- Open Access
Expression profiling of cell-intrinsic regulators in the process of differentiation of human iPSCs into retinal lineages
- Jen-Hua Chuang†1, 3,
- Aliaksandr A. Yarmishyn†1, 3,
- De-Kuang Hwang2, 5,
- Chih-Chien Hsu1, 5,
- Mong-Lien Wang1, 3, 5,
- Yi-Ping Yang1, 3,
- Ke-Hung Chien4,
- Shih-Hwa Chiou1, 3, 5,
- Chi-Hsien Peng†2, 6Email author and
- Shih-Jen Chen2, 4, 5Email author
© The Author(s). 2018
- Received: 9 November 2017
- Accepted: 16 March 2018
- Published: 11 May 2018
Differentiation of human induced pluripotent stem cells (hiPSCs) into retinal lineages offers great potential for medical application. Therefore, it is of crucial importance to know the key intrinsic regulators of differentiation and the specific biomarker signatures of cell lineages.
In this study, we used microarrays to analyze transcriptomes of terminally differentiated retinal ganglion cell (RGC) and retinal pigment epithelium (RPE) lineages, as well as intermediate retinal progenitor cells of optic vesicles (OVs) derived from hiPSCs. In our analysis, we specifically focused on the classes of transcripts that encode intrinsic regulators of gene expression: the transcription factors (TFs) and epigenetic chromatin state regulators. We applied two criteria for the selection of potentially important regulators and markers: firstly, the magnitude of fold-change of upregulation; secondly, the contrasted pattern of differential expression between OV, RGC and RPE lineages.
We found that among the most highly overexpressed TF-encoding genes in the OV/RGC lineage were three members of the Collier/Olfactory-1/Early B-cell family: EBF1, EBF2 and EBF3. Knockdown of EBF1 led to significant impairment of differentiation of hiPSCs into RGCs. EBF1 was shown to act upstream of ISL1 and BRN3A, the well-characterized regulators of RGC lineage specification. TF-encoding genes DLX1, DLX2 and INSM1 were the most highly overexpressed genes in the OVs, indicating their important role in the early stages of retinal differentiation. Along with MITF, the two paralogs, BHLHE41 and BHLHE40, were the most robust TF markers of RPE cells. The markedly contrasted expression of ACTL6B, encoding the component of chromatin remodeling complex SWI/SNF, discriminated hiPSC-derived OV/RGC and RPE lineages.
We identified novel, potentially important intrinsic regulators of RGC and RPE cell lineage specification in the process of differentiation from hiPSCs. We demonstrated the crucial role played by EBF1 in differentiation of RGCs. We identified intrinsic regulator biomarker signatures of these two retinal cell types that can be applied with high confidence to confirm the cell lineage identities.
- Human induced pluripotent stem cells
- Retinal ganglion cells
- Optic vesicles
- Retinal pigment epithelium
- DNA microarrays
The retina is the complex sensory tissue located at the back of an eye and is responsible for transducing light signals into nerve impulses that are conducted by the optic nerve into the visual cortex to form visual perception. The retina is organized into 10 anatomical layers and is composed of several cell types arranged in the anterior–posterior direction, such as retinal ganglion cells (RGCs); bipolar, amacrine and horizontal interneuron cells; Müller glia cells; rod and cone photoreceptor cells; and retinal pigment epithelium (RPE) cells. A number of pathological conditions is associated with the degeneration of different retinal cell types, the most prevalent of them are age-related macular degeneration (AMD), which results from the death of RPE, and glaucoma, associated with the death of RGCs. The RGCs and RPE cells are terminally differentiated cell lineages that cannot be regenerated, and therefore their progressive loss eventually leads to irreversible blindness.
