Generation of highly enriched populations of optic vesicle-like retinal cells from human pluripotent stem cells

Abstract

The protocol outlined below is used to differentiate human pluripotent stem cells (hPSCs) into retinal cell types through a process that faithfully recapitulates the stepwise progression observed in vivo. From pluripotency, cells are differentiated to a primitive anterior neural fate, followed by progression into two distinct populations of retinal progenitors and forebrain progenitors, each of which can be manually separated and purified. The hPSC-derived retinal progenitors are found to self-organize into three-dimensional optic vesicle-like structures, with each aggregate possessing the ability to differentiate into all major retinal cell types. The ability to faithfully recapitulate the stepwise in vivo development in a three-dimensional cell culture system allows for the study of mechanisms underlying human retinogenesis. Furthermore, this methodology allows for the study of retinal dysfunction and disease modeling using patient-derived cells, as well as high-throughput pharmacological screening and eventually patient-specific therapies.

Keywords: development; differentiation; human pluripotent stem cells (hPSCs); optic vesicle; retina.

Figure 1. Overview of retinal differentiation protocol

hPSCs can be directed to differentiate into all cell types of the retina in a step-wise process. From cultures of undifferentiated hPSCs, cells are directed to differentiate by the generation of embryoid bodies. By 7 total days of differentiation, embryoid bodies are plated and adherent cultures maintained for a total of 16 days. At this point, neurospheres are generated and retinal progenitor populations may be enriched by 20 days of differentiation. Maintained culture of these retinal neurospheres will yield all of the major cell types of the neural retina within the first 70 days of differentiation. Alternatively, optic vesicle-like cultures at day 16 of differentiation may be utilized to generate retinal pigmented epithelium through the maintenance of adherent cultures.

Figure 2. Characterization of undifferentiated hPSCs

hPSCs displayed a typical undifferentiated morphology, including tightly packed colonies of cells and clearly defined edges (A). RT-PCR analysis demonstrated the expression of characteristic pluripotency makers in hPSCs, while lacking mesodermal, endodermal and ectodermal markers (B). Immunocytochemistry further demonstrated widespread expression of pluripotency-associated transcription factors (C–E) as well as cell surface markers (F–H). See Tables 1 and 2 for a listing of primers and antibodies used for RT-PCR and immunocytochemistry.

Figure 3. Induction of hPSCs to a Neural Progenitor Fate

After ten days of differentiation and plating (A), differentiating hPSCs were analyzed by RT-PCR and demonstrated expression of the neural markers PAX6 and SOX1. An anterior neural, eye-field fate was further indicated by the expression of OTX2. RAX, SIX3, and LHX2 (B). Immunocytochemistry demonstrated the widespread expression of many of these transcription factors (C–E).

Figure 4. Differentiation of hPSCs to Retinal Pigmented Epithelium

hPSC-derived RPE-like cells expressed typical RPE associated markers when screened by RT-PCR (A). Under brightfield microscopy, these cells displayed proper morphological features distinct to RPE, including a hexagonal shape and areas of pigmentation (B). Immunocytochemical analysis revealed the features typical of the retinal pigmented epithelium (C).

Figure 5. Identification, Enrichment, and Characterization of Retinal Progenitor Cells

After 30 days of differentiation, hPSCs were isolated into two morphologically distinct and readily identifiable populations (A). Retinal neurospheres, characterized by a bright ring surrounding the outer layer (B) and a non-retinal neural population displaying a larger, more uniform appearance (C). RT-PCR analysis revealed striking differences between these two neurosphere populations. While both populations expressed neural associated transcription factors (PAX6, NeuroD1, and FABP7), retinal neurospheres expressed CHX10, RAX, and SIX6, characteristic of retinal progenitors which were absent from non-retinal neural populations. Conversely, the non-retinal neural cells expressed forebrain-associated transcription factors including SOX1, DLX1, and EMX1, which were absent from retinal neurospheres (D). Immunocytochemical analysis revealed that retinal neurospheres widely expressed retinal progenitor markers such as CHX10 and PAX6 (E), but largely lacked the expression of the forebrain-associated marker SOX1 (F). hPSC-derived retinal progenitors also remained highly proliferative within the first 30 days of differentiation (G). Non-retinal neural populations displayed typical features of emerging forebrain neurons, including the expression of βIII-Tubulin and OTX2 (H), as well as the forebrain-associated DLX5, but lacked the retinal progenitor marker CHX10 (I). Non-retinal neural cells also retained the expression of both PAX6 and SOX1 (J).

Figure 6. Differentiation of hPSCs to Retinal Neurons

Within 90 total days of differentiation, hPSC-derived retinal cells displayed typical neuronal morphologies under DIC microscopy (A). Analysis by RT-PCR illustrated an array of retinal-associated transcription factors including those associated with ganglion cells (BRN3 and Islet1), as well as those associated with photoreceptors (CRX and NeuroD4). Furthermore, proteins associated with phototransduction could also be identified, including Red/Green Opsin, Arrestin, and Transducin (B). Immunocytochemistry analysis confirmed the expression of BRN3-positive retinal ganglion cells extending Map2-positive dendrites (C) as well as photoreceptor-like phenotypes including the expression of CRX, OTX2 and Recoverin (D, E).