Reviewer #1:
In this manuscript, Fernandez et al examine the impact of defective telomere length maintenance on type II alveolar epithelial cells, which are thought to be central to the pathogenesis of pulmonary fibrosis in dyskeratosis congenita (DC) and related telomere biology disorders. Murine models have been used to address how telomere dysfunction in AT2 cells drives pulmonary fibrosis however these models have limitations. Therefore, the investigators' study of human AT2 cells/organoids derived from induced pluripotent stem cells (iAT2 cells) in the presence and absence of a known DC pathogenic variant provides an exceptional model. In addition, the investigators use expression profiling to uncover decreased canonical WNT signaling in iAT2 cells with telomere dysfunction and then demonstrate rescue of telomere dysfunction and iAT2 cell growth with the addition of a GSK3 inhibitor, a canonical WNT agonist. The data appear to be of high quality, the approaches and interpretation appropriate, with some noted exceptions below. Given the importance of the problem (dysfunctional telomere-induced pulmonary fibrosis) and the apparent benefit of GSK3 inhibition of iAT2 cell growth and telomere dysfunction, which extends the work published by this group previously on intestinal organoids (might enhanced canonical WNT signaling more broadly affect other tissues with telomere-induced senescence?), this work is significant.
A few aspects of the studies dampen the ability to draw certain conclusions. For example, the authors use iPSCs that are 5 vs 25 passages after introduction (or not) of the DKC1 A386T mutation for the generation of iAT2 cells. They then show iAT2 DKC1 mutant organoids generated from the later passage iPSCs have an apparent growth defect as early as Day 50 but that those generated from the earlier passage iPSCs do not at Day 70 [with caveats the images are of different quality (comparing Fig. 1B and Fig. S3D) and quantitative data (similar to Fig. 1C) are lacking for the iAT2 organoids generated from the early passage iPSCs]. They argue that progressive telomere shortening is the cause of the growth defects. If this is the case, then the iAT2 cells generated from the earlier passages should eventually show growth defects with progressive telomeres shortening, which was not shown.
The telomere length analysis of the iAT2 cells at Day 50 and Day 70 are not markedly different, and neither the % p21 + nor TIF+ cells is shown for Day 50. Therefore, the conclusion that it is the accumulation of short uncapped telomeres in the DKC1 mutant iAT2 cells that alters gene expression and induces senescence at Day 70 ignores the extent of these changes at Day 50.
The statement that CHIR99021 (when present in the medium from Day 49-70) rescued the growth defect seems generous; the effect is partial and the assay is for organoid formation efficiency only. Moreover, it is most likely prohibiting the further accumulation of senescent cells rather than rescuing cells that were not previously growing.
It is striking that prolonged CHIR99021 treatment (ie, through to Day 70) resulted in increased telomerase activity, and more so in mutant compared to wild type cells. First, how reproducible was this effect? I appreciate that the authors have not explored this for this manuscript, however, TERT expression does not rescue DKC1 mutants but TERC does. Were TERC levels increased? Also, given this robust increase, it is striking that no difference is detected in TeSLA assays given the proportion of very short detected telomeres that would presumably be substrates for telomerase. It is noteworthy that, in the protocol to derive iAT2 cells, CHIR99021 is present in the media prior to Day 28. This raises the question of whether there is rescue of telomerase in the cells exposed to CHIR99021 in the interval of iAT2 specification?