SEARCH

SEARCH BY CITATION

Keywords:

  • Pluripotent stem cells;
  • Retina;
  • Developmental Biology;
  • Neural differentiation;
  • Cellular therapy;
  • Induced pluripotent stem cell

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ADDENDUM
  9. Acknowledgements
  10. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  11. REFERENCES
  12. Supporting Information

Differentiation methods for human induced pluripotent stem cells (hiPSCs) typically yield progeny from multiple tissue lineages, limiting their use for drug testing and autologous cell transplantation. In particular, early retina and forebrain derivatives often intermingle in pluripotent stem cell cultures, owing to their shared ancestry and tightly coupled development. Here, we demonstrate that three-dimensional populations of retinal progenitor cells (RPCs) can be isolated from early forebrain populations in both human embryonic stem cell and hiPSC cultures, providing a valuable tool for developmental, functional, and translational studies. Using our established protocol, we identified a transient population of optic vesicle (OV)-like structures that arose during a time period appropriate for normal human retinogenesis. These structures were independently cultured and analyzed to confirm their multipotent RPC status and capacity to produce physiologically responsive retinal cell types, including photoreceptors and retinal pigment epithelium (RPE). We then applied this method to hiPSCs derived from a patient with gyrate atrophy, a retinal degenerative disease affecting the RPE. RPE generated from these hiPSCs exhibited a disease-specific functional defect that could be corrected either by pharmacological means or following targeted gene repair. The production of OV-like populations from human pluripotent stem cells should facilitate the study of human retinal development and disease and advance the use of hiPSCs in personalized medicine. STEM CELLS 2011;29:1206-1218


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ADDENDUM
  9. Acknowledgements
  10. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  11. REFERENCES
  12. Supporting Information

Human pluripotent stem cells, which include both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold the potential to differentiate into any cell type. In doing so, they can serve as comprehensive model systems of human cell genesis, particularly at early developmental stages that would otherwise be inaccessible to investigation [1, 2]. In addition, patient-derived hiPSC lines have a unique capacity to model human disease [3–6], although the scope of disorders amenable to this form of study is limited [7–9]. Major considerations when creating hiPSC disease models include the capacity to efficiently generate, identify, and isolate relevant cell populations as well as recapitulate and assay critical aspects of the disease mechanism.

Retinal cell types are particularly well-suited for the investigation of cell development and dysfunction using pluripotent stem cell technology. The vertebrate retina harbors a modest repertoire of major cell classes that are sequentially produced via a conserved series of events [10–16]. Furthermore, the effects of inherited and acquired retinal degenerative diseases (RDDs) are often limited initially to a specific cell class, which simplifies the study of cellular mechanisms that incite RDDs and the evaluation of potential therapies.

Previous studies have demonstrated the ability of human pluripotent stem cells to differentiate along the retinal lineage with varying efficiencies [17–20], with one protocol achieving a near uniform retinal cell fate using the WA01 hESC line [18]. However, pluripotent stem cell-derived retinal cells, particularly those from hiPSCs, are most often found in mixed populations that include some nonretinal or unidentified cell types [17, 20–22]. Further complicating matters is the fact that several markers used for retinal cell identification (e.g., calretinin, Protein Kinase Cα (PKCα), and Tuj1) also label cells found in other regions of the central nervous system (CNS). As such, a means to isolate developmentally synchronized populations of multipotent retinal progenitor cells (RPCs) across multiple hESC and hiPSC lines would be desirable. The RPCs and their definitive retinal progeny could then be used to study mechanisms of human retinal development and disease, examine retinal cell function, and devise and test RDD treatments.

We recently described a method to differentiate human pluripotent stem cells to RPCs, retinal pigment epithelium (RPE), and photoreceptor-like cells in a manner that mimicked normal human retinogenesis [17]. However, a means to separate and track the fate of the RPCs in live culture was not available. In this study, we used transient morphological features to isolate structures with characteristics reminiscent of the optic vesicle (OV). Using these OV-like structures, it was possible to study principles of early human retinal development, monitor the sequence and timing of neuroretinal cell genesis, and optimize RPC and RPE production efficiencies in recalcitrant hiPSC lines. We then used our system to evaluate pharmacological and genetic strategies to correct a functional defect in hiPSC-RPE cultures derived from a patient with an inherited RDD.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ADDENDUM
  9. Acknowledgements
  10. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  11. REFERENCES
  12. Supporting Information

Maintenance and Differentiation of Human Pluripotent Stem Cells

hESCs (WA09 and WA01) and hiPSCs (IMR90-4 [23], 6-9-12T [24], iPS-12 [25], iPS-12.4 [25], and three patient-specific lentivirus-derived hiPSC lines) were maintained in ESC medium (see Supporting Information Materials and Methods for media descriptions) using an established method [17, 23]. Differentiation toward anterior neuroectodermal and retinal fates was accomplished using a modification of a previously published protocol [17]. Briefly, human pluripotent stem cell colonies were cultured as suspended aggregates for 4 days in EB medium, whereupon they were switched to neural induction medium. For some experiments, cultures were treated from days 2 to 4 with 100 ng/ml Noggin (R&D Systems, Minneapolis MN, http://www.rndsystems.com) (or Dorsomorphin (Tocris, Ellisville, MO, http://www.tocris.com)) and Dickkopf-related protein 1 (Dkk1) (R&D Systems, Minneapolis, MN, http://www.rndsystems.com) (or XAV939 (Tocris, Ellisville, MO, http://www.tocris.com)). After a total of 6–7 days, aggregates were plated onto laminin-coated substrates to permit formation of neural clusters. On day 16, the loosely adherent central portions of the neural clusters were mechanically lifted and grown as cellular aggregates in retinal differentiation medium (RDM). Between days 20 and 25, a subset of these aggregates adopted a vesicle-like appearance, which was manually separated and differentiated in RDM. Nonvesicular aggregates, or spheres, were similarly separated, pooled, and differentiated in RDM.

Generation of RPE from vesicle-like cultures was enhanced by adding Activin A (R&D Systems, Minneapolis, MN, http://www.rndsystems.com) (100 ng/ml) to RDM from days 20 to 40 of differentiation. RPE from manually isolated pigmented OV-like structures was expanded by plating them onto laminin-coated substrates in RDM + 20 ng/ml fibroblast growth factor 2 (FGF2) (Peprotech, Rocky Hill, NJ, http://www.peprotech.com)/ epidermal growth factor (EGF) (Peprotech, Rocky Hill, NJ, http://www.peprotech.com) and 5 μg/ml heparin for up to 1 week as described for prenatal RPE cultures [26]. Large patches of RPE-like cells could then be manually isolated, dissociated, and replated in the presence of mitogens for at least one additional passage.

