Combined PI3K/Akt and Smad2 Activation Promotes Corneal Endothelial Cell Proliferation

Abstract

Purpose: The purpose of this study was to develop a culture method for expansion of corneal endothelial cells (CEC) based on the combined activation of PI3K/Akt and Smad2.

Methods: Morphology, proliferation, and migration of cultured rabbit and nonhuman primate CEC were examined in the presence of the PI3K/Akt activators IGF-1 and heregulin beta in combination with the Smad2 activator activin A. Phenotypic characterization of CEC was performed at the RNA and protein levels. Cell pump function and transepithelial electric resistance were used for in vitro functional assessment of CEC. Finally, ex vivo-expanded rabbit CEC were transplanted into a model of endothelial damage in rabbit corneas.

Results: Treatment of rabbit and nonhuman primate CEC in vitro with IGF-1, heregulin beta, and activin A induced an upregulation of PI3K/Akt and Smad2 signaling pathways and an increase in proliferation and migration of CEC expressing ZO-1, connexin-43, and Na⁺/K⁺-ATPase. Cell pump function evaluation revealed the complete functionality of cultured CEC. Injection of rabbit CEC successfully produced recovery of normal corneal thickness in a rabbit model of endothelial dysfunction.

Conclusions: We demonstrated that the combined activation of PI3K/Akt and Smad2 results in in vitro expansion of phenotypic and functional CEC. Expanded cells were able to contribute to restoration of corneal endothelium in a rabbit model. These findings may represent a new therapeutic approach for treating corneal endothelial diseases.

Corneal endothelium is a single layer of cells on the inner surface of the cornea. These cells are important for preserving corneal transparency by transporting fluid out of the stroma and into the aqueous humour. Primate and human corneal endothelial cells (CEC) in vivo are arrested in the G₁ phase of the cell cycle and appear to be actively maintained in a nonproliferative state.^1,2 Actually, when the endothelium is damaged, tissue integrity is maintained by migration and enlargement of the endothelial cells bordering the lesion and not by cell mitosis.¹ Therefore, when a significant loss of CEC occurs as a result of corneal dystrophy, trauma, or disease (glaucoma or diabetes), barrier function is lost, and more fluid enters the cornea than can be removed through the activity of the ionic “pump.” Then, corneal edema and development of bullous keratopathy may occur.³ This condition is characterized by the formation of bullae in the corneal epithelium, resulting in corneal clouding and loss of visual acuity. 

Currently, over 50% of all corneal transplantations performed in the United States are performed for visual loss due to endothelial dysfunction.⁴ Penetrating keratoplasty has been the only surgical treatment for patients with corneal endothelial disorders, but recently, endothelial keratoplasty has become a viable and less aggressive alternative technique.⁵ However, both of these transplantation techniques have disadvantages, such as nonimmunologic graft failure, allograft endothelial rejection, or a global shortage of donor corneas. As an alternative to corneal transplantation, a cell therapy approach based on the transplantation of cultivated CEC would provide a new therapeutic option for the treatment of corneal endothelial dysfunction.^6,7 

Over the past few years, we and other groups have developed culture media with which to expand human CEC ex vivo.^6,8 Unfortunately, primary cultured human CEC tend to become senescent or to undergo a transition to a mesenchymal phenotype within days or after several passages depending on the donor age and culture conditions.⁹ 

In the present study, we developed a culture method for expanding CEC based on the effect of the combined activation of PI3K/Akt and Smad2. In vitro and in vivo characterization of the growth and function of rabbit and nonhuman primate CEC indicated the potential for this method to generate large numbers of CEC that could be applied clinically. Moreover, we explored the molecular mechanisms that were responsible for the induction of CEC proliferation by this novel strategy. 

Materials and Methods

Isolation and Culture of Corneal Endothelial Cells

All studies adhered to the Declaration of Helsinki. Ten primate and 10 rabbit corneas were obtained at the time animals were euthanized for other research purposes at Centro de Investigación en Farmacobiologia Aplicada (University of Navarra, Spain). All tissues were maintained at 37°C in Dulbecco's modified Eagle medium (DMEM) supplemented with fetal bovine serum (FBS; 10%; Biochrom, Berlin, Germany) and 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA) for less than 3 days before the study. 

