Differentiation and characterization of human iPSC-derived microglia.

(A) Human iPSCs were cultured in a 6-well plate. Scale bar = 200µm. (B) Embryoid bodies formation was enabled in AggreWell™800 plate at day eight in culture medium mTeSR1 plus BMP4, VEGF, and SCF. Scale bar = 200µm. (C) Image of a myeloid precursor cluster following 1-month culture of embryoid bodies in TheraPEAK™ X-vivo™-15 Serum-free Hematopoietic Cell Medium with added M-CSF and IL3. Scale bar = 50µm. (D) Image of microglial cells in maturation culture for two weeks with DMEM/F12 plus nonessential amino acids, glutamine, IL34, CSF1, TGFb2, and CX3CL1. Scale bar = 50µm. (E) Immunohistochemical staining for Iba11 and human CD34, CX3CR1, P2RY12, CD11b, CD68. Scale bar = 100µm. (F) Cell counts and colocalization analysis of (F) CD34- and Iba11- positive cells and (G) positivity for myeloid cell markers CX3CR1, CD11b, activation marker CD68, and microglia marker P2ry12 in differentiated microglia.

Profiling of genes differentially expression between differentiated microglial cells vs. myeloid progenitor cells (MPC) using bulk RNAseq analysis.

(A) Volcano plot showing representative genes that were either upregulated (red) or downregulated (green) in differentiated microglia vs. MPCs. (B) Heat map showing increased expression of microglia-enriched genes in differentiated microglia vs. MPCs. (C) Histogram comparing the expression levels of microglia-enriched genes expression in terms of Fragments Per Kilobase of transcript per Million mapped reads (FPKM). * P<0.05. (D) Histogram comparing expression levels of myeloid cell lineage genes in human iPSC-derived MPC and microglia cells using FPKM. * P<0.05. (E) Graphic signaling pathway analysis with IPA analysis highlighting IL6 and IL1b as signaling hubs in differential gene expression patterns in differentiated microglia vs. MPCs.

Inflammation responses of hiPSC-derived microglial cells following Lipopolysaccharide (LPS) stimulation.

(A) Ingenuity Pathway Analysis (IPA) differentially-expressed genes (fold change >2-fold, p < 0.05) between LPS-treated and control hiPSC-derived microglial cells., demonstrating activation of core pathways involving IL6, IL1a, IL1b, and IFNG. (B) Assessment of mRNA expression of selected genes for inflammatory cytokines using QRT-PCR demonstrated increased expression following LPS (0.1µg/ml) stimulation for 6hrs. These changes corresponded to increases in the protein expression levels of inflammatory cytokines following 24 hours of LPS stimulation as measured with a Multiplex kit (Millipore) on celllysate (C) and in conditioned media (D). The data in (C) and (D) are presented as means ± SEM.* p<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

Human iPSC-derived microglia demonstrate robust phagocytosis.

(A) pHrodo™ Red E. coli bioparticles were incubated in Human iPSC-derived microglia were incubated for 1 hour in either labelled pHrodo™ Red E. coli (A), pHrodo™ Red zymosan bioparticles (B), DiI-labeled bovine photoreceptor outer segments (POS) (C), and colabeled with anti-human P2RY12 antibody (green) and DAPI. Scale bar = 40µm. (D) A high-magnification view of a POS-containing intracellular vesicle within a labeled microglial cell is shown. Scale bar = 30µm.

Human iPSC-derived microglial cells xenotransplanted into recipient mouse retina in vivo demonstrate recapitulation of endogenous distribution, cellular morphology, and stable integration for up to 4 months.

(A) Schematic diagram shows the timeline for transplantation experiments. Two-month old adult transgenic Rag2-/-;IL2rg-/-;hCSF1+/+ mice were fed a PLX-5622-containing diet for 10 days before switching to standard chow. Two days following the resumption of standard chow, human iPSC-derived microglial cells expressing either tdTomato or EGFP were xenotransplanted into the subretinal space via subretinal injection (5000 cells in 1µl injection volume). Retinas were harvested for analysis 120 and 240 days following transplantation. (B) and (C) Retinas isolated post-transplantation were analyzed in flat-mounted tissue with confocal imaging. Transplanted hiPSC-derive microglia were visualized through their expression of tdtomato (TdT) (B) or EGFP (C), while endogenous mouse microglia were visualized using immunostaining for mouse Tmem119 (mTmem119). Imaging analysis was performed in separate layers of the retina including the ganglion cell layer (GL), inner plexiform layer (IPL), and outer plexiform layer (OPL). Scale bar = 100µm. (D) The retinal section showed human iPSC-derived microglial cells integrated into whole retinal layers (top panel) and positively stained with human P2ry12 and TMEM119 microglia signature markers. Scale bar = 100µm. The microglia cell number in GL, IPL, and OPL of host mouse retina were counted: mouse microglial cells (Iba11+, tdT-) and grafted human microglial cells (Iba11+,tdT+) were shown in (E), (F) and (G), respectively. *** P<0.001, **** P<0.0001. (H) and (I) showed tdT (H) or EGFP (I) labeled human iPSC-derived microglial cells in the IPL and OPL of the flat-mount retina with human CD11b staining. These results demonstrated that the infiltration of grafted hiPSC-derived microglial cells into the mouse retina is general in nature and not cell-line specific. Scale bar = 100µm.

