Cell Transplantation, Vol. 24, pp. 287–304, 2015 Printed in the USA. All rights reserved. Copyright © 2015 Cognizant Comm. Corp.

0963-6897/15 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096368913X675719 E-ISSN 1555-3892 www.cognizantcommunication.com

Propagation of Human Corneal Endothelial Cells: A Novel Dual Media Approach Gary S. L. Peh,* Zhenzhi Chng,† Heng-Pei Ang,* Terence Y. D. Cheng,† Khadijah Adnan,* Xin-Yi Seah,* Benjamin L. George,* Kah-Peng Toh,* Donald T. Tan,*‡§ Gary H. F. Yam,* Alan Colman,† and Jodhbir S. Mehta*‡¶ *Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore, Singapore †A*STAR Institute of Medical Biology, Singapore, Singapore ‡Singapore National Eye Centre, Singapore, Singapore §Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore ¶Duke Medical School of Medicine, National University of Singapore, Singapore, Singapore

Corneal endothelium-associated corneal blindness is the most common indication for corneal transplantation. Restorative corneal transplant surgery is the only option to reverse the blindness, but a global shortage of donor material remains an issue. There are immense clinical interests in the development of alternative treatment strategies to alleviate current reliance on donor materials. For such endeavors, ex vivo propagation of human corneal endothelial cells (hCECs) is required, but current methodology lacks consistency, with expanded hCECs losing cellular morphology to a mesenchymal-like transformation. In this study, we describe a novel dual media culture approach for the in vitro expansion of primary hCECs. Initial characterization included analysis of growth dynamics of hCECs grown in either proliferative (M4) or maintenance (M5) medium. Subsequent comparisons were performed on isolated hCECs cultured in M4 alone against cells expanded using the dual media approach. Further characterizations were performed using immunocytochemistry, quantitative real-time PCR, and gene expression microarray. At the third passage, results showed that hCECs propagated using the dual media approach were homogeneous in appearance, retained their unique polygonal cellular morphology, and expressed higher levels of corneal endothelium-associated markers in comparison to hCECs cultured in M4 alone, which were heterogeneous and fibroblastic in appearance. Finally, for hCECs cultured using the dual media approach, global gene expression and pathway analysis between confluent hCECs before and after 7-day exposure to M5 exhibited differential gene expression associated predominately with cell proliferation and wound healing. These findings showed that the propagation of primary hCECs using the novel dual media approach presented in this study is a consistent method to obtain bona fide hCECs. This, in turn, will elicit greater confidence in facilitating downstream development of alternative corneal endothelium replacement using tissue-engineered graft materials or cell injection therapy. Key words: Cornea; Human corneal endothelial cells (hCECs); Cell culture; Cell transplantation; Tissue engineering

INTRODUCTION

edema, corneal clouding, and a loss of visual acuity, which will eventually result in corneal blindness. Restorative corneal transplant surgery, either by full thickness penetrating keratoplasty or selective replacement of the ineffective corneal endothelium by endothelial keratoplasty (EK) with a functional donor corneal endothelium, is the only option to restore vision (36,53). Selective transplantation of the donor corneal endothelium using less invasive sutureless keyhole EK options, such as Descemet’s stripping endothelial keratoplasty (DSEK) (45) and Descemet’s membrane endothelial keratoplasty (DMEK) (38), involves the transplantation of a very thin corneal endothelial layer

The corneal endothelium is the innermost layer of the human cornea. This cellular monolayer is an important “barrier and pump” that regulates corneal hydration to keep the cornea transparent (4). It is known that corneal endothelial cells (CECs) are arrested in the G1 phase of the cell cycle and do not undergo active cellular regeneration within the eye (26,27). Hence, in situations where they become damaged or the cell density falls below a critical threshold, the functional dynamics of the corneal endothelium will be compromised (41). This leads to a cascade of pathological events, beginning with stromal

Received September 3, 2013; final acceptance November 20, 2013. Online prepub date: November 21, 2013. Address correspondence to Adj. Assoc. Professor Jodhbir Mehta, Singapore National Eye Centre, 11 Third Hospital Avenue, #08-00, Singapore 168751, Singapore. Tel: +65 6322 4571; Fax: +65 6323 1903; E-mail: [email protected]