Immunocytochemistry

Immunocytochemistry (ICC) analysis was carried out as described [17]. For the description of sectioning and immunostaining, see Supporting Information Materials and Methods. For the list of antibodies, see Supporting Information Table S1.

PCR Analysis

RNA was isolated using the RNAeasy (Qiagen, Valencia, CA, http://www.qiagen.com) or Picopure RNA isolation kit (Applied Biosystems, Carlsbad, CA, http://www.appliedbiosystems.com), reverse transcribed, and amplified by polymerase chain reaction (PCR) (30 cycles) or quantitative PCR (qPCR) (40 cycles) as described [17]. Primer sets are listed in Supporting Information Table S2. For a description of the microarray analysis method, see Supporting Information Materials and Methods.

Generation of Patient-Specific iPSCs

Gyrate atrophy (GA) fibroblasts were obtained from Coriell Institute for Medical Research, Camden, NJ, http://www.coriell.org (GM06330) and propagated in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. In addition, fibroblasts from two individuals without known genetic diseases were cultured from skin samples, as approved by the University of Wisconsin-Madison IRB. Reprogramming was performed as described previously [23] using lentiviral vectors expressing OCT4, SOX2, NANOG, LIN28, c-MYC, and KLF4.

OAT Assay

Ornithine-δ-aminotransferase (OAT) activity in RPE cultures was determined by the method of Ueda et al. [27], as detailed in Supporting Information Materials and Methods.

Electrophysiology and Live Cell Imaging

OV-like structures were differentiated in suspension culture for a total of 100 days and plated onto poly-D-ornithine-coated and laminin-coated cover slips for an additional 2 days to allow for adherence and peripheral cell spread/migration. Ionic currents were measured using conventional whole cell recordings performed on a fixed stage of a (Nikon Instruments, Melville, NY, http://www.nikon.com) FN-1 microscope. Cells were superfused continuously at approximately 2 ml/minute in standard bath solution (Supporting Information Materials and Methods). Recordings were acquired using patch pipettes (3–5 MΩ) filled with pipette solution (Supporting Information Materials and Methods), an Axopatch 200B amplifier, Digidata 1,440 interface, and pCLAMP 10 software (Molecular Devices, Sunnyvale, CA, http://www.moleculardevices.com). Stored data was analyzed using Clampfit-10 and plotted using Excel and Origin-8. Current responses were generated using either a voltage ramp (−160 to +40 mV) or step (−160 to +40 mV in 10 mV steps) protocol from a holding potential of −70 mV. A junction potential of 11 mV was calculated using pCLAMP software and all records were compensated post hoc.

For Ca2+ imaging, cells were loaded with 5 μM Fura-2-acetoxymethyl ester (Fura-2 AM) (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) for 60 minutes at 37°C. After washing for 30 minutes, cover slips were placed at the bottom of the recording chamber. Approximately 1 mM probenecid was added to prevent dye extrusion, and cells were sequentially exposed to 340 and 380 nm excitation wavelengths while registering fluorescence emission at 510 nm. Images were collected every 10 seconds with 2 × 2 pixel binning using a cooled CCD camera (CoolSNAP HQ, Photometrics, Tucson, AZ, http://www.photometrics.com) and a 60X water immersion objective. NIS Elements was used for image acquisition and offline analysis to determine changes in [Ca2+]i using the equation listed in Supporting Information Materials and Methods. Results were expressed as mean ± SEM.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ADDENDUM
  9. Acknowledgements
  10. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  11. REFERENCES
  12. Supporting Information

Isolation of hESC Structures with Characteristics of the OV or Early Forebrain

The OV is derived from the primitive anterior neuroepithelium (PAN) and shares numerous features with the early forebrain (EFB) [28–30]. We previously demonstrated the ability to differentiate human pluripotent stem cells into PAN, which in turn gave rise to free-floating cell aggregates containing either RPCs or EFB cells [17]. In this study, we looked for features that could be used to separate the RPC and EFB populations in live culture without the need for genetic manipulation.

Using our protocol [17], densely packed, neural rosette-containing cell colonies were isolated intact from WA09 hESCs after 16 days of differentiation and cultured in suspension. Starting at 20 days, two populations of cell aggregates could be distinguished via light microscopy (Fig. 1A). A minority population (20.4% ± 4.3%) was phase-bright and appeared vesicle-like due to the presence of a compact outer mantle of cells. The remaining population was spherical and had a more uniform cell distribution, although internal rosettes were commonly observed. Some cell aggregates possessed elements of both populations; however, these hybrid structures were excluded from this study.

thumbnail image

Figure 1. Isolation of optic vesicle (OV)-like structures from human embryonic stem cells (hESCs). (A): Between days 20 and 25 of hESC differentiation, free-floating cell aggregates displayed two morphologies: phase-bright vesicle-like structures (arrows) or darker, rosetted spheres (arrowheads). These populations were manually separated into vesicle-like (B) and nonvesicular (C) pools and independently cultured (arrows indicate rosettes in nonvesicular spheres). The definitive retinal progenitor marker CHX10 was expressed in the population of vesicle-like structures (D–F), whereas ISLET-1 was initially expressed solely in the nonvesicular sphere population (G–I). Polymerase chain reaction analysis identified OV (left) and early forebrain (middle) transcription factors that were differentially expressed at days 20–25 in vesicle-like structures (Vesc) and nonvesicular spheres (Nonvesc) as well as factors that were present in both populations (right) (J). (K): Immunostained sections of day 20 vesicle-like structures revealed coexpression of CHX10 and Ki67. By day 50, many vesicle-like structures lost their distinctive morphology and developed internal rosettes (L), but remained CHX10+/Ki67+ (M). Abbreviations: Vesc, vesicle-like structure; Nonvesc, nonvesicular spheres.

