Characterization and Transplantation of CD73-Positive Photoreceptors Isolated from Human iPSC-Derived Retinal Organoids

Abstract

Photoreceptor degenerative diseases are a major cause of blindness for which cell replacement is one of the most encouraging strategies. For stem cell-based therapy using human induced pluripotent stem cells (hiPSCs), it is crucial to obtain a homogenous photoreceptor cell population. We confirmed that the cell surface antigen CD73 is exclusively expressed in hiPSC-derived photoreceptors by generating a fluorescent cone rod homeobox (Crx) reporter hiPSC line using CRISPR/Cas9 genome editing. We demonstrated that CD73 targeting by magnetic-activated cell sorting (MACS) is an effective strategy to separate a safe population of transplantable photoreceptors. CD73+ photoreceptor precursors can be isolated in large numbers and transplanted into rat eyes, showing capacity to survive and mature in close proximity to host inner retina of a model of photoreceptor degeneration. These data demonstrate that CD73+ photoreceptor precursors hold great promise for a future safe clinical translation.

Keywords: cell sorting; cell therapy; iPSC; neurodegeneration; organoids; photoreceptor; retina; transplantation.

Graphical abstract

Figure 1

Analysis of CD73 Expression in hiPSC-Derived Retinal Organoids (A and B) qRT-PCR analysis of NT5E (coding for CD73) and PR markers during differentiation between D50 and D200 (mean ± SD; n = 5 organoids from N = 3 differentiations per time point). Gene expression at each time point is indicated relative to organoids at D50. (C) Percentage of CD73+ cells in organoids between D85 and D200 of differentiation analyzed by flow cytometry using CD73-FITC antibody (mean ± SD; n = 10 organoids from N ≥ 3 differentiations D85 versus D180 ^∗p < 0.05, D85 versus D200 ^∗∗p < 0.01, multiple comparisons Kruskal-Wallis test). (D) Schematic summarizing temporal expression of CD73 and mature PR markers in organoids. (E) Endogenous mCherry staining (red) and CRX immunolabeling (green) on solvent-cleared D75 organoid generated from the AAVS1:CrxP_H2BmCherry hiPSC line. (F) Immunostaining showing the expression of CD73 in mCherry+ PRs in section of D100 AAVS1:CrxP_H2BmCherry retinal organoid. (G) Dissociated mCherry+ cells (red) from D140 AAVS1:CrxP_H2BmCherry retinal organoids co-expressing RECOVERIN (gray) and CD73 (green). Nuclei were counterstained with DAPI (blue) in the first left panel. Scale bars, 100 μm (E), 25 μm (F and G).

Figure 2

Selection of hiPSC-Derived PRs by Targeting of CD73 (A) Representative CD73-PE flow cytometry analysis plot (specific staining in blue, isotype control staining in red) on unsorted, and MAC-sorted CD73+ and CD73– fractions from D120 organoids showing the percentage of CD73+ cells. (B) Immunofluorescence analysis of PR markers CRX and RECOVERIN in dissociated cells from D120 organoids (unsorted fraction) and in CD73+ and CD73– fractions after MACS. (C) Immunolabeling of RECOVERIN+ cells in unsorted, and sorted CD73+ and CD73– fractions from D140 AAVS1:CrxP_H2BmCherry retinal organoids. (D) Exclusive expression of CRX and PAX6 in dissociated cells from D120 organoids (unsorted) and after MACS targeting CD73 (positive). (E) qRT-PCR expression analysis of PR-specific genes in cells from unsorted (gray), and CD73+ (red) and CD73– (blue) fractions after MACS at D120 (mean ± SD; n = 25 organoids from N = 6 differentiations, ^∗p < 0.05, ^∗∗p < 0.01, multiple comparisons Kruskal-Wallis test). Gene expression is indicated relative to organoids at D120. (F) Quantitative analysis by flow cytometry of apoptotic (PI+) cells on unsorted and CD73+ fractions in dissociated cells from: unfrozen retinal organoids (unsorted and CD73+ fresh), freeze-thawed CD73+ cells, freeze-thawed retinal organoids (unsorted and CD73+ freeze-thawed organoids) (^∗p < 0.05, multiple comparisons Kruskal-Wallis test). (G) Co-expression of CRX and RECOVERIN in unsorted or sorted CD73+ cells from dissociated freeze-thawed organoids. Scale bars, 50 μm (B), 25 μm (C, D, and G).

