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. 2014 Jun 10;111(23):8518-23.
doi: 10.1073/pnas.1324212111. Epub 2014 May 27.

From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium

Sacha Reichman  1 , Angélique Terray  1 , Amélie Slembrouck  1 , Céline Nanteau  1 , Gaël Orieux  1 , Walter Habeler  2 , Emeline F Nandrot  1 , José-Alain Sahel  3 , Christelle Monville  2 , Olivier Goureau  4
Affiliations

From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium

Sacha Reichman et al. Proc Natl Acad Sci U S A. .

Abstract

Progress in retinal-cell therapy derived from human pluripotent stem cells currently faces technical challenges that require the development of easy and standardized protocols. Here, we developed a simple retinal differentiation method, based on confluent human induced pluripotent stem cells (hiPSC), bypassing embryoid body formation and the use of exogenous molecules, coating, or Matrigel. In 2 wk, we generated both retinal pigmented epithelial cells and self-forming neural retina (NR)-like structures containing retinal progenitor cells (RPCs). We report sequential differentiation from RPCs to the seven neuroretinal cell types in maturated NR-like structures as floating cultures, thereby revealing the multipotency of RPCs generated from integration-free hiPSCs. Furthermore, Notch pathway inhibition boosted the generation of photoreceptor precursor cells, crucial in establishing cell therapy strategies. This innovative process proposed here provides a readily efficient and scalable approach to produce retinal cells for regenerative medicine and for drug-screening purposes, as well as an in vitro model of human retinal development and disease.

Keywords: cones; retinal ganglion cells; rods.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Efficient generation of RPE cells and NR-like structures from confluent hiPSCs. (A) Schematic diagram showing the stages of the differentiation protocol. (B and C) Neuroepithelial-like structure derived from hiPSC-2 at D7 and D14. (D) qRT-PCR analysis of NOGGIN and DKK1 in hiPSC-2 at D0, D7, and D14. Data are normalized on hiPSC-2 single colonies. (E) qRT-PCR analysis of eye-field transcription factors NRL, CRX, NEUROD1, and pluripotency marker OCT4 in NR-like structures at D14. Data are normalized to hiPSC-2 at D0. (F–P) Immunofluorescence staining of cryosectioned NR-like structures at D14 for PAX6 and RAX (F–H), Ki67 and LHX2 (I–K), or MITF and VSX2 (L–P). (Scale bars: B, C, F, I, and L, 100 µm; G, H, J, and M–P, 50 µm.)
Fig. 2.
Fig. 2.
Amplification and characterization of hiRPE cells. (A) Schematic illustration outlining the differentiation protocol to amplify hiRPE (arrow 1) or to maturate NR-like structures (arrow 2). (B and C) Phase-contrast images of hiRPE cell monolayer after 45 d. (D) ZO-1 and MITF immunostaining of hiRPE cell monolayer after 45 d. (E) qRT-PCR analysis of mature RPE markers in hiRPE cells at passage 0 (P0), P1, and P2. Data are normalized to control RNA isolated from human adult RPE cells. (F) Evaluation of RPE cell phagocytic activity; ratio of FITC/DAPI fluorescence in hiRPE cell cultures at P1 and in control RPE-J cell line after 3 h incubation with FITC-labeled POS. Binding and uptake of POS were assayed as described in SI Materials and Methods. (Scale bars: B and C, 100 µm; D, 50 µm.)
Fig. 3.
Fig. 3.
Characterization of early differentiating cells in floating NR-like structures. (A–C) Morphology of representative isolated floating NR-like structures at D15, D21, and D28. (D and E) qRT-PCR analysis of eye-field and photoreceptor-specific transcription factors in NR-like structures from D14 to D42. Data are normalized to NR-like structures at D14. (F–N) Immunohistochemistry analysis of cryosectioned NR-like structures at D21 or D28 for MITF (F), VSX2 (F and G), PAX6 (G, I, and N), OTX2 (H and N), BRN3A (H), CRX (L and M), CALRETININ (J), LIM1 (K), and Ki67 (I). (Scale bars: A–C and F–H, 100 µm; I–N, 50 µm.)
Fig. 4.
Fig. 4.
Differentiation of all retinal cell types from floating NR-like structures. (A–E) qRT-PCR analysis of selected neural retinal cell types in NR-like structures at different times. Data are normalized to NR-like structures at D14 and at D35 for both R/G and BLUE OPSIN. (F–Q) Immunohistochemical analysis of cryosectioned NR-like structure at different stages of differentiation using markers for RGCs (BRN3A, PAX6, and CALRETININ), amacrine cells (PAX6, AP2, and CALRETININ), horizontal cells (LIM1, PAX6, and CALRETININ), photoreceptors (OTX2, RECOVERIN, CRX, CD73, RHODOPSIN, and R/G OPSIN), bipolar cells (PKCα), Müller glial cells (GS and SOX9), and for mitotic progenitors (Ki67). (Scale bars: F–N, 50 µm; O–Q, 25 µm.)
Fig. 5.
Fig. 5.
Acceleration of photoreceptor precursor generation from floating NR-like structures by Notch inhibition. (A) Schematic illustration of the experiment with DAPT treatment either from D21 to D28 or from D28 to D35. (B–I) Immunohistochemical analysis of cryosectioned NR-like structures at D28 or D35, with (C, E, G, and I) or without (B, D, F, and H) DAPT treatment. (J and K) Quantification of photoreceptor precursors (CRX and RECOVERIN) and mitotic progenitors (Ki67) at D28 (J) and D35 (K) with or without (control) DAPT. Values represent the mean percentage of positive cells ± SEM (n = 4, *P < 0.05). (L) qRT-PCR analysis of maturing photoreceptor markers and GLAST1 (marker for Müller glial cells) in D35 NR-like structures treated with DAPT. Data are normalized to NR-like structures at D35 without DAPT treatment. (Scale bars: 100 µm.)
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