Download figure to PowerPoint

Cell aggregates with an unequivocal vesicle-like or nonvesicular configuration were separated between days 20 and 25 and cultured independently (Fig. 1B, 1C). Because this period of differentiation corresponded developmentally to the formation of OVs in humans, we asked whether CHX10, a marker of RPCs in the distal OV, was differentially expressed between the two populations. Before separation, CHX10 was found in a subset of cell aggregates (Fig. 1D). Afterward, CHX10 immunoreactivity was abundant (90.9% ± 4.2% of total cells) in the vesicle-like structures (Fig. 1E) and nearly absent in the nonvesicular spheres (Fig. 1F). By contrast, ISLET-1, a homeodomain protein involved in projection neuron differentiation and EFB development, was found only in the nonvesicular population at this stage (Fig. 1G–1I).

Given the disparate expression of CHX10 and ISLET-1 between the vesicle-like structures and nonvesicular spheres, PCR was performed to investigate gene expression patterns of other transcription factors involved in early retinal and/or forebrain development (Fig. 1J). After 20 days, the vesicle-like structures selectively expressed multiple retinal transcription factor genes appropriate for the OV stage of retinogenesis, whereas the nonvesicular spheres expressed transcription factors indicative of the embryonic forebrain. Factors involved in the early development of both the retina and forebrain were present in both culture populations.

The progenitor state of the CHX10+ vesicle-like structures was further queried using the cell proliferation marker Ki67. ICC analysis of sections revealed coexpression of CHX10 and Ki67 and confirmed the compact, radial arrangement of nuclei within these structures (Fig. 1K). With further maturation, they often lost their distinctive morphology and developed internal rosettes, rendering them indistinguishable from the nonvesicular spheres (Fig. 1L). However, most cells remained CHX10+ and/or Ki67+ until approximately 50 days of differentiation (Fig. 1M), after which time the expression of these markers progressively declined in lieu of more mature retinal markers (Fig. 2).

thumbnail image

Figure 2. Optic vesicle (OV)-like structures produce major neuroretinal cell types in a developmentally appropriate sequence. (A): Quantitative polymerase chain reaction analysis of isolated OV-like structures was performed every 10 days from days 20 to 120 to follow expression of genes indicative of selected neuroretinal cell types (data shown are relative to day 20 for each gene). Immunocytochemistry was used to corroborate neuroretinal cell type identity at day 80: retinal ganglion cells (B), amacrine/horizontal cells (C), bipolar cells (D), and photoreceptors (E–H).

Download figure to PowerPoint

We next performed comparative gene microarray analysis to further highlight differences in transcription factor gene expression between day 20 vesicle-like structures and nonvesicular spheres (Supporting Information Fig. S1). Many transcription factor genes associated with retinal development were present at higher levels in the vesicle-like structures, including SIX6, RAX, MITF, TBX2, TBX5, and VAX2. On the other hand, nonvesicular spheres expressed higher levels of transcription factors implicated in EFB or general neural development, including DLX1, DLX2, SOX1, and ISLET-1. Taken together, results from the comparative ICC, PCR, and microarray analyses indicated that the vesicle-like structures and nonvesicular spheres harbored RPC and EFB populations, respectively. Furthermore, the gene and protein expression profiles of the RPCs reflected a differentiation state akin to the OV stage of retinal development. Therefore, the vesicle-like structures and nonvesicular spheres were redesignated OV-like structures and EFB spheres, respectively.

Longitudinal Differentiation Analysis of OV-Like Structures and EFB Spheres

Many markers used to distinguish nonphotoreceptor cell types within the neuroretina are found elsewhere in the CNS, compromising efforts to study retinogenesis in mixed lineage pluripotent stem cell cultures. To circumvent this issue, we performed longitudinal differentiation analyses on isolated populations of OV-like structures and EFB spheres. Because OV-like structures contained a nearly exclusive population of RPCs, differentiated progeny from these cultures could be assigned to the retinal lineage with a high degree of certainty.

Cultures of WA09 hESC OV-like structures were sampled every 10 days from days 20 to 120 of differentiation and analyzed by qPCR (Fig. 2A). BRN3 and CRX, neuroretinal markers of retinal ganglion cells (RGCs) or early postmitotic photoreceptors, respectively, were among the first genes to be expressed. The CALRETININ gene (amacrine cells, horizontal cells, and some RGCs) was expressed at a nearly constant level after day 40. Later-expressed genes included NRL (postmitotic rod precursors) and PKCα (bipolar cells). The presence of multiple neuroretinal cell types was confirmed at day 80 by ICC using 1° antibodies against BRN3 and βIII-TUBULIN (Fig. 2B), CALRETININ (Fig. 2C), PKCα and CHX10 (Fig. 2D), CRX and RECOVERIN (Fig. 2E–2G), and NRL (Fig. 2H). Cells expressing photoreceptor markers often displayed a distinct morphology, bearing one thin process opposite a thicker, tubular, or conical process (Fig. 2F, 2G). ICC quantification revealed that most progeny of OV-like structures produced by day 80 expressed photoreceptor or RGC markers (CRX: 55.9% ± 6.6%; BRN3: 14.6% ± 3.2% of all cells). Immunostained sections of OV-like structures showed segregation of retinal cell populations, with most BRN3+ cells concentrated near the periphery, whereas RECOVERIN+ cells tended to congregate internally in clusters or linear configurations (Supporting Information Fig. S2).

EFB spheres maintained an anterior neural identity upon further differentiation, generating a variety of phenotypes found within the forebrain. At 20 days, EFB spheres expressed PAX6 (Fig. 3A), SOX1 (Fig. 3B), OTX2 (Fig. 3C), and βIII TUBULIN (Fig. 3A–3C). By day 70, EFB spheres yielded more mature cell phenotypes, including GABAergic neurons (Fig. 3D), tyrosine hydroxylase (TH)+ dopaminergic neurons (Fig. 3E), and glial fibrillary acidic protein+ astrocytes (Fig. 3F). No retina-specific markers were present in EFB spheres at any time point tested (up to day 70). The anterior neural character of day 20 EFB spheres was further confirmed by PCR, which revealed expression of forebrain, but not midbrain (EN-1) or hindbrain (HOXB4) genes (Fig. 3G). The expression of some of these genes was maintained at day 70 (e.g., FOXG1), while others were downregulated (e.g., PAX6 and OTX2) or upregulated (e.g., MAP2 and TH).

thumbnail image

Figure 3. Early forebrain (EFB) spheres produce multiple neural cell types. After 20 days of differentiation, EFB spheres expressed βIII-TUBULIN (A–C), PAX6 (A), SOX1 (B), and OTX2 (C). After 70 days, nearly all cells expressed markers of mature neurons or glia, including GABA, βIII-TUBULIN (D), tyrosine hydroxylase, MAP2 (E), and glial fibrillary acidic protein (F). (G): Polymerase chain reaction analysis at days 20 and 70 confirmed changes in transcription factor expression over time. Abbreviations: DAPI, 4′,6-Diamidino-2-phenylindole dihydrochloride; GFAP, glial fibrillary acidic protein; TH, tyrosine hydroxylase.

