Direct conversion of human fibroblasts into retinal pigment epithelium-like cells by defined factors
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The generation of functional retinal pigment epithelium (RPE) is of great therapeutic interest to the field of regenerative medicine and may provide possible cures for retinal degenerative diseases, including age-related macular degeneration (AMD). Although RPE cells can be produced from either embryonic stem cells or induced pluripotent stem cells, direct cell reprogramming driven by lineage-determining transcription factors provides an immediate route to their generation. By monitoring a human RPE specific Best1::GFP reporter, we report the conversion of human fibroblasts into RPE lineage using defined sets of transcription factors. We found that Best1::GFP positive cells formed colonies and exhibited morphological and molecular features of early stage RPE cells. Moreover, they were able to obtain pigmentation upon activation of Retinoic acid (RA) and Sonic Hedgehog (SHH) signaling pathways. Our study not only established an ideal platform to investigate the transcriptional network regulating the RPE cell fate determination, but also provided an alternative strategy to generate functional RPE cells that complement the use of pluripotent stem cells for disease modeling, drug screening, and cell therapy of retinal degeneration.
Keywordsretinal pigment epithelium fibroblasts direct conversion
age-related macular degeneration
embryonic stem cell
fluorescence activated cell sorting
induced pluripotent stem cell
mouse embryonic fibroblast
retinal pigment epithelium
The retinal pigment epithelium (RPE) is a pigmented monolayer of epithelium residing outside of the neurosensory retina where it supports metabolic and cellular processes of retinal photoreceptors. Dysfunction and degeneration of RPE lead to photoreceptor loss in many sight-threatening diseases, including the leading causes of blindness in the developed world, age-related macular degeneration (AMD) (Khandhadia et al., 2012). Currently, treatments available for these diseases are limited and do not offer a cure for the cell loss. Therefore, the generation of functional RPE cells is of great therapeutic interest to the field of regenerative medicine and offers possible cures for retina degeneration diseases (Schwartz et al., 2012).
Induced RPE differentiation has been achieved previously using embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) (Carr et al., 2009; Lu et al., 2009; Zhu et al., 2013). Although differentiating RPE cells from hESCs is promising (Zhang et al., 2013), the application of hESCs for therapeutic purposes remains challenging due to un-eased ethical concerns. iPSC technology holds the potential to be used to generate patient specific cells for autologous cell transplantation, which has created enormous expectations and circumvented some ethical debates (Takahashi and Yamanaka, 2006; Takahashi et al., 2007). However, differentiation of RPE cells from iPSCs is inefficient, time-consuming, and variable among different iPSC lines (Buchholz et al., 2009). Moreover, iPSCs present several safety concerns such as the genetic and epigenetic aberrations they are carrying, as well as the potential risk for tumor formation (Panopoulos et al., 2011). Recently, advances in direct cell lineage conversion have suggested a potential solution to these issues. The fact that one somatic cell type can be readily converted into another implicates access to abundant resources of any clinically relevant cell type. Moreover, as direct lineage conversion bypasses the pluripotent state, it could theoretically reduce the risk of tumorigenicity after transplantation (Ben-David and Benvenisty, 2011). Using distinct sets of transcription factors, direct lineage conversion technology has been applied to produce various cell types, including neurons, hepatocytes and neural stem cells (Vierbuchen et al., 2010; Huang et al., 2011; Kim et al., 2011; Pang et al., 2011; Sekiya and Suzuki, 2011; Giorgetti et al., 2012; Liu et al., 2012; Zhang et al., 2012).
Here we report the successful development of a RPE-specific Best1::GFP reporter, which faithfully represented human RPE lineage commitment during hESC differentiation. Using this reporter system, we show that a defined set of transcription factors can reprogram human fibroblasts into Best1::GFP+ colonies. These Best1::GFP+ cells exhibit specific morphological and molecular features of RPE lineage and are capable of pigmentation. Our study not only provided a powerful system to study the nature of cellular identity and plasticity of RPE lineage, but also offered a new path to produce functional RPE cells for regenerative therapy and drug development in the future.
