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. 2022 May 14;23(10):5499.
doi: 10.3390/ijms23105499.

Impaired LEF1 Activation Accelerates iPSC-Derived Keratinocytes Differentiation in Hutchinson-Gilford Progeria Syndrome

Xiaojing Mao  1 , Zheng-Mei Xiong  1   2 , Huijing Xue  1 , Markus A Brown  1 , Yantenew G Gete  1 , Reynold Yu  1 , Linlin Sun  1   3 , Kan Cao  1
Affiliations

Impaired LEF1 Activation Accelerates iPSC-Derived Keratinocytes Differentiation in Hutchinson-Gilford Progeria Syndrome

Xiaojing Mao et al. Int J Mol Sci. .

Abstract

Hutchinson-Gilford progeria syndrome (HGPS) is a detrimental premature aging disease caused by a point mutation in the human LMNA gene. This mutation results in the abnormal accumulation of a truncated pre-lamin A protein called progerin. Among the drastically accelerated signs of aging in HGPS patients, severe skin phenotypes such as alopecia and sclerotic skins always develop with the disease progression. Here, we studied the HGPS molecular mechanisms focusing on early skin development by differentiating patient-derived induced pluripotent stem cells (iPSCs) to a keratinocyte lineage. Interestingly, HGPS iPSCs showed an accelerated commitment to the keratinocyte lineage than the normal control. To study potential signaling pathways that accelerated skin development in HGPS, we investigated the WNT pathway components during HGPS iPSCs-keratinocytes induction. Surprisingly, despite the unaffected β-catenin activity, the expression of a critical WNT transcription factor LEF1 was diminished from an early stage in HGPS iPSCs-keratinocytes differentiation. A chromatin immunoprecipitation (ChIP) experiment further revealed strong bindings of LEF1 to the early-stage epithelial developmental markers K8 and K18 and that the LEF1 silencing by siRNA down-regulates the K8/K18 transcription. During the iPSCs-keratinocytes differentiation, correction of HGPS mutation by Adenine base editing (ABE), while in a partial level, rescued the phenotypes for accelerated keratinocyte lineage-commitment. ABE also reduced the cell death in HGPS iPSCs-derived keratinocytes. These findings brought new insight into the molecular basis and therapeutic application for the skin abnormalities in HGPS.

Keywords: HGPS; LEF1; WNT signaling; epidermal development; keratins; lamin A; progeria; skins.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
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.
Figure 2
Figure 2
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.
Figure 3
Figure 3
Cell cycle and apoptosis during iPSCs-keratinocytes differentiation (A) Quantification of the G2/M phase cell cycle partitioning during normal and HGPS iPSCs-keratinocytes induction. Data are presented as mean ± SD (n = 3). # p < 0.1, ** p < 0.01, ns, not significant, two-way ANOVA followed by Tukey’s multiple comparisons test. (B) Representative flow cytometry plots showing PI–annexin V apoptosis assay during normal and HGPS iPSCs-keratinocytes induction. The gates were set according to the positive and negative controls as per the manufacturer’s instructions. The cells in the lower right quadrant were quantified as early apoptotic populations. (C) Percentage of early apoptotic cells by PI–annexin V flow cytometry analysis during normal and HGPS iPSCs-keratinocytes induction. Data are presented as mean ± SD (n = 3). # p < 0.1, ns, not significant, Two-way ANOVA followed by Tukey’s multiple comparisons test.
Figure 4
Figure 4
LEF1 down-regulation in early HGPS iPSCs-keratinocytes differentiation. (A) Western blot analysis of total and non-phospho (active) β-catenin expression during normal and HGPS iPSCs-keratinocytes induction. Experiments were repeated at least three times, and representative data are shown as indicated. (B) Western blot analysis of nuclear and cytosolic non-phospho (active) β-catenin level in early-differentiating (week 1) normal and HGPS iPSCs. Regulator of Chromosome Condensation 1 (RCC1) and S6 were used as a nuclear marker and the overall loading control, respectively. Experiments were repeated at least three times, and representative data are shown as indicated. (C) Quantitative RT-PCR analysis of the relative expression of WNT transcription factors LEF1, TCF7, TCF7L1, and TCF7L2 before (day 0) and after induction with RA and BMP-4 (day 5) in normal and HGPS iPSCs differentiation. Data were normalized to endogenous ACTB mRNA and to the average of Normal day 0. Data are presented as mean ± SD (n = 3). * p < 0.05, ** p < 0.01, ns, not significant, two-way ANOVA followed by Tukey’s multiple comparisons test. (D) Quantitative RT-PCR analysis of the relative expression of LEF1 during normal and HGPS iPSCs-keratinocytes induction. Data were normalized to endogenous ACTB mRNA and to the average of Normal week 1. Data are presented as mean ± SD (n = 3). ** p < 0.01, *** p < 0.001, ns, not significant, two-way ANOVA followed by Sidak’s multiple comparisons test. (E) Western blot analysis of LEF1 expression during normal and HGPS iPSCs-keratinocytes induction. Experiments were repeated at least three times, and representative data are shown as indicated.
Figure 5
Figure 5
LEF1 regulates keratinocytes differentiation through K8/K18. (A) Schematic representation showing putative LEF1 binding sites at K8/K18 gene locus. The position of seven putative LEF1 binding sites and a non-specific locus (negative) were indicated by black arrowheads. (B) Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) analysis of the DNA binding activity of LEF1 at the K8/K18 locus. A known LEF1 binding site at the MYC gene was used as a positive control. The binding sites with LEF1 enrichment four-fold greater than the non-specific locus (above the dashed line) were considered to have strong LEF1 binding. Data are presented as mean ± SD (n = 3). (C) Representative Western blot result showing LEF1 protein expression 48 h after LEF1 siRNA knockdown in normal iPSCs differentiation. (D) Quantification of LEF1 siRNA knockdown Western blot analysis in (C). Data are presented as mean ± SD (n = 3). * p < 0.05, ns, not significant, one-way ANOVA followed by Tukey’s multiple comparisons test. (E) Quantitative RT-PCR analysis of the relative expression of K8 and K18 48 h after LEF1 siRNA knockdown in normal iPSCs differentiation. Data are normalized to endogenous ACTB mRNA and to the average of untransfected samples. Data are presented as mean ± SD (n = 3). * p < 0.05, ** p < 0.01, ns, not significant, one-way ANOVA followed by Tukey’s multiple comparisons test.
Figure 6
Figure 6
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.
Figure 7
Figure 7
Working model of epidermal development in HGPS. (A) Schematic representation of the keratinocyte-related markers succession during iPSCs-keratinocytes induction. (B) Schematic diagram showing the molecular mechanism for the early commitment to the keratinocyte lineage in HGPS iPSCs differentiation.
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