All cell culture studies were performed at the Cell Therapy Laboratory, Clínica Universidad de Navarra, Spain. The tissue was rinsed three times with DMEM medium containing 1% penicillin-streptomycin. Descemet's membrane as well as CEC were stripped from the posterior surface of the corneal tissue under a dissecting microscope and digested at 37°C for 16 hours with 1 mg/mL collagenase A (Roche, Indianapolis, IN, USA) in medium 1, which was made of DMEM supplemented with 10% FBS, bFGF (2 ng/mL; R&D Systems, Minneapolis, MN, USA) and 1% penicillin-streptomycin.⁸ After digestion, CEC formed aggregates that were collected by centrifugation at 1800 rpm for 3 minutes to remove the digestion solution. The resultant CEC aggregates were cultured in medium 1 (DMEM supplemented with 10% FBS), bFGF (2 ng/mL), and 1% penicillin-streptomycin¹⁰; or transferred to medium 2, a modified medium consisting of knockout DMEM supplemented with 10% FBS, bFGF (2 ng/mL), 2-mercaptoethanol (0.1 mM; Gibco), l-glutamine (2 mM; Gibco), and 1% penicillin-streptomycin; or to medium 3, consisting of medium 2 plus heregulin beta (10 ng/mL; Sigma-Aldrich Corp., St. Louis, MO, USA), activin A (10 ng/mL; Miltenyi Biotec, Bergisch Gladbach, Germany), and IGF-I (200 ng/mL; Sigma-Aldrich Corp.). Cells were plated in 48-well plates coated with fibronectin-collagen coating mixture (Athena Environmental Sciences, Inc., Baltimore, MD, USA) for 10 to 13 days until they were confluent and then passaged at 1:2 ratios using 0.25% trypsin/0.02% EDTA. 

Morphology Analysis of Corneal Endothelial Cells

Cells were cultured in media 1, 2, and 3 for 10 days, and then the shapes of the cells were assessed using phase-contrast microscopy. Corneal endothelial cells of the two species were characterized with respect to their elongation ratios.¹¹ To quantify this value, the long and short axes of 30 cells from three fields were measured using ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA) and divided between themselves. 

Proliferation Assay of Corneal Endothelial Cells

Cell growth curves were derived over a 72-hour time period by seeding 5000 cells per well in a 48-well plate. First, media 1, 2, and 3 from rabbit and primate CEC were compared. The effects of the addition of each growth factor (IGF-1, heregulin beta, and activin A) to medium 2 were also assessed. On 3 consecutive days, cells were harvested by trypsinization, and cell numbers were counted with a hemocytometer under a microscope. 

Wound-Healing Assay of Corneal Endothelial Cells

Cell migration was assessed in a wound-healing assay. Rabbit and primate CEC were cultured in media 1, 2, and 3 in 48-well plates coated with fibronectin-collagen coating. Scrape wounding of confluent cell monolayers was performed using a sterile plastic pipette tip. The detached cells were rinsed away with phosphate-buffered saline (PBS), and new medium was added. Then, phase-contrast photomicrographs of the wounds were taken every 6 to 8 hours at three different locations. The width of the wound area was measured using ImageJ software, and the percentage of wound closure was calculated for each condition until the wound was completely covered by cells. 

Western Blot Analysis

Rabbit CEC were seeded at a density of 5.0 × 10³ cells/cm² in a T-25 cell culture flask (Nunc, Rochester, NY, USA) for 24 hours. Then, to analyze the activation of PI3K/Akt signaling pathway, CEC were subjected to serum starvation for an additional 24 hours, and then CEC were isolated after 1 hour in the presence of different combinations of medium 2 plus IGF-1, heregulin beta, and activin A at different concentrations. CEC proteins were extracted in a cold lysis buffer composed of 50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease and phosphatase inhibitor cocktails. Equal amounts of protein extracts (25 mg) were subjected to Western blot analysis using Akt1 (1:500 dilution; Cell Signaling, Boston, MA, USA), phosphorylated Akt (1:500 dilution; Cell Signaling), Smad 2 (1:500 dilution; Millipore Corp., Darmstadt, Germany), and phosphorylated Smad 2 and 3 (1:500 dilution; Millipore Corp.) antibodies. Results were visualized by enhanced chemiluminescence reagents and recorded (ChemiDoc XRS gel imaging system; Bio-Rad, Hercules, CA, USA). 

Immunostaining

The following antibodies were used: ZO-1 (rabbit anti-ZO-1 antibody; Invitrogen, Carlsbad, CA, USA) Na⁺/K⁺-ATPase (mouse anti-Na⁺/K⁺-ATPase a-1 [clone C464.6] antibody; Millipore Corp.), connexin 43 (mouse monoclonal anti-connexin 43, C8093; Sigma-Aldrich Corp.), NANOG (goat anti-NANOG antibody; Everest Biotech, Oxfordshire, UK), OCT4 (anti-OCT4; clone 7F9.2; product MAB4419; Millipore Corp.), SOX2 (rabbit anti-SOX2; product AB5603; Millipore Corp.), and p75 (antibody to nerve growth factor receptor; product AB-N07; Advanced Targeting Systems, San Diego, CA, USA). 

