter driving Rluc and RFP. (b) Brightfield and fluorescence microscopy showing RFP expression in pES cells and ES cells. (c) BLI of pES cells and ES cells shows a robust correlation between cell number and Rluc activity.
Figure 2
Figure 2
The characterization of reporter gene-labeled cells. (a, b) Cell proliferation activity between wild-type cells and labeled cells shows no significant difference in vitro. (c) The pluripotency of the cells was detected by ALP staining, and undifferentiated cells appear red, whereas MEF cells appear colorless. ALP staining of each group exhibited the transfected report genes does not affect the pluripotency of pES cells and ES cells.
Figure 3
Figure 3
Visualization of the teratoma growth and VEGFR2 expression in vivo. (a) Rluc imaging of teratoma progression of Rluc signals showed that the teratoma formed in pES-RR group was smaller than that in ES-RR group. Quantification analysis demonstrated there was a significant difference between the two groups. (b) Fluc imaging and quantification analysis of VEGFR2 expression in transgenic mice revealed enhanced angiogenesis in ES cell-derived teratomas. P < 0.05 compared with the ES-RR group.
Figure 4
Figure 4
Analysis of teratoma angiogenesis. (a) Angiogenesis observed in pES-RR group and ES-RR group by CD31 immunofluorescence staining. (b) Microvessel density (MVD) of teratoma tissue was measured by computer image analyzing system. (c) The expression of CD34/VEGF/VEGFR2 was significantly lower in pES-RR group compared with ES-RR group, whereas there was no significant difference in the expression of ANG-1/ANG-2/Tie-2 between these two groups. P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.
Figure 5
Figure 5
Differentiation potential between two cell lineages. (a) RT-PCR analysis of the expression of stemness-related genes of EBs from two groups in D6 and D12, which displayed no significant difference. (b) Teratoma formation of pES-RR and ES-RR. Histological analysis of teratoma identifying the presence of derivatives of all three germ layers: neural tissue (ectoderm), vascular tissue (mesoderm), and glandular tissue (endoderm).

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