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. 2016 Jul 29;6:30742.
doi: 10.1038/srep30742.

cGMP Production of Patient-Specific iPSCs and Photoreceptor Precursor Cells to Treat Retinal Degenerative Blindness

Free PMC article

cGMP Production of Patient-Specific iPSCs and Photoreceptor Precursor Cells to Treat Retinal Degenerative Blindness

Luke A Wiley et al. Sci Rep. .
Free PMC article


Immunologically-matched, induced pluripotent stem cell (iPSC)-derived photoreceptor precursor cells have the potential to restore vision to patients with retinal degenerative diseases like retinitis pigmentosa. The purpose of this study was to develop clinically-compatible methods for manufacturing photoreceptor precursor cells from adult skin in a non-profit cGMP environment. Biopsies were obtained from 35 adult patients with inherited retinal degeneration and fibroblast lines were established under ISO class 5 cGMP conditions. Patient-specific iPSCs were then generated, clonally expanded and validated. Post-mitotic photoreceptor precursor cells were generated using a stepwise cGMP-compliant 3D differentiation protocol. The recapitulation of the enhanced S-cone phenotype in retinal organoids generated from a patient with NR2E3 mutations demonstrated the fidelity of these protocols. Transplantation into immune compromised animals revealed no evidence of abnormal proliferation or tumor formation. These studies will enable clinical trials to test the safety and efficiency of patient-specific photoreceptor cell replacement in humans.