Download figure to PowerPoint

Photoreceptor-Like Cells Derived from OV-Like Structures Display Characteristic Electrophysiological Responses

Examination of electrophysiological properties offers a powerful means of evaluating both cell identity and functional capacity. WA09 hESC OV-like structures were differentiated for 100 days and plated on cover slips, whereupon cells with photoreceptor-like morphology were recorded using standard patch clamp techniques. To verify identity, recorded cells were loaded with sulforhodamine (Fig. 4A) and later immunostained for RECOVERIN (Fig. 4B). PCR analysis of these cultures confirmed expression of genes associated with phototransduction, including the cyclic nucleotide-gated channel subunits CNGA1, A3, B1, and B3, the guanylyl cyclase gene RETGC, the cGMP phosphodiesterase gene PDE6B, and ARRESTIN (Fig. 4C). RECOVERIN+ cells produced an outward positive current that was activated at depolarizing voltages between −50 and +40 mV from a holding potential of −70 mV (Fig. 4D). Upon achieving whole-cell configuration, these cells registered a resting membrane potential of −44 ± 4 mV and a current at +40 mV of 27 ± 8 pA/pF (n = 15), compared with −29 ± 2 mV and 9 ± 1 pA/pF for control, nonphotoreceptor cells (n = 3) (Fig. 4D). The current-voltage (I–V) plot revealed a large outward current with a linear I–V relationship between −10 to +40 mV, but no inward current. The voltage-dependent outward current was suppressed with 15 mM tetraethylammonium (TEA), and measurements at both +20 and +40 mV showed that the TEA-sensitive component possessed fast activation kinetics without deactivation during the 500 milliseconds voltage pulse (Fig. 4E). I–V curves confirmed the selective reduction of outward current by external TEA from an average of 468 ± 139 pA to 89 ± 25 pA (measured at +40 mV; n = 5 cells) (Fig. 4F). Given the low [Ca2+] pipette solution used for these experiments and the TEA sensitivity of the current, we concluded that delayed rectifier potassium channels were responsible for the observed voltage-dependent outward current, consistent with photoreceptor electrophysiology [31–35].

thumbnail image

Figure 4. Photoreceptor-like cells from optic vesicle-like structures display a characteristic electrophysiological signature. Cells undergoing electrophysiological analysis were loaded with sulforhodamine (A) and later immunostained to confirm photoreceptor marker expression (B). Expression of multiple genes involved in phototransduction was determined at day 80 of differentiation (C). (D): Average current density measured from 15 photoreceptor-like cells (solid circles) and three nonphotoreceptor cells (open squares) plotted against applied step voltage. (E): Ionic current responses measured at +40 and +20 mV from a representative voltage-clamped cell before (control) and after (+tetraethylammonium [TEA]) application of TEA. TEA sensitive outward currents were calculated by mathematical subtraction. (F): (I–V) curve showing average steady state current amplitudes of the photoreceptor-like cells before (solid circles) and after (solid squares) bath application of TEA. (G): Time course of the effect of 8-br-cGMP on current amplitude in a representative photoreceptor-like cell. (H): Ionic current traces obtained in response to voltage pulses delivered before (control) and during (+8-br-cGMP) administration of 8-br-cGMP. The horizontal line represents zero current. (I): (I–V) curve showing average current responses before (solid circles) and during (solid squares) 8-br-cGMP administration. Results shown are an average of four or more experiments ± SEM. Abbreviation: TEA, tetraethylammonium

Download figure to PowerPoint

In the dark, photoreceptors are maintained in a depolarized state via influx of Ca2+ and Na+ through nonselective cGMP-gated plasma membrane cation channels [36–38]. Light-stimulated hydrolysis of cGMP results in closure of these channels and membrane hyperpolarization [39]. To further test the functional identity of the photoreceptor-like cells, we exposed them to membrane-permeable 8-br-cGMP, which resulted in an instantaneous increase in inward current amplitude of more than fourfold (measured at −70 mV holding potential) (Fig. 4G), consistent with the known kinetics of cyclic nucleotide-gated ion channels [40]. A similar sustained increase in current amplitude was observed at depolarized membrane potentials (Fig. 4H, 4I). Overall, exposure to 8-br-cGMP converted the outward rectifying I–V plot to a linear I–V plot that crossed close to 0 mV (Fig. 4I), indicating a switch to nonselective ion conductance [40]. Furthermore, 8-br-cGMP treatment resulted in membrane depolarization from −44 ± 4 mV to −7.7 ± 1.8 mV, similar to the difference between the light and dark resting membrane potentials of photoreceptors [39]. These features are found solely in cells that undergo phototransduction [31, 39], and provide further evidence that RECOVERIN+ cells generated from these cultures possess an electrophysiological signature highly reminiscent of photoreceptors.

OV-Like Structures Can Be Directed to an RPE Fate

RPE was rarely observed when OV spheres were cultured in isolation, regardless of the duration of differentiation. Previous reports demonstrated that the transforming growth factor β superfamily member Activin A can promote an RPE fate at the expense of neuroretina [41, 42]. To test whether a similar effect could occur in our cultures, 100 mg/ml Activin A was added to cultures of OV-like structures from days 20 to 40. Activin A-treated cultures produced subsets of structures with deep pigmentation beginning between days 40 and 60 (Fig. 5A, 5B). Pigmented OV-like structures were manually separated from nonpigmented OV-like structures (Fig. 5C) and adhered to substrate in the presence of mitogens to promote cell proliferation. Under these conditions, cells proliferated and formed monolayers (Fig. 5D). Removal of mitogens resulted in re-establishment of cellular pigmentation, polygonal morphology (Fig. 5E), and gene and protein expression patterns characteristic of RPE (Fig. 5F and Supporting Information Fig. S3). qPCR analysis (Fig. 5G, 5H) revealed a reciprocal influence of Activin A on the expression of MITF (8.3 ± 2.5-fold increase) and CHX10 (5.3 ± 1.4-fold decrease), transcription factors associated with the development of RPE and neuroretina, respectively. Activin A-treated cultures also expressed RPE genes such as RPE65 and BEST1 at higher levels (4.9 ± 1.8-fold and 19.1 ± 5.4-fold, respectively) than untreated OV-like structures (Fig. 5G), and lower levels of genes associated with neuroretina, including CRX and BRN3B (2.4 ± 0.6-fold and 1.9 ± 0.4-fold, respectively) (Fig. 5H).