The pluripotent stem cells, such as human embryonic stem cells (hESCs), offer a great potential for application in medicine as they can be directed to differentiate into virtually any cell type of an organism. This makes them an excellent tool for producing relevant cell lineages for tissue repair by transplantation, as well as for in vitro disease modeling and drug testing. The advent of technology for reprogramming somatic cells into induced pluripotent stem cells (iPSCs) by forced overexpression of stemness-associated transcription factors (TFs) further expanded the opportunities for medical application of stem cells [1, 2]. Firstly, the use of human iPSCs (hiPSCs) is not subject to the ethical concerns associated with hESCs, which are obtained from early human embryos. Secondly, using patient-derived hiPSCs allows personalized therapeutic approaches, including the reduction of risks of immune rejection by autologous transplantation of hiPSC-derived cells, as well as feasibility of gene repair for correction of inherited genetic diseases.
Stem cell-based therapy is a promising means for restoration of vision in retinal degenerative disease patients by generating and replacing missing retinal cells [3, 4]. Indeed, various studies have shown that under specifically defined culturing conditions, ESCs and iPSCs can be induced to differentiate along retinal lineages . For example, RPE cells can spontaneously differentiate from ESCs and iPSCs if FGF2 is removed from the culture medium [6, 7]. Subsequently, the efficiency of RPE differentiation was significantly improved by manipulating a number of signaling pathways that contribute to RPE specification during retinogenesis in vivo, which included the use of Wnt and Nodal antagonists [8, 9], TGF-β family member Activin A , and a combination of BMP4 antagonist Noggin, FGF2, retinoic acid and sonic hedgehog . In a protocol by Buchholz et al. , combined application of IGF1, Noggin, Activin A, DKK1, FGF2 and nicotinamide significantly accelerated the time of differentiation and achieved an efficiency of 80%. The clinical trials performed with hESC-derived RPE cells have shown their ability to improve vision in AMD patients and demonstrated a good safety profile . Unlike RPE cells, RGCs require a more complex differentiation procedure, which involves formation of 3D cell aggregates, such as embryoid bodies (EBs) and later optic vesicles (OVs), which are analogous to those formed during eye development in vivo . The modulation of signaling pathways, such as Wnt, IGF1 and TGF-β, using small molecules and growth factors added in a stepwise manner at appropriate times leads to specification of the RGC cell type [5, 14]. In a protocol by Riazifar et al. , use of the chemical inhibitor of Notch signaling pathway significantly enhanced efficiency of RGC differentiation from hESCs and hiPSCs.
Differentiation protocols are based on recapitulating the signaling pathways that normally lead to specifications of these cell types in vivo. These extrinsic cues regulate the action of intrinsic regulators, such as TFs and regulators of chromatin state, which in turn execute lineage-specific gene expression programs. At the early stages of eye development, the antagonistic expression of TFs MITF and CHX10 in different regions of OVs specifies the developmental fates toward the RPE and neural retina, respectively [16–18]. Within neural retina, specification of the RGC lineage is initiated by basic helix–loop–helix (bHLH) family TF ATOH7, which in turn regulates expression of TFs BRN3B and ISL1 [19–23]. Different TFs, such as BRN3A, EBF1, EBF3, TBR2, ONECUT1 and ONECUT2, are expressed at the downstream stages of transcriptional cascade [23–26]. By the end, the combinatorial expression of different cell-intrinsic regulators specifies the identity of RGCs and their subtypes . Whereas the process of differentiation of retinal lineages from ESCs or iPSCs mimics development in vivo, differences between these differentiation programs may exist. In order to generate the hiPSC-derived cells that are suitable for medical application, the differentiated cells should conform to the high standards of purity and safety. For this purpose, the expression of signature of markers can be useful.
In this study, we used microarray analysis to characterize the transcriptomes of RPE cells, RGCs and intermediate RGC precursors of OVs, differentiated from the same hiPSC line. We pursued two major objectives: to identify novel potentially important intrinsic regulators of RGC and RPE cell lineage specification in a process of differentiation from hiPSCs; and to identify intrinsic regulator biomarker signatures of these two retinal cell types that can be applied with high confidence to confirm the cell lineage identities. We analyzed two classes of intrinsic regulators: TFs and components of developmentally important epigenetic complexes. We used stringent criteria of the fold-change cutoff values and selected the most drastically differentially regulated genes. Secondly, we applied the criterion of contrasted expression pattern of these between samples to identify the lineage-specific genes as potential candidates for specific biomarkers.
