(A): Gene network model for photoreceptor differentiation from human retinal stem cells (hRSCs). CHX10VP16 increases the population of competent immature cells by blocking proliferation, and facilitates the coexpression of subtype specification factors such as OTX2 and/or CRX which serve to bias these cells to adopt a photoreceptor cell fate. (B): Reverse transcription polymerase chain reaction (RT-PCR) lineage analysis and apoptosis in CHX10VP16/OTX2/CRX transfected hRSC progeny. RT-PCR analysis of genes associated with neural (NESTIN, PAX6), mesodermal (GATA-1, BRACHYURY), and endodermal (GATA-4) identity in CHX10VP16/OTX2/CRX transfected hRSC progeny (right). Human embryoid body (hEB) samples were used as positive controls (left). The fate changes seen through transcriptional modulation are only within the retinal lineage. (C): Apoptosis in CHX10VP16/OTX2/CRX transfected hRSC progeny. Human retinal stem cell (hRSC) spheres transfected with control or CHX10VP16/OTX2/CRX were dissociated and subjected to immunocytochemistry for active caspase3 (an apoptotic cell marker). The apoptotic cell number was not significantly increased in CHX10VP16/OTX2/CRX-transfected hRSC progeny versus control (t = 0.24, p >.05). Abbreviations: hEB, human embryoid body; hRSC, human retinal stem cell; RPE, retinal progenitor.

Transduction of OTX2 and CRX together with modulation of CHX10 produce the most potent induction of photoreceptor differentiation from human retinal stem cell progeny. (A,B): Results of the in vitro differentiation assay with clonal hRSC derived spheres transfected with control (green fluorescent protein [GFP]), CRX, NRL, NEUROD, OTX2, NEUROGENIN2, or MASH1-expressing lentiviral vectors. Rho1D4 was used as a rod photoreceptor marker and human cone arrestin as a cone photoreceptor marker. The y-axis indicates the percentage of photoreceptor marker and GFP coexpressing cell number/GFP expressing cell number. OTX2 or CRX transduction significantly increased the numbers of differentiated rod (A), and cone (B) photoreceptor (analysis of variance and a Dunnette’s multiple comparison test, *p < .05). (C,D): Photoreceptor differentiation was significantly promoted by coexpression of OTX2/CRX, CHX10VP16/OTX2, and CHX10VP16/OTX2/CRX compared with control ((C) rods, and (D) cones) (analysis of variance and Dunnette’s multiple comparison test, *p < .05). (E): Rho1D4 positive cells (red) or (F) human cone arrestin positive cells (red) coexpress GFP from the control (left) or from the CHX10VP16/OTX2/CRX0expression vector (right) as illustrated by the merged field (yellow, marks by arrowheads). Many more human retinal stem cell progeny differentiated into (E) rod or (F) cone photoreceptors with CHX10VP16/OTX2/CRX-transduction compared with control-GFP.

Molecular interaction of CHX10, OTX2, and CRX in human retinal stem cell (hRSC) progeny. (A–C): Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis. OTX2, CHX10, or CRX mRNA levels were normalized to GAPDH mRNA (the control sample was set to 1.0). (A): OTX2 mRNA level was significantly downregulated in CHX10-transduced hRSC progeny, whereas OTX2 mRNA was increased in CHX10VP16 transduced progeny (analysis of variance and Dunnette’s multiple comparison test, *p < .05). (B): CHX10 expression is not affected in OTX2 transduced hRSC progeny. (C): CRX mRNA levels were significantly increased in OTX2 or CHX10VP16-transduced hRSC progeny (analysis of variance and a Dunnette’s multiple comparison test, *p < .05). (D): Partial genomic map (5′ region) for the human OTX2 gene. Exons (Ex1,2, and 3), transcriptional start sites (TS) and the initiator codon (ATG) are indicated. A putative CHX10-binding area (CHX10BA) is located in intron2. (E): Chromatin immunoprecipitation analysis. Anti-Chx10 antibody specifically precipitates the chromatin containing the end of intron two of OTX2 (CHX10BA), but not the 3′UTR region (control), from hRSC progeny. Preimmune serum does not precipitate these regions. (F): Luciferase assay. OTX2 genomic fragments 1-4 shown in (D) were cloned into a luciferase reporter and cotransfected into hRSC progeny with or without the CHX10 expression vector. The activity of the βactin minimal promoter-luciferase reporter without genomic OTX2 was taken as 100%. Suppression of the reporter activity by CHX10 required the CHX10 binding area (CHX10BA) present in fragments 2 and 4 (analysis of variance and a Dunnette’s multiple comparison test, *p < .05). Abbreviations: TS, transcriptional start sites.