Establishment of a human RPE specific reporter system
Monitoring the Best1::GFP expression represents a direct way to visualize the dynamics of RPE differentiation. In both the “Activin A” and “RA plus SHH” treatment groups, Best1::GFP+ cells were initially observed at around day 21 of differentiation, and the percentage of Best1::GFP+ cells increased to approximate 50% after 40 days of differentiation (Fig. 2C). Consistently, time-course studies showed that the levels of Best1 mRNA were strongly upregulated after 3 weeks of differentiation from H9 hESCs (Fig. 2D). Interestingly, Best1::GFP was highly expressed in non-pigmented and lightly pigmented RPE cells, while when cells were heavily pigmented, GFP fluorescence diminished or even became undetectable (Fig. 2E c–c″, d–d″). Since endogenous Best1 protein was still expressed in highly pigmented cells (Fig. 2E d–d″), the absence of GFP fluorescence in these cells may be attributed to the absorption of fluorescence by melanin. To further characterize the mature status of Best1::GFP+ cells that followed the protocol with Activin A treatment, we performed qPCR analysis at day 40. The results demonstrated that Best1::GFP+ cells did express the early eye field genes including Pax6, Lhx2, Six6 and Rax (Zuber et al., 2003) (Fig. 2F). Two key genes for RPE differentiation, Mitf and Otx2 (Boulanger et al., 2000; Steingrimsson et al., 2004), were markedly upregulated. Other mature RPE hallmark genes were also highly expressed in Best1::GFP+ cells, including Best1, pigment epithelium derived factor (Pedf), RPE-specific protein 65 kDa (Rpe65), cellular retinaldehyde-binding protein (Cralbp) involved in vitamin A metabolism, tyrosinase (Tyr) and tyrosinase related protein 2 (Tyrp2), both involved in pigment synthesis (Martinez-Morales et al., 2004; Strauss, 2005) (Fig. 2F). These gene expression patterns of Best1::GFP+ cells are similar to those of human primary RPE cells (Fig. 2F). Collectively, these data demonstrate the successful establishment of a human RPE specific reporter system, which faithfully represented RPE lineage commitment during ESC differentiation.
Definition of transcription factor pool
Best1::GFP+ cells generated by direct reprogramming of human fibroblasts
Characterization of HFF-derived Best1::GFP+ cells
Encouraged by the first report of the positive implication from prospective clinical trials transplanting hESCs derived RPE cells into two patients (Schwartz et al., 2012), RPE cells generated from pluripotent stem cells have attracted increasing attention for the promise of regenerative medicine (Zhang et al., 2013). Current methods differentiating RPE cells from human pluripotent cells include spontaneous differentiation, monolayer differentiation or 3D culture using mouse and human ESCs (Meyer et al., 2009; Osakada et al., 2009; Nakano et al., 2012). Although several of these recent methods have significantly advanced the yield and accelerated differentiation, all methods to date result in a mixture of RPE cells and neural retina cells, and thus require stringent selection before any therapeutic application. However, the only method of RPE selection described so far is the manual picking and expansion of pigmented cells, a time and labor consuming process. In this study, we generated a RPE specific reporter, which faithfully represents RPE cell differentiation and allows for easy cell sorting.
Lineage conversion of one somatic cell type to another is an alternative approach for generating specific cell types bypassing a pluripotent state. This approach has been successful in various cases, from neural lineages to cardiomyocytes and hepatocytes (Sancho-Martinez et al., 2012; Yi et al., 2012). Here we show that a small set of transcription factors can convert adult human fibroblasts into Best1::GFP positive cells, which bear many morphological and molecular features of RPE such as the expression of Mitf, Best1, Cralbp, and Tyrp2. Moreover, these cells are able to obtain pigmentation, a typical characteristic of functional RPE cells.