For OCT4, NANOG, SOX2, and p75NTR immunostaining, CEC from passages 2 to 3 were cultured for 24 to 48 hours to avoid cell confluency. For ZO-1, connexin 43, and Na⁺/K⁺-ATPase immunostaining, CEC from passages 2 to 3 were cultured for at least 15 days until they reached high confluency. Cells were fixed with 10% formalin for 10 minutes and permeabilized with Triton X-100 1% for 15 minutes. Cells were then preincubated in blocking 5% bovine serum albumin (BSA) and subsequently incubated overnight at 4°C in diluted primary antibody (anti-ZO-1, 1:50 dilution; anti-connexin 43, 1:500 dilution; Nanog, 1:20 dilution; OCT4, 1:100 dilution; and SOX2, 1:500 dilution). Cells were then incubated with secondary antibodies Alexa Fluor goat anti-rabbit (ZO-1, Cx43, SOX2; 1:200 dilution), Alexa Fluor goat anti-mouse (OCT4), and Alexa Fluor rat anti-goat (NANOG) for 1 hour at room temperature, after which cells were washed and mounted with 4′,6-diamino-2-phenylindole (DAPI). For Na⁺/K⁺-ATPase staining, cells were fixed in cold methanol for 10 minutes and then washed with PBS and incubated with Triton X-100 for 2 minutes. After two rinses with PBS, the primary antibody diluted in PBS (1:50) was incubated with the cells for 1 hour at room temperature. Cells were washed twice with PBS and incubated with the secondary antibody Alexa Fluor 594 goat anti-mouse for 1 hour at room temperature, washed, and mounted with DAPI. Negative controls were prepared by using nonimmune immunoglobulin G (IgG) from the same species, subtype, and concentration. Cells were observed using an inverted fluorescence microscope equipped with an epifluorescence attachment (Eclipse TS100; Nikon, Tokyo, Japan). 

Explanted rabbit corneas were fixed with methanol for 10 minutes and washed with PBS. Corneas were permeabilized for 10 minutes with Triton X-100 0.1% and then preincubated in 20% goat serum and BSA 5% in PBS for 1 hour, and with the primary antibody diluted in blocking buffer (Na⁺/K⁺-ATPase, 1:50 dilution) for 3 hours at 4°C. After two rinses with PBS, corneas were incubated with secondary antibodies at 1:200 dilution in blocking solution (ZO-1 in Alexa Fluor 488 goat anti-rabbit and Na⁺/K⁺-ATPase in Alexa Fluor 594 goat anti-mouse) for 1 hour at room temperature, washed with PBS, and mounted with DAPI. 

Total RNA Isolation and Real-time PCR

Stripped Descemet's membrane from 3 primate donor corneas were divided into 2 equal sections. One section (Descemet membrane fragments and CEC) was frozen at −80°C until RNA isolation, and the other section was used for isolation and expansion of CEC in medium 3. This approach was followed to minimize interindividual differences. Confluent primary cultures were trypsinized and pelleted. Total RNA was extracted from both sections (frozen Descemet membrane fragments and nonconfluent CEC from passages 2 and 3). RNA extraction was performed using MagMAXTM-96 Total RNA isolation kit (Thermo Fisher, Waltham, MA, USA) according to the manufacturer's protocol. RNA quantity was determined by spectrophotometric absorbance of the sample at 260 nm measurement, and purity was determined based on the ratio of 260:280 nm (A260:A280) using a NanoDrop spectrophotometer (Thermo Fisher). Total RNA sample was also analyzed on the model 2100 bioanalyzer (Agilent, Palo Alto, CA, USA). Samples were frozen at −80°C until use in microarrays. 

After extraction, RNA was checked for stability and DNA contamination by running a sample with an RNA loading dye on a 1% agarose gel, and visualization was done by staining with ethidium bromide. Lack of genomic DNA contamination was confirmed by PCR amplification of RNA samples that had not been reverse-transcribed. Only RNA samples with the A260:A280 ratio were used for further experiments. 

