Figure 2.
Figure 2. High occurrence of NRTS during hESC and mESC differentiation. (a) Experimental design scheme representing how NRTS was assessed in EBs 7 d after differentiation for H9 hESCs that have a population doubling time of 24 h. The first label (BrdU for single labeling or CldU for double labeling) was added to EBs for 8 h between days 6 and 7. This was followed by a 16-h chase without any label. 24 h after addition of the first label, when cells have completed one division and started a second one, EBs were dissociated into single cells, plated at very low density, and allowed to complete the second division (in the presence of IdU for the double-labeling method). Paired cells were then fixed and stained for BrdU or CldU and IdU. (b) Theoretical predicted outcomes of the experiment described in a. (c) H9 hESC pairs that are symmetric (top) or asymmetric (bottom) for BrdU in the single-labeling method. Paired-cell assays were performed on EBs 7 d after differentiation. Representative photographs of cell pairs immunostained for BrdU are shown alongside bright-field (BF) and DAPI nuclear counterstaining images. (d) Quantification of asymmetric pairs during H9 hESC differentiation. Paired-cell assays were performed at different days after EB formation as indicated. Cell pairs were immunostained for BrdU. More than 200 pairs were analyzed per time point and the percentage of pairs showing asymmetric BrdU (i.e., NRTS) was quantified. Data represent means ± SEM (n = 3 independent experiments). (e and f) H9 hESC (e) and R1 mESC (f) pairs that are symmetric or asymmetric for CldU in the double-labeling method. Paired-cell assays were performed on EBs 7 d after differentiation. Cell pairs were immunostained for CldU and IdU. Bright-field images and DAPI nuclear counterstaining are shown. Representative photographs of symmetric (top), asymmetric (middle), and IgG control (bottom) pairs are displayed for each cell line. (g and h) CldU (g) and IdU (h) fluorescence intensity distribution among daughter cells from R1-derived EB pairs experiment using the CldU/IdU double-labeling method. CldU intensity was normalized to IdU intensity for each cell, and IgG background was subtracted from the CldU and IdU signals. The scatter graphs show the quantification of immunofluorescence intensity of visually symmetric (with a distribution from equal to 60:40, as indicated) and visually asymmetric (with a distribution of 90:10 or greater, as indicated) pairs of cells (n = 58 representative pairs from three to four independent experiments). (i) Quantification of the occurrence of NRTS in H9- and R1-derived EBs 7 d after differentiation and IMR-90 human fetal fibroblasts using the CldU/IdU double-labeling method. NRTS frequency was also quantified in R1-derived EBs after the first division in which the CldU label is expected to be segregated to both daughter cells. NRTS occurrence in undifferentiated R1 mESCs was assessed using the BrdU single-labeling method and quantified among Oct4 double-positive pairs. More than 100 pairs were analyzed for each cell line per experiment, and the percentage of cells showing asymmetric CldU or BrdU (i.e., NRTS) was quantified (all cells had symmetric IdU in the double-labeling method). Data represent means ± SEM (n = 3–6 independent experiments).

High occurrence of NRTS during hESC and mESC differentiation. (a) Experimental design scheme representing how NRTS was assessed in EBs 7 d after differentiation for H9 hESCs that have a population doubling time of 24 h. The first label (BrdU for single labeling or CldU for double labeling) was added to EBs for 8 h between days 6 and 7. This was followed by a 16-h chase without any label. 24 h after addition of the first label, when cells have completed one division and started a second one, EBs were dissociated into single cells, plated at very low density, and allowed to complete the second division (in the presence of IdU for the double-labeling method). Paired cells were then fixed and stained for BrdU or CldU and IdU. (b) Theoretical predicted outcomes of the experiment described in a. (c) H9 hESC pairs that are symmetric (top) or asymmetric (bottom) for BrdU in the single-labeling method. Paired-cell assays were performed on EBs 7 d after differentiation. Representative photographs of cell pairs immunostained for BrdU are shown alongside bright-field (BF) and DAPI nuclear counterstaining images. (d) Quantification of asymmetric pairs during H9 hESC differentiation. Paired-cell assays were performed at different days after EB formation as indicated. Cell pairs were immunostained for BrdU. More than 200 pairs were analyzed per time point and the percentage of pairs showing asymmetric BrdU (i.e., NRTS) was quantified. Data represent means ± SEM (n = 3 independent experiments). (e and f) H9 hESC (e) and R1 mESC (f) pairs that are symmetric or asymmetric for CldU in the double-labeling method. Paired-cell assays were performed on EBs 7 d after differentiation. Cell pairs were immunostained for CldU and IdU. Bright-field images and DAPI nuclear counterstaining are shown. Representative photographs of symmetric (top), asymmetric (middle), and IgG control (bottom) pairs are displayed for each cell line. (g and h) CldU (g) and IdU (h) fluorescence intensity distribution among daughter cells from R1-derived EB pairs experiment using the CldU/IdU double-labeling method. CldU intensity was normalized to IdU intensity for each cell, and IgG background was subtracted from the CldU and IdU signals. The scatter graphs show the quantification of immunofluorescence intensity of visually symmetric (with a distribution from equal to 60:40, as indicated) and visually asymmetric (with a distribution of 90:10 or greater, as indicated) pairs of cells (n = 58 representative pairs from three to four independent experiments). (i) Quantification of the occurrence of NRTS in H9- and R1-derived EBs 7 d after differentiation and IMR-90 human fetal fibroblasts using the CldU/IdU double-labeling method. NRTS frequency was also quantified in R1-derived EBs after the first division in which the CldU label is expected to be segregated to both daughter cells. NRTS occurrence in undifferentiated R1 mESCs was assessed using the BrdU single-labeling method and quantified among Oct4 double-positive pairs. More than 100 pairs were analyzed for each cell line per experiment, and the percentage of cells showing asymmetric CldU or BrdU (i.e., NRTS) was quantified (all cells had symmetric IdU in the double-labeling method). Data represent means ± SEM (n = 3–6 independent experiments).

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