Figure 3

Characterization of CD73+ Cells from Mature Retinal Organoids (A) qRT-PCR expression analysis of PR-specific genes in cells from unsorted (gray), and CD73+ (red) and CD73– (blue) fractions after MACS at D200 (mean ± SD; n = 25 organoids from N = 6 differentiations, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗p < 0.001, multiple comparisons Kruskal-Wallis test). Gene expression is indicated relative to organoids at D200. (B) qRT-PCR time course analysis of CNG channel subunits in organoids between D50 and D200 (mean ± SD; n = 5 organoids from N = 3 differentiation per time point). Gene expression at each time point is indicated relative to organoids at D50. (C) Representative examples of 2-Photon Fura2-AM fluorescence images (calcium imaging) obtained before (left) and during (right) application of 8-Br-cGMP on dissociated retinal cells at D190. White arrows 1 and 2 represent two responsive cells. (D) Infra-red (IR) image showing CD73-PE staining on dissociated retinal cells at D190. White arrows indicate responsive cells denoted 1 and 2 from image (C). (E) Fluorescence traces after application of cGMP analog on cells 1 and 2 displayed in (C and D), expressed as percentage change from baseline fluorescence (Δf/f%). (F) Percentage of CD73+ cells among the 8-Br-cGMP-responsive cell population (n = 19 from N = 5). Scale bars, 10 μm (C and D).

Figure 4

Safety Assessment of hiPSC-Derived Retinal Cells (A) Immunostaining showing the expression of pluripotency markers OCT4, NANOG, and SSEA-4 in iPSC colonies (hiPSCs) but not in dissociated cells (unsorted) from D120 retinal organoids. (B) Immunolabeling for Ki67 protein, associated with cell proliferation, in a representative iPSC colony and dissociated cells from D120 retinal organoids. Note loss of Ki67 expression in retinal cells. (C) Table recapitulating the tumorigenicity test after subretinal transplantation into NUDE rats of dissociated hiPSCs and D120 unsorted or MAC-sorted CD73+ cells. Observations were made 8 weeks after the transplantation. (D) Example of eyeballs of NUDE rats 8 weeks after subretinal transplantation of hiPSCs (black arrow) compared with non-transplanted eye. Bottom panel illustrates H&E staining of cross-section of the same hiPSC-transplanted eye. (E) Representative confocal images showing the presence of human S121+ cells in the SRS of NUDE rat 8 weeks after transplantation of D120 unsorted cells or MAC-sorted CD73+ cells. Nuclei were counterstained with DAPI (gray). (F) Absence of double Ki67+/HNA+ cells in unsorted and sorted CD73+ grafted cells 8 weeks after transplantation. Nuclei were counterstained with DAPI (gray). Scale bars, 1 cm (D) (top panel), 1 mm (D) (bottom panel), 50 μm (A and B), 20 μm (E and F). SRS, subretinal space; rPR, rat photoreceptors.