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instead of using a full-thickness cornea. With the rapid advancements in EK techniques over the past decade, surgical outcomes of such selective replacement of the dysfunctional corneal endothelial layer have significantly improved (49,53). However, as current corneal endothelial transplant is a one-to-one donor-to-recipient surgery, the shortage of available donor corneal graft tissues remains a problem. This is an increasingly pertinent multifaceted global issue driven further by (a) the process involved in the stringent assessments of donor corneal tissues that may render a potential donor cornea unsuitable for transplantation (41), (b) a potential need for regrafting procedures following graft failures as a result of infection or nonimmunologic/immunological endothelial decompensation (5,37), and (c) a global aging population that reduces the potential donor pool while potentially increasing the demand for corneal transplantation (41). Although human corneal endothelial cells (hCECs) are not known to be proliferative within the eye, the limited in vitro expansion of isolated CECs has been demonstrated in several laboratories, including ours (2,10,24,34, 44). Consistency in the culture and expansion of primary hCECs is a significant issue, affected by factors such as donor-to-donor variation (63); different isolation protocols, which may affect the overall yield of hCECs; as well as the use of different complex serum-supplemented culture media as reported in various studies (41,44). Although a robust and clearly described culture methodology is still lacking, a great amount of clinical interest has been generated for the development of alternative approaches in the treatment or in reversing the effect of corneal endothelial decompensation using cultivated hCECs. Potentially, thin tissue-engineered constructs of approximately 100 mm developed from expanded hCECs can be used as alternative graft material for selective replacement of the dysfunctional corneal endothelium using advanced EK surgical techniques. The use of a thin tissue-engineered corneal endothelial construct is a very attractive alternative as current DSEK and DMEK surgical approach enables the delivery of such thin graft material into the anterior chamber of the eye. It has also been postulated that cultured hCECs can be injected into the anterior chamber in patients afflicted by corneal endothelial dysfunction as an alternative form of treatment (31). Nevertheless, either approach will require a robust culture system that enables the isolation and propagation of hCECs in vitro with relative consistency despite known donor-to-donor variations. In our previous study, we showed that two serumsupplemented media, coded in that report as M2 (63) and M4 (15), were able to consistently support the proliferation of primary hCECs isolated from pairs of donor corneas (44). However, the unique cellular polygonal morphology of the cultivated CECs could not be maintained beyond the second passage in a majority of the

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established cultures, and cells became fibroblast-like (44). This phenomenon has been reported by Zhu and colleagues in cultures of hCECs that were exposed to growth factors such as basic fibroblast growth factor (bFGF), which could activate canonical Wnt signaling, resulting in an endothelial-to-mesenchymal transition (EMT) (64). We have discovered the use of a serum-supplemented culture medium (referred to as M5 in this study), which is able to preserve the cellular morphology of primary hCECs in vitro (unpublished observation). For this study, in order to prevent EMT of hCECs expanded in proliferative medium M4, we assessed the incorporation of M5 in a dual media culture system as a novel approach for the propagation of isolated hCECs. Expression of key markers indicative of the human corneal endothelium was examined for cells grown to P3 in M4 alone and were compared pairwise to CECs from the same donors expanded to P3 using the dual media approach. Finally, microarray analysis was performed on P3 hCECs that were expanded using the dual media system to compare gene expression profiles of the proliferating cells before the switch to M5 and those exposed to M5 for 7 days. MATERIALS AND METHODS Materials Ham’s F12, Medium 199 (M199), human endothelial serum-free medium (SFM), fetal bovine serum (FBS), Dulbecco’s phosphate-buffered saline (PBS), TrypLE™ Express (TE), gentamicin, amphotericin B, penicillin G, streptomycin sulfate, TotalPrep™ 96 RNA Amplification Kit, and Ambion® proprietary MEGAscript® Kit were purchased from Life Technologies (Carlsbad, CA, USA). Insulin, transferrin, selenium (ITS), ascorbic acid, trypan blue (0.4%), calcium chloride, chondroitin sulfate, paraformaldehyde (PFA), bovine serum albumin (BSA), Triton X-100, normal goat serum, and chloroform were purchased from Sigma (St. Louis, MO, USA). Fibronectin, collagen, and albumin (FNC) coating mix was purchased from United States Biologicals (Swampscott, MA, USA). Collagenase A was obtained from Roche (Mannhein, Germany). Research-Grade Human Corneoscleral Tissues This study was approved by the institutional review board of the Singapore Eye Research Institute/Singapore National Eye Centre according to the tenets of the Declaration of Helsinki, and written consent was acquired from the next of kin of all deceased donors regarding eye donation for research. A total of 21 pairs of research-grade human cadaver corneal tissues deemed unsuitable for transplantation were obtained from Lions Eye Institute for Transplant and Research, Inc. (Tampa, FL, USA). Human corneoscleral tissues with endothelial cell counts of at least 2,000 cells per mm2 were procured and preserved in Optisol-GS (Bausch & Lomb, Rochester, NY, USA) at

