Three-Dimensional Neuroepithelial Culture from Human Embryonic Stem Cells and Its Use for Quantitative Conversion to Retinal Pigment Epithelium

<div><p>A goal in human embryonic stem cell (hESC) research is the faithful differentiation to given cell types such as neural lineages. During embryonic development, a basement membrane surrounds the neural plate that forms a tight, apico-basolaterally polarized epithelium before closing to form a neural tube with a single lumen. Here we show that the three-dimensional epithelial cyst culture of hESCs in Matrigel combined with neural induction results in a quantitative conversion into neuroepithelial cysts containing a single lumen. Cells attain a defined neuroepithelial identity by 5 days. The neuroepithelial cysts naturally generate retinal epithelium, in part due to IGF-1/insulin signaling. We demonstrate the utility of this epithelial culture approach by achieving a quantitative production of retinal pigment epithelial (RPE) cells from hESCs within 30 days. Direct transplantation of this RPE into a rat model of retinal degeneration without any selection or expansion of the cells results in the formation of a donor-derived RPE monolayer that rescues photoreceptor cells. The cyst method for neuroepithelial differentiation of pluripotent stem cells is not only of importance for RPE generation but will also be relevant to the production of other neuronal cell types and for reconstituting complex patterning events from three-dimensional neuroepithelia.</p> </div

Characterization of the hESC-derived RPE sheets.

<p>(A) Immunostaining of ZO-1 (red) on hESC-derived RPE cells at Day 30 indicated the presence of tight junctions. Pigmented cells displayed polygonal shape. (B–D) Electron microscopic analyses of hESC-derived RPE cells at Day 50. hESC-derived RPE cells had abundant apical microvilli (mv), melanin granules (mel) in the apical half and the nucleus (nu) in their basal half. A basement membrane (bm) was visible. Tight junctions (tj) and desmosomes (de) could be found. (E) The transepithelial resistance (TER) of hESC-derived RPE progenitor or RPE cells increases during differentiation. (F) Cross-sections through the pigmented cell sheet immunostained for RPE65 and BESTROPHIN. hESC-derived RPE cells at Day 40 expressed the mature RPE cell markers RPE65 and BESTROPHIN. (G) hESC-derived RPE cells exited the cell cycle by Day 30 as demonstrated by RPE cultures pulse labeled with EdU at Day 7, 15 and 30. EdU incorporation by proliferating cells was observed at Day 7 and 15, but rarely observed at Day 30.</p

Transplantation of hESC-derived RPE cells effectively rescues photoreceptors in RCS rats.

<p>(A, B) Sections of transplanted retina in the central region adjacent to the injection site shown in DIC image (A) and immunostained to identify human nuclei (red) and DAPI (blue) (B). Donor RPE cells (red) were localized in the sub-retinal space integrated into the host RPE monolayer and generated monolayer-like structures (A, B, arrows). The ONL overlying the donor RPE monolayer was well preserved and contained 5–6 rows of nuclei (B, arrow heads). (C) Electron microscopic analyses of transplanted RPE cells. The dotted line outlined one RPE cell. Typical polarized RPE morphology with foldings at the basal membrane, a nucleus located at the basal side (labeled by N) and apically located melanin-containing melanosomes (some labeled by M) were observed. (D, E) Representative fluorescence images of central or peripheral region in the hESC-derived RPE cell transplanted retina. Dashed box showed the ONL. Following transplantation of donor RPE cells, central regions of the retina showed an increased ONL thickness with an average of 5–6 rows of nuclei (D) in contrast to the periphery with 1–2 rows of nuclei (E). (F) Quantitative data for the effect of different transplanted cells on ONL thickness. (G) Quantitative data for the protected area of ONL in the human RPE and fibroblast cell transplanted retina. In panels (F) and (G), 6 RCS rats transplanted with human ESC-derived RPE cells, 5 with human fibroblasts, 5 sham-injected, and 6 untreated controls were quantified. Data were presented as means±SD. GCL: ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer. n.s.: not significant, *: P<0.05, **: P<0.01, ***: P<0.001. Nuclei were counterstained with DAPI. Scale bars, 10 µm (A, B), 5 µm (C), 20 µm (D, E).</p

Efficient generation of polarized neural progenitors from hESCs in a Matrigel-based 3D neuroepithelial cyst model.

<p>(A) Schematic of the experiment. (B) hESC clumps found at Day 0 formed neural tube-like cysts with a single lumen by Day 5. (C) By Day 1, hESC-derived cysts were positive for SOX2 and NESTIN. The apical localization of CD133 indicates apicobasal polarity is firmly established. (D) Immunostaining of PAX6 and ZO-1 during cyst growth in Matrigel. PAX6 was strongly expressed in Day 5 cysts that display clear properties of a pseudostratified epithelium. (E) M-phase cells stained with Phospho-Histone H3 (PH3) antibody only localized at the apical side of the cysts and S-phase cells labeled with EdU at the basolateral side, indicating that luminal mitosis occurred within the cysts. Nuclei were counterstained with Hoechst. Scale bar, 50 µm.</p

Directed differentiation of hESC-derived cysts to RPE using transwell filters.

<p>(A) Schematic of RPE differentiation protocol. UD: undifferentiated. Ac+/Ac−: with or without Activin A. (B) Top view of transwell filters at 30 days of culture showing the appearance of a pigmented cell sheet in the presence of Activin A (100 ng/ml) but not its absence. Arrows point to a few pigmented foci in the non-Activin A treated sample. (C) Immunostaining of PAX6 and MITF at Day 15 and Day 22 during RPE differentiation. Expression of PAX6 remained stable, while MITF was up-regulated by Day 22. Scale bars, 1 mm (B), 50 µm (C).</p

hESC-derived cysts enter and maintain retinal identity.

<p>(A) RT-PCR analyses for genes involved in retinal identity on undifferentiated hESCs (UD) and hESC-derived cysts at representative time points. Retinoic acid (RA) treated cysts were used as positive control for <i>HOX B1</i> and <i>HOX C5</i>. (B) Immunocytochemical analysis of hESC-derived cysts at Day 5. Upper row: cysts stained for PAX6 (green) and RX (red). Lower row: cysts stained for OTX2 (green) and CHX10 (red). Day 5 cysts were positive for PAX6, RX and OTX2 but few CHX10⁺ cells were detected at Day 5 indicating that cells achieved an optic vesicle identity. (C) Immunocytochemical analyses of hESC-derived cysts at Day 15. Upper row: cysts stained for PAX6 (green) and RX (red). Lower row: cysts stained for OTX2 (green) and CHX10 (red). CHX10⁺ cells were detected at Day 15. (D and E) Involvement of IGF-1/insulin signaling in optic vesicle stage neuroepithelium formation during hESC differentiation in Matrigel. hESC-derived cysts at Day 5 were co-stained with PAX6 and RX antibodies (upper row) or OTX2 and ZO-1 antibodies (lower row). Nuclei were counterstained with Hoechst. Scale bar, 50 µm.</p