Reverse transcription to cDNA using Prime Script RT reagent kit (Takara, Kyoto, Japan). Real-time PCR was performed using a StepOne real-time PCR detection system (Applied Biosystems, Darmstadt, Germany) with an SYBR Premix ExTaq kit (Takara, Dalian, China), according to the manufacturer's instructions. The amplification program included an initial denaturation step at 95°C for 10 minutes, followed by denaturation at 95°C for 10 seconds, and annealing and extension at 60°C for 30 seconds for 40 cycles. SYBR green fluorescence was measured after each extension step, and the specificity of amplification was evaluated by melting curve analysis. The primers used to amplify specific gene products from primate CEC cDNA are shown in Table 1. Results of the relative quantitative real-time PCR were analyzed by using the comparative threshold method and normalized to β-actin as the internal control. 

Table 1

 

Primers Used for Real-Time Quantitative PCR in This Study

 

Measurement of Na⁺/K⁺-ATPase Pump Function and Transepithelial Electric Resistance

The pump function and transepithelial electric resistance (TEER) of rabbit and primate CEC were measured using a Millicell ERS-2 epithelial volt-ohm meter (Millipore, Billerica, VA, USA). Fresh rabbit and primate corneas deprived of epithelium served as positive controls, and empty wells served as negative controls. Fresh corneas were placed with the endothelial side up on top of Transwell (Corning, Inc., Corning, NY, USA) cell culture inserts in a 24-well plate. Rabbit and primate CEC were seeded at 100,000 cells/cm² on Transwell cell culture inserts in a 24-well plate. All Transwell inserts were incubated for 48 hours in medium 3 at 37°C with 5% CO₂. Overnight equilibration of the electrodes was performed to eliminate any offset before voltage measurements. Voltage and TEER measurements were expressed as millivolts and ohms/centimeter squared, respectively. After the steady-state levels of potential difference were measured, 1 mM Ouabain (Sigma-Aldrich Corp.) a Na⁺/K⁺-ATPase inhibitor, was added to the Transwell cell culture inserts, and the potential difference was measured again. 

Transplantation of Cultured CEC Into a Rabbit Model by Cell Injection Therapy.

A total of eight New Zealand rabbits weighing 2.0 to 2.4 kg were used in this study. All animals were treated in agreement with the Helsinki Convention on the use of animals in research and approved by the Committee for Animal Research of the University of Navarra. All animals were first anesthetized with an intramuscular injection of ketamine HCL (15 mg/kg) and medetomidine (0.25 mg/kg). Deep anesthesia was then achieved by inhalation of isoflurane (4%), and each animal received 2 drops of oxybuprocaine and tetracaine prior to inducing corneal endothelial damage. A 4-mm descemetorrhexis was performed in the central right cornea, and the anterior chamber was washed with PBS three times in all rabbits. Then, 50,000 cells labeled with 1,1′-dictadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil) (Molecular Probes, Eugene, OR, USA) were injected into the anterior chamber of the right eye in four rabbits. Briefly, passage 2 CEC were trypsinized, marked with Dil according to the manufacturer's protocol and resuspended in 0.5 mL of PBS in an insulin syringe. Thereafter, all rabbits were kept in the eye-down position for 2 hours under deep anesthesia (isoflurane 3%) to facilitate cell attachment by gravitation. After surgery local analgesia with pranoprofen (0.01%) was administered two times per day for 48 hours, azithromycin (Azydrop; Laboratoires Théa, Clermont-Ferrand, France) eye drops were administered two times per day for 3 days and dexamethasone eye drops were administered two times per day for 5 days. Each eye was checked twice a week by external examination, and photographs were taken on days 7, 14, 21, 28, and 35 after surgery. Central corneal thickness was measured using an ultrasound pachymeter (DGH Pachette 3, Exton, PA), and intraocular pressure was measured with a tonometer (TonoPen; Reichert Ophthalmic Instruments, NY, USA) on days 0, 7, 14, 21, 28, and 35 after surgery, in both eyes. An average of three readings was derived. 

Statistical Analysis

Data were analyzed using Prism 6 software (GraphPad Software, La Jolla, CA, USA) for Macintosh (Apple, Cupertino, CA, USA). Data are means ± SD. The statistical significance (P value) in mean values was determined using ANOVA and Student's t-test. The Shapiro-Wilk test was used to test for normality. Values shown on the graphs are means ± SD. A P value of <0.05 was considered statistically significant. 

Results

Culture and Expansion of CEC in Specific Media

We initially compared media 1 and 2 (which included two basic components, knockout DMEM and 2-mercaptoethanol) in rabbit and primate CEC. In contrast to medium 1, rabbit and primate CEC cultured in medium 2 formed compact and confluent monolayers with significantly lower elongation ratios (P < 0.005) (Fig. 1). Cell proliferation was also higher, and migration assay showed a faster wound closure (Figs. 2, 3). These results indicate that the combination of knockout DMEM and 2-mercaptoethanol is clearly beneficial for the shape and growth of rabbit and primate CEC. Based on these results, medium 2 was used as the basal medium with which to analyze the effect of PI3K/Akt and Smad2 activators on CEC. 