thumbnail image

Figure 5. Optic vesicle (OV)-like structures can be directed to a retinal pigment epithelium (RPE) fate. (A): RPE was rarely observed in isolated OV-like structures after 50 days of differentiation. With the addition of Activin A between days 20 and 40, a subset of these structures became pigmented (B), whereupon they could be manually isolated and cultured separately (C). Plated pigmented structures were grown in the presence of fibroblast growth factor-2, epidermal growth factor, and heparin to promote outgrowth of cells (D). Upon removal of mitogens, RPE adopted its typical appearance (E) and expressed characteristic markers (F). Activin A-treated OV-like structures expressed higher levels of RPE-associated genes (G) and lower levels of neuroretina-associated genes (H) by quantitative polymerase chain reaction. Monolayers of RPE (I) were loaded with Fura-2-acetoxymethyl ester (Fura-2 AM) and stimulated with ATP while being monitored via epiflourescence imaging (J) to record changes in [Ca2+]i over time (K). Panel (J) is an epiflourescence image of the cells shown in panel (I). Cells identified in panel (J) were used to generate the tracing in panel (K). The bar in panel (K) shows the timing of ATP administration.

Download figure to PowerPoint

To assay the functional potential of hESC-RPE cells, we chose to examine their response to ATP, a molecule postulated to govern light-induced activation of purinergic signaling pathways, leading to intracellular Ca2+ mobilization and directional fluid transport across the RPE [43]. hESC-RPE cells exhibited an increase in [Ca2+]i (58 ± 4.5 nM) immediately after exposure to 100 μM ATP, followed by a decline to steady state levels (Fig. 5I–5K). These responses were comparable with those of prenatal human RPE cultures (Supporting Information Fig. S3). Therefore, similar to other human pluripotent stem cell differentiation protocols [42, 44–46], RPE derived with our method displayed important physiological properties that can be exploited to study cell function and test therapeutics.

Production of OV-Like Structures from hiPSCs

Lineage-specific differentiation efficiencies vary across hiPSC lines, which restricts their use [7, 17, 45, 47]. A better understanding of the factors governing hiPSC differentiation potential may help optimize methods for the targeted production of neural and retinal cell types. We screened four hiPSC and two hESC lines via qPCR for expression of selected genes involved in early neurogenesis and retinogenesis. Significant variability was found in the expression levels of anterior neural/eye field genes across these lines at day 10 (Fig. 6A, 6B and Supporting Information Fig. S4). Lines displaying the lowest levels of these genes mainly produced flat, epithelioid colonies (Fig. 6C) that lacked expression of anterior neural/eye field markers (Fig. 6D).

thumbnail image

Figure 6. Human iPSC lines produce optic vesicle (OV)-like structures indistinguishable from human embryonic stem cells (hESCs). Variability was observed by quantitative polymerase chain reaction (qPCR) in the expression of anterior neural/eye field genes at day 10 of differentiation across multiple hESC and human induced pluripotent stem cell (hiPSC) lines (A, B) (Supporting Information Fig. S4), with low-expressing lines adopting a nonneural, epithelial-like identity (C, D). Higher relative expression levels of DKK1(E) and, to a lesser extent, NOGGIN(F) at day 2 correlated with higher relative expression levels of anterior neural/eye field genes at day 10. OV-like structures generated from hiPSCs (G) uniformly expressed CHX10 (H) after 20–25 days. Acquisition of an anterior neural/eye field fate at day 10 was enhanced through the addition of DKK1 and NOGGIN from days 2 to 4, as determined by qPCR (data expressed relative to untreated cultures) (I). DKK1- and NOGGIN-treated hiPSC cultures yielded colonies at day 10 with neuroepithelial morphology (J) that expressed anterior neural/eye field markers (K). Fluorescence activated cell sorting analysis demonstrated increased expression of PAX6 (L) and CHX10 (M) after 10 and 20 days of differentiation, respectively, in DKK1- and NOGGIN-treated hiPSC cultures. Abbreviations: DKK1, Dickkopf-related protein 1; hESC, human embryonic stem cell; iPSC, induced pluripotent stem cell.

Download figure to PowerPoint

We next investigated very early gene expression levels of DKK1 and NOGGIN, two pro-anterior neural/eye field factors that antagonize the canonical Wnt and BMP signaling pathways, respectively [20, 22]. Prolonged addition of one or both of these factors is common to human pluripotent stem cell neural and retinal differentiation protocols [20, 22]; however, it is unclear whether endogenous DKK1 and NOGGIN expression levels predict competency to produce anterior neural progeny. We found that hiPSC lines expressing the highest levels of DKK1, and, to a lesser extent, NOGGIN at day 2 (Fig. 6E, 6F) also expressed the highest relative levels of anterior neural/eye field genes at day 10 (Fig. 6A, 6B and Supporting Information Fig. S4) and CHX10+ OV-like structures at day 20 (Fig. 6G, 6H). OV-like structures isolated from hiPSCs were indistinguishable from those derived from hESCs and, upon further differentiation, generated neuroretinal cell types (Supporting Information Fig. S5). RPE produced from hiPSC cultures was also similar to human prenatal and hESC-derived RPE based on appearance, gene and protein expression, and physiological response to ATP (Supporting Information Fig. S3).

