Fig 2: Differentiating normal and HGPS-derived iPSCs into keratinocytes. (A) Schematic representation of the protocol for differentiating normal and HGPS patient-derived iPSCs into a keratinocyte lineage. RA and BMP-4 were added on day 1 and day 3. Cells were maintained in keratinocyte growth medium for more than 28 days (4 weeks). Representative phase-contrast images of normal and HGPS iPSCs at different time points during keratinocyte induction were shown next to the timeline (scale bar = 200 µm). (B) Lamin A/C and progerin expression in normal and HGPS iPSCs differentiated cells at week 4 indicated by immunofluorescence staining (scale bar = 25 µm). (C) Basal layer keratinocyte markers ?Np63 and K14 expression in normal and HGPS iPSCs differentiated cells at week 4 as well as in primary keratinocytes indicated by immunofluorescence staining. Representative keratin 14 filaments from single selected cells were shown in the upper-right corner (scale bar = 25 µm). The fluorescence intensities of protein markers were auto-adjusted to demonstrate their cellular distributions.

Fig 3: ABE corrects the HGPS mutation in iPSCs-derived keratinocytes. (A) Schematic representation of the Adenine base editing in HGPS patient-derived iPSCs-keratinocytes induction. (B) LMNA c.1824 nucleotide identity in HGPS iPSCs derived-keratinocytes untreated or treated with ABE7.10max-VRQR lentivirus (mock and HGPS mutation-targeting) at differentiation week 4. (C) Lamin A/C and progerin expression in mock-corrected and HGPS-corrected iPSCs-derived keratinocytes after week 4 (~day 35, passage 1) indicated by immunofluorescence staining (scale bar = 25 µm). (D) Quantitative RT-PCR analysis of the relative expression of Lamin A and progerin in mock-corrected and HGPS-corrected iPSCs-derived keratinocytes at week 4. Data were normalized to endogenous ACTB mRNA and to the average of mock-corrected cells. Data are presented as mean ± SD (n = 3). ** p < 0.01, ns, not significant, unpaired two-tailed t-test. (E) Western blot analysis of Lamin A/C and progerin expression in mock-corrected and HGPS-corrected iPSCs-derived keratinocytes at week 4. Experiments were repeated at least three times, and representative data are shown as indicated. (F) Quantitative RT-PCR analysis of the relative expression of ?Np63, K14, K8, K18, K1, and LEF1 in mock-corrected and HGPS-corrected iPSCs-derived keratinocytes at week 4. Data were normalized to endogenous ACTB mRNA and to the average of mock-corrected cells. Data are presented as mean ± SD (n = 3). ** p < 0.01, **** p < 0.0001, ns, not significant, unpaired two-tailed t-test. (G) Percentage of early apoptotic cells by PI–annexin V flow cytometry analysis in mock-corrected and HGPS-corrected iPSCs-derived keratinocytes at week 4. Data are presented as mean ± SD (n = 3). # p < 0.1, unpaired two-tailed t-test.

Fig 4: Obesity enhances mammary epithelial cell progenitor activity. a Mammary epithelial cell colony stained for cytokeratin (CK)8 (purple) and CK14 (brown) grown on an NIH 3T3 cell feeder layer. Colonies were quantified from n = 8 mice/group in duplicate. b Correlation scatterplot with best fit line representing the average (Avg) number of mammary epithelial progenitor colonies per well with respect to mouse weight gain (n = 16 mice). c Mammary epithelial cell round colony formation on collagen gel. Colonies were quantified from five mice per group, in duplicate. d Correlation scatterplot with best fit line depicting the average number of mammary epithelial round colonies on collagen gels with mouse weight gain (n = 10 mice). e Mammosphere formation in suspension on ultra-low adherence plates. Primary and secondary generation mammospheres were quantified from eight mice per group in triplicate. f Correlation scatterplot with best fit line representing frequency of mammosphere formation from breast epithelial cells isolated from reduction mammoplasty tissue with respect to body mass index (BMI) quantified from 15 patient samples in triplicate. g Correlation scatterplot with best fit line representing average adherent colony formation from 15 patient samples in triplicate with respect to BMI. h Correlation scatterplot with best fit line representing average floating colony formation from 15 patient samples in triplicate with respect to BMI. Bars represent mean ± s.d. Magnification bar = 100 µm. Freq, frequency; HFD, high-fat diet; Con, Control diet

Fig 5: The expression of keratinocyte markers during iPSCs-keratinocytes differentiation. (A) Quantitative RT-PCR analysis of the relative expression of Lamin A, progerin, ?Np63, K14, K8, and K1 during normal and HGPS iPSCs-keratinocytes induction. Data were normalized to endogenous ACTB mRNA and to the average of Normal week 4. Data are presented as mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant, two-way ANOVA followed by Tukey’s multiple comparisons test. (B) Western blot analysis of Lamin A/C (including progerin in HGPS-differentiated cells indicated by the red arrow), ?Np63, K14 (indicated by red arrow), and K8 expression during normal and HGPS iPSCs-keratinocytes induction. Primary keratinocytes were used as a positive control. All experiments were repeated at least three times, and representative data are shown as indicated.