Although we cannot rule out the possibility that other RPE-inducing factors have been overlooked, our study indeed established a platform to study the transcriptional network that regulates the conversion from human fibroblasts to RPE cells. Consistent with the previous reports that Mitf and Otx2 are crucial for RPE development and function (Martinez-Morales et al., 2004; Bharti et al., 2006), our data showed that Mitf and Otx2 are necessary for Best1::GFP+ colony formation. Notably, mature RPE cells are polarized, highly pigmented cells which have very limited ability for proliferation, a feature which is not suitable to be used for cell transplantation (Salero et al., 2012). On the other hand, Best1::GFP+ colonies we converted from adult fibroblasts showed a progenitor-like status which is expandable. Developing an inducible expression system capable of shutting down some progenitor genes’ expression at certain time points would be crucial for controlling the switch between the proliferation and maturation status of these Best1::GFP+ cells. In addition to transcription factors, culture condition has also been highlighted as a critical factor determining the lineage conversion process (Efe et al., 2011; Kurian et al., 2013). Here in this report, we provided a matrigel based culture condition combined with RA and SHH treatment that supports the generation of pigmented cells from the Best1::GFP+ cells. Although we have not tested whether the RPE cells we obtained are fully functional, e.g. by cell transplantation in animal models, with further methodological optimization, this approach would facilitate the functional RPE cells’ generation from human fibroblasts. In summary, our findings provide a powerful system not only for studying the molecular nature of cell identity and plasticity, but also for developing therapeutic strategies for retinal degenerative diseases.
Material and methods
H9 and H1 hESCs (WiCell Research) were maintained on a layer of mitotically inactivated mouse embryonic fibroblasts (MEFs) in hESC medium (CDF12 medium): DMEM/F12 (Invitrogen) supplemented with 0.1 mmol/L non-essential amino acids (Invitrogen), 1 mmol/L GlutaMAX (Invitrogen), 20% Knockout Serum Replacement (Invitrogen), 55 μmol/L β-mercaptoethanol (Invitrogen) and 10 ng/mL bFGF (Joint Protein Central). hESCs were also cultured in mTeSR medium (StemCell Technologies). Plates were coated with Matrigel (BD Biosciences), and medium was changed daily.
Human primary RPE cells (HRPE) (Lonza) used as a positive control were cultured in basic RPE medium consisting of DMEM/F-12 supplemented with 0.1 mmol/L non-essential amino acids, 1 mmol/L GlutaMAX, 1% N2 supplement (Invitrogen) and 5% Knockout Serum Replacement (Invitrogen).
RPE differentiation from hESCs
hESCs were dissociated as cell clumps and plated on 1% ESC qualified-Matrigel for 1 h. Attached aggregates of cells were covered with 2% ESC qualified-Matrigel diluted in N2B27 medium consisting of DMEM/F-12 supplemented with 0.1 mmol/L non-essential amino acids, 1 mmol/L GlutaMAX, 1% N2 supplement (Invitrogen) and 2% B27 (Invitrogen). After overnight incubation, fresh medium without Matrigel was added and changed every other day.
For RA plus SHH treatment, Retinoic acid (RA) (500 nmol/L, Sigma) was supplemented into N2B27 medium from day 7, then gradually reduced to 200 nmol/L by day 10. From day 11 to day 14, medium was changed into N2B27 medium supplemented with Sonic Hedgehog (SHH) (25 nmol/L, Prospec). Later, medium was changed into basic RPE medium consisting of DMEM/F-12 supplemented with 0.1 mmol/L non-essential amino acids, 1 mmol/L GlutaMAX, 1% N2 supplement and 5% Knockout Serum Replacement.
For Activin A treatment, the medium was changed to DMEM supplemented with 0.1 mmol/L non-essential amino acids, 1 mmol/L GlutaMAX, 20% Knockout Serum Replacement (Invitrogen), and Activin A (100 nmol/L, Prospec) from day 10 to day 20. Later medium was changed into the basic RPE medium as described above.
Production of lentivirus and retrovirus
For generating lentiviral reporters, DNA fragments as described in Fig. 1A were obtained by PCR amplification from H1 hESC-derived genomic DNA template and cloned into pGreenZeo lentiviral vector (System bioscience). Corresponding packaging plasmids are pMDL, pCMV_VSVG and pRSV_REV. For transcription factors mediated lineage conversion, the human cDNAs listed in Fig. 3A were cloned into the pMX-gateway vector (Clontech). Coding region for mCherry was cloned as control. Corresponding packaging plasmids are pCMV-GAG-Pol and pCMV-VSV-G. HEK293T cells were seeded at a density between 6.0–8.5 × 104 cells/cm2 and transfected by Lipofectamine 2000 (Invitrogen) 16 h later. Individual supernatants containing virus were harvested at 48 h and 72 h post-transfection and filtered with 0.45 μm PVDF membrane (Millipore).