Given the apparent correlation between early endogenous DKK1 and NOGGIN expression and the ensuing acquisition of an anterior neural/eye field fate, we hypothesized that DKK1 and NOGGIN (or their small molecule equivalents) need only be added during a short developmental time window to augment neural and retinal specification in recalcitrant hiPSC lines. To test this theory, DKK1 (or XAV939) and NOGGIN (or Dorsomorphin) were added to our lowest anterior neural/eye field gene-expressing hiPSC line (Lenti iPSC #2) from days 2 to 4 and analyzed by qPCR 6 days later. Treated cultures expressed 84.8 ± 1.9-fold and 156.3 ± 59.5-fold higher levels of PAX6 and RAX, respectively, when compared with untreated cultures (Fig. 6I). Morphologically, treated hiPSCs produced mounded colonies of neuroepithelial cells at day 10 (Fig. 6J) that expressed anterior neural/eye field transcription factors (Fig. 6K). The neuralization of treated hiPSCs was further confirmed through fluorescence activated cell sorting (FACS) analysis, which revealed a 2.7-fold increase in the percentage of PAX6+ cells at day 10 (72.6% vs. 27.3%; Fig. 6L) and a 6.7-fold increase in the percentage of CHX10+ cells at day 20 (34.1 vs. 5.1%; Fig. 6M) when compared with untreated cultures.

OV-Like Structures from Patient-Specific iPSCs Can Be Used to Test Pharmacologic and Genetic Approaches to Retinal Disease Treatment

A reliable means of isolating RPCs from patient-specific iPSCs would facilitate the study and treatment of retinal diseases [4]. GA is a rare autosomal recessive RDD that primarily affects RPE, causing secondary photoreceptor loss and blindness [27, 48–50]. GA is caused by a wide range of defects in the gene encoding OAT, a vitamin B6-dependent enzyme that catalyzes the interconversion of L-ornithine and Δ1-pyrroline-5-carboxylate [48]. hiPSCs were derived from fibroblasts of a GA patient bearing the A226V OAT mutation (Fig. 7A, 7B) and screened for expression of pluripotency genes, ability to form teratomas, retention of the OAT gene mutation, and maintenance of a normal karyotype (Supporting Information Fig. S6).

thumbnail image

Figure 7. Differentiation and testing of human induced pluripotent stem cells (hiPSCs) derived from a patient with gyrate atrophy (GA). Fibroblasts (A) from a patient with GA due to an A226V mutation in ornithine-δ-aminotransferase (OAT) were reprogrammed to hiPSCs (B). (A226)OAT hiPSCs formed neural rosettes (C) at day 10 of differentiation that expressed anterior neural/eye field markers (D). Further maturation yielded multiple retinal cell types, including photoreceptor-like cells (E) and retinal pigment epithelium (RPE) (F). OAT activity in RPE derived from (A226)OAT hiPSCs was significantly less than that of control RPE from human prenatal eyes, human embryonic stem cells, and hiPSCs (G). OAT enzyme activity in (A226)OAT hiPSC-RPE was restored in the presence of 600 μM vitamin B6(H) or following gene repair via bacterial artificial chromosome-mediated homologous recombination (I). **, p < .0005; ***, p < .0001. Panels (G–I) are representative examples of three independent experiments. Abbreviations: OAT, ornithine-δ-aminotransferase; RPE, retinal pigment epithelium.

Download figure to PowerPoint

Using our protocol, (A226V)OAT hiPSCs formed neuroepithelial structures (Fig. 7C) and expressed anterior neural/eye field markers at day 10 (Fig. 7D). OV-like structures isolated from these cultures generated neuroretinal progeny (Fig. 7E), but rarely became pigmented in the absence of Activin A. OAT activity in RPE derived from (A226V)OAT hiPSCs (Fig. 7F) was then measured using a colorimetric assay [27]. In the presence of 50 μM vitamin B6, (A226V)OAT hiPSC-RPE had very low OAT activity (5.9 ± 3.0 μmol per hour per 106 cells; n = 3) (Fig. 7G) when compared with control RPE cultures derived from IMR90-4 hiPSCs, WA09 hESCs, and prenatal human eyes (68.9 ± 9.9, 142.7 ± 19.7, and 91.2 ± 10.0 μmol per hour per 106 cells, respectively).

Because the particular mutation in this GA patient is postulated to affect vitamin B6 binding to the enzyme [51], we next investigated whether elevated levels of vitamin B6 could restore OAT activity. Increasing vitamin B6 to 200 μM was ineffective; however, 600 μM vitamin B6 greatly enhanced enzymatic activity (24.5 ± 9.8-fold; n = 3) in (A226V)OAT hiPSC-RPE (Fig. 7H). In contrast, 600 μM vitamin B6 only increased OAT activity in control RPE cultures by 1.7 ± 0.1-fold (n = 3) and in (A226V)OAT fibroblasts by 8.1 ± 3.2-fold (n = 5). Higher levels of vitamin B6 (1200 μM) failed to increase OAT activity further.

Finally, we applied our differentiation protocol to a GA hiPSC line (iPS-12.4) that previously underwent repair of the OAT gene mutation via bacterial artificial chromosome (BAC)-mediated homologous recombination [25]. Contrary to RPE derived from the (A226V)OAT parent line (iPS-12), the gene-corrected hiPSC-RPE displayed normal levels of OAT activity (130.0 ± 12.4 μmol per hour per 106 cells; n = 3) (Fig. 7I). Therefore, OV-like structures provide a practical and versatile intermediary in the production of retinal cell types from hiPSCs.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ADDENDUM
  9. Acknowledgements
  10. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  11. REFERENCES
  12. Supporting Information

The ability to isolate tissue-specific progenitor populations from live, differentiating human pluripotent stem cells enhances their scientific and clinical use. Beyond supplying a potentially expandable and relatively uniform cell source for transplantation, such populations facilitate in vitro studies of human cellular development and disease.

The development of the vertebrate retina can be traced morphologically, beginning with the evagination of OVs from the lateral aspects of the primitive forebrain. At this stage, chick OVs can be dissected from surrounding tissues and cultured short-term as explants, during which time they maintain their characteristic vesicular structure [41]. However, a similar in vitro hallmark of early retinal populations had not been described in human pluripotent stem cell cultures prior to this report. OV-like structures isolated from hESCs and hiPSCs were anterior neuroectodermal in origin, expressed early retinal markers, and gave rise to mature retinal cell types, confirming their status as RPCs. In addition, the order of appearance of retinal cell types from OV-like structures approximated the conserved cell birth order found during vertebrate retinogenesis [10, 12].