Generation of human induced pluripotent stem cells
Sendai reprogramming vectors (SeV) reprogramming was performed using the CytoTune-iPS 2.0 Sendai Reprogramming Kit (Life Technologies) following the manufacturer’s protocol. Peripheral blood mononuclear cells (PBMCs) were obtained from a healthy male Han Chinese donor and were seeded in 24-well plates (5 × 105 cells/well) in complete PBMC medium. PBMCs were transduced with a mix of SeV vectors encoding OCT3/4, SOX2, KLF4 and c-MYC at a multiplicity of infection (MOI) of 3. The medium was changed every other day, and on day 7 post transduction, 1.25 × 105 cells were plated onto a 10-cm dish previously coated with a mouse embryonic fibroblast (MEF) feeder layer. The day after, the medium was switched to hES medium and the cells were fed every other day for 1 week before switching to the daily feeding. Once the colonies emerged, they were picked by mechanical dissection and transferred to a fresh feeder.
Differentiation of hiPSCs to RGCs
The protocol of differentiation of hiPSCs to RGCs was adapted with modifications from Riazifar et al. . The confluent hiPSCs were scraped off into small aggregates and transferred to nonadherent dishes containing MTeSR1 medium (STEMCELL Technologies), where they were cultivated for 10 days to generate embryoid bodies (EBs). In the course of cultivation, the medium was replaced every second day with 3:1 (v:v) MTeSR1:human embryonic stem (hES) medium (day 2), 1:1 (v:v) MTeSR1:hES medium (day 4), 1:3 (v:v) MTeSR1:hES medium (day 6) and hES medium (day 8). The hES medium was composed of DMEM/F12 supplemented with 1 mM nonessential amino acids, 1 mM l-glutamate, 1 mM penicillin and 1 mM streptomycin. On the 10th day, EBs were transferred to the plates coated with 0.1% gelatin and were cultivated in hES medium supplemented with 10% FBS and 10 ng/ml IGF for 7 days until neural rosettes (NRs) appeared. Following that, the NRs were scraped off into suspension and cultivated for 12 days in hES medium supplemented with 10% FBS, 2.5 μM retinoic acid (RA) and 10 μM N-(N-(3,5-difluorophenacetyl)-l-alanyl)-S-phenylglycine t-butyl ester (DAPT). At this stage the optic vesicles (OVs) have been formed, a sample of which was harvested for RNA isolation and microarray analysis. The OVs were stimulated to differentiate into RGCs by culturing for 1 day in DMEM/F12 containing N-2 supplement (Gibco), on the following day the medium was replaced with Neurobasal medium/B-27 (Gibco) supplemented with DAPT and BDNF, and on the 5th day the medium was replaced with Neurobasal/B-27. On the 15th day the differentiated RGCs were harvested for RNA isolation.
Differentiation of hiPSCs to RPE cells
The protocol of differentiation of hiPSCs to RPE cells was adapted from Buchholz et al. . hiPSCs were passaged onto Geltrex-coated dishes in DMEM/F12 supplemented with N-2 and B-27. From days 0 to 2 this base medium was supplemented with 10 ng/ml IGF1, 50 ng/ml Noggin and 10 ng/ml DKK1. From days 2 to 4 it was supplemented with 10 ng/ml IGF1, 10 ng/ml Noggin, 10 ng/ml DKK1 and 5 ng/ml bFGF. From days 4 to 6 it was supplemented with 10 ng/ml IGF1 and 10 ng/ml DKK1. From days 6 to 8 it was supplemented with 100 ng/m Activin A and 10 μM SU5402. From days 6 to 14 it was supplemented with 100 ng/ml Activin A. On day 14 the foci of the melanin-stained differentiated RPE cells were separated manually from undifferentiated cells and passaged onto a new dish. One more passage was repeated and by day 40 the RPE cells were harvested for RNA extraction and microarray analysis.
