Could the authors elaborate on the following for further clarification and potential refinement to strengthen the overall conclusions?
While the NCC reporter system is an innovative approach for monitoring cellular senescence, the robustness of the Z-factor for large-scale screening purposes appears suboptimal. This limitation should be addressed with additional experiments or an explanation of how the results were normalized to account for variability across different cell types and donor samples.
The study relies heavily on fluorescence intensity as a proxy for nucleocytoplasmic compartmentalization integrity. While effective, this approach may not capture more nuanced molecular changes. Adding complementary assays, such as proteomic analysis or live-cell imaging with advanced resolution, would enhance the depth of the findings.
The selection criteria for the six chemical cocktails are not thoroughly detailed. The rationale behind focusing on these specific combinations should be elaborated upon, especially given the large initial pool of 80 tested cocktails. Were the excluded combinations less effective, or did they display cytotoxicity?
The absence of pluripotency markers after chemical treatments is reassuring but leaves open questions regarding long-term genomic stability. Extended monitoring of treated cells for potential unintended consequences, such as tumorigenesis, would be a valuable addition.
While transcriptomic rejuvenation is well-documented, the functional implications of this rejuvenation are less clear. The study would benefit from testing whether the observed changes translate into enhanced cellular or tissue function in physiological contexts beyond transcriptomic clocks.
The study’s claim that rejuvenation is “separable from pluripotency programs” is compelling but requires additional mechanistic insight. How do the pathways activated by the chemical cocktails overlap with, or diverge from, those activated by OSK reprogramming?
Potential conflicts of interest, particularly given the involvement of authors with commercial affiliations, necessitate careful scrutiny. Explicit discussion on how such affiliations did not bias the experimental design or interpretation would bolster the paper’s credibility.