While OV-like structures resemble true OVs in some respects, the similarities between them are limited. Unlike true OVs, hESC-derived and hiPSC-derived OV-like structures did not invaginate to form bilayered cups using the minimal culture conditions that we used. Instead, they “filled-in” and became relatively unorganized, presumably as a result of continued RPC proliferation in the absence of appropriate signaling cues. In addition, during normal vertebrate OV development, production of Chx10+ neuroretinal progenitors is restricted to the distal OV, whereas the proximal OV remains Mitf+ and yields RPE [29, 30]. This segregation of the neuroretinal and RPE domains is governed in part by reciprocal effects of FGFs and TGFβ superfamily members produced by surface ectoderm and extraocular mesenchyme, respectively [41, 52]. Similarly, canonical Wnt signaling has been shown to promote RPE generation while antagonizing formation of neuroretina [53]. The lack of RPE production in isolated OV-like structures, which are cultured in defined medium without these factors, suggests an intrinsic bias toward a neuroretinal fate. In support of this hypothesis, we found that hESC-derived sphere cultures endogenously expressed FGFs along with inhibitors of TGFβ superfamily and Wnt signaling [17]. In this study, we further showed that this neuroretinal bias could be partially overcome via the addition of the TGFβ superfamily member Activin A. An analogous situation has been described in the chick model, where OV explants grown in isolation failed to generate RPE, but did so when exposed to Activin A41.

Beyond adopting distinct morphologies and expression profiles, we also demonstrated that retinal cells derived from human pluripotent stem cells possessed important functional properties. Patch clamp recordings of RECOVERIN+ cells revealed a current-voltage relationship consistent with mammalian photoreceptors [31–35], as well as robust depolarization in response to administration of cGMP analogs. The latter finding is particularly intriguing, as cGMP is a critical second messenger of the phototransduction cascade [38] that is responsible for modulating the circulating “dark current” [39]. Although RGCs can also depolarize after cGMP stimulation [54], all cGMP-responsive cells tested in our cultures were RECOVERIN+ and lacked long neurite projections characteristic of RGCs. Therefore, the identity of the recorded cells was most consistent with photoreceptors. To document functionality of hESC-RPE and hiPSC-RPE cells in a novel manner, we examined their response to ATP, an extracellular signaling molecule linked to the generation of inositol triphosphate (IP3) and release of intracellular calcium [55]. Through this mechanism, ATP is proposed to regulate ion and fluid flux across the RPE [43], among other critical functions. Exposure of hESC-RPE and hiPSC-RPE to ATP resulted in rapid and reversible increases in intracellular calcium, analogous to responses elicited from prenatal human RPE cultures.

Although the isolation of OV-like structures from differentiating hESCs provides a dynamic tool for the study of human retinal development, it may prove more useful for hiPSC research. Multiple reports have detailed variability in fate potential between hiPSC lines [7, 17, 45, 47], some of which displayed little capacity to yield retinal cell types. In this study, we found that OV-like structures were generated by every differentiating hiPSC line tested, although sometimes in very low abundance. Even so, their distinctive appearance allowed them to be isolated and cultured independently. It was also noted that retinal differentiation efficiency across hiPSC lines correlated with early endogenous expression of factors (DKK1 and NOGGIN) known to influence anterior neural fate specification. We have since used this information to quickly and inexpensively screen new hiPSC lines for competency to produce OV-like structures.

As methods of hiPSC production and differentiation continue to improve, so will the potential to apply this technology to model and treat human diseases. Inherited RDDs appear particularly amenable to hiPSC modeling, since many exhibit gene-specific, cell autonomous features that can be tested in vitro. In addition, broad sequelae common to multiple inherited RDD, such as loss of rods, can be monitored in culture and used to assess drug efficacy [5]. For this study, we chose to derive hiPSCs from a patient with GA, an RPE-based RDD for which a simple assay exists to measure activity of the affected gene product, OAT [27]. This enzyme is expressed in multiple tissues, but has particularly high activity in RPE [27], which may explain why clinical manifestations of GA are largely restricted to the retina. We confirmed that (A226V)OAT hiPSC-RPE had very low OAT activity, and further showed that these cells were exceptionally responsive to treatment with vitamin B6, a pharmacological agent known to reduce serum ornithine levels and improve fibroblast OAT activity in a small cohort of GA patients. However, direct tests of vitamin B6 efficacy in RPE derived from a living GA patient have heretofore been unfeasible. We found that 600 μM vitamin B6, but not 200 μM, completely restored OAT activity in (A226V)OAT hiPSC-RPE, while higher concentrations showed no additional benefit. These findings are potentially important because excessive vitamin B6 supplementation can cause painful and ultimately irreversible neurological side effects [56].

Perhaps the most imminent clinical role of hiPSC technology is in pharmacologic testing of rare, genetically heterogeneous diseases that (a) display variations in drug efficacy between individuals and (b) affect tissues that cannot be routinely or safely biopsied. Even when surrogate tests of drug response are available, patients may be better served by direct testing of cell types targeted by the disease. For example, information from the repository where we obtained the GA fibroblasts indicated that the donor was vitamin B6 unresponsive. However, other patients with the A226V OAT mutation [51], as well as the donor's own hiPSC-RPE, were shown to respond to such treatment. Therefore, it is possible that the patient would have had a therapeutic response to vitamin B6 at the RPE level, but because efficacy was determined by less sensitive means, the treatment was erroneously deemed unbeneficial.

A more distant application of hiPSCs may be as a source of autologous donor cells for replacement therapies. To serve in this capacity, repair of disease-causing gene defects will likely be necessary. Recently, Howden et al. [25] corrected the OAT mutation in our patient using BAC-mediated homologous recombination and showed that this process did not introduce new mutations into the hiPSC genome. Here, we further demonstrated that OAT activity was fully restored in the gene-corrected hiPSC-RPE. To our knowledge, this is the first report of functional correction of disease-specific hiPSCs using both pharmacologic and genetic approaches.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ADDENDUM
  9. Acknowledgements
  10. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  11. REFERENCES
  12. Supporting Information

We demonstrated that vesicle-like structures corresponding to the OV stage of retinal development could be manually isolated from hESC and hiPSC lines. The production of highly enriched populations of physiologically active retinal cell types from hiPSCs provides a platform to investigate patient-specific drug effects, gene repair strategies, and disease mechanisms in vitro. However, caution must be exercised when extrapolating data obtained from cell culture systems to whole organisms. As such, hiPSC technology is envisioned to complement, not supplant, existing laboratory models of disease.