In vivo human retinal stem cell (hRSC) transplantation into mouse eye. (A): Human retinal stem cell progeny (green fluorescent protein [GFP] positive) are immunostained with a photoreceptor marker Rom1 (red), which marks a outer segment protein in transplanted human (double labeled with two markers shown as yellow) and host (red) CD1 retinal cells (scale bar: 100 μm). GCL; retinal ganglion layer, INL; inner nuclear layer, ONL; outer nuclear layer, OS; outer segment. (B): Mouse retina section (adjacent to the one shown in A) stained with cresyl violette to show the retinal location of the transplanted human cells shown in (A) (scale bar: 100 μm). (C): High-power image of a single hRSC-derived phtotoreceptor (GFP positive) integrated into the host retina. The human donor cell shows the morphology of a photoreceptor (scale bar: 20 μm). (D): Transplanted hRSC progeny transfected with CHX10VP16/OTX2/CRX show improved integration and survival compared with control transfection at 1, 3, and 5 weeks after transplantation. (E,F): Improved photoreceptor differentiation in hRSC progeny transfected with CHX10VP16/OTX2/CRX compared with control transfected cells at 5 weeks after transplantation (rods (E) and cones (F)) (analysis of variance [ANOVA] and Dunnette’s multiple comparison test, *p < .05). Rho1D4 was used as a rod photoreceptor marker and human cone arrestin as a cone photoreceptor marker. The y-axis indicates the percentage of photoreceptor marker and GFP coexpressing cell number/GFP expressing cell number. (G): At the lowest flash intensities (−3.2 and −2.8) the CHX10VP16/OTX2/CRX group shows a higher response than the nontransplanted and GFP-only vector-treated groups (ANOVA and a Dunnette’s multiple comparison test, *p < .05). Inset shows that a significant correlation of maximal b wave response and surviving human photoreceptor cell number (PhR numb) was seen. For reasons of space within this inset, the two data points for the control animals represent the data from four animals. (H): As a within-animal control in the transplantation model, the differences in the minimal spatial frequency detected between the transplanted eye (right) and nontransplanted eye (left) in each individual mice were estimated. Abbreviations: GCL, retinal ganglion layer; hRSCs, human retinal stem cells; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segment.

Modulation of CHX10 expression is important for induction and early maturation of photoreceptors from human retinal stem cell (hRSC) progeny. (A): Schematic illustration of effecter vectors encoding CHX10, CHX10VP16 or VP16. CHX10VP16 encodes human CHX10 fused to amino acids 410-490 of the VP16 activation domain. All genes were introduced into the pMXIE expression vector. (B): The expression vector pMXIE-CHX10 represses activation. NG108 cells were cotransfected with equimolar amounts of control effector plasmid or pMXIE-CHX10 along with GAL4-HSF1 activator and HD4-pG5EC chloramphenicol acetyl transferase (CAT) reporter containing four homeodomain binding sites and five GAL4 DNA binding sites. One hundred percent CAT activity is taken as that obtained in the presence of control effector plasmid was set to 1.0. The y-axis indicates the percentage of reporter transcription with CHX10 transfection/reporter transcription with control transfection. *p < .05 indicates statistically significance with Student’s t test. (C): Transcription is activated by pMXIE-CHX10VP16. NG108 cells were cotransfected with equimolar amounts of control effector plasmid or pMXIE-CHX10VP16 along with HD4-pG5EC CAT reporter. The y-axis indicates fold activity of reporter transcription with VP16 or CHX10VP16 transfection/reporter transcription with control transfection set to 1.0. *p < .05 indicates statistically significance with analysis of variance (ANOVA) and a Dunnette’s multiple comparison test. (D): Schematic of replication-defective self-inactivating lentiviral vectors containing an internal ribosome entry site (IRES) sequence followed by enhanced green fluorescent protein (GFP) (CSEIE). CHX10 or CHX10VP16 cDNA were cloned into CSEIE, which directs the expression of the cloned genes together with GFP from the internal promoter, EF1α. Control vector expresses only GFP. (E): Human retinal stem cells-derived sphere transfected with CHX10 (left) and CHX10VP16 (right). Spheres ubiquitously express GFP, but some of the cells in the clonal sphere are pigmented, thus obscuring GFP and producing a mottled GFP appearance in the spheres. Scale bar: 100 μm. (F): Sphere diameters that are a proxy for total cell number generated by proliferation were measured in control, CHX10, or CHX10VP16-induced retinal stem cell (RSCs) colonies. We measured more than 30 spheres in each group in at least three independent experiments. Sphere diameters were significantly increased in CHX10-induced clonally-derived RSC colonies compared with control. On the other hand, sphere diameters were significantly decreased in CHX10VP16-induced clonally-derived RSC colonies (analysis of variance and Dunnette’s multiple comparison test, *p < .05). (G): PAX6/NESTIN double labeling cells, which indicate undifferentiated retinal cells, were measured in the in vitro differentiation assay with hRSC colonies transfected with control, CHX10,or CHX10VP16. With CHX10 transduction, most of hRSC progeny maintained an undifferentiated state. In contrast, CHX10VP16 transduction significantly decreased the number of undifferentiated cells (ANOVA and Dunnette’s multiple comparison test, *p < .05). The y-axis indicates the percentage of PAX6/NESTIN double labeling cells /total cell number after neomycin selection. (H,I): CHX10 transduction abolished photoreceptor cell differentiation, while CHX10VP16 transduction significantly increased rod (H) and cone (I) photoreceptor differentiation. Rho1D4 was used as a rod photoreceptor marker and human cone arrestin as a cone photoreceptor marker. CHX10 transduction abolished photoreceptor cell differentiation, whereas CHX10VP16 transduction significantly increased rod and cone photoreceptor differentiation (Student’s t test, *p < .05). The y-axis indicates the percentage of photoreceptor marker and GFP coexpressing cell number/GFP expressing cell number. Abbreviations: EF, elongation factor; GFP, green fluorescent protein; IRES, internal ribosome entry; LTR, long terminal repeat.