Figure 1

 

Morphology analysis of rabbit and primate CEC in media 1, 2, and 3. Rabbit and primate confluent CEC in media 1, 2, and 3 (A). Elongation ratios of rabbit (B) and primate (C) CEC in media 1, 2, and 3. Error bars are ±SD (**P < 0.05; n = 3).

Figure 2

 

Proliferation analysis of rabbit and primate CEC. Cell growth curves showed that medium 3 induced a higher proliferation rate than media 1 and 2 in rabbit (A) and primate (B) CEC. Additionally, the combination of the three main components (IGF-1, heregulin beta, and activin A) were superior each of those components separately in terms of proliferative capacity, in both the rabbit (C) and primate (D) CEC. Error bars are ±SD (n = 3).

Figure 3

 

Wound-healing assay in rabbit and primate CEC. Representative images obtained at 0, 6, and 12 hours after scrape wounding of rabbit (A) and primate (B) CEC in media 1 (M1), 2 (M2), and 3 (M3). Percentage of remaining wound area is represented in the adjacent graphs in both the rabbit (C) and primate (D) CEC. Error bars indicate ±SD (**P < 0.05; n = 3).

Next we examined the effect of combined PI3K/Akt and Smad2 activators in rabbit and primate CEC cultures. IGF-1 and heregulin beta activated PI3K/Akt signaling via phosphorylation of Akt, while the addition of activin A activated Smad2 signaling pathway via phosphorylation of Smad2. Additionally, Smad2 was down-regulated by IGF-1 and heregulin beta (Figs. 4A, 4B). In both species, CEC were smaller and exhibited a more compact and hexagonal shaped in medium 3 (Fig. 1). The elongation ratio was slightly lower when CEC were cultured in medium 3, although differences from medium 2 were not statistically significant. Rabbit and primate CEC proliferation was examined in the presence of isolated and combined IGF-1, heregulin beta, and activin A. Comparison analysis revealed that the use of combined PI3K/Akt and Smad2 activators achieved the highest proliferative capacity in both rabbit and primate CEC (Fig. 2). Culture of CEC in medium 3 was also associated with a faster wound closure in primate CEC, although not in rabbit CEC (Fig. 3). 

Figure 4

 

Western blot analysis in rabbit CEC. Western blot analysis showed that activation of PI3K/Akt was higher when IGF-1 and heregulin beta were added (A). On the other hand, activation of Smad2 was also enhanced when activin A was added, although it was regulated by IGF-1 and heregulin beta (B).

Combined Activation of PI3K/Akt and Smad2 Signaling Promotes CEC Expansion by a Cell Dedifferentiation Process

To understand the process by which activation of PI3K/Akt and Smad2 pathways induced CEC expansion and proliferation, we analyzed the expression of markers associated with stem cells and neural crest cells. Interestingly, culture of CEC in medium 3 was associated with the expression of OCT4, NANOG, and SOX2, as well as neural crest stem cell marker p75NTR (Figs. 5A–H) markers of undifferentiated stem cells. NANOG and SOX2 exhibited a combined nuclear a cytoplasmic staining pattern in most of the cells, which was confirmed using two different antibodies against NANOG and SOX2 (data not shown). On the other hand, when rabbit and primate CEC reached confluence and were maintained at high density for at least 1 week, stem cell marker expression levels became negative in CEC (Figs. 5I–P). However, cytoplasmic expression of SOX2 was observed in some rabbit CEC, although characteristic SOX2 nuclear expression was negative. 

Figure 5

 

Stem cell and neural crest stem cell marker expression levels in growing rabbit and primate CEC. Immunofluorescence staining showed expression of stem cell markers OCT4 (A, E), NANOG (B, F), and SOX2 (C, G) and neural crest stem cell marker p75NTR (D, H) in rabbit and primate nonconfluent (A–H) CEC. Stem cell marker OCT4 showed its characteristic nuclear staining. NANOG and SOX2 exhibited a combined nuclear and cytoplasmic staining. Expression of stem cell markers OCT4 (I, M), NANOG (J, N), and SOX2 (K, O) and neural crest stem cell marker p75NTR (L, P) was negative in rabbit and primate confluent (I–P) CEC. SOX2 cytoplasmic expression was positive in some rabbit CEC at confluence.

