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Keywords:

  • Induced pluripotent stem cells;
  • Retinal ganglion cell;
  • Rod photoreceptors;
  • Cone photoreceptors;
  • Retina;
  • Stem cells

Abstract

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

The direct reprogramming of somatic cells to a pluripotent state holds significant implications for treating intractable degenerative diseases by ex vivo cell therapy. In addition, the reprogrammed cells can serve as a model for diseases and the discovery of drugs and genes. Here, we demonstrate that mouse fibroblast induced pluripotent stem cells (iPSCs) represent a renewable and robust source of retinal progenitors, capable of generating a wide range of retinal cell types that includes retinal ganglion cells (RGCs), cone, and rod photoreceptors. They respond to simulated microenvironment of early and late retinal histogenesis by differentiating into stage-specific retinal cell types through the recruitment of normal mechanisms. The depth of the retinal potential of iPSCs suggests that they may be used to formulate stem cell approaches to understand and treat a wide range of retinal degenerative diseases from glaucoma to age-related macular degeneration (AMD). STEM CELLS 2010;28:695–703


INTRODUCTION

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

The identification and characterization of stem cells have led to the formulation of different approaches to potentially treat intractable degenerative changes in the retina that lead to vision loss [1, 2]. These approaches are underpinned by two defining properties of stem cells: the ability to self-renew and the plasticity to differentiate along multiple sublineages.

Despite the success in isolating and maintaining embryonic retinal stem cells/progenitors and demonstration of their differentiation into specific retinal cell types, both in vitro [3–9] and in vivo [10–16], barriers are anticipated that may make their clinical use for ex vivo cell therapy rather impractical. First, embryonic retinal stem cells/progenitors are not available in sufficient quantity to sustain clinical use because of the scarce sources of cells and because these cells possess limited self-renewal potential. Second, they do not address the important issues of immune rejection. And third, there are ethical issues for their clinical use because of the embryonic/fetal origin. These barriers are significant enough to prompt the examination of alternate sources of retinal progenitors to support ex vivo cell therapy.

Embryonic stem (ES) cells have been identified as a viable source of retinal progenitors [17–21]. However, with ES cells, the issue of immune rejection remains. Recently, a significant breakthrough toward the use of stem cells in regenerative medicine was made by direct reprogramming of differentiated somatic cells to a pluripotent state by the ectopic expression of defined transcription factors [22]. These cells have demonstrated their potential to differentiate into specific neuronal types, including dopaminergic [23] and motor [24] neurons. Although the retinal potential of induced pluripotent stem cells (iPSCs) has been demonstrated recently [25–27], the range of retinal cell types they are capable of generating the information necessary for iPSC-based therapy for glaucoma, age-related macular degeneration (AMD), and retinitis pigmentosa (RP) in which different retinal cells degenerate, remains unknown. Further, whether or not neurally-induced iPScs, like retinal progenitors, respond to stage-specific cues of the developing retina, which is a measure of fidelity of retinal induction, remains to be determined.

Retinal cells are generated by multipotent progenitors in an evolutionarily conserved temporal sequence during which RGCs, cone photoreceptors (cones), horizontal cells, and most amacrine cells are born during early retinal histogenesis, whereas bipolar cells, Muller cells, rod photoreceptors (rods), and a significant number of amacrine cells are born during late retinal histogenesis [28–30]. Here, we demonstrate that mouse fibroblast iPS cells [31] are a robust renewable source of multipotential retinal progenitors that have a range of retinal potential that encompasses generations of both early- and late-born retinal neurons in response to stage-specific developmental cues. The iPSCs respond to simulated microenvironment of early retinal histogenesis and generate RGCs and cones, cell types that tend to degenerate in glaucoma and AMD, respectively. Given the simulated microenvironment of late retinal histogenesis they differentiate into rods that specifically degenerate in RP. In both cases, the acquisition of phenotype-specific properties was accompanied by the expression of cell-type-specific regulators, suggesting the recruitment of normal mechanism for their differentiation along retinal sublineages.

MATERIALS AND METHODS

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

Cell Culture.

All experiments in this study were conducted using mouse Nanog-iPS cell line iPS-MEF-Ng-20D-17 (RIKEN, Kobe, Japan, http://www2.brc.riken.jp/.) induced from mouse embryonic fibroblasts by retroviral transfection of Oct3/4, Sox2, Klf4, and c-Myc [31]. The iPS cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 15% fetal bovine serum (FBS), 0.1 mM nonessential amino acid (NEAA), 0.1 mM 2-mercaptoethanol and 1,000U/ml mouse leukemia inhibitory factor on feeder layers of mitomycin-C-treated mouse embryonic fibroblast cells (ATCC).

