PAX6-positive microglia evolve locally in hiPSC-derived ocular organoids

Abstract

Microglia are the resident immune cells of the central nervous system (CNS). They govern the immunogenicity of the retina, which is considered to be part of the CNS; however, it is not known how microglia develop in the eye. Here, we studied human-induced pluripotent stem cells (hiPSCs) that had been expanded into a self-formed ectodermal autonomous multi-zone (SEAM) of cells that partially mimics human eye development. Our results indicated that microglia-like cells, which have characteristics of yolk-sac-like linage cells, naturally develop in 2D eye-like SEAM organoids, which lack any vascular components. These cells are unique in that they are paired box protein 6 (PAX6)-positive, yet they possess some characteristics of mesoderm. Collectively, the data support the notion of the existence of an isolated, locally developing immune system in the eye, which is independent of the body's vasculature and general immune system.

Keywords: ESC; development; iPSC; microglia; neuronal immunity; ocular organoid; pluripotent stem cell.

Figure 1

Spontaneously generated PPM cells in a 2D SEAM of human eye development (A) A self-formed ectodermal autonomous multi-zone (SEAM) derived from hiPSCs contains cells of ectodermal lineage that mimic anterior and posterior eye development in vivo. CNS, central nervous system; NE, neuroectoderm; OC, optic cup; NR, neuroretina; NC, neural crest; LE; lens; OSE, ocular surface ectoderm; SE, surface ectoderm; CE, corneal epithelium; EK, epidermal keratinocyte. (B) Schematic showing the expression pattern of microglia-related genes in the SEAM. (C–E) Immunostaining of PAX6 (red) in zones 1–3 of a 4-week, cultivated 201B7 hiPSC-derived SEAM along with staining of nuclei (blue) and TMEM119 (C, green), CD11b (D, green), and CX3CR1 (E, green). (F and G) Immunostaining after 7 weeks of differentiation for retinal culture: (F) CD11b (green) and MITF retinal pigment epithelium cells (red) and (G) TMEM119 (green) and CHX10 neuro-retinal cells (red). Images are representative of three independent experiments. Scale bar, 200 μm.

Figure 2

Immunostaining of differently derived SEAMs and expression of immune cell markers (A and B) hES KhES-1-derived SEAMs after 4 weeks of culture immunostained with (A) TMEM119 (green) and PAX6 (red) and (B) CD11b (green) and PAX6 (red). (C and D) 1383D2 hiPSC-derived SEAMs after 4 weeks of culture immunostained with (C) TMEM119 (green) and PAX6 (red) and (D) CD11b (green) and PAX6 (red). Images are representative of multiple independent experiments. Nuclei are in blue. Scale bar, 200 μm. (E) Gene expression analysis of immune-related markers in SEAMs after 4 weeks of culture (six independent experiments). Error bars show standard deviation (SD).

Figure 3

Developmental and immune cell markers in a SEAM, and isolation and analysis of PPM cells (A) Gene expression analysis of development-related markers in a SEAM after 1 week of culture (six independent experiments). (B) Flow cytometric analysis of CD11b and CD45 in differentiated hiPSCs after 27–28 days of culture, with PPM cells indicated (box). Data are representative of seven independent cell-sorting experiments. (C) RNA-seq data of PPM cells and human immortalized microglia. Both cell types were stimulated for 24 h. (D and E) Gene expression analysis of (D) microglia-related markers and (E) PAX6 in PPM cells and human immortalized microglia cells (data from four independent experiments). ^∗p < 0.05 by the Mann-Whitney rank-sum test. (F) IL-6 and IL-8 levels after stimulation of PPM cells in 10–16 independent experiments. (G) Gene expression analysis of VEGFA and TGFB2 after stimulating PPM cells (data from seven to ten independent experiments). Error bars show SD.

Figure 4

Single-cell RNA-seq analysis of CD11b + cells in a SEAM (A) Uniform manifold approximation and projection (UMAP) plot visualizing 1,498 CD11b + cells and colored based on 11 cluster types. (B) Scatterplot showing the gene expression of PPM cluster_0 and 1 identified in CD11b + scRNA-seq and of a YSMP cluster in scRNA-seq of embryonic macrophages. (C) Feature plots of PAX6, PTRG, and MYCN expression. The expression levels of each cell type are colored in the UMAP plot. (D and E) Immunostaining of (D) PAX6 (green) and PRTG (red) and (E) MYCN (red). CD11b + cells were sorted by FACS and cytospin after 4 weeks of culture. Images represent three independent experiments. Nuclei are in blue. Scale bar, 25 μm.