Figure 1
Figure 1. Generation of cGMP, clinical-grade patient-specific iPSCs.
(A) Schematic depicting the timeline and stepwise procedure for cGMP-compliant fibroblast isolation from patient skin biopsies and subsequent generation, clonal expansion and analysis of patient-derived iPSCs. (B) Photograph of one of two cGMP processing suites within the Steven W. Dezii Translational Vision Research Facility equipped with an ISO class 5 BioSpherix Xvivo Closed Incubation System. (C–F) Light micrographs of two independent patient-derived cell lines (DB-005 (C,E) and DB-006 (D,F)). Fibroblasts can be seen migrating from and growing around a fragment of patient skin (C,D). Typical iPSC colonies with large nuclear to cytoplasm ratio generated from each fibroblast line are also shown (E,F and insets in each). (G,H) Data generated by hPSC Scorecard Analysis Software demonstrating each independent patient line primarily expresses genes that participate only in self-renewal, not in formation of endoderm, mesoderm or endoderm. (I) Graph comparing the algorithm scores for expression of genes involved in self-renewal. Compared to the internal reference data set provided by the software, each patient line falls within the average. (J) Graph showing the correlation coefficient comparing all gene expression data for each patient line. The two lines are highly correlated to one another (r2 = 0.97). These data demonstrate how similar each line is to one another, speaking to the consistency of our cGMP protocol for the generation of patient-specific iPSC lines. (K) Schematic showing the timeline and stepwise procedure for cGMP-compliant three-dimensional differentiation of patient iPSCs and the production of iPSC-derived retinal organoids.
Figure 2
Figure 2. Early 3D organoid development in patient-specific iPSCs.
Immunocytochemical analysis of 3D organoids after 3–5, 6–8 and 9–10 weeks of differentiation. (A–D) After 3–5 weeks of differentiation, 3D spheres have evaginated loops comprised of polarized F-actin-positive structures (A and inset; Phalloidin; green) and proliferating, Ki67-positive neural epithelia (A; red). At this stage, spheres express SOX2 (B; green), PAX6 (B; red) and OTX2 (B; gray) and have the appearance of developing organoids. Many cells positive for SOX2 (C; green) also express VSX2/CHX10 (C and inset; gray). There are isolated pockets of presumptive RPE cells that co-express MITF (D and inset; green) and PAX6 (D and inset; red). (E–H) After 6–8 weeks, organoids are multilayered structures (E and inset) that continue to express SOX2 (E; green). Some organoids begin to develop pockets of photoreceptor precursor cells within neural rosette-like structures that express OTX2 (F; gray), independently of PAX6 (F; red). Areas of pigmented cells that express MITF (G and inset; green) independently of PAX6 (G; red) are also observed. There is a subpopulation of cells that express the neuronal factor, HuC/D (H; green), an early marker of amacrine and ganglion cells and cell proliferation continues at a high rate (H; Ki67; red). (I–L) After 9–10 weeks, 3D organoids begin to look morphologically similar to a mature retina (inset of I) and in some cases have a laminated structure with an outer layer of MITF-positive, RPE cells (I; green) with underlying PAX6-expressing cells (I; red) and clusters of OTX2-positive photoreceptor precursor cells (I; gray). Within neural rosettes, SOX2-positive cells (J; green) now lack VSX2/CHX10 (J and inset; gray) and have fewer Ki67-positive cells (J; red). OTX2 (K; gray) is robustly expressed independently of PAX6 (K and inset; red) throughout neural rosettes that are now more widespread and larger in size (K). Many cells throughout the organoids are now positive for the pan neuronal marker, TUJ1 (L; red), but do not yet express the rod photoreceptor-specific factor, NRL (L and inset; gray). Small panels to the right of B show individual fluorophores. Scale bars = 50 μm, except for D = 100 μm.
Figure 3
Figure 3. Development of clinical-grade photoreceptor precursor cells.
Morphological and immunocytochemical analysis of 3D organoids after 11–12 and 13–16 weeks of differentiation. (A–C) After 11–12 weeks of differentiation, organoids have many areas of retinal-like folds (A; light micrograph) and retain highly organized F-actin-positive structures (inset in A; green). Organoids express the phototransduction molecule, recoverin (B; red) and the photoreceptor precursor-specific transcription factor, CRX (B; gray), a marker of committed photoreceptor precursor cells that is functionally downstream of OTX2 (Fig. 2). Some recoverin-positive cells within organoids at this stage also begin to express NRL, a rod photoreceptor-specific transcription factor (C; gray). Recoverin labeling can also be observed along neuronal-like processes of photoreceptor precursor cells. (D–G) Organoids after 13–16 weeks of differentiation are typically full of neural rosettes (D; light micrograph) and the majority of OTX2-positive cells (inset of D; gray) are now post-mitotic, Ki67-negative cells (inset of D; red). By this stage of development, many neuronal cells that are TUJ1-positive (E; red) are now positive for NRL (E; gray). Many recoverin-positive cells (F and inset; red) within neural rosettes co-express NRL (F and inset; gray) and NR2E3 (F and inset; green), which is functionally downstream of NRL in rod photoreceptor development and is transcriptionally regulated by NRL. Some organoids retain their lamination and resemble mature retina with independent layers of recoverin-positive photoreceptors (G; red) and HuC/D-positive inner retinal amacrine- and ganglion-like cells (G; green). 3D reconstrutction of a z-stack through a laminated eyecup further demonstrates the separate layers of photoreceptor-like cells and inner retinal neuron-like cells (G, inset). Small panels to the right of (B,C,E,F) show individual fluorophores. Scale bars A,D = 400 μm; B,C,EG = 50 μm.
Figure 4
Figure 4. cGMP patient-specific organoids recapitulate photoreceptor cell lineage.
Immunocytochemical analysis of 3D organoids from a patient with mutations in NR2E3 and a patient with normal NR2E3 alleles. (A) NR2E3 organoids develop normally and are initially indistinguishable from control organoids (Fig. 2). At 5–7 weeks post-differentiation NR2E3 organoids contain polarized (Phalloidin; green) neural epithelial cells that express PAX6 (inset; red) and OTX2 (inset; gray) and are actively proliferating (Ki67; red). (B) After 13 weeks of differentiation, NR2E3 organoids have cells that express the inner retinal-specific marker, HuC/D (green) and the early photoreceptor-specific lineage transcription factor, RORβ (red and inset). (C) After 15–16 weeks of differentiation, presumptive photoreceptors within NR2E3 mutant organoids are CRX- (gray and inset) and recoverin-positive (red). (D) Also around 15–16 weeks, NR2E3 organoids show robust labeling for SW Opsin, which is specific for blue (S) cones (D and insets; red). These SW Opsin-positive cells are NRL-negative (D and insets; gray) and have strands of SW Opsin emanating from the cell nucei towards outersegment-like cone-shaped membranes (D; insets). (E) Quantification of the percentage of SW Opsin-positive cells in two patients with NR2E3-associated enhanced S-cone syndrome (NR2E3-1 vs Control; p < 0.01 and NR2E3-2 vs Control; p < 0.001) compared to an unaffected control eyecup at 15–16 weeks post-differentiation. (F) Age-matched organoids from a patient with normal NR2E3 alleles are not positive for SW Opsin (E). Further, an eyecup from an individual with normal NR2E3 alleles after 21 weeks of differentiation has only a few SW Opsin-positive cells (E and inset; red) amongst many NRL-positive presumptive rod photoreceptor cells (E; gray). Scale bars: A and inset of E,F = 100 μm; all others = 50 μm.

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