Neural and Retinal Induction.

The iPSCs were differentiated into neurons by a previously described method [32]. Briefly, embyoid bodies (EBs) generated from iPSCs [33] were cultured in neural induction medium (DMEM-F12, N2 supplement, glutamine, B27 supplement, insulin, transferrin, sodium selenite, fibronectin (ITSFn) and Noggin [100 ng/ml]) for 10 days at 37°C (induction phase). The resulting colonies were trypsinized and cultured with neural expansion medium (neural induction medium + 20 ng/ml of fibroblast growth factor-2 (FGF2) (R&D Systems Inc., Minneapolis, MN, http://www.rndsystems.com) on Poly-D-lysine (PDL) and laminin-coated coverslips for 25 days (the expansion phase). The expanded cells were cultured in differentiation retinal culture medium (RCM) (DMEM/F12, N2 supplement, glutamine) containing equal volume of conditioned medium from postnatal day-1 rat retinal cells for photoreceptor differentiation or E14 rat retinal cells for retinal ganglion cells (RGC) differentiation for 10 days (differentiation phase).

FACS Analysis.

The uninduced and neurally—induced iPSCs were harvested using 0.05% trypsin/EDTA. Cells were strained using a 40-mm cell strainer and resuspended in DMEM-F12 medium. Fluorescence-activated cell sorting (FACS) analysis was performed for green fluorescent protein (GFP).

Polymerase Chain Reaction Analysis.

Polymerase chain reaction (PCR) analysis was done as previously described [34]. Briefly, total RNA was extracted from cells using the MiniRNeasy Kit (Qiagen, Hilden, Germany, http://www1.qiagen.com) according to the manufacturer's instructions. Complementary DNA synthesis was carried out on 5 μg of the total RNA/sample using the Superscript III RT kit (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) following the manufacturer's instructions. Transcripts were amplified and their levels quantified using gene-specific primers (Table 1, online supporting information) and a Quantifast SYBR Green PCR Kit (Qiagen Inc., Valencia, CA) on a RotorGene 6,000 (Corbett Robotics, San Francisco, CA, http://www.corbettlifescience.com). Measurements were performed in triplicate. A reverse-transcription-negative blank of each sample and a no-template blank served as negative controls. Amplification curves and gene expression were normalized to the housekeeping gene GAPDH, used as an internal standard.

Microarray Analysis.

Total RNA was isolated from uninduced and induced iPSCs and used to synthesize biotin-labeled cRNA probe using a Gene Chip 30 IVT Express Kit (Affymetrix, Santa Clara, CA, http://www.affymetrix.com). Fragmented cRNA probes were hybridized to Genechip Mouse genome 430 2.0 arrays (Affymetrix) at 45°C for 16 hours. The arrays were scanned using an Affymetrix GCS3000 7G device, and images were analyzed using GCOS software (Affymetrix , CA, USA, http://www.affymetrix.com/). Normalization and expression values were calculated using log scale robust multiarray analysis, implemented in BioConductor [35].

Retinal Transplantation.

Intravitreal transplantation of iPSCs was carried out as previously described [36]. Briefly, 50,000 to 100,000, carboxyfluorescein diacetate (CFDA)-labeled, neurally induced, and expanded iPSCs that had been cultured for 2 to 3 days in PN1 CM were injected in 1 μl volume into the vitreous cavity of PN1 C57Bl6 mouse pups using a glass micropipette. Eyes were enucleated after 2 and 4 weeks for immunohistochemical analysis.

Immunocytochemical Analysis.

Immunocytochemical analysis was carried out for the detection of cell-specific markers as previously described [17]. Briefly, paraformaldehyde-fixed cells or cryostat sections were incubated in Phosphate buffered saline containing 5% normal goat serum (NGS) and 0, 0.2, or 0.4% Triton-X100 followed by an overnight incubation in antibodies at 4°C. The list of antibodies used is given in Table 2 (online supporting information). Cells were examined for epifluorescence following incubation in IgG conjugated to Cy3. Images were captured using a cooled CCD-camera (Princeton Instruments NJ, USA, http://www.princetoninstruments.com/.) and Openlab software (Improvision, MA, USA, http://www.improvision.com/).

Electrophysiologic Analysis.