Total RNA was purified from the cell lysates of hiPSCs, OVs, RGCs and RPE cells using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. RNA was analyzed using GeneChip Human Genome U133 Plus 2.0 microarrays (Affymetrix) according to the manufacturer’s instructions. Two biological replicates were analyzed for each cell type. The microarray data analysis was performed in the R (v.3.2.2) environment using Bioconductor packages (http://www.bioconductor.org). The raw intensity data were background corrected, log2 transformed and quantile normalized using the Robust Multi-array Average (RMA) algorithm implemented by the affy package . The differentially expressed genes (DEGs) were further identified using the limma package . The probe sets that demonstrated Benjamini–Hochberg adjusted p < 0.01 and log2 fold-changes of more than 1 or less than −1 (OV vs hiPSC, RGC vs hiPSC and RPE vs hiPSC) were selected for further analysis as DEGs. The microarray data were deposited in the NCBI Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) and are accessible via the GEO accession number GSE96853. PANTHER software (version 11.1) was used for statistical overrepresentation analysis (binomial test) of GO-Slim terms, PANTHER Pathways and PANTHER Protein Classes in the lists of DEGs .
Quantitative real-time PCR
cDNA was synthesized from the total RNA samples using SuperScript III Reverse Transcriptase (Invitrogen) and random hexamer primers and was used as a template for SYBR Green qRT-PCR reactions run on the StepOnePlus Real-Time PCR System (Applied Biosystems). The primers used in this study are presented in Additional file 1.
For immunofluorescent staining, the cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. After two washes with PBS, the cells were incubated with blocking buffer (10% goat serum and 0.3% Triton X-100 in PBS) for 1 h. Primary antibodies were diluted in blocking buffer and then added to the cells for 2 h at room temperature. The following primary antibodies were used: Bestrophin, MITF, PMEL17, AMPA R2 and Tuj1 (Abcam), ZO-1 (Thermo Fisher Scientific), Math5 (Millipore) and Brn3a (Santa Cruz). The cells were then subjected to three 3-min washes with PBS and incubated with secondary antibodies Alexa Fluor 488 or Alexa Fluor 594 of goat anti-rabbit or goat anti-mouse IgG (Thermo Fisher Scientific) diluted in blocking buffer containing DAPI (Thermo Fisher Scientific) for 1 h at room temperature. After three 3-min washes with PBS, the cells were visualized using confocal microscopy (Zeiss LSM 700).
Western blot analysis
For western blot analysis, the following primary antibodies were used: PEDF, Islet1 and CECR2 (Santa Cruz), Protein S, Capthesin D, MerTK, AMPA R1, AMPA R2, Brn3a, EBF1 and EBF3 (Abcam), Myosin IIA (Sigma Aldrich), MAP2 (Cell Signaling) and GAPDH (GeneTex). The secondary antibodies used were goat anti-rabbit or goat anti-mouse IgG-HRP (Cell Signaling).
The LKO.1-puro-based shRNA constructs targeting EBF1 were obtained from National RNAi Core Facility (Taipei, Taiwan). The constructs were cotransfected with two viral packaging plasmids, pCMV-d8.91 and pMD.G, into HEK 293 T cells. The virus-containing medium was collected 24, 48 and 72 h after transfection and concentrated by centrifuging at 25,000 rpm for 2 h. The titer of virus was calculated following the method provided by RNAi core (RIU Method) (http://rnai.genmed.sinica.edu.tw/file/protocol/4_1_EstimationLentivirusTiterRIUV1.pdf). The hiPSCs were continuously infected by the virus for 2 weeks followed by puromycin selection until RGC isolation from OVs.
