Brief summary of the study - a sentence summarizing the study and general comments that apply across the full paper
Major comments - Comments on the validity or strength of the methodology, experiments and analyses, strength of the conclusions
Minor comments - Clarifications to statements in the text, interpretation of the results, presentation of the data/figures
Comments on reporting - information on the statistical analyses or availability of data.
Please add comments on the preprint below via comments. You can add comments on the full paper, sections or only individual fragments. Any comments added here will be reviewed for inclusion in the public review section if relevant, but will not be posted publicly in any way that can identify the commenter for individual comments.
Introduction section: “[...], such as lesion formation or progression. Understanding microglial dysfunction in MS is crucial for developing targeted therapies.”
Introduction section: “ In recent years, pioneering studies have reported methods to produce microglia-like cells (iMGLs) from iPSCs, addressing the previously unmet need for in vitro models of microglial phenotypes in neurological disorders”
Introduction section: “These findings suggest that patient-specific iMGLs represent an exceptional tool for modelling microglial dysfunction in MS and may inform therapeutic strategies that focus on targeting microglia.”
Comments:
(Minor) It seems to me like the characterization of the iMGLs and their validation as in vitro model is the main point of the manuscript. Highlighting the need for this model more, instead of the current focus on transcriptomic findings from other models, could showcase this better.
Materials and methods section: “The inclusion criteria were a confirmed MS diagnosis according to the 2010 McDonald criteria42, a previous PET scan using the [11C]PK11195 radioligand and female sex. ”
Materials and methods section: “IPSCs were passaged enzymatically with TrypLE™ Select Enzyme and Defined Trypsin Inhibitor (both from Thermo Fisher Scientific) in the presence of 10 µM ROCK inhibitor (ROCKi, Y-27632, StemCell Technologies) twice a week.”
Materials and methods section: “ On Days 0 and 1, the cells were maintained in E8 flex -medium containing 5 ng/ml BMP4, 25 ng/ml activin A (both from Peprotech) and 1 µM CHIR 99021 (Axon).”
Comments:
please explain that CHIR 99021 is Glycogen synthase kinase 3β inhibitor
Similarly, please identify DMEM/F-12 as (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12), (FGF-2) as Fibroblast growth factor 2, (VEGF) as Vascular endothelial growth factor, transforming growth factor-β receptor inhibitor, (TPO) asThyroid peroxidase, (SCF) as Stem cell factor, (M-CSF) as macrophage-colony stimulating factor in the same section.
Materials and methods section: “This analysis revealed that the cell line was a significant confounding factor influencing the samples’ expression profiles.”
Materials and methods section: “This step provided insights into the biological functions and pathways affected by the experimental conditions.”
Results section: “The iPSCs were successfully generated from PBMCs using Sendai virus reprogramming, and newly established iPSC lines were characterized based on their expression of pluripotency markers and trilineage differentiation capacity (Supplementary Fig. 1)”
Figure 2 (A): “The data are presented as single datapoints and medians. No significant differences were observed between the cell lines using the Kruskal–Wallis test with Dunn’s post hoc test.”
Comments:
In Figure 2D, while the authors appropriately used the Kruskal-Wallis test with Dunn's post hoc test to compare Iba1-positive cell percentages across cell lines, the addition of an effect size measure would strengthen the statistical reporting. For non-parametric comparisons across multiple groups, including either epsilon-squared (ε²) or eta-squared (η²) would help readers better understand the magnitude of any differences between groups, beyond just statistical significance. This would provide additional context for interpreting the biological relevance of the findings, even though no significant differences were observed between cell lines.
Figure 2 (H): “The grey values indicate the statistical significance of differences between the HC VEH and LPS groups, the green values between the MS VEH and LPS groups, and the black values between the HC and MS groups. ”
Figure 3 (B): “Violin plot showing the quantification of the nuclear localization of NF-κB p65 from the immunofluorescence images. ”
Results section: “Remarkably, the DEG analysis confirmed the transcriptomic difference between MS and HC iMGLs in the basal state by identifying of 1,027 DE genes (using a cut-off of logFC > ± 1 and adjusted p value < 0.05; Fig. 4A). ”
Results section: “These terms included “immune receptor activity” (PIGR, FPR1, IL21R, FPR3, FCER1A, CTSH, and FCGR2B), “antigen processing and presentation of exogenous peptide antigen via MHC class II” (HLA-DRA, HLA-DPA1, HLA-DRB1, HLA-DQB1, CD74, and HLA-DRB5), “”
Discussion section: “suggesting that CCL2 contributes to microglial activation in the basal state. Prior reports of EAE and cuprizone mouse models of MS suggest that microglia are one of the primary sources of CCL2 in the brain”
Discussion section: “Many of the upregulated genes that were detected in our MS iMGLs have also been linked to phagocytosis in transcriptional studies of postmortem tissues from pwMS suggesting that dysregulated phagocytosis contributes to MS pathology.”
The authors declare that they have no competing interests.