Electrophysiologic analysis was carried out as previously described [34]. Cells were plated on coverslips, placed in a chamber, and perfused on the stage of an upright, fixed stage microscope (Olympus BHWI, Olympus, Tokyo, Japan, http://www.olympus-global.com) with oxygenated Ames medium. Experiments were performed at room temperature. Patch pipettes were pulled on a Narishige PB-7 vertical puller from borosilicate glass pipettes (1.2 mm O.D., 0.95 mm I.D.) and had tips of 1-2 m O.D. with tip resistances of 6-12 M. Pipettes were filled with a solution containing (in mM): KCH3SO4, 98; KCl, 44; NaCl, 3; HEPES, 5; EGTA, 3 mM; MgCl2, 3; CaCl2, 1; glucose, 2; Mg-ATP, 1; GTP, one (pH 7.2). Electrophysiologic recordings were obtained using an Axopatch 200B or multiclamp amplifier (Axon Instruments/Molecular Devices Corp., Union City, CA, http://www.moleculardevices.com), and responses were acquired using a Digidata 1,322 interface and PClamp 9.2 software (Axon Instruments). Cells were voltage clamped at a steady membrane potential of −70 mV. Capacitative and leak currents were subtracted using a P/8 protocol.

RESULTS

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

Neural Induction.

Mouse iPSCs, generated from mouse fibroblasts by retrovirus-mediated inductions of OCT4, KLF4, SOX2, and cMYC [31] were cultured on mitomycin-treated fibroblasts for 5 days in the presence of leukemia inhibitory factor (LIF) until the formation of colonies consisting of GFP+ cells. The GFP+ iPSCs were subjected to established neural induction protocol [32], which had been used previously to generate retinal progenitors from the mouse ES cells [17, 37] (Fig. 1A). FACS analysis of iPSCs following neural induction revealed a decrease in the number of cells expressing GFP compared with those before induction, suggesting the attenuation of Nanog promoter activities in induced cells (Fig. 1B, 1C). To determine whether neural induction similarly affects the activities of endogenous genes of pluripotency, we examined the expression of transcripts corresponding to Oct4, Nanog, Lin28, and Sox2, all of which, with the exception of Sox2, were undetectable in induced mouse iPSCs (Fig. 1D, 1E). Transcripts corresponding to mesodermal (Brachyury) and endodermal (Alpha fetoprotein) makers were not detected (data not shown). In contrast, transcripts corresponding to neurogenic genes, Mash1, Otx2, and Nestin, with the exception of Mash1, which were not detected in uninduced iPSCs, were expressed in neurally-induced iPSCs. Together, these observations suggested that neural induction attenuated the expression of genes for pluripotency and programmed cells along neural lineage. To test the fidelity of neural induction, we cultured induced iPSCs in the neuronal differentiation medium for 10 days. Cells were detected with typical neuronal morphology expressing β-tubulin (Fig. 1F) and neurofilament (Fig. 1G), markers corresponding to immature and mature neurons, respectively. The proportion of β-tubulin+ cells was significantly greater than neurofilament+ cells (58.39 ± 6.082 vs. 30.88 ± 3.375; p = .0021). Electrophysiologic analyses of cells with neuronal morphology revealed the presence of sustained outward currents caused by outwardly rectifying K+ channels and fast inward Na+ currents (12 of 24 cells) (Fig. 1H), blocked by bath application of tetrodotoxin (TTX) (1μMB, n = 4 cells) (Fig. 1I), and restored by wash (Fig. 1J), characteristics of functional neurons. Together, these observations suggested that iPSC-derived neural progenitors differentiated into neurons as ascertained by biochemical and electrophysiologic properties.

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Figure 1. Neural induction of induced pluripotent stem cells (iPSCs). A schematic representation of neural induction protocol is given in (A). Fluorescence-activated cell sorting analysis revealed a decrease in Nanog-GFP+ cells among induced iPSCs (C), compared with uninduced cells (B). Polymerase chain reaction (PCR) analyses of gene expression. (D) Reverse transcription polymerase chain reaction (RT-PCR). (E) Quantitative PCR revealed levels of transcripts corresponding to pluripotency genes (Oct4, Nanog, and Lin28) decreased and those corresponding to neural progenitors (Sox2, Mash1, Otx2, and Nestin) increased in induced iPSCs (D, lane 2; E), compared with that in uninduced controls (D) lane 1; (E). Immunocytochemical analysis revealed a subset of induced iPSCs expressing immunoreactivities corresponding to β-tubulin (F) and neurofilament (G). Whole cell recording of induced iPSCs with neuronal morphology revealed fast-acting inward currents caused by voltage-dependent sodium channels (H), blocked by TTX (I) and restored by washout (J). (Scale bars, 50 μm in panels (F) and (G). Abbreviations: DMEM, Dulbecco's Modified Eagle's Medium; FBS, fetal bovine serum; GFP, green fluorescent protein; IMDM, Iscove's modified Dulbecco's medium; TTX, tetrodotoxin. ITSFn, Insulin Transferrin sodium selenitefibronectin

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Retinal Induction (Neural Expansion Phase).