For patch clamp recording, the cell culture medium was replaced with artificial cerebrospinal fluid (125 mM NaCl, 25 mM NaHCO3, 1.25 mM NaH2PO4, 2.5 mM KCl, 25 mM glucose, 2 mM CaCl2 and 1 mM MgCl2). Cell-attached and whole-cell recordings were performed with patch pipettes (3–5 MΩ) pulled from borosilicate glass tubing (outer diameter 1.5 mm, inner diameter 0.86 mm; Harvard Apparatus, Holliston, MA, USA) filled with internal solution (142 mM K-gluconate, 2 mM KCl, 0.2 mM EGTA, 4 mM MgATP, 10 mM HEPES, 7 mM Na2-phosphocreatine, pH 7.3). Signals were recorded with MultiClamp 700B amplifiers or Axopatch 200B amplifiers (Molecular Devices, Union City, CA, USA). Data were filtered at 5 kHz and sampled at 10 kHz with a Digidata 1440A interface (Molecular Devices) controlled by pCLAMP version 10.2 (Molecular Devices). The recording temperature was 22–24 °C.
Differentiation of human induced pluripotent stem cells into optic vesicles and retinal ganglion cells
Differentiation of hiPSCs into RPE cells
Identification of genes differentially expressed in OV, RGC and RPE lineages
Functional enrichment analysis
Differentially regulated transcription factors
Validation of differentially expressed TF-encoding genes by qRT-PCR
Knockdown of EBF1 leads to defective RGC differentiation
Differentially regulated epigenetic factors
In recent years, several protocols have been developed to differentiate retina cell lineages from pluripotent stem cells, such as ESCs and iPSCs, which are based on recapitulating the pathways that lead to specification of retinal cell types during the natural development of an eye in an embryo . The bifurcation of the neural retina lineage from primordial retinal pigment epithelium is the key event that occurs in the early stages during eye development that gives rise to retinal neurons and RPE . These extrinsic cues regulate the action of intrinsic regulators, such as transcription factors (TFs) and regulators of chromatin state, which in turn execute lineage-specific gene expression programs. Some of these intrinsic regulators of normal eye development are well known, such as BRN3A, BRN3B and ATOH7 during RGC development and MITF during RPE development. These factors are widely used as markers to confirm specificity of cell lineages derived from iPSCs. However, their expression is not limited to the given cell type only, and therefore it is important to analyze markers in combination to define a particular cell type with high specificity. In addition, development of lineages from iPSCs can be different and involve different intrinsic regulator networks as compared to normal eye development, which may result in similar, but not equivalent, cell types. Therefore, in this study we focused on characterizing the expression patterns of various intrinsic regulators in the process of differentiation of hiPSCs into RGCs and RPE cells in order to expand the repertoire of markers for these hiPSC-derived cell types and reveal new potential key regulators of their differentiation.
In this study, we followed the most recent and efficient protocols to differentiate hiPSCs into RGCs and RPE cells [7, 15]. The identity of these cell lines was confirmed by demonstrating expression of their well-known markers by western blotting analysis and immunostaining (Figs. 1 and 2). Gene expression profiling revealed that differentiation was associated with drastic differential gene expression (Fig. 3). Functional enrichment analysis demonstrated several general patterns, such as overrepresentation of genes involved in DNA replication and cell cycle in the lists of underexpressed DEGs, which is consistent with these processes being the signatures of actively proliferating pluripotent stem cells (Fig. 4b, d, f). On the other hand, overexpressed genes common to all lineages were mostly enriched in TF-encoding genes and genes encoding cell adhesion proteins, particularly the cadherins (Fig. 4a, c, e), which is consistent with the crucial roles of TFs as intrinsic regulators of differentiation, and cadherins as key determinants of tissue morphogenesis . Remarkably, TF-encoding genes were mostly overrepresented in the upregulated transcriptome of OVs, which may reflect the heterogeneity of cell types and their different transcriptional programs, constituting this intermediate organoid. To narrow down the lists of potential key regulators of retinogenesis, we focused our microarray analysis on expression patterns of the intrinsic regulators, which we classified into two classes: TFs and epigenetic regulators. We applied two criteria for the selection of potentially important regulators and markers: fold-change of upregulation, and the contrasted pattern of differential expression between OV, RGC and RPE lineages.