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4°C. Corneoscleral tissues were processed within 14 days of preservation. The age of the donors used in this study ranged from 2 to 37 years old (Table 1), and no significant growth differences were observed, specifically for CECs of donors isolated for cell expansion. Isolation and Growth of Human Corneal Endothelial Cells Isolation of hCECs using a two-step peel and digest method was performed as previously described (44). Briefly, after peeling off the Descemet’s membrane (DM), the pieces of DM were exposed to 2 mg/ml collagenase (in M5 medium) for 2–4 h to dislodge the CECs from the DM, which resulted in tightly packed CEC clusters. The CEC clusters were further dissociated in 1× TE for 2 min to further dissociate the clusters into smaller clumps and single cells. For initial comparative studies, isolated CEC clumps were divided equally into two conditions: either M4 alone or M5 alone. The formulation of M4 [Ham’s F12/M199, 5% FBS, 20 mg/ml ascorbic acid, 1% ITS, 10 ng/ml bFGF (R&D Systems, Minneapolis, MN, USA), and 1% antibiotic/ antimycotic] was reconstituted as previously published (15). The basal medium of M5, human endothelial-SFM, was supplemented with 5% FBS and 1% antibiotic/antimycotic.

In subsequent studies wherein the dual media culture approach was utilized, isolated hCECs were first established in M5 medium overnight to allow for cell adherence and stabilization. The culture medium was subsequently replaced with M4 to promote the proliferation of the adhered CECs. When the growth of CECs reached 80% to 90% confluence (approximately 2 weeks), M4 medium was withdrawn, and M5 medium was reintroduced to the CECs for at least 7 days before passage. Confluent CECs were passaged via 1× TE dissociation. For cellular expansion, the dissociated CECs were plated at the seeding density of 104 cells per cm2, as described previously (43). A Nikon TS1000 phase contrast microscope with a Nikon DS-Fi1 digital camera (Tokyo, Japan) was used to capture cellular morphology at every passage. All cultures were incubated in a humidified atmosphere at 37°C and 5% CO2. Cell Proliferation Assay Proliferation rate of hCECs cultured in M4 alone or M5 alone was determined by Click-iT™ ethynyl deoxyuridine (EdU) Alexa Fluor 488 imaging kit (Invitrogen/Life Technologies) as per the manufacturer’s instructions. Briefly, passaged hCECs were seeded onto a FNC-coated slide at a lower density of 5 × 103 cells per cm2 and cultured for 24 h

Table 1. Donor Information Experiment Serial Number 01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20 21

Age

Sex

Days to Culture

Cell Count (OS/OD)

COD

A

B

14 23 23 33 37 27 25 19 16 32 19 27 18 23 24 32 27 18 15 29 2

M M F M M F M M M M M M M M M M F M M M F

12 8 7 7 10 10 8 8 11 12 8 9 12 12 10 7 6 7 8 11 12

2907/3215 3058/3077 3012/3049 2865/2976 2646/2674 3175/2967 3195/2874 3344/3195 3448/3584 2174/2618 3378/3257 2725/2506 3344/3509 2907/ 2732 2703/2639 3021/2427 2203/2037 3040/2907 3215/3096 3205/2899 4348/4425

Acute cardiac crisis Blunt trauma Overdose Acute cardiac crisis Acute cardiac crisis Stroke Cardiopulmonary arrest Anoxia MVA Overdose MVA Acute cardiac crisis MVA Acute Cardiac Crisis Overdose Sepsis Seizures MVA Overdose Sepsis GI bleed

• • • • • • • • • • • • • • • •

• • • • •

C

D

E

• • • • • • • • • • • •

OS, oculus sinister (left eye); OD, oculus dexter (right eye); COD, cause of death; MVA, motor vehicle accident. Donor age ranged from 2 to 37 years old with a median age of 23 years old. Days taken from death of donor to the initiation of CEC culture ranged from 6 to 12 days with a median of 9 days. Experiment A: Morphological assessment and growth profile; Experiment B: Cell proliferation – Click-iT ethynyl deoxyuridine (EdU); Experiment C: Real-time PCR analysis; Experiment D: Immunofluorescence staining; Experiment E: Microarray analysis.