Next, we examined whether the iPSC-derived neural progenitors possess the properties of retinal progenitors (Fig. 2). First, to promote the expansion of retinal progenitors, we cultured the induced iPSCs in FGF2 and Noggin for another 25 days. Next, we compared the transcription profiles of induced iPSCs with that of uninduced iPSCs to examine the extent of retinal imprinting (Fig. 2). The correlation of global gene expression (R2 = 0.76) suggested that the expression pattern in induced iPSCs had diverged from uninduced iPSCs (Fig. 2B). More importantly, the expression of eye field genes Rx, Pax6, Six6, Lhx2 and Chx10 [38] that characterize retinal progenitors were upregulated in induced iPSCs, compared to uninduced iPSCs. Corroborating reverse transcription polymerase chain reaction (RT-PCR) and quantitative PCR (Q-PCR) analysis demonstrated that eye field gene transcripts, undetectable in uninduced cells, were expressed in induced iPSCs (Fig. 2C, 2D). To further characterize retinal features of induced iPSCs, we carried out immunocytochemical analyses, which revealed a subset of cells expressing immunoreactivities corresponding to PAX6, RX, and CHX10. The proportion of induced iPSCs expressing PAX6, RX and CHX10 were 40.15 ± 2.415%; 28.45 ± 3.392%; 17.9 ± 2.205% respectively (Fig. 2F, 2H, 2J, 2K). Double immunocytochemical analysis revealed that 22.15 ± 5.75% of induced iPSCs coexpressed PAX6 and RX (Fig. 2H, 2K). The comparative transcription profiles and immunocytochemical analysis suggested that the induction-expansion protocol altered global gene expression of iPSCs, conferring retinal progenitor properties on them.

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Figure 2. Enrichment of retinal progenitors in the iPSCs population. A schematic representation of enrichment protocol is given in (A). A comparative global gene expression analysis of induced and expanded (retinal progenitors) and uninduced iPSCs revealed the divergence (R2 = 0.74) in expression profile between the two populations and upregulation of eye field genes, Rx, Pax6, Chx10, Six6, and Lhx2 (B). Polymerase chain reaction (PCR) analyses of gene expression. (C) Reverse transcription polymerase chain reaction (RT-PCR) and (D) quantitative PCR revealed that levels of transcripts corresponding to pluripotency genes (Oct4 and Nanog) decreased, and these corresponded to eye field genes (Rx, Pax6, Chx10, Six3, Six6, and Lhx2) upregulated in induced and expanded iPSCs (C) lane 2; (D), compared with those in uninduced controls (C) lane 1; (D). Immunocytochemical analysis of induced and expanded iPSCs revealed a subset of cells expressing immunoreactivities corresponding to PAX6 (F, K), RX (H, K) (PAX6+ RX+ cells in the inset), and CHX10 (J, K), which is characteristic of retinal progenitors (E, G, I) located in the periphery (arrowheads) of E14 retina. (Scale bars, 400 μm in panels (E, G, I) and 40 μm in panels (F, H, J). Abbreviations: DMEM, Dulbecco's Modified Eagle's Medium; FGF2, fibroblast growth factor-2; iPSCs, induced pluripotent stem cells; ITSFn, Insulin Transferrin sodium selenitefibronectin.

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Differentiation into Early-Born Retinal Neurons: Generation of Retinal Ganglion Cells.