Conversion of human fibroblasts into Best1::GFP+ cells
Human foreskin fibroblasts (HFF-1, HFF-693) were plated on Matrigel-coated six-well plates at 75,000 cells per well. The next day, cells were infected with an equal ratio of a combination of eight retroviruses encoding PAX6, RAX, CRX, MITF-A, OTX2, NRL, KLF4 and c-MYC (8F) as well as pGZ-BEST1-GFP lentivirus. The plates were infected by spinfection of the cells at 1850 r/min for 1 h at room temperature in the presence of polybrene (4 μg/mL) and put back in the incubator without medium change. 24 h later, the medium was switched to CDF12 medium with medium changes every day. Cells were covered with 2% ESC qualified-Matrigel diluted in N2B27 medium at day 7. After overnight incubation, fresh RPE medium without Matrigel was added and changed every other day. At day 21, Best1::GFP+ colonies were picked and cultured in Matrigel coated 12 well plates, followed by 10 days treatment with 100 ng/mL Activin A or 500 nmol/L RA plus 25 ng/mL SHH in base RPE medium.
Cells were fixed using 4% paraformaldehyde in PBS at 4°C for 1 h. After fixation, cells were exposed to 0.3% Triton X-100 in PBS for 5 min at RT. Cells were blocked with 5% BSA in PBS for 30 min and incubated with primary antibody for 1 h at RT or overnight at 4°C. Washing was conducted with PBS followed by incubation with a corresponding secondary antibody for 1 h at RT. DAPI, was used to stain nuclei. Primary antibodies were obtained from the following sources: Mouse anti-Pax6 (1:1000, DSHB); Mouse anti-MITF (1:50, Millipore); Rabbit anti-ZO-1 (1:200, Sigma); Mouse anti-Best1 (1:200, Millipore).
Quantitative PCR (qPCR)
Total cellular RNA was isolated using Trizol reagent (Invitrogen), or RNeasy Micro Kit (Qiagen) for a small amount of cell samples according to the manufacturer’s recommendations. Total RNA was treated with 2 μg of DNase 1 (Invitrogen) and used for cDNA synthesis using iScript reverse transcription supermix (Bio-Rad). Real-time PCR was performed using the SYBR Green PCR Master Mix (Applied Biosystems). The expression levels of respective genes were normalized to corresponding GAPDH values and are shown as fold change relative to the value of the control sample. All the assays were performed in triplicate. Primer sequences are listed in Table S1.
Flow cytometry analysis and cell sorting
H9 hESCs were infected with lentiviral Best1::GFP reporter and then underwent RPE differentiation. Cells were harvested using TrypLE (Invitrogen), washed once with PBS and resuspended with 1× PBS/10% FCS medium. A minimum of 10,000 cells in the living population were analyzed by using a BD LSRII flow cytometry machine equipped with five different lasers and the BD FACSDiva software. Percentages of Best1::GFP+ cells are presented after the subtraction of isotype background and refer to the total living population analyzed. Results are representative of at least three independent experiments with a minimum of two technical replicates per experiment. For cell sorting, cells were treated as described above and sorted with a BDAria II FACS sorter (BD Biosystems).
G.H.L. is supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA01020312), National Natural Science Foundation of China (Grant Nos. 81271266, 31222039, and 31201111), the Thousand Young Talents program of China, National Laboratory of Biomacromolecules (2013kf05, and 2013kf11), and State Key Laboratory of Drug Research (SIMM1302KF-17). JCIB was supported by CIBER-BBN and grants from MINECO, Fundacion Cellex, G. Harold and Leila Y. Mathers Charitable Foundation, The Leona M. and Harry B. Helmsley Charitable Trust and The Ellison Medical Foundation.
Compliance With Ethics Guidelines
All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (Center of Regenerative Medicine in Barcelona, Spain) and with the Helsinki Declaration of 1975, as revised in 2000 (5). Informed consent was obtained from all patients for being included in the study.
Kejing Zhang, Guang-Hui Liu, Fei Yi, Nuria Montserrat, Tomoaki Hishida, Concepcion Rodriguez Esteban, Juan Carlos Izpisua Belmonte declare that they have no conflict of interest.
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