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in their respective culture condition. The cells were then incubated in the respective medium containing 10 mM EdU for another 24 h. After incubation, the cells were rinsed once with PBS followed by fixation with 4% PFA for 15 min on ice. The cells were then washed twice with 3% BSA in PBS and permeabilized using 0.5% Triton X-100 for 20 min at room temperature and washed twice with 3% BSA. The samples were incubated in a reaction cocktail containing 1× reaction buffer, CuSO4, and Alexa Fluor azide for 30 min in the dark. Samples were rinsed with PBS and mounted in Vectashield containing 4¢,6-diamidino-2phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). A Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss, Jena, Germany) was used to examine the labeled proliferative cells. At least 250 nuclei were analyzed for each experimental set. Morphometry Analysis of Cellular Circularity Cells of the healthy human corneal endothelium are generally hexagonal in shape (60,62). Hence, an important morphological characteristic of hCECs expanded in vitro is the maintenance of their polygonal/hexagonal cellular shape in culture. The roundness or circularity of a cell was determined as previously described using the forArea mula: circularity ¼ 4pðPerimeter 2 Þ, where a value approaching 1.0 is equivalent to a cell with a cellular profile nearing that of a perfect circle. Therefore, hexagonal CECs will have a profile closer to 1.0, whereas an increasingly elongated fibroblast-like CEC will have a circularity value closer to zero (42). Digital micrographs of hCECs cultured using M4 alone or using the dual media approach were taken at the third passage at confluence. Morphometric data of the area and perimeter of randomly selected cells from phase contrast images were manually outlined by point-to-point tracing of the cell borders using ImageJ software (NIH, Bethesda, MD, USA) (50). At least 100 cells from each condition (n = 3) were analyzed. Gene Expression Analysis Quantitative RT-PCR was performed on hCECs cultured to the third passage using either M4 alone or the dual media approach. Briefly, total RNA of confluent cells was extracted using the Qiagen RNeasy kit (Qiagen, Hilden, Germany) and purified using Turbo DNA-free™ DNase treatment (Life Technologies). The RNA sample was reverse transcribed to cDNA using the SuperScript First-Strand Synthesis System (Life Technologies). Corneal endothelial-related genes of interest were studied (Table 2), and primers were designed to accommodate the Roche Universal Probe Library system using the Roche ProbeFinder (Version 4.9) to create the most optimal realtime PCR assay (Roche). Quantitative PCR was carried out on a Lightcycler 480 system (Roche), with 45 cycles of DNA denaturation (95°C; 10 s), annealing (60°C; 30 s),