Next, we examined the ability of the cells to respond to environment-simulating early and late retinal histogenesis [39] and to differentiate into stage-specific retinal neurons. We cultured iPSC-derived retinal progenitors in the presence of conditioned medium (CM) from E14 rat retinal cells, which are known to elaborate RGC-promoting activities that influence late retinal stem cells/progenitors to generate RGCs [3, 40] (Fig. 3). Examination of gene expression at the end of the differentiation period showed the expression of transcripts corresponding to the regulators of RGC differentiation [41], Ath5, Wt1, Brn3b, Rpf1, and Irx2, which were undetectable in iPSC-derived neural progenitors at the beginning of the differentiation period (Fig. 3B, 3C). In contrast and as expected, the levels of transcripts corresponding to progenitor markers Sox2, Rx, and Chx10 decreased in cells at the end of the differentiation period compared with those in the beginning (Fig. 3B, 3C). Immunocytochemical analysis revealed a subset of cells expressing immunoreactivities corresponding to ATH5 (26.72 ± 3.653%), BRN3b (14.3 ± 3.439%), and RPF1 (12.57 ± 1.391%), regulators that identify developing and subsets of mature RGCs [41] (Fig. 3E, 3G, 3I, 3J).

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Figure 3. Differentiation of iPSCs along RGC lineage. A schematic of differentiation protocol is given in (A). Polymerase chain reaction (PCR) analyses of gene expression. Reverse transcription polymerase chain reaction (RT-PCR) (B) and quantitative PCR (C) revealed levels of transcripts corresponding to retinal progenitor genes (Sox2, Rx, and Chx10) decreased and those corresponding to regulators of RGCs genes (Ath5, Wt1, Brn3b, Rpf1, and Irx2) upregulated in induced iPSCs cultured in E14CM (B) lane 2; (C), compared with induced and expanded progenitors (B) lane 1; (C). Immunocytochemical analysis of iPSCs cultured in E14CM revealed a subset of cells expressing immunoreactivities corresponding to ATH5 (E, J), BRN3b (G, J), and RPF1 (I, J), which is characteristic of nascent RGCs (D, F, H) located in the center (arrowheads) of E14 mouse retina. CFDA labeled induced pluripotent stem cells differentiated under E14CM expressed BRN3b and elaborated processes toward superior colliculus (SC) (K; inset and M) and not in the presence of inferior colliculus (IC) explants. Whole cell recording of iPSC-derived RGC in contact with superior colliculus cells revealed fast acting inward currents caused by voltage-dependent sodium channels (O), blocked by TTX (P) and restored by washout (Q). (Scale bars, 400 μm in panels (D, F, H) and 40 μm in panels (E, G, I). Abbreviations: DMEM, Dulbecco's Modified Eagle's Medium; FGF2, fibroblast growth factor-2; IC, inferior colliculus; iPSCs, induced pluripotent stem cells; RGC, retinal ganglion cell; SC, superior colliculus; TTX, tetradotoxin.

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Next, to determine whether iPSC-derived cells demonstrated target selectivity of RGCs and neuronal physiology, the former, labeled with CFDA, were cocultured with explants of mouse superior colliculus (SC), a target of retinal RGCs [42]. Controls consisted of iPSC-derived RGCs cocultured with mouse inferior colliculus (IC) explants. A subset of CFDA-labeled cells expressed BRN3b (18.29 ± 3.53%) and extended processes toward the SC explants (Fig. 3K–3M). Those cocultured with IC explants failed to elaborate processes at all (Fig. 3N). Whole cell recordings of CFDA-labeled cells that elaborated processes toward SC explants displayed prominent voltage-dependent sodium currents (6 of 6 cells) that were sensitive to TTX (5 of 5 cells) and recoverable after washout (5 of 5 cells) (Fig. 3O–3Q). Taken together, these results demonstrated that iPSC-derived neural progenitors could respond to cues elaborated during early retinal histogenesis and differentiate along RGC lineage with target selectivity of retinal RGCs, displaying characteristics of neurons.

Differentiation into Late-Born Retinal Neurons; Generation of Photoreceptors.

Next, we cultured iPSC-derived neural progenitors in the presence of conditioned medium (CM) from PN1 rat retinal cells (PN1CM), which are known to elaborate rod-promoting activities capable of influencing ES cells to differentiate along rod photoreceptor lineage [17] (Fig. 4A). Examination of gene expression in cells at the end of the differentiation period revealed the expression of transcripts corresponding to the photoreceptor regulators Crx and Nrl and mature markers Rhodopsin and Rhodopsin kinase, [43–44] which were either undetectable (Nrl, Rhodopsin, and Rhodopsin kinase) or expressed at lower levels (Crx) in iPSC-derived retinal progenitors at the beginning of the differentiation period (Fig. 4B, 4C). Immunocytochemical analysis revealed a subset of cells expressing immunoreactivities corresponding to cone-rod homeobox (CRX) (22.96 ± 4.535%), NRL (15.34 ± 3.082%), rhodopsin (9.18 ± 0.844%), and rhodopsin kinase (16.12 ± 2.527%) (Fig. 4E, 4G, 4I, 4K, 4L). To demonstrate the specificity of rod differentiation, iPSC-derived retinal progenitors preincubated in PN1CM were transplanted intravitreally in the eyes of PN1 C57Bl6 mouse pups [36]. Given that the transplantation was carried out in animals with no genetic or mechanical injury, very few transplanted cells were found incorporated in the host retinas [36]. Those that were found in the outer nuclear layer (<0.2% of total cells) were scattered and expressed immunoreactivities corresponding to RHODOPSIN, suggesting their differentiation along rod photoreceptor lineage in vivo. However, the grafted rhodopsin-positive cells did not display morphologic features of rod photoreceptors such as the elaboration of the outer segments, suggesting an immature stage of differentiation. That the grafted cells were not apoptotic was demonstrated by the absence of cleaved caspase-3 immunoreactivities in these cells (online supporting information Fig. 1). The retinas remained free of any tumor formation a month after transplantation.