For analysis of TFs, we used annotation by the AnimalTF database . Interestingly, the topmost overexpressed TFs in OV retinal progenitor cells and RGCs were three closely related paralogs of the Collier/Olfactory-1/Early B-cell (COE) family of TFs (EBF1, EBF2 and EBF3), which demonstrated on average 300–800-fold upregulation in OV and RGC lineages, as compared to the original hiPSCs, but at the same time were mostly downregulated in RPE cells. This is consistent with the study by Trimarchi et al. , who demonstrated by single cell microarray profiling that Ebf3 is one of the most highly specific biomarkers of the mouse RGCs. Later, it was shown that all four members of the COE family are expressed in mouse RGCs, and transcription of the Ebf1, Ebf2 and Ebf3 genes is regulated by BRN3B and is required but not sufficient for RGC differentiation . Here, we knocked down EFB1 and demonstrated that it is required for differentiation of hiPSCs into RGCs. We show that it acts in the early stages of OV morphogenesis, as a clearly defined neural retina precursor layer cannot form (Fig. 7c). Moreover, EBF1 acts upstream of RGC-specifying TFs BRN3A and ISL1, as their levels are downregulated upon EBF1 knockdown (Fig. 7a).
Of prominent interest are the TFs with particularly high overexpression in OVs, lower overexpression in differentiated RGCs and, at the same time, significant downregulation in RPE cells, as they are very likely to be important for the early stages of RGC differentiation from the OV RPCs. In our study, such an expression pattern was validated for the TF-encoding genes DLX1, DLX2 and INSM1 (Fig. 6a). The important role of DLX1 and DLX2 in RGC differentiation has been well characterized previously . In a recent study it was found that mouse Brn3b expression is directly regulated by DLX1 and DLX2, thus emphasizing their role in the early stages of RGC development . Given the fact that BRN3B is directly downstream of ATOH7, DLX1 and DLX2 may complement its function in initiation of RGC lineage specification. INSM1 encodes a zinc finger TF with a repressive mode of action, which has been characterized as an important regulator of neuroendocrine system development . It was shown to be required for retinal differentiation in zebrafish, where it acts upstream of ath5/atoh7 and is negatively regulated by Notch signaling . The latter observation is consistent with our data, as Notch inhibitor DAPT was used in our protocol of RGC differentiation. INSM1 has a repressive action on cell cycle and is believed to regulate the transition stage between late progenitors and nascent neurons , which is consistent with the observed expression pattern of drastic upregulation in OVs and reduced overexpression in mature RGCs (Figs. 5d and 6).
RPE cells had significantly less differentially expressed TFs, according to our highly stringent criteria, than OVs and RGCs (Fig. 5d). As expected, the most highly overexpressed TF in RPE cells was MITF, the master regulator of RPE differentiation . Interestingly, among the TF genes most highly overexpressed in RPE cells, but not in OVs or RGCs, were two paralogs, BHLHE41 and BHLHE40 (Fig. 5d). These members of the bHLH family TFs were extensively characterized as important circadian rhythm regulators; however, they were also shown to be involved in various developmental processes . Interestingly, BHLHE40 and BHLHE41 are known to negatively regulate TWIST1 , which is consistent with our data, as TWIST1 was in the top 10 of the most highly overexpressed TF-encoding genes in OVs and RGCs, but not in RPE cells.
The transcriptional activity of lineage-specific TFs is tightly linked to the chromatin state of the genome. The pluripotent stem cells undergo genome-wide chromatin remodeling during differentiation. In general, the genomes of ESCs/iPSCs are characterized by a fairly open chromatin state that is permissive for global transcription; at the same time, lineage-specific regulatory genes are maintained in a transcriptionally silenced chromatin configuration [46, 47]. In the process of differentiation, chromatin undergoes global change into a more compact, heterochromatic configuration, whereas lineage-specific regulators are transcriptionally activated [46, 47]. The chromatin state is regulated by various mechanisms that include DNA modifications (e.g., cytosine methylation), post-translational modifications of histones (e.g., methylation, acetylation and ubiquitination) and ATP-dependent remodeling of chromatin. In our study, we investigated differential expression of components of PcG (including PRC1 and PRC2) and TrxG complexes (including COMPASS/MLL and SWI/SNF), two groups of chromatin-modifying complexes that have opposing effects on transcriptional activity: the former repressive, the latter activating. In contrast to TFs, the patterns of regulation of components of epigenetic complexes were more similar between RGCs and RPE cells, which may reflect the less specific but more general requirement for epigenetic complexes to create the correct chromatin environment for the action of lineage-specific TFs. Most of the components of COMPASS/MLL complexes, that perform activating chromatin methylation H3K4-me3, were downregulated upon differentiation, which is consistent with a more permissive chromatin environment in hiPSCs.