To investigate whether characteristic CEC markers were still maintained after cell expansion with medium 3, rabbit CEC were subjected to immunostaining for tight junction ZO-1, gap junction connexin-43, and Na⁺/K⁺-ATPase. Results showed that ZO-1, connexin-43, and Na⁺/K⁺-ATPase were all present after rabbit CEC expansion (Figs. 6A–C). On the other hand, the expression of characteristic CEC markers (ATP1A1, COL8A2, COL4A2, SCL4A4, and CDH2) was clearly positive in cultured primate CEC (Fig. 6D). These results, altogether suggest an expansion of CEC in the presence of IGF-1, heregulin beta, and activin A is associated with a partial dedifferentiation of the cells. 

Figure 6

 

Expression of characteristic corneal endothelial cell markers in rabbit and primate confluent CEC. Immunostaining for CEC markers ZO-1 (A), gap junction connexin-43 (B), and Na⁺/K⁺-ATPase (C) was positive in rabbit confluent CEC. RT-PCR analyses revealed high expression levels of characteristic CEC markers (ATP1A1, COL8A2, COL4A2, SCL4A4, and CDH2) in both in vivo (control) and expanded (cultured) primate CEC (D). Error bars indicate ±SD (**P < 0.05).

Evaluation of Na⁺/K⁺-ATPase Pump Function and Transepithelial Electric Resistance

In order to assess whether CEC were functional, we examined the activity of the Na⁺/K⁺-ATPase pump. The mean potential differences of rabbit and primate CEC at 0, 5, and 10 minutes were 83.3% of that for rabbit and primate donor corneas denuded of epithelium. After the Na⁺/K⁺-ATPase inhibitor ouabain was added, the potential difference and short-circuit currents reached 0 mV for all test samples within 5 min. These results indicate that the pump function of CEC (mainly depending on Na⁺/K⁺-ATPase) was satisfactory. The potential differences of empty Transwell inserts and primate donor corneas denuded of epithelium and endothelium (data not shown) were 0 mV at each time of assessment (Fig. 7). TEER measurements are shown in Table 2. 

Figure 7

 

Analysis of Na⁺/K⁺-ATPase pump function. Confluent rabbit and primate CEC cultured in medium 3 were analyzed. The mean potential differences after 0, 5, and 10 minutes were similar to the ranges for rabbit (A) and primate (B) donor corneas denuded of epithelium, indicating that the rabbit (A) and primate (B) CEC had adequate transport activity. After the Na⁺/K⁺-ATPase inhibitor ouabain was added to the chamber, the potential difference fell to 0 mV in all cases.

Table 2

 

Transepithelial Electrical Resistance Measurements of Rabbit and Primate Corneal Endothelial Cells

 

In Vivo Transplantation of Cultured Rabbit CEC

Finally, the functional capacity of CEC in vivo was determined. Rabbit CEC were transplanted by cell injection into the anterior chamber of four rabbits. Eyes transplanted with CEC recovered normal corneal thickness and almost total transparency within 3 weeks after surgery, whereas nontreated eyes remained with severe corneal edema at 5 weeks after surgery (Figs. 8A, 8C, 8D). Intraocular pressure remained constant in both groups, although an IOP elevation was observed in two of the nontreated rabbits at weeks 3 and 4 that recovered to normal values at week 5 (Fig. 8B). Flat-mount studies revealed multiple Dil-labeled cells surrounded by nonlabeled cells in the recipient cornea of treated rabbits (Fig. 8E). Furthermore, immunostaining for Na⁺/K⁺-ATPase was also positive in central corneas of treated rabbits (Fig. 8F). 

Figure 8

 

Transplantation of ex vivo-expanded rabbit CEC. Treated rabbits (n = 4) with CEC injection in the anterior chamber recovered normal corneal thickness at day 35, whereas nontreated rabbits (n = 4) remained with abnormal corneal thickness (A). Intraocular pressure remained stable in both groups (B). Severe corneal edema was persistent at day 35 in nontreated rabbits (C). In contrast, corneal transparency was almost totally recovered in treated rabbits (D). Flat-mount studies revealed several Dil-labeled cells attached to the recipient cornea of treated rabbits (E). Additionally, immunostaining for Na⁺/K⁺-ATPase was positive in central corneas (F). Error bars indicate ±SD (**P < 0.05).

Discussion

At present, more than 40,000 corneal transplantations are performed each year in the United States, and approximately 40% of them were indicated due to CEC failure. Indeed, the number of endothelial keratoplasties performed worldwide has been continuously increasing in the last 5 years. Accordingly, in order to meet the increasing demand for donor corneas, novel strategies to treat corneal endothelial disorders should be explored. In fact, transplantation of in vitro expanded CEC was reported by Mimura et al.¹² and Ishino et al.¹⁰ more than 10 years ago. However, numerous strategies have been reported in order to expand CEC in vitro,⁶ but none of them has become widely accepted. In fact, corneal endothelial cell therapy-based treatments are not yet clinically available. 