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and extension (72°C; 1 s), using a premade mastermix containing DNA polymerase (Probes master 480; Roche). Samples were run in triplicate, and the gene expression levels were normalized with the endogenous levels of GAPDH (glucose 6-phosphate dehydrogenase), and relative fold changes were analyzed using the comparative CT method (35). Immunocytochemistry Confluent passage 2 (P2) hCECs were passaged and plated at a high density of 2,000 cells per mm2 on FNCcoated glass coverslips (7 mm in diameter) and maintained for approximately 7 days in M5 medium before fixation with 100% ice-cold ethanol for 5 min or 4% PFA for 15 min at 4°C. Samples were rinsed and blocked in 5% normal goat serum in PBS for 30 min at room temperature. Subsequently, samples were labeled with primary antibodies for 1 h at room temperature. Primary antibodies used in this study were mouse IgG1 anti-sodium-potassium-transporting adenosine triphosphatase (Na+K+/ATPase, 5 µg/ml; Santa Cruz Biotechnology, Dallas, TX, USA), mouse IgG1 antizona occludens 1 (ZO-1, 5 µg/ml; BD Biosciences Pharmingen, Franklin Lakes, NJ, USA), mouse IgG1 anti-cluster of differentiation 200 (CD200, 20 µg/ml; BD Biosciences Pharmingen), and mouse IgG1 anti-glypican 4 (GPC4, 20 µg/ ml; Novus Biologicals, Littleton, CO, USA). The samples were then washed twice with PBS, 5 min each, and labeled with AlexaFluor 488-conjugated goat anti-mouse IgG secondary antibody (1:750; Life Technologies) for 1 h at room temperature in dark. After two brief PBS washes, they were mounted in Vectashield containing DAPI and visualized under a fluorescence microscope. Microarray Analysis hCECs from two donors (D17 and D18) were cultivated to the third passage using the dual media approach. Confluent cells in the proliferative M4 medium (D17p and D18p) were compared to the same batches of confluent cells that were maintained in M5 medium (D17m and D18m) for an additional 7 days. Samples were extracted in 1 ml TRIzol reagent (Invitrogen). Samples were homogenized using a handheld homogenizer (VWR, Radnor, PA, USA) before the addition of 200 µl chloroform. After vigorous shaking, samples were centrifuged at 12,500 relative centrifugal force (rcf) for 15 min at 4°C. The upper aqueous phase was transferred to a new tube and mixed with an equal volume of 70% ethanol. The resulting solution was transferred to a Qiagen RNeasy column, and the RNA purification procedures were performed as per the manufacturer’s protocol with a RNAse-free DNase digestion step incorporated. An Agilent RNA 6000 Nano Kit and Agilent 2100 Bioanalyzer (Santa Clara, CA, USA) were used to determine the quality and integrity of RNA before microarray analysis. Subsequently, total RNA was prepared for microarray analysis

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Table 2. List of Primer Sequences Gene

Accession No.

ATP1A1

NM_000701.7

SLC4A11

NM_032034.3

COL8A1

NM_001850.4

THY1

NM_006288.3

COL1A1

NM_000088.3

ANGPTL7

NM_021146.2

SCNN1A

NM_001159576.1

SERPINA3

NM_001085.4

PIP5K1B

NM_003558.2

IFITM1

NM_003641.3

LAMC2

NM_005562.2

DIRAS3

NM_004675.2

ESM1

NM_007036.4

DDIT4L

NM_145244.3

GAPDH

NM_002046.4

Primer Sequence

UPL Probe

FOR: gaagctcatcattgtggaagg REV: agtcattcacaccgtcacca FOR: acccatgcggggtaaagt REV: taccgcacccctgtcact FOR: ccaactcacccttgaagtcat REV: ggctggtttctgtctcttcag FOR: aggacgagggcacctacac REV: gccctcacacttgaccagtt FOR: gggattccctggacctaaag REV: ggaacacctcgctctcca FOR: aacaaccaaattgacatcatgc REV: ggtagagggaagagcagtcg FOR: caaccaggtctcctgcaac REV: gaaagtatagcagtttccatacatcg FOR: actccagacagacggctttg REV: attctctccattctcaactctgc FOR: gcgcaactggtcttggtag REV: ggctctgcagtcacatctca FOR: cacgcagaaaaccacacttc REV: tgttcctccttgtgcatcttc FOR: cagaagcccagaaggttgat REV: acactgagaggctggtccat FOR: ttggctccaaggaacagaag REV: gaaggcgcggaggataag FOR: catggatggcatgaagtgtg REV: ggtgccgtagggacagtct FOR: cccagagagcctgctaagtg REV: ttgctttgatttggacagaca FOR: agccacatcgctcagacac REV: gcccaatacgaccaaatcc

76 3 8 22 67 46 31 42 3 60 21 29 30 67 60

Forward and reverse primer sequences used in the amplification of the indicated corneal endothelial genes of interest, including its accession number and UPL probe number. ATP1A1, adenine triphosphatase (ATPase), Na+/K+ transporting, a 1 polypeptide; COL8A1, collagen, type VIII, a 1; THY1, thymocyte antigen 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. See Tables 4 and 5 for other definitions.