The heterogeneous composition of various chromatin modifier complexes may define their specificity and therefore may lead to different lineage specification. For example, PCGF homologs, CBX proteins and RYBP define functionally distinct PRC1 Family Complexes . Six different classes of PRC1 complexes are defined by the identity of their six PCGF subunits, PRC1.1–PRC1.6 . Here, we show that the most overexpressed PCGF subunit was PCGF5, thus making the PRC1.5 complex the most prevalent variant in retinal lineages, which is consistent with its association with neuronal function . At the same time, PCGF6 was downregulated in differentiated retinal lineages, which is consistent with its role in embryonic stem cell maintenance .
Interestingly, among the components of SWI/SNF chromatin remodeling complexes, ACTL6B demonstrated a significantly contrasted expression pattern: it was significantly downregulated in RPE cells but, on the other hand, it was highly upregulated in the OV/RGC lineage. Given the fact that ACTL6B is a component of the neuronal-specific SWI/SNF complex (nBAF) that participates in postmitotic neural development and dendritic outgrowth , it may play a similar role in RGC development.
To summarize, we identified novel potentially important intrinsic regulators of RGC and RPE cell lineage specification in a process of differentiation from hiPSCs. In addition, we identified intrinsic regulator biomarker signatures of these two retinal cell types that can be applied with high confidence to confirm the cell lineage identities. We show that EBF1 is required for differentiation of hiPSCs into RGCs via the OV stage, and it acts upstream of the crucial regulators of RGC specification BRN3A and ISL1. This study identified a number of novel potential crucial regulators of retinal lineage specification for future characterization.
This study was funded by the Ministry of Science and Technology (MOST) (105-2633-B-009-003, and 105-3011-B010-001), Academia Sinica (Genomic Research Center and IBMS-CRC106-P02) and MOST (MOST 104-0210-01-09-02, and 105-0210-01-13-01), Taipei Veterans General Hospital (V105C-077, and V104E14-001-MY3-2), the Department of Health Cancer Center Research of Excellence (MOHW105-TDU-B-211-134003, and MOHW105-TDU-B-211-133017), TVHG and NTU joint project (102-108) , NRPB Human iPSC Alliance-Core Service (MOST 105-2325-B-010-005), VGH, TSGH, NDMC, AS Joint Research Program (VTA105-V1-5-1), and National Health Research Institutes (NHRI-106), Taiwan. This paper was particularly supported by the Ministry of Education through the SPROUT Project- Center for Intelligent Drug Systems and Smart Bio-devices (IDS2B) of National Chiao Tung University, and Cancer Progression Center of National Yang-Ming Univeristy, Taiwan.
Availability of data and materials
The microarray data were deposited in the NCBI Gene Expression Omnibus and are accessible through GEO accession number GSE96853.
JHC performed the bulk of experimental work and contributed to the draft of the manuscript; AAY performed microarray data analysis, qRT-PCR validation and drafted the manuscript; DKH, CCH, MLW, YPY and KHC participated in the study design and manuscript draft; SHC, CHP and SJC conceived the study and participated in its design and coordination. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All of the experimental procedures with hiPSCs were performed with the donor’s consent and under the supervision and approval of Taipei Veterans General Hospital Institutional Review Board under IRB authorization number 2014–06-006A.
Consent for publication
The authors declare that they have no competing interests.
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