Human iPSC-derived Microglia Cells Integrated into Mouse Retina and Recapitulated Features of Endogenous Microglia Cells

Reviewer #1 (Public Review):

Summary:
This paper reported a protocol of using human-induced pluripotent stem cells to generate cells expressing microglia-enriched genes and responding to LPS by drastic upregulation of proinflammatory cytokines. Upon subretinal transplantation in mice, hiPSC-derived cells integrated into the host retina and maintained retinal homeostasis, while they responded to RPE injury by migration, proliferation and phagocytosis. The findings revealed the potential of using hiPSC-derived cell transplantation for microglia replacement as a therapeutic strategy for retinal diseases.

Strengths:
The paper demonstrates a method of consistently generating a significant quantity of hiPSC-derived microglia-like cells for in vitro study or for in vivo transplantation. RNAseq analysis offers an opportunity for comprehensive transcriptome profiling of the derived cells. It is impressive that following transplantation, these cells integrated into the retina well, migrated to the corresponding layers, adopted microglia-like morphologies, and survived long term without generating apparent harm. The work has laid a foundation for future utilization of hiPSC-derived microglia in lab and clinical applications.

Weaknesses:
1. The primary weakness of the paper concerns its conclusion of having generated "homogenous mature microglia", partly based on the RNAseq analysis. However, the comparison of gene profiles was carried out only between "hiPSC-derived mature microglia" and the proliferating myeloid progenitors. While the transcriptome profiles revealed a trend of enrichment of microglia-like gene expression in "hiPSC-derived mature microglia" compared to proliferating myeloid progenitors, this is not sufficient to claim they are "mature microglia". It is important that one carries out a comparative analysis of the RNAseq data with those of primary human microglia, which may be done by leveraging the public database. To convincingly claim these cells are mature microglia, questions need to be addressed including how similar the molecular signatures of these cells are compared with the fully differentiated primary microglia cell or if they remain progenitor-like or take on mosaic properties, and how they distinguish from macrophages.

2. While the authors attempted to demonstrate the functional property of "hiPSC-derived mature microglia" in culture, they used LPS challenge, which is an inappropriate assay. This is because human microglia respond poorly to LPS alone but need to be activated by a combination of LPS with other factors, such as IFNγ. Their data that "hiPSC-derived mature microglia" showed robust responses to LPS indeed implicates that these cells do not behave like mature human microglia.

3. The resolution of Figs. 4 - 6 is so low that even some of the text and labels are hardly readable. Based on the morphology shown in Fig. 4 and the statement in line 147, these hiPSC-derived "cells altered their morphology to a rounded shape within an hour of incubation and rapidly internalized the fluorescent-labeled particles". This is a peculiar response. Usually, microglia do not respond to fluorescent-labeled zymosan by turning into a rounded shaped within an hour when they internalize them. Such a behavior usually implicates weak phagocytotic capacity.

4. Data presented in Fig. 5 are not very convincing to support that transplanted cells were immunopositive for "human CD11b (Fig.5C), as well as microglia signature markers P2ry12 and TMEM119 (Fig.5D)" (line 167). The resolution and magnification of Fig. 5D is too low to tell the colocalization of tdT and human microglial marker immunolabeling. In the flat-mount images (C, I), hCD11b immunolabeling is not visible in the GCL or barely visible in the IPL. This should be discussed.

5. Microglia respond to injury by becoming active and lose their expression of the resting state microglial marker, such as P2ry12, which is used in Fig. 6 for detection of migrated microglia. To confirm that these cells indeed respond to injury like native microglia, one should check for activated microglial markers and induction of pro-inflammatory cytokines in the sodium iodate-injury model.

Reviewer #2 (Public Review):

Summary:
Ma et al. employed a myeloid progenitor/microglia differentiation protocol to produce human-induced pluripotent stem cell (hiPSC)-derived microglia in order to examine the potential of microglial cell replacement as a treatment for retinal disorders. They characterized the iPSC-derived microglia by gene expression and in vitro assay analysis. By evaluating xenografted microglia in the partly microglia-depleted retina, the function of the microglia was further assessed.

Strengths:

Overall, the study and the data are convincing, and xenografted microglia were also tested in a RPE injury paradigm.

Weaknesses:

Gene expression analysis of mature microglia cells should be better interpreted and it would be beneficial to compare the iPSC-derived microglia gene set to a human microglial cell line (for example, HMC3) instead of myeloid progenitor cells.
The way that the manuscript has been written, unfortunately, is not optimal. I recommend that the entire manuscript be edited and proofread in English. The text contains spelling and grammar mistakes, and the manuscript is inconsistent in several parts. The manuscript should also be revised for a scientific paper format.