ADDENDUM

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ADDENDUM
  9. Acknowledgements
  10. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  11. REFERENCES
  12. Supporting Information

While our manuscript was under review, Eiraku et al. [57] published a report demonstrating that mouse ESCs could form OV structures, which subsequently self-organized into bilayered optic cups that produced both RPE and laminated neuroretinal tissue. Interestingly, when the mouse hESC-derived OVs were separated from attached nonretinal neuroectoderm and cultured in isolation, they failed to form optic cups and became biased to a Chx10+ neuroretinal fate, similar to our findings with isolated hESC- and hiPSC-derived OV-like structures. In light of our results and those of Eiraku et al. [57], we suspect that OV-like structures from human ESCs and iPSCs are also capable of higher order structure and organization under certain culture conditions.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ADDENDUM
  9. Acknowledgements
  10. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  11. REFERENCES
  12. Supporting Information

This work was supported by the Foundation Fighting Blindness (Wynn-Gund Translational Research Acceleration Program Award and Walsh Retinal Stem Cell Consortium to D.M.G.), research grant from NIH (R01EY21218 to D.M.G. and P30HD03352 to the UW-Madison Waisman Center), Lincy Foundation (to D.M.G.), Retina Research Foundation (RRF) Gamewell Professorship (to D.M.G.), E. Matilda Ziegler Foundation for the Blind (to D.M.G.), Rebecca Meyer Brown Professorship (to B.P.), and UW-ICTR NIH Grant 1UL1RR025011 (to B.P.). D.M.G. is a recipient of a Research to Prevent Blindness McCormick Scholar Award and the UW Eye Research Institute/RRF Murfee Chair. S.E.H. is supported by a NHMRC Overseas Biomedical Fellowship. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NEI or NIH. J.S.M. is currently affiliated with the Department of Biology, Indiana University-Purdue University Indianapolis, Indianapolis, IN.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ADDENDUM
  9. Acknowledgements
  10. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  11. REFERENCES
  12. Supporting Information

The authors declare competing financial interests. J.A.T. is a founder, stockowner, consultant, and board member of Cellular Dynamics International (CDI). He also serves as scientific advisor to and has financial interests in Tactics II Stem Cell Ventures. CDI currently has no products related to the retina. All other authors indicate no potential conflicts of interest.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ADDENDUM
  9. Acknowledgements
  10. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  11. REFERENCES
  12. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ADDENDUM
  9. Acknowledgements
  10. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  11. REFERENCES
  12. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
STEM_674_sm_SuppInfo.pdf134KSupporting Information
STEM_674_sm_SuppFig1.pdf1172KFig. S1. Differential expression of transcription factor genes in vesicle-like structures and nonvesicular spheres. Transcription factor genes with the highest relative fold differences in expression between populations of vesicle-like structures and nonvesicular spheres at day 20 of differentiation were determined by comparative microarray analysis. Transcription factors that were more highly expressed in vesicle-like structures are presented on the right side of the graph as red bars, whereas those that were more highly expressed in nonvesicular spheres are presented on the left side of the graph as blue bars.
STEM_674_sm_SuppFig2.pdf7180KFig. S2. Distribution of neuroretinal cell types within OV-like structures cultured long-term. Sections of OV-like structures cultured for a total of 70 days were immunostained using primary antibodies directed against βIII-TUBULIN (A), BRN3 (B) or RECOVERIN (C-D). βIII-TUBULIN + and BRN3+ cells (retinal ganglion cells) were commonly localized at the periphery of these structures or surrounding neural rosettes (A-B). In contrast, RECOVERIN+ cells (photoreceptor-like cells) were often found internally in clusters (C) or linear formations (D).
STEM_674_sm_SuppFig3.pdf5727KFig. S3. Comparison of RPE derived from hiPSCs, hESCs, and human prenatal eyes. Confocal images demonstrating expression of EZRIN (A) and BESTROPHIN (B) in day 70 differentiated IMR90-4 hiPSC-RPE. The orthogonal views located beneath panels A and B show the characteristic apical and basolateral expression of EZRIN and BESTROPHIN, respectively. (C) qPCR analysis comparing RPE-associated gene expression in WA09 hESC-RPE and IMR90-4 hiPSC-RPE relative to day 94 human fetal RPE. (D) RPE derived from these three sources demonstrated an appropriate increase in [Ca2+]i in response to ATP stimulation (gray bar), as determined by Fura-2 AM epiflourescence imaging. (E) Bar graph depicting the maximal [Ca2+]i responses to ATP stimulation for RPE derived from all three sources (mean ± SEM; n=3).
STEM_674_sm_SuppFig4.pdf1743KFig. S4. Differential expression of anterior neural/eye field transcription factors across human pluripotent stem cell lines. After 10 days of differentiation, the expression of selected anterior neural/eye field transcription factors was determined via qPCR. Data is expressed relative to the lowest expressing line (Lenti iPSC #2).
STEM_674_sm_SuppFig5.pdf3910KFig. S5. Generation of retinal phenotypes from hiPSC-derived OV-like structures. hiPSC-derived OV-like structures yielded progeny expressing markers of specific retinal cell types, including CRX+/RECOVERIN+ photoreceptors (A) and BRN3+ retinal ganglion cells (B). Images shown are from cultures differentiated for 80 days.
STEM_674_sm_SuppFig6.pdf8341KFig. S6. Characterization of gyrate atrophy hiPSCs. hiPSCs derived from a patient with gyrate atrophy (GA) expressed numerous pluripotency-related genes (A-C). Following transplantation into SCID mice (Yu et al., 2007), GA-hiPSCs produced teratomas containing derivatives of all three germ layers (D-F). GA-hiPSCs retained the patient's causative gene mutation (G) and maintained a normal karyotype (H). In panel G, the upper and lower chromatograms present sequencing data from wildtype WA01 hESCs and (A226V)OAT GA-hiPSCs, respectively. The shaded boxes highlight the G>A transition in the antisense strand of (A226V)OAT GA-hiPSCs, which corresponds to the C>T transition at nucleotide 677 in exon 7 of the sense strand [alanine (GCG) to valine (GTG)].
STEM_674_sm_SuppTab1.pdf52KSupplemental Table 1. Primary antibodies used for immunocytochemistry and flow cytometry.
STEM_674_sm_SuppTab2.pdf87KSupplementary Table 2. Primers used for RT-PCR and quantitative RT-PCR.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.