In the present work, we propose a highly efficient method for expansion of CEC based on the combined activation of PI3K/Akt and Smad2 signaling pathways. Using this strategy, we have been able to expand rabbit and primate CEC for several passages and to recover corneal transparency in rabbits with corneal endothelial decompensation by injecting CEC into the anterior chamber. 

A few years ago, Singh et al.¹³ described a signaling mechanism where simultaneous PI3K/Akt and Smad2 activity promoted human pluripotent cells self-renewal by restraining differentiation signaling and activating specific target genes, such as OCT4 and NANOG. On the other hand, it has been shown that the using FGF-2, ROCK inhibitors, or mesenchymal stem cell-derived conditioned medium induces PI3K/Akt signaling to promote CEC proliferation,^14–17 whereas Smad2 signaling activation has been related to corneal endothelial fibrosis and epithelium-to-mesenchyme transition.^18,19 Interestingly, in human pluripotent cells, Singh et al.¹³ demonstrated that PI3K/Akt suppresses Erk and Wnt signaling, allowing Smad2,-3 to activate a specific subset of target genes required for self-renewal. In contrast, in the absence of PI3K/Akt signaling, Erk and Wnt pathways are activated and effectors such as β-catenin and Snail can permit Smad2,-3 to activate genes that direct early differentiation and enothelial-to-mesenchymal transition (EMT). However, as far as we know, this combination has never been used to expand a population of differentiated cells. 

In fact, when activin A (a potent activator of Smad2 signaling pathway) was added to our basal medium (medium 2) without IGF-1 and heregulin beta (activators of PI3K/Akt signaling pathway), CEC became fibroblastic, and proliferation was completely arrested after one passage (data not shown). On the other hand, when IGF-1 was added to the basal medium without activin A, CEC morphology was better preserved, and proliferation was increased in primate but not in rabbit CEC (Figs. 2C, 2D). Heregulin beta did not have any significant effect on proliferation when used alone, but it helped to improve CEC morphology in confluence when used in combination with IGF-1. However, when activin A was combined with IGF-1 and heregulin beta, we observed a marked increase in the proliferation rate of rabbit and primate CEC (Figs. 2C, 2D). Statistically, this increase was not significant in primate CEC, as there were important differences between each animal in terms of absolute proliferation rate values. However, when CEC isolated from the same animal were compared from each medium component, medium 3 was clearly superior. Moreover, when these three components were combined, a high percentage of CEC adopted a smaller cell size and started to proliferate very actively until they reached cell confluence (Fig. 1). Next, after 1 week in confluence, CEC adopted a hexagonal shape (Fig. 1), expressed characteristic CEC markers (Fig. 6), and were fully functional (Fig. 7). 

Interestingly, levels of activin A and Smad2 signaling were regulated when IGF and heregulin were added to the culture medium (Fig. 4). In fact, Singh et al.¹³ determined that, in pluripotent stem cells, the status of PI3K/Akt signaling regulates the threshold levels of activin A and Smad2,-3 signaling to activate different subsets of target genes to impact cell fate decisions. Therefore, we hypothesize that this mechanism may explain the reason why Smad2 activation can trigger EMT in CEC if PI3K/Akt signaling is not highly activated. However, further studies with siRNA knockdown of Smad2 would provide clearer information about the mechanistic pathways involved in the effects of medium 3. 

Recently, Zhu et al.²⁰ reported a novel strategy to expand human CEC by reprogramming adult human CEC to neural crest-like progenitors through activation of the miR302b-Oct4-Sox2-Nanog network. However, they observed that disruption of intercellular junctions altered CEC morphology and transformed them into fibroblastic-like cells due to EMT. Therefore, this strategy might not be suitable to expand CEC after passage 1. In contrast, we observed that expression of stem cell markers was maintained for several passages with medium 3. Specifically, OCT4 immunofluorescence showed its characteristic nuclear staining pattern, and NANOG and SOX2 showed combined nuclear and cytoplasmic expression in both rabbit and primate CEC. Additionally, we found a faint expression of the neural crest stem cell marker p75NTR. NANOG cytoplasmic staining has been previously reported, for example, in monkey-derived embryonic stem cells²¹ or in porcine umbilical cord matrix cells.²² Similarly, cytoplasmic staining of SOX2 has also been found in oocytes.^23,24 Altogether, we support a model where the combined PI3K/Akt and Smad2 activation is able to promote cell proliferation for several passages by reprogramming mature CEC to precursor CEC. It is possible that medium 2 could also induce the expression of neural crest markers or stem cell markers in CEC. However, our aim was not to formally compare all 3 media but rather to develop a specific medium that would allow expansion of CEC and also to understand the mechanism that supported this growth. Furthermore, the fact that preliminary studies performed with human CEC indicate a limited proliferation of CEC when cultured in medium 2, led us to focus on the analysis of CEC expanded in medium 3. Whether the addition of IGF-1, heregulin beta, and activin A is solely responsible for expression of neural crest and stem cells markers remains to be definitively proven. 