Figure 5

Transplantation of hiPSC-Derived Retinal Cells into a Rat Model of Retinal Degeneration (A) Identification of donor human cells 1 week after transplantation by using three different human-specific antibodies (red): STEM 121 (left), MTCO2 (center), and HNA (right). (B) Confocal mosaic image of D120 unsorted cell-transplanted eye showing the distribution of human S121+ cells in the SRS 4 weeks after transplantation. Arrowheads delimitate the spread of the grafted cells; the site of injection is indicated with an arrow. (C) Most transplanted human retinal cells (S121+ cells) coexpress RECOVERIN. Note that RECOVERIN staining is lower in rat cells than in human cells. (D) Example of RECOVERIN immunostaining in a non-grafted retina from P23H rat at 10 weeks of age. RECOVERIN antibody specifically labels rat PRs and part of the bipolar cells within the INL. (E) Size of nuclei area of transplanted human cells and endogenous rat PRs 4 weeks after transplantation (mean ± SD, n = 30 nuclei measured, N = 3 grafted retinas; ^∗∗∗∗p < 0.0001, Mann-Whitney test). (F) Some transplanted human S121+ retinal cells express the transcription factor PAX6. Nuclei were counterstained with DAPI (gray). Scale bars, 20 μm (A, C, D, and F), 500 μm (B). SRS, subretinal space; ONL, outer nuclear layer, INL, inner nuclear layer; RGC, retinal ganglion cell layer.

Figure 6

Transplantation of hiPSC-Derived CD73+ Photoreceptors into the P23H Rat Model of Retinal Degeneration (A) Human S121+ cells co-expressing PR marker REVOVERIN located in close proximity to the rat INL. The residual rat PRs are delimited by the white dotted line. (B) Absence of double S121/PAX6-positive cells. Transplanted CD73+ cells labeled by S121 are in close proximity with host PAX6+ cells within the INL. Arrowheads indicate residual rat PR nuclei. (C) Labeling of human cone PRs by human-specific CONE ARRESTIN antibody (hCAR) and rod PRs by RHODOSPIN antibody. Arrowheads indicate human RHODOSPIN+ cells. Residual rat Rhodopsin+ PRs are delimited by the white dotted line. (D) Human S121+ cells co-expressing cone PR marker R/G OPSIN. Residual S121– rat PRs (arrowheads) can be recognized by their nuclei features (smaller size and brightness). (E) Some human HNA+ cells express blue cone marker BLUE OPSIN (arrowheads). (F) Staining for synaptic protein RIBEYE and human CONE ARRESTIN (hCAR) showing localized punctate RIBEYE expression in human cone PRs directly in contact with the INL. (G) Rat PKCα+ bipolar cells are in close proximity to human S121+ cells inserted within the host ONL. The area delimited by the line indicates the location of high-magnification images presented in (G'). All observations were made 4 weeks after transplantation. Nuclei were counterstained with DAPI (gray). Scale bars, 20 μm (A–D), 10 μm (E–G′). SRS, subretinal space; rPR, rat photoreceptors; INL, inner nuclear layer; RGC, retinal ganglion cell layer.

Figure 7

Long-Term Survival of Transplanted CD73+ Cells (A) CD73+ human PRs, co-expressing S121 and RECOVERIN, are intercalated within the rat retina. (B) Example of the distribution of human S121+ cells in close proximity to rat PKCα+ bipolar cells. (C) Transplanted PRs co-expressing human CONE ARRESTIN (hCAR) and human marker HNA. The residual rat PRs are delimited by the white dotted line. (D) Staining for human and rat rod PRs with RHODOPSIN and RECOVERIN antibodies. (E) RIBEYE protein expression in the rat outer plexiform layer close to human cone PRs (hCAR+ cells). The area delimited by the line indicates the location of the high-magnification image presented in (E'). (F) RIBEYE protein expression in close proximity to the synaptic terminals of rat PKCα+ bipolar cells. The area delimited by the line indicates the location of the high-magnification image presented in (F'). All observations were made 10 weeks after transplantation. Nuclei were counterstained with DAPI (gray). Scale bars, 50 μm (A and B), 20 μm (C–E and F), 5 μm (E′ and F′). SRS, subretinal space; rPR, rat photoreceptors; INL, inner nuclear layer; RGC, retinal ganglion cell layer.