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. 2024 Nov 8;12:RP90695. doi: 10.7554/eLife.90695

Figure 5. Xenotransplanted human-induced pluripotent stem cell (iPSC)-derived microglial cells into recipient mouse retina in vivo demonstrate recapitulation of endogenous distribution, cellular morphology, and stable integration for up to 4 months.

(A) The 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 1 µl injection volume). Retinas were harvested for analysis 120 and 240 days following transplantation. (B, C) The retinas isolated from post-transplantation were analyzed in flat-mounted tissue with confocal imaging. Transplanted human-induced pluripotent stem cell (hiPSC)-derived 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 = 100 µm. (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 = 100 µm. The microglia cell number in GL, IPL, and OPL of host mouse retina were counted: mouse microglial cells (Iba1+, tdT−) and grafted human microglial cells (Iba1+, tdT+) were shown in (E), (F), and (G), respectively. ***p < 0.001, ****p < 0.0001, 3-6 biological replicates were performed. (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 integrated into the mouse retina is general in nature and not cell line specific. Scale bar = 100 µm.

Figure 5.

Figure 5—figure supplement 1. Homeostatic human-induced pluripotent stem cell (hiPSC)-derived microglial cells in the mouse retina do not affect local retinal cells.

Figure 5—figure supplement 1.

Entire section images showed GFAP, GS, RBPMS, and Iba1 staining for astrocytes, Müller cells, ganglion cells, and microglia cells in the retina after 4 months of xenotransplantation. Scale bar = 300 µm.
Figure 5—figure supplement 2. Homeostatic human-induced pluripotent stem cell (iPSC)-derived microglia cells in the mouse retina do not affect local retinal cells.

Figure 5—figure supplement 2.

The high-magnification images showed GFAP, GS, RBPMS, and Iba1 staining for astrocytes, Müller cells, ganglion cells, and microglia cells in the retina after 4 months of transplantation. Scale bar = 100 µm.
Figure 5—figure supplement 3. Homeostatic human-induced pluripotent stem cell (iPSC)-derived microglia cells in the mouse retina do not affect local retinal cells.

Figure 5—figure supplement 3.

Entire section images showed arrestin, calbindin, and PKCα staining for cone photoreceptors, horizontal and some amacrine cells, and bipolar cells in the retina after 4 months of xenotransplantation. Scale bar = 300 µm.
Figure 5—figure supplement 4. Homeostatic human-induced pluripotent stem cell (iPSC)-derived microglia cells in the mouse retina do not affect local retinal cells.

Figure 5—figure supplement 4.

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 4 months of xenotransplantation. Scale bar = 100 µm.
Figure 5—figure supplement 5. Homeostatic human-induced pluripotent stem cell (iPSC)-derived microglia cells in the mouse retina do not take over local retinal microglia cells.

Figure 5—figure supplement 5.

The section images showed Red Fluorescence Protein (RFP) and mouse CD11b staining to determine the tdT+ human microglia cells and local mouse microglia cells in the retina after 4 months of xenotransplantation. The results showed that the tdT+ human microglia cells colocalized with RFP staining (Far red) but not mouse CD11b (green) staining. Scale bar = 100 µm.
Figure 5—figure supplement 6. Homeostatic human-induced pluripotent stem cell (iPSC)-derived microglia cells in the mouse retina do not take over local retinal microglia cells.

Figure 5—figure supplement 6.

The hight magnification images showed RFP and mouse CD11b staining in the retina after 4 months of xenotransplantation. The results showed that the tdT+ human cells colocalized with RFP staining (Far red) but not 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 = 50 µm.