Outer Segment Formation of Transplanted Photoreceptor Precursor Cells

<div><p>Transplantation of photoreceptor precursor cells (PPCs) into the retina represents a promising treatment for cell replacement in blinding diseases characterized by photoreceptor loss. In preclinical studies, we and others demonstrated that grafted PPCs integrate into the host outer nuclear layer (ONL) and develop into mature photoreceptors. However, a key feature of light detecting photoreceptors, the outer segment (OS) with natively aligned disc membrane staples, has not been studied in detail following transplantation. Therefore, we used as donor cells PPCs isolated from neonatal double transgenic reporter mice in which OSs are selectively labeled by green fluorescent protein while cell bodies are highlighted by red fluorescent protein. PPCs were enriched using CD73-based magnetic associated cell sorting and subsequently transplanted into either adult wild-type or a model of autosomal-dominant retinal degeneration mice. Three weeks post-transplantation, donor photoreceptors were identified based on fluorescent-reporter expression and OS formation was monitored at light and electron microscopy levels. Donor cells that properly integrated into the host wild-type retina developed OSs with the formation of a connecting cilium and well-aligned disc membrane staples similar to the surrounding native cells of the host. Surprisingly, the majority of not-integrated PPCs that remained in the sub-retinal space also generated native-like OSs in wild-type mice and those affected by retinal degeneration. Moreover, they showed an improved photoreceptor maturation and OS formation by comparison to donor cells located on the vitreous side suggesting that environmental cues influence the PPC differentiation and maturation. We conclude that transplanted PPCs, whether integrated or not into the host ONL, are able to generate the cellular structure for effective light detection, a phenomenon observed in wild-type as well as in degenerated retinas. Given that patients suffering from retinitis pigmentosa lose almost all photoreceptors, our findings are of utmost importance for the development of cell-based therapies.</p> </div

Transplantation of rhoEGFP–positive photoreceptors into the degenerated retina of heterozygous P347S mice.

<p>Low and high magnification views (A, A′) of a degenerated retina from a heterozygous P347S mouse, 3 weeks post-transplantation of CD73-enriched PPCs isolated from double-transgenic (rhoEGFP, actinDsRed) reporter animals. Cell nuclei are stained with DAPI (blue). Note, that transplanted cells (cell body, magenta; outer segment, green) are found in a sheet-like structure in the sub-retinal space (SRS), i.e. above the host outer nuclear layer (ONL), which is reduced to only 1–2 cell rows. Electron microscopy analyses of rhoEGFP–positive structures reveal the formation of ultra-structurally normal OSs containing morphologically native disc membrane staples (A″, A′″). In light adapted animals (B–B′″), transducin (B″, white and B′″, magenta) is expressed in OSs of transplanted, sub-retinal PPCs at low levels (B′ and B″, arrows), whereas high levels of transducin could be detected in IS-like structures next to rhoEGFP-positive OSs (B′ and B″, arrowhead). In contrast, dark adapted animals show significantly higher levels of transducin in rhoEGFP-positive OSs (C′ and C″, arrows), indicated also by white overlay staining in C′″. This illustrates, that sub-retinal PPCs show the native translocation of transducin under different light conditions, which is a prerequisite for functionality. INL, inner nuclear layer. Scale bars: A: 50 µm, A′: 10 µm, A″, A′″: 500 nm, B–B′″ and C–C′″: 10 µm.</p

Transplanted photoreceptors remaining in the sub-retinal space generate outer segments.

<p>Upon transplantation into an adult wild-type mouse (A), many CD73-enriched photoreceptor precursor cells (PPC) isolated from double-transgenic (rhoEGFP, actinDsRed) reporter animals remain in the sub-retinal space (SRS) at the top of the host outer nuclear layer (ONL, dashed line) and develop outer segment (OS)-like structures that are labeled with rhoEGFP (green, arrows). The cell body of transplanted cells is observed by DsRed-expression (magenta) whereas cell nuclei are stained with DAPI (blue). An electron microscopy overview (B) of transplanted PPCs found in the SRS demonstrates that several of them (asterisks) lay above the host photoreceptor OSs, which are delimitated by a dashed line. Magnifications of the boxed areas in panel B reveal that the non-integrated PPCs developed as well organized disc membrane staples, which are positive for rhoEGFP (B′, B″, black dots). Scale bars: A: 10 µm, B: 5 µm, B′ and B″: 2 µm.</p

Differences in outer segment formation between sub-retinal and vitreal located transplanted photoreceptor precursor cells.

<p>CD73-enriched photoreceptor precursor cells (PPC) isolated from double-transgenic (rhoEGFP, actinDsRed) reporter animals were simultaneously transplanted into the sub-retinal (SRS) and vitreous (VS) space of 4-weeks old P347S hosts (A; B is the same image as A but with increased green fluorescence channel saturation level). Cell nuclei are highlighted with DAPI staining (blue). Donor cells (magenta) were detected in the SRS (e.g. boxed area in A) and VS (A and B, arrow) showing survival of transplanted cells in both locations. Whereas sub-retinally injected PPCs showed strong EGFP expression (green; e.g. boxed area in A), vitreally injected donor cells appeared almost devoid of EGFP signal (A, arrow). However, by increasing the saturation level of the green fluorescence signal channel in image A, faint EGFP signal could be detected in the vitreally located donor cells (B; B′ is a separate image with adjusted shutter time showing a magnification of the boxed area in B) demonstrating that donor cells located in the SRS had higher rhoEGFP expression levels than those in the VS (compare different cell populations in each individual panel A and B). Moreover, almost every single sub-retinal located donor PPC showed rhoEGFP labeled OSs (A′), in contrast to those located in the VS that generated only in few cases OS-like protrusions (B′, arrows). ONL, outer nuclear layer; INL, inner nuclear layer. Scale bars: 50 µm.</p

Vitreal-located transplanted photoreceptor precursor cells develop disturbed outer segment-like structures with misaligned disc membranes.

<p>CLEM analysis on resin sections allowed examination of vitreally-transplanted rhoEGFP–positive photoreceptor precursor cell aggregates. Cell nuclei are highlighted by DAPI staining (blue). A representative example is shown with fluorescence light microscopic- (A) and transmission electron microscopic-illumination (A′). The donor cells in A and A′ are shown at higher magnification in the respective panels B and B′. RhoEGFP-enriched structures (A, B; green, arrows) next to the cell bodies suggest the formation of OSs, and three of these (C, D and E) are displayed in the corresponding panels at a high-resolution level by TEM-analysis. These structures appear either as rhoEGFP-enriched cell debris (C) or rhoEGFP–positive OS-like structures with an abnormal organization/integrity including misaligned disc membranes (D, E). Additionally, mislocation of rhoEGFP to the plasma membrane of the soma was observed (F). Scale bars: A, A′: 20 µm, B, B′: 10 µm, C: 1 µm, D–F: 500 nm.</p

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

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

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

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