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Figure 4. Differentiation of iPSCs along rod photoreceptor lineage: A schematic of differentiation protocol is given in (A). Polymerase chain reaction (PCR) analyses of gene expression. (B) Reverse transcription polymerase chain reaction (RT-PCR); (C) quantitative PCR revealed levels of transcripts corresponding to retinal progenitor genes (Sox2, Rx, and Chx10) decreased and those corresponding to regulators of photoreceptor genes (Crx and Nrl) and rod-specific genes (Rhodopsin and Rhodopsin kinase) upregulated in induced iPSCs cultured in PN1CM (B), lane 2; (C), compared with induced and expanded progenitors (B) lane 1, (C). Immunocytochemical analysis of iPSCs cultured in PN1CM revealed a subset of cells expressing immunoreactivities corresponding to CRX (E, L), NRL (G, L), rhodopsin (I, L), and rhodopsin kinase (K, L) which is characteristic of nascent rods (D, F, H, J) located in outer nuclear layer (ONL) of PN5 mouse retina. Induced iPSC, preincubated in PN1CM and labeled with CFDA were transplanted intravitreally in PN1 C57Bl6 mouse retina, incorporated in the ONL and expressed immunoreactivities corresponding to Rhodopsin (M). (Scale bars, 50 μm in panels (D, F, H, J) and 40 μm in panels (E, G, I, K, M). Abbreviations: DMEM, Dulbecco's Modified Eagle's Medium; FGF2, fibroblast growth factor-2; iPSCs, induced pluripotent stem cells; ONL, outer nuclear layer; RCM, retinal culture medium.

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Next, we examined whether iPSC-derived retinal progenitors have the potential to generate cones (Fig. 5). Although cones are specified during early retinal histogenesis, the expression of CRX, a general photoreceptor regulator [44] in induced iPSCs and its upregulation in differentiation conditions, suggested that, in addition to rods, the induced iPSCs might differentiate into cones. We observed both transcripts and immunoreactivities corresponding to GNAT2 and S-OPSIN in cells subjected to differentiation conditions characteristic of late histogenesis. To examine the premise of cone differentiation further, induced iPSCs were cultured in the presence of E14 retinal cell conditioned medium (E14CM), which simulated the environment of early retinal histogenesis. Transcripts corresponding to Gnat2 and S-opsin were observed upregulated, compared to neurally-expanded progenitors (Fig. 5B, 5C). Examination of cells in differentiation conditions revealed a subset expressing S-OPSIN (11.28 ± 1.467%) and GNAT2 (12.36 ± 1.653%) (Fig. 5E, 5G, 5H).

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Figure 5. Differentiation of iPSCs along cone photoreceptor lineage: A schematic of differentiation protocol is given in (A). Polymerase chain reaction (PCR) analyses of gene expression. (B) Reverse transcription polymerase chain reaction (RT-PCR); (C) Quantitative PCR revealed levels of transcripts corresponding to retinal progenitor genes (Sox2, Rx, and Chx10) decreased and those corresponding to cone photoreceptor genes (Gnat2 and s-opsin) upregulated in induced iPSCs cultured in E14CM (B) lane 2; (C), compared with induced and expanded progenitors (B) lane 1, (C). Immunocytochemical analysis of iPSCs cultured in E14CM revealed a subset of cells expressing immunoreactivities corresponding to S-OPSIN (E, H) and GNAT2 (G, H), which is characteristic of cones (D, F) located in the outer nuclear layer (ONL) of PN5 retina. Scale bars, 50 μm in panels (D, F) and 40 μm in panels (E, G). Abbreviations: DMEM, Dulbecco's Modified Eagle's Medium; FGF2, fibroblast growth factor-2; iPSCs, induced pluripotent stem cells; ONL, outer nuclear layer.