When CEC reached confluence, they adopted a characteristic CEC hexagonal shape as far as maintained in high density for at least 1 week. Additionally, at this stage, we observed a negative expression of stem cell and neural crest stem cell markers (Fig. 5) and a positive expression of characteristic CEC markers (Fig. 6). Intriguingly, a positive cytoplasmic expression of SOX2 was observed in some rabbit CEC at confluence (Fig. 5K). 

Corneal endothelial cell pump function was evaluated by measuring potential differences and TEER, using an epithelial volt-ohm meter. To our knowledge, TEER assay has been previously used to assess CEC function by Goldberg's group (Bartakova A, et al. IOVS 2014;55:ARVO E-Abstract 2038). Although this method is easier to set up than the classic Ussing chamber system,²⁵ it would be interesting to compare both methods. The epithelium volt-ohm meter allows measurement of the potential difference and TEER in a 24-well plate by introducing a probe that has an outer and an inner electrode. The inner electrode is placed inside a Transwell insert that contains the confluent endothelial cell layer, while the outer electrode is placed outside the insert. Both electrodes are submerged in endothelial culture medium. This configuration allows the epithelium volt-ohm meter to measure the potential differences generated by the confluent endothelial cell layer. However, in order to validate this simple and practical method for assessing CEC function, we used rabbit and primate donor corneas denuded of epithelium as positive controls, and empty Transwell inserts and primate donor corneas denuded of epithelium and endothelium as negative controls. Additionally, Na⁺/K⁺-ATPase inhibitor ouabain was also used to reconfirm that Na⁺/K⁺-ATPase pumps were responsible for maintaining potential difference. 

Finally, expanded rabbit CEC in medium 3 were transplanted by cell injection into the anterior chamber as described by Mimura et al.²⁶ After 35 days of surgery, treated rabbits had a normal corneal thickness, and corneal transparency was almost totally recovered, whereas nontreated rabbits had a significant central corneal edema (Fig. 8). A caveat that should be kept in mind is that, unlike human CEC, rabbit CEC have the potential to proliferate and, in that sense, the model of damaged endothelium in rabbit does not faithfully reproduce human disease. Nevertheless, this rabbit injury model has proven to be useful for the assessment of CEC therapy strategies.^10,12,27,28 An alternative worth exploring before translation into patients might be the use of a nonhuman primate model which would have limitations because of cost and potential ethical difficulties. 

For this pilot study, we decided to inject only 50,000 Dillabeled cells in each eye, which is significantly less than the number previously reported by other authors (Kunzevitzky NJ, et al. IOVS 2014;55:ARVO E-Abstract 2040; Kitano J, et al. IOVS 2013;54:ARVO E-Abstract 1692).²⁶ Interestingly, we observed very good clinical results after 35 days of treatment, and no secondary effects were appreciated. However, a higher cell number injection would probably have been more beneficial and would have helped a faster corneal transparency recovery. Nevertheless, we plan to perform additional studies to define a detailed and safe protocol for this cell therapy treatment. 

In conclusion, we have presented a novel strategy for the expansion of CEC based on the simultaneous activation of PI3K/Akt and Smad2 signaling pathways. This strategy has proved to be successful in rabbit and primate CEC, and we are currently testing its efficacy in human CEC. Expanded rabbit and primate CEC were fully functional after several passages, and they were able to restore corneal endothelial dysfunction in a rabbit model. Consequently, this new strategy may represent a safe and efficient approach for treating corneal endothelial disorders and has a strong potential to replace current transplantation techniques. 

Acknowledgments

ALS is a recipient of a postdoctoral fellowship from Fundación Alfonso Martin Escudero (Spain). 

Disclosure: A.L. Sabater, None; E.J. Andreu, None; M. García-Guzmán, None; T. López, None; G. Abizanda, None; V.L. Perez, None; J. Moreno-Montañés, None; F. Prósper, None