Migration and proliferation of hiPSC-derived microglia in the mouse retina after sodium iodate-induced RPE cell injury.

(A) The schematic diagram shows the procedure of the experiment. After eight months post-transplantation of hiPSC-derived microglia, recipient animals were administered NaIO3 (30mg/kg body weight, intraperitoneal injection) to induce RPE injury. Retina were harvested at 3 and 7 days after NaIO3 administration and microglia numbers in the subretinal space monitored in retina and RPE-choroid flat mounts. (B) RPE-choroid flat-mounts demonstrate an increase of hiPSC-derived microglia (tdTomato+ and P2RY12+) in the subretinal space in response to RPE injury. A subset of subretinal microglia labelled for Ki67 indicating active proliferation. Scale bar = 60µm. (C) and (D) showed the number of P2ry12+&tdtomato+ human microglial cells in IPL (C) and OPL (D) decreased; some of them showed Ki67+ staining, Scale bar = 60µm. The cell count results showed in (F) and (G). (E) The retinal flat mount showed the number of P2ry12+&tdtomato+ human microglial cells in IPL and OPL that were repopulated, and the cells stopped dividing with lost the Ki67 staining at seven days after NaIO3 injection. The cell numbers were shown in (F) and (G). Scale bar = 60µm.

Dyshomeostasis human iPSC-derived microglial cells in the mouse retina phagocytose dead photoreceptor cells/debris after RPE cell injury.

(A) Dyshomeostasis human microglial cells (tdtomato+) accumulated in the photoreceptor cell layer after 3 days of NaIO3-induced RPE cell injury compared with no NaIO3 administration. The photoreceptor cells stained with cone arrestin (green), autofluorescent showed in magenta. Scale bar = 60µm. (B) High magnificent and side view image showed human microglial cells (red) co-labeled with photoreceptor cells arrestin staining (green) after 3 days of NaIO3 injury. The yellow triangle showed the colocalized tdT+ human microglia cell and arrestin+ cone photoreceptor cell. Scale bar =40µm. (C) The number of human microglial cells in the photoreceptor layer; (D) The mean gray value of autofluorescence in each human microglia cell. **** P<0.0001.

Immunocytochemistry staining with human PU.1 and TREM2(A). Scale bar = 100µm. (B) PU.1 and TREM2 positive cells are 99.7% and 99.4%, respectively, in entire DAPI+ cell counts.

Analysis results of the canonical pathway of completed differentiated hipsc-derived microglia vs. myeloid progenitor cells.

Hierarchical cluster analysis on microglia enriche genes among hiPSC-derived microglial cells (hiPSC-MG) and human adult brain microglia cells (AMG), fetal brain microglia cells (FMG), inflammatory monocytes (IM), monocytes(M). The human microglia gene panel combined our mouse microglia-enriched genes and human microglia-enriched genes (Abud et al., 2017; Muffat et al., 2016; Douvaras et al., 2017; Böttcher et al., 2019; Van et al., 2019). The total microglia enriched gene list contains 203 genes (Suppl. Table 1), which used to be extracted from the gene profile of hiPSC-MG and downloaded human adult microglia(AMG), fetal microglia (FMG), inflammatory monocyte (IM) and monocytes (M) (GSE 178846, Abud et al., 2017). 188 genes (Suppl. table 1) were obtained from both gene profiles. All the gene counts were normalized with 4 human cell housekeeping genes C1orf43, RAB7A, REEP5 and VCP (Eisenberg et al., 2013). The hierarchical cluster was analyzed by JMP (JMP Statistical Discovery LLC.). Results showed that hiPSC-MG is more comparable with AMG and FMG.

The correlation analysis of human microglia enriched genes among hiPSC-derived microglia cells (hiPSC-MG), human adult brain microglia cells (A), fetal brain microglia cells (B), inflammatory monocytes (C), monocytes (D) respectively. The images and the analysis results (Prism, GraphPad) showed hiPSC-MG correlated significantly with FMG (r=0.7358, p<0.0001) and AMG (r=0.7057, p<0.0001).

Correlation comparison of entire gene profiles between male (A)/female (B) hiPSC-derived microglial cells and male (A)/female (B) human brain microglia cells (GSE 111972, van der Poel et al. 2019; Suppl. table 2) respectively. The results showed they are significantly correlative (r=0.8055(M), r=0.8326(F), P<0.0001).

