PREreview of Microglia from patients with multiple sclerosis display a cell-autonomous immune activation state

Brief summary of the study - a sentence summarizing the study and general comments that apply across the full paper

• Hyvärinen, Lotila et al. apply previously published protocols to generate induced pluripotent stem cells from MS patient PBMCs and subsequently differentiate these into microglia-like cells (iMGLs). They then investigate whether microglia derived from iPSCs of patients with MS exhibit cell-autonomous immune activation. Using RNA-seq, Western Blotting, cytokine secretion, and phagocytosis assays, they demonstrate that these MS iMGLs show elevated activation and inflammation status compared to iMGLs from healthy donors. This indicates that these MS iMGLs may replicate the microglia phenotype observed in the central nervous system of patients with multiple sclerosis.

Major comments  - Comments on the validity or strength of the methodology, experiments and analyses, strength of the conclusions

• It would be highly interesting to show that the authors can extract cells from the blood for iPSC and subsequent iMGL derivation to recapitulate the microglial phenotype in MS patients. So far, however, the manuscript only shows that their iMGLs from both healthy and MS donors both mimic an MS microglia cluster equally well (Fig. 2G). The MS iMGLs having increased inflammation markers is shown extensively, but this is not sufficient as evidence for disease representation - limiting the application of the hereby presented MS iMGLs as MS microglia models. If the authors could show that their MS iMGLs transcriptomically match published MS microglia RNA datasets better than healthy iMGLs, this would majorly improve the manuscript and its impact.

• The authors describe in detail which genes are expressed differentially between MS and healthy iMGLs in unstimulated and stimulated settings. However, the impact of these individual genes, and a comparison whether this differential expression is also seen in patient microglia is lacking.

Minor comments - Clarifications to statements in the text, interpretation of the results, presentation of the data/figures

• Assuming the MS iMGLs recapitulate the situation in the patient’s CNS, can the authors speculate on how this could be mediated? Are the (epi)genetic alterations mediating microglial activation in MS conserved throughout the whole body? Would the authors expect the same results from iPSCs derived from other cell types, e.g. skin fibroblasts?

Comments on reporting - information on the statistical analyses or availability of data.

• Adding a list of abbreviation could be helpful for the readers

• A section that includes the limitations of the study could enrich the article

Suggestions for future studies

• A more diverse cohort of MS patients (including primary progressive MS and early-stage MS patient samples) would help anaylsis across disease subtypes and progression.

• Using single-cell RNA-seq to MS and HC iMGLs to understand microglial subpopulations in more detail

Conflicts of interest of reviewers

• We declare no conflicts of interests

Inline commenting section

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.

1. Introduction section: “[...], such as lesion formation or progression. Understanding microglial dysfunction in MS is crucial for developing targeted therapies.”

   • Comments:

   • I wonder if you thought about the microenvironment of microglial, such as its interacting cells? Is it looking different? 

2. 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”

   • Comments:

   • please revise this expression as the references used were published in 2019 (ref.37) and 2017 (ref. 38-40)

3. 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.

4. 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. ”

   • Comments:

   • What is the reason for specifically selecting female patients?

5. 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.”

   • Comments:

   • please identify this abbreviation as rho-associated protein kinase

6. 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.

7. Materials and methods section: “This analysis revealed that the cell line was a significant confounding factor influencing the samples’ expression profiles.”

   • Comments:

   • It would be helpful to clarify this sentence further for people who are not experts in RNA-seq analysis.

8. Materials and methods section: “This step provided insights into the biological functions and pathways affected by the experimental conditions.”

   • Comments:

   • please explain what the authors mean by experimental conditions

9. 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)”

   • Comments:

   • please clarify that the newly generated lines are 5 lines as presented in suppl.fig. 1 and supp. Table 1

10. 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.

11. 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. ”

    • Comments:

    • This abbreviation should be explained here or previously elsewhere

12. Figure 3 (B):  “Violin plot showing the quantification of the nuclear localization of NF-κB p65 from the immunofluorescence images. ”

    • Comments:

    • We suggest using box-and-whisker plots to more effectively visualize medians and quartiles (e.g. especially for VEH groups)

13. 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). ”

    • Comments:

    • please identify DEG as differentially expressed gene

14. 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), “”

    • Comments:

    • please identify this as major histocompatibility complex (MHC)

15. 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”

    • Comments:

    • please identify this abbreviation as experimental autoimmune encephalomyelitis (EAE)

16. 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.”

    • Comments:

    • What could be the implication of these upregulated genes in context of MS?

Competing interests

The authors declare that they have no competing interests.