Differentiation and characterization of human iPSC-derived microglia cell. (A) Human iPSC were cultured in a 6-well plate. Scale bar = 200µm. (B) Embryoid bodies formation in AggreWell™800 plate at day eight in culture medium mTeSR1 plus BMP4, VEGF, and SCF. Scale bar = 200µm. (C) Myeloid precursor cluster at the 1-month culture of embryoid bodies under TheraPEAK™ X-vivo™-15 Serum-free Hematopoietic Cell Medium with M-CSF and IL3. Scale bar = 50µm. (D) Microglia cells maturation culture for two weeks with DMEM/F12 plus nonessential amino acids, glutamine, IL34, CSF1, TGFb2, and CX3CL1. Scale bar = 50µm. (E) Immunocytochemistry staining with Iba1 and human CD34, CX3CR1, P2ry12, CD11b, CD68. Scale bar = 100µm. (F) The cell counts and collaboration analysis of CD34 and Iba1 positive cells. (G) Percentage analysis of myeloid cell marker CX3CR1, CD11b, activation marker CD68, and microglia cell signature marker P2ry12.

Comparison of the gene expression profile of mature microglia cells vs. myeloid progenitor cells (MPC) with bulk RNA seq. (A) The volcano plot shows the gene expression in mature human microglia cells and MPC. The MPC and microglia representative genes were showed in green and red color. (B) The heat map shows mature microglia-enrich gene expression compared to MPC. (C) Histogram showed the comparison of microglia-enrich gene expression in human iPSC-derived MPC and microglia cells with FPKM. (D) Histogram showed the comparison of myeloid cell lineage gene expression in human iPSC-derived MPC and mature microglia cells with FPKM. (E) Graphic signaling pathway analysis with IPA showed the hub genes are IL6 and IL1b in comparison of mature microglia vs. myeloid progenitors.

Inflammation responses of Human iPSC-derived microglia cells under Lipopolysaccharide (LPS) stimulation. (A) Graphic signaling pathway analysis with IPA on comparison of LPS-treated and non-LPS-treated microglia cells showed the increased core pathways are IL6, IL1a, IL1b, and IFNG. (B) The QPCR results showed proinflammatory factors expression increased after LPS (0.1ug/ml) stimulation for 6hrs. (C) The inflammatory cytokines in protein level without/with LPS (0.1ug/ml) stimulation for 24hrs in human iPSC-derived microglia cell lysate. (D) Human iPSC-derived microglia cells released inflammatory cytokines in the medium without/with LPS (0.1ug/ml) stimulation for 24hrs. (C) and (D) results were measured with a Multiplex kit (Millipore), Graphical data in (C) and (D) are presented as means ± SEM).

Phagocytosis evaluation of Human iPSC-derived microglia cells. (A) pHrodo™ Red E. coli Bioparticles were incubated in Human iPSC-derived microglia cells for 1 hour. After washing with PBS, Human iPSC-derived microglia cells were stained with anti-human P2ry12 antibody (green) and DAPI. Scale bar = 40µm. (B) pHrodo™ Red Zymosan Bioparticles were incubated in human iPSC-derived microglia cells for 1 hour. After washing with PBS, Human iPSC-derived microglia cells were stained with anti-human P2ry12 antibody (green) and DAPI. Scale bar = 40µm. (C) DiI (Vybrant Cell-Labeling Solutions; Invitrogen) labeled bovine photoreceptor outer segments (POS) were added to the culture medium and incubated for 1 hour. After washing with PBS, Human iPSC-derived microglia cells were stained with anti-human P2ry12 antibody (green) and DAPI. Scale bar = 40µm. (D) and (E) are highly magnificent showed of POS-DiI in human iPSC-derived microglia cells. Scale bar = 30µm (D) and 15µm (E).

Human iPSC-derived microglia cells integrated into mouse retina and formed stable homeostasis after subretinal injection for 4 months. (A) The schematic diagram shows the timeline of the experiments. The Plx5622 diet was administrated to 2-month-old Reg2-/-;IL2rg-/-;hCSF1+/+ mice for ten days, then switched with a normal chow; at day 12, the human iPSC-derived microglia cells which were labeled with tdTomato or EGFP were xenotransplanted into mouse subretinal space. The samples were harvested four months after transplantation. (B) The retinal flat mount showed microglia cells in the ganglion cell layer (GL), inner plexiform layer (IPL), and outer plexiform layer (OPL) with Iba1 (green) and mouse TMEM119 (Far-red) staining. The human microglia cells showed tdTomato positive. Scale bar = 100µm. (C) The retinal flat mount showed human CD11b staining only in tdTomato+ human microglia cells. Scale bar = 100µm. (D) The retinal section showed human iPSC-derived microglia 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 microglia cells (Iba1+, tdT-) and grafted human microglia cells (Iba1+,tdT+) were shown in (E), (F) and (G), respectively. (H) and (I) showed EGFP-labeled human iPSC-derived microglia cells in the IPL and OPL of the flat-mount retina with mouse TMEM119 staining (H) and human CD11b staining (I). These results demonstrated that the infiltration of grafted hiPSC-derived microglia cells into the mouse retina is general in nature and not cell-line specific. Scale bar = 100µm.

Migration and proliferation of homeostatic human iPSC-derived microglia cells in the mouse retina after RPE cells injury. (A) The schematic diagram shows the procedure of the experiment. After eight months of xenotransplantation, the NaIO3 (30mg/kg body weight) was injected into the mice through I.P. injection. The samples were harvested at 3 and 7 days after NaIO3 administration. (B) RPE-choroid flat-mount showed the microglia cells in the subretinal space. The tdtomato+ human microglia cells were stained with human P2ry12(green); the dividing cells were labeled with Ki67(Far red). The results indicated that tdtomato+ human microglia cells were attracted to the subretinal space in NaIO3-treated mice retina compared to non-NaIO3 injection (G). Some tdtomato+ & P2ry12+ human microglia cells started dividing as shown Ki67+ (F). Scale bar = 60µm. (C) and (D) showed the number of P2ry12+&tdtomato+ human microglia 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 microglia 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 microglia cells in the mouse retina phagocytose dead photoreceptor cells/debris after RPE cell injury. (A) Dyshomeostasis human microglia 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 autofluorescent in each human microglia cell. **** P<0.0001.