Compromised Chondrocyte Differentiation Capacity in TERC Knockout Mouse Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer
<p>Characterizations of nuclear transfer embryonic stem cells (ntESCs) with different Terc genotypes. (<b>A</b>) Genotyping of <span class="html-italic">Terc</span><sup>+/+</sup> (WT), <span class="html-italic">Terc</span><sup>+/−</sup> (HT) and <span class="html-italic">Terc</span><sup>−/−</sup> (KO) ntESCs. (–): negative control without template DNA. The 280 bp band indicates the KO allele and the 150 bp band indicates the wild-type allele. (<b>B</b>) Telomerase activity is assayed by Telomerase Repeated Amplification Protocol (TRAP) assay for each <span class="html-italic">Terc</span> genotype. Positive control is human 293T cells (293T), and the heat-inactivated 293T cells (Heated 293T) is used as negative control. Error bar indicates the standard deviation (SD). (<b>C</b>) Detection of pluripotent markers by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). (–): negative control without template cDNA. (<b>D</b>) Western blot analysis of pluripotency by detecting NANOG, octamer-binding transcription factor 4 (OCT4), Sal-like protein 4 (SALL4), and SRY (sex determining region Y)-box 2 (SOX2) in ntESC lines. TUBULIN is used as the internal control. Mouse embryonic fibroblast (MEF) is used as the negative control for pluripotent markers. (<b>E</b>) Immunofluorescent staining shows the positive signal of SOX2 and SALL4 in ntESC lines. 4′,6-diamidino-2-phenylindole (DAPI) is used for nuclei stain. (<b>F</b>) Growth curve of ntESCs in six-days of culture in embryonic stem cell (ESC) medium (<span class="html-italic">n</span> = 3). *** indicates significant difference between groups (<span class="html-italic">p</span> < 0.001). ns: no significant differences. (<b>G</b>) Telomere length analysis by telomere restriction fragment (TRF) in three genotypes of ntESCs. Red line indicates the medium length of genomic DNA. (<b>H</b>) Comparison of telomere length by telomere to single-copy gene ratio (T/S ratio). * indicates significant difference between groups (<span class="html-italic">p</span> < 0.05) analyzed by unpaired student <span class="html-italic">t</span>-test.</p> "> Figure 2
<p>Decreased size of embryoid bodies (EBs) and loss of cartilage cells in teratomas from <span class="html-italic">Terc<sup>−</sup></span><sup>/−</sup> ntESCs. (<b>A</b>) Morphology of EBs derived from <span class="html-italic">Terc</span><sup>+/+</sup> (WT), <span class="html-italic">Terc</span><sup>+/−</sup> (HT), and <span class="html-italic">Terc</span><sup>−/−</sup> (KO) ntESCs. Spontaneous differentiation is performed in ESC medium without leukemia inhibitory factor (LIF). (<b>B</b>) Formation efficiency and diameter of EBs derived from three genotype<span class="html-italic">s</span> of ntESC. The size of EBs from <span class="html-italic">Terc</span><sup>−/−</sup> is significantly smaller than other genotypes (triplicate experiments). ns = no significance. ** = <span class="html-italic">p</span> < 0.005, *** = <span class="html-italic">p</span> < 0.001. (<b>C</b>) Detection of three germ layers markers in EBs. Endoderm markers: SRY (Sex-Determining Region Y)-Box 17 (<span class="html-italic">Sox17</span>) and GATA binding protein 4 (<span class="html-italic">Gata4)</span>. Mesoderm markers: <span class="html-italic">Brychury (Bry)</span> and heart and neural crest derivatives expressed 1 (<span class="html-italic">Hand1</span>). Ectoderm markers: paired box 6 (<span class="html-italic">Pax6</span>) and SRY (Sex-Determining Region Y)-Box 1 (<span class="html-italic">Sox1</span>). Glyceraldehyde-3-phosphate dehydrogenase <span class="html-italic">(Gapdh)</span> is used for the internal control. Triplicates of experiments are shown. (+): Positive control, cDNA of the Institute of Cancer Research (ICR) mouse embryo at E7.5 (<span class="html-italic">n</span> = 3). (-): negative control without template cDNA (<b>D</b>) Quantification of RT-PCR analysis of markers of three germ layers. Error bar indicates the SD, *: <span class="html-italic">p</span> < 0.05; ns: no significant differences. (<b>E</b>) Size of teratomas from WT, HT, and KO ntESCs is analyzed. Three individual clones of each genotype of ntESCs are assayed as triplicate, ns: no significant differences. (<b>F</b>) Teratomas are derived from the WT, HT, and KO ntESCs by injection into immune-deficient Nu mice. Hematoxylin and eosin (H&E) staining of teratomas demonstrated typical cell types of three germ layers, including endoderm (ciliated epithelium), ectoderm (neuron like), and mesoderm (cartilage), indicated by blue arrows. All lineages of cells are observed in <span class="html-italic">Terc</span><sup>+/+</sup> and <span class="html-italic">Terc</span><sup>+/−</sup> groups, but loss of cartilage from mesoderm in KO group. Scale bar = 50 μm.</p> "> Figure 3