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Next we compared the relative response of induced iPSCs to E14CM and PN1CM in generating early and late retinal cell types. We observed that the number of induced iPSCs expressing immunoreactivities corresponding to early-born retinal cells, RGCs (BRN3b) and cones (GNAT2) was significantly higher (∼3 fold; p < .05; p < .001, respectively) in E14 CM than in PN1CM (Fig. 6A–6D, 6K). The preferential increase in the number of BRN3b+ and GNAT2+ cells in E14 CM was corroborated by a significant increase in the levels of Brn3b and Gnat2 transcripts in induced iPSCs in E14CM than in PN1CM (Fig. 6L). Similarly, induced iPSCs expressed higher levels of transcripts corresponding Calbindin, a marker of horizontal cells, another early-born retinal cells, in E14CM than in PN1CM, but the increase was not statistically significant (Fig. 6L). In contrast, the number of induced iPSCs expressing immunoreactivities corresponding to late-born retinal cells, bipolar cells (PKC) was significantly higher (∼3.5-fold; p < .05) in PN1CM than in E14CM (Fig. 6E, 6F, 6K). RHODOPSIN KINASE+ cells were detected only in PN1CM (Fig. 6G, 6H, 6K). The preferential increase in the number of PKC+ and RHODOPSIN KINASE+ cells was corroborated by a significant increase in the levels of transcripts corresponding to mGluR6, another bipolar cell marker, and Rhodopsin kinase (Fig. 6L). Although no significant difference was observed in the number of cells expressing immunoreactivities corresponding to Glutamine synthetase (GS), a Muller cell marker, the levels of transcripts corresponding to gs were significantly higher in induced iPSCs in PN1 CM than in E14 CM (Fig. 6I–6L). Levels of transcripts corresponding to Syntaxin1, a marker of amacrine cells, a significant number of which is generated during late retinal histogenesis, were significantly higher in induced iPSCs in PN1CM than in E14CM (Fig. 6L). Taken together, these observations suggested that induced iPSCs possess the potential to generate early- and late-born retinal cells by responding to the microenvironments specific to different stages of retinal development.

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Figure 6. Relative responses of iPSCs to E14CM and PN1CM in generating early- and late-born retinal cells. Immunocytochemical analysis of iPSCs cultured in E14CM and PN1CM revealed that the number of BRN3b+ cells (RGCs) (A, B, K) and GNAT2+ cells (cones) (C, D, K) were higher in E14CM than in PN1CM. In contrast, the number of PKC+ cells (bipolar cells) (E, F, K) was higher in PN1CM than in E14 CM, and RHODOPSIN KINASE+ cells (rods) (G, H, K) were observed only in PN1 CM. No significant difference was observed in GS+ cells (Muller cells) (I, J, K) in PN1CM and E14CM. Q-PCR analysis of iPSCs cultured in E14CM and PN1CM revealed that levels of transcript corresponding to Brn3b (RGCs) and Gnat2 (cones) were significantly higher in E14CM than in PN1 CM, Calbindin (horizontal cells) did not show any significant difference, and in contrast, those of syntaxin (amacrine cells), mGlur6 (bipolar cells), rhodopsin kinase (rods), and gs (Muller cells) were significantly higher in PN1CM than in E14CM (L). Neurally-induced iPSCs respond to conditions simulating early retinal histogenesis and generate RGCs and cones, cell types selectively degenerated in glaucoma and AMD (M). Induced iPSCs also respond to conditions simulating late retinal histogenesis and differentiate into rods that degenerate in retinitis pigmentosa (RP). Induced pluripotent stem cells-derived retinal cells can be used for ex vivo therapy or as a model for understanding retinal degeneration and screening of drugs and genes. Scale bars, 40 μm in panels (A–J); *p < .05; ** p < .01; ***p < .001. Abbreviations: AMD, age-related macular degeneration; iPSCs, induced pluripotent stem cells; RGC, retinal ganglion cell; RP, retinitis pigmentosa.