Inflammatory cytokines were produced synthetically by IFNγ and LPS in hiPSC-derived microglial cells. hiPSC-derived microglia cells were cultured in 6-well plates for 14 days, 20ng/ml of human IFNγ and 0.5ug/ml of LPS were added to the wells, respectively, or 20ng/ml of human IFNγ and 0.5ug/ml of LPS were added to the wells together. After 6 hours of incubation, the cells were washed and harvested into a 1.5ml Eppendorf tube for RNA isolation with a NucleoSpin RNA/Protein Mini kit (Macherey-Nagel, #740933.50). The cDNA was synthesized with PrimeScript™ 1st strand cDNA Synthesis Kit (Takara, #6110A). qRT-PCR was performed using a SYBR green RT-PCR kit (Affymetrix), using the Bio-Rad CFX96 Touch™ Real-Time PCR Detection System under the following conditions: denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, and then 60 °C for 45 s. Threshold cycle (CT) values were calculated and expressed as fold-induction determined using the comparative CT (2ΔΔCT) method. Ribosomal protein S13 (RPS13) and GAPDH were used as internal controls. Oligonucleotide primers are provided in Suppl. Table 4.

Floating myeloid progenitor cells were cultured in a 2-well slide chamber overnight, and then the DiI-labeled bovine photoreceptor outer segments were added to the chamber and incubated for 1 hr. The cells were fixed with 4% PFA for 20 minutes and stained with Iba1 and hP2ry12, respectively. The DiI-labeled bovine photoreceptor outer segments can only be engulfed by Iba1 or hP2ry12-positive microglia cells. Scale bar = 16µm.

The morphology of hiPSC-derived microglia cells under 1-hour zymosan treatment with different concentrations. After 1 hour of treatment with zymosan, 5ug/ml concentration didn’t change the microglia cell morphology, but over 20ug/ml concentration changed the microglia morphology to an ameboid round shape.

Homeostatic hiPSC-derived microglial cells in the mouse retina do not affect local retinal cells. Entire section images showed GFAP, GS, RBPMS, and Iba1 staining for astrocytes, Müller cells, ganglion cells, and microglia cells in the retina after four months of xenotransplantation. Scale bar = 300µm.

Homeostatic human iPSC-derived microglia cells in the mouse retina do not affect local retinal cells. The partial section high magnification images showed GFAP, GS, RBPMS, and Iba1 staining for astrocytes, Müller cells, ganglion cells, and microglia cells in the retina after four months of transplantation. Scale bar = 100µm.

Homeostatic human iPSC-derived microglia cells in the mouse retina do not affect local retinal cells. Entire section images showed arrestin, calbindin, and PKCα staining for cone photoreceptors, horizontal and some amacrine cells, and bipolar cells in the retina after four months of xenotransplantation. Scale bar = 300µm.

Homeostatic human iPSC-derived microglia cells in the mouse retina do not affect local retinal cells. The partial section of high magnification images showed arrestin, calbindin, and PKCα staining for cone photoreceptors, horizontal and some amacrine cells, and bipolar cells in the retina after four months of xenotransplantation. Scale bar = 100µm.

Homeostatic human iPSC-derived microglia cells in the mouse retina do not take over local retinal microglia cells. The section images showed RFP and mouse CD11b staining to determine the tdT+ human microglia cells and local mouse microglia cells in the retina after four months of xenotransplantation. The results showed that the tdT+ human cells colocalized with RFP staining (Far red) but not with mouse CD11b (green) staining. Scale bar = 100µm.

Homeostatic human iPSC-derived microglia cells in the mouse retina do not take over local retinal microglia cells. The magnification images showed RFP and mouse CD11b staining in the retina after four months of xenotransplantation. The results showed that the tdT+ human cells colocalized with RFP staining (Far red) but not with mouse CD11b(green) staining. The triangle marker indicated that the local mouse microglia cells were only stained with mouse CD11b but not colocalized with tdT+ human microglia cells. Scale bar = 50µm.

hP2rY12 staining on the retina after eight months of xenotransplantation. The images showed the tdtomato+ human microglia cells colocalized with hP2rY12 staining in GL, IPL, and OPL in the mouse retina. Scale bar = 300µm.

High magnification of hTMEM119 staining on the retina after eight months of xenotransplantation. The images showed the tdtomato+ human microglia cells colocalized with hTMEM119 staining in GL, IPL, and OPL in the mouse retina. Scale bar = 300µm.

The inflammation, phagocytosis, adhesion and migration, neurotrophic factors, and microglia signature gene expression in hiPSC-derived microglia cells of grafted retinas. We compared gene coding sequences between humans and mice, chose a human-specific sequence to make the oligos (Suppl. Table 5), and ran a qRT-PCR on 8-month hiPSC-derived microglia cells grafted retinas with/without NaIO3-treated retinas. The results revealed that hiPSC-derived microglia cells expressed more inflammatory factors and phagocytosis genes and promoted cell migration but decreased microglia cell signature genes and neurotrophic factors in NaIO3-treated retina.

The heat map of 42 candidate genes from 34 loci associated with AMD expressed in retinal microglia cells. The microglia gene expression data are from microarray data previously published (Ma et al., 2013). The candidate genes came from the published paper (Den et al., 2022). The gene list is in Supplement Table 6.

The heat map of 209 genes associated with AMD (Fritsche et al., 2016) expressed in retinal microglia cells. The microglia gene expression data are from microarray data previously published (Ma et al.,2013). The gene list is in Supplement Table 7.