<p><span class="html-italic">Terc</span> depletion abolishes the chondrogenesis in vitro. (<b>A</b>) Morphology of chondrocyte differentiation of <span class="html-italic">Terc</span><sup>+/+</sup> (WT), <span class="html-italic">Terc</span><sup>+/−</sup> (HT), and <span class="html-italic">Terc</span><sup>−/−</sup> (KO) ntESCs at different time point. Both <span class="html-italic">Terc</span><sup>+/+</sup> and <span class="html-italic">Terc</span><sup>+/−</sup> continues the differentiation procedure till 30 day (red arrows), but the differentiates of <span class="html-italic">Terc</span><sup>−/−</sup> ntESCs show massive cell death around 10–15 days (blue arrow). Fewer remained cells in <span class="html-italic">Terc</span><sup>−/−</sup> group. (<b>B</b>) After 30 days of differentiation, the cells differentiate into fibroblastic morphology and more chondrocyte-like cells with positive signal stained with Alcian Blue (red arrow indicated). (<b>C</b>) Cartilage associated genes are evaluated in differentiated ntESCs. The early cartilage marker SRY (Sex-Determining Region Y)-Box 9 (<span class="html-italic">Sox9</span>) and late marker Collagen II (<span class="html-italic">Col2a1</span>) expressed at 20 and 30 days of differentiation, indicating the specific induction of mesoderm-chondrocyte from ntESCs. Scale bar = 100 μm. (+): the positive control cDNA for detecting <span class="html-italic">Sox9</span> and <span class="html-italic">Col2a1</span> collected from mouse testis and cartilage tissue respectively. 1 and 2 indicate the cDNA of testis and cartilage detected with internal control <span class="html-italic">Gapdh</span> primer. (-): negative control without template cDNA. (<b>D</b>) Quantification of <span class="html-italic">Sox9</span> and <span class="html-italic">Col2a1</span> expression level at 20 and 30 days (20D and 30D) of differentiation, calculated from the result in (<b>C</b>) for triplicate. Error bar indicates the standard deviation. Ns = no significant differences. * = <span class="html-italic">p</span> < 0.05. (<b>E</b>) Real-time PCR analysis confirms the relative expression level of <span class="html-italic">Sox9</span> in differentiated chondrocytes. Cycle threshold (Ct) value for detecting <span class="html-italic">Sox9</span> was normalized with <span class="html-italic">Gapdh</span> and wild-type at 20 and 30 days, respectively. Ns = no significant differences. * = <span class="html-italic">p</span> < 0.05.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Shortened Telomeres and Slowed Cell Growth in Terc−/− ntESCs
2.2. Compromised Spontaneous In Vitro and In Vivo Differentiation Capacity in Terc−/− ntESCs
2.3. Compromised In Vitro Differentiation Capacity to Chondrocytes in Terc−/− ntESCs
3. Discussion
4. Materials and Methods
4.1. Culturing ntESCs
4.2. In Vitro Differentiation
4.3. Gene Analysis
4.4. Teratoma Assay
4.5. Terminal Restriction Fragment (TRF) Analysis for Telomere Lengths
4.6. Telomerase Activity Measurement
4.7. Quantitative Real-Time PCR for Telomere Assay
4.8. Western Blot
4.9. Immunofluorescent Staining and Confocal Microscope
4.10. Genotyping
4.11. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
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Chang, W.-F.; Wu, Y.-H.; Xu, J.; Sung, L.-Y. Compromised Chondrocyte Differentiation Capacity in TERC Knockout Mouse Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer. Int. J. Mol. Sci. 2019, 20, 1236. https://doi.org/10.3390/ijms20051236
Chang W-F, Wu Y-H, Xu J, Sung L-Y. Compromised Chondrocyte Differentiation Capacity in TERC Knockout Mouse Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer. International Journal of Molecular Sciences. 2019; 20(5):1236. https://doi.org/10.3390/ijms20051236
Chicago/Turabian StyleChang, Wei-Fang, Yun-Hsin Wu, Jie Xu, and Li-Ying Sung. 2019. "Compromised Chondrocyte Differentiation Capacity in TERC Knockout Mouse Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer" International Journal of Molecular Sciences 20, no. 5: 1236. https://doi.org/10.3390/ijms20051236