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DISCUSSION

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

The direct reprogramming of somatic cells to a pluripotent state holds significant implications for treating intractable degenerative diseases by ex vivo cell therapy [45]. In addition, the reprogrammed cells can serve as a model for diseases and discovery of drugs and genes. The initial test of these premises requires reproducible differentiation of iPSCs into cell types that are vulnerable because of disease and injury. In the retina, degeneration of specific cell types is associated with different blinding diseases. In glaucoma, retinal ganglion cells degenerate [46, 47], whereas in AMD, cones are vulnerable to the disease process, secondary to RPE degeneration [48]. In RP, rods degenerate first and cones are eventually affected [49, 50]. Therefore, if iPSCs were to be relevant in understanding and formulation of approaches to treat retinal degeneration, their retinal potential needs to be examined in the range of cell types that they could generate. Here, we demonstrate that iPSCs generated from mouse fibroblasts [31] could serve as a renewable source of retinal progenitors, which respond to a simulated environment of developing retina to generate neurons that are born during early histogenesis, that is, retinal ganglion cells and cones and late retinal histogenesis, that is, rods. The cell type-specific differentiation involved activation of cell type-specific regulatory genes, suggesting the involvement of a normal mechanism that requires interactions between cell-intrinsic and cell extrinsic factors during retinal development [51]. These observations constitute the first demonstration of the depth of the retinal potential of iPSCs and the range of retinal cell types that they are capable of generating. This suggests that these cells may support stem cell approaches to treat and understand the range of retinal degeneration encountered in glaucoma, AMD, and RP (Fig. 6M).

The implications of these observations are several. First, iPSCs-derived from patients' somatic cells can be used for ex vivo cell therapy to treat retinal degeneration, which is predicated upon successful functional engraftment in animal models. This approach will address the immunologic barrier with which cell therapy is beset. The other barrier inherent with ES and iPS cells is their potential to form teratomas. Our results suggest that this barrier may be surmounted if these cells are precommitted along a somatic lineage. This notion is supported by an earlier observation that transplantation of ES cells predifferentiated along oligodendrocytic lineage did not cause teratomas and rescued disease phenotype in an animal model of motor neuron diseases [52]. Similarly, we did not observe teratomas when we transplanted iPSCs predifferentiated along photoreceptor lineage in the retina. Second, iPSCs represent a naïve model for retinal differentiation and degeneration by recapitulation of developmental mechanism under the influence of extrinsic factors and small molecules. For example, iPSC-derived RGCs can serve as an excellent model for formulating approaches to promote de novo generated RGCs to connect with their targets. The challenge in addressing degenerative changes in glaucoma is not whether vulnerable RGCs could be replaced by ex vivo cell therapy approach, but whether nascent RGCs axons can navigate toward their centrally located targets and connect. The coculture model system of iPSC-derived RGCs and SC/IC explants holds promise to yield information for devising such approaches, and may also identify small molecules that can promote survival of vulnerable RGCs. Also, iPSCs derived from patients suffering from retinitis pigmentosa in whom mutations responsible for the disease is well characterized will yield valuable information on underlying mechanisms of photoreceptor degeneration and thus identify molecular targets for therapeutic intervention.

CONCLUSION

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

Our observations demonstrate that iPSCs represent a renewable, reliable, and robust source of retinal progenitors with the depth of potential approximating progenitors isolated from retina, generating early-born neurons (RGCs and cones) and late-born cells (rods) when exposed to stage-specific cues of the developing retina. The iPSC-derived RGCs and rods demonstrate target specificity and integrate in the ONL of the host retina, respectively. This wide range of retinal potential suggests that iPSCs may be used to formulate stem cell approaches to understand and treat a wide range of retinal degenerative diseases including glaucoma, AMD, and RP.

Acknowledgements

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

We thank Dr. Shinya Yamanaka for providing mouse fibroblast iPSCs, Dr. Anand Swaroop for Nrl antibody, Dr. Rakesh Singh for cleaved caspase-3 antibody, and Fariha Ahmed for technical help. This research was supported by the Lincy Foundation, the Pearson Foundation, the Nebraska Department of Health and Human Services, and Research to Prevent Blindness.

References

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

Supporting Information

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

Additional supporting information available online.

FilenameFormatSizeDescription
STEM_320_sm_suppinfofigure1.tif10626KSupplementary figure 1: Immunohistochemical analysis of survival of transplanted cells in PN1 C57Bl6 mouse 1 month post-transplantation. Immunohistochemical analysis of CFDA+ve transplanted cell revealed absence of cleaved caspase 3 expression (arrow) suggesting lack of apoptosis. An apoptotic cell expressing cleaved caspase 3 (arrow head). (Scale bar −40 μm).
STEM_320_sm_suppinfo.doc173KSupporting Information

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