using a TotalPrep™ 96 RNA Amplification Kit according to the manufacturer’s protocol. Briefly, total RNA was reverse transcribed with oligo(dT) primer bearing a T7 promoter using ArrayScript™ reverse transcriptase. The cDNA underwent second-strand synthesis and cleanup to become a template for in vitro transcription with T7 RNA Polymerase. Ambion® proprietary MEGAscript® Kit was used to generate biotinylated antisense cRNA. The labeled strands were hybridized in triplicate onto HumanHT-12 v4 Expression BeadChips (Illumina, San Diego, CA, USA) using the Illumina IntelliHyb Seal. Array data were analyzed using Partek Genomic Suite 6.5 beta software (Partek, Inc., St. Louis, MO, USA). Data was first imported and normalized using robust multiarray averaging. Statistical testing using ANOVA was performed to identify genes that were differentially expressed. The lists of differentially expressed gene transcripts between the two culture conditions were filtered based on a selection criterion of ≥2 relative fold change at

a false discovery rate of £5%. Raw data of the array have been deposited at Gene Expression Omnibus under accession number GSE50212. Pathway Analyses Differentially expressed genes were imported to the Database for Annotation, Visualization and Integrated Discovery (DAVID) Functional Annotation Bioinformatics Microarray Analysis v6.7 (http://david.abcc.ncifcrf. gov). Functional gene clusters in association with biological events were identified. The likelihood of event presentation in Gene Ontology Consortium Annotation Categories (GO biological process, cell component, and molecular functions) was examined. Statistics All numeric data obtained were expressed as mean ± standard deviation (SD). Statistical analyses were performed using SPSS Statistics 17.0 (IBM, Chicago, IL, USA) as

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follows: the analyses of cell proliferation between hCECs cultured in M4 alone or M5 alone using Click-iT EdU assay (Fig. 1A and B); comparisons of cell numbers obtained from culture in M4 alone and M5 alone of the five donors (Fig. 1C); comparisons of cell sizes and cell circularity (Table 3) of hCECs propagated in M4 alone and the dual media approach and between each passage were evaluated using paired Student’s t tests. For all gene expression analysis, results were analyzed using independent Student’s t tests. Results with a value of p < 0.05 were deemed to be statistically significant. RESULTS Morphology and Proliferation of Human Corneal Endothelial Cells Grown in M4 and M5 A total of five pairs of donor corneas (Table 1; serial numbers 01 to 05) were used for this experiment. hCECs

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were harvested and cultured in either M4 alone or M5 alone. At confluence (P0), striking morphological differences were observed in CECs that were grown in M4 (Fig. 1A) and M5 (Fig. 1B). Assessment of cell proliferation at the first passage using Click-iT EdU assay showed that hCECs grown in M4 alone (Fig. 1A inset; 21.1 ± 8.8%) were significantly more proliferative (*p < 0.05) than cells grown in M5 alone (Fig. 1B, inset; 6.9 ± 4.5%). As assessed by paired Student’s t tests, confluent P0 hCECs grown in M4 alone yielded significantly more cells compared to those grown in M5 alone (**p < 0.01) (Fig. 1C). As such, with the exception of Donor 3, insufficient hCECs from M5 culture were obtainable for continual cell expansion. Although we were able to expand hCECs grown in M4 culture to the third passage and beyond, the unique polygonal/hexagonal cellular morphology was lost, and cells became elongated and fibroblast-like (Fig. 1D).

Figure 1. Human CECs established and propagated in M4 alone and in M5 alone. Representative sets of photomicrographs showing morphology of confluent human corneal endothelial cells (hCECs) (passage 0; P0) established in (A) M4 alone; with percentages of proliferative hCECs (at the first passage; inset) as assessed by the Click-IT ethynyl deoxyuridine (EdU) assay, which is significantly different (*p < 0.05) when compared to (B) percentages of proliferative hCECs cultured in M5 alone (at the first passage; inset). (C) Total cell numbers obtained for the passage of five independent sets of donor-matched hCECs cultured in M4 alone compared to M5 alone, **p < 0.01. (D) Representative photomicrograph showing a typical fibroblast-like morphology of cultivated hCECs at the third passage cultured using M4 alone.

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Table 3. Cell Circularity of Cultured hCECs at P1, P2, and P3 at Confluence Passage 1 2 3 3

Culture System

Cell Size ± SD (mm2)

Cell Circularity ± SD

M4 alone Dual media M4 alone Dual media M4 alone Dual media Dual mediaH

3,410.73 ± 1,196.30 3,630.60 ± 969.10 3,355.72 ± 1383.58 4,954.47 ± 1,227.25* 3,476.74 ± 1,128.29 4,839.94 ± 1,614.22** 828.06 ± 256.20

0.68 ± 0.14 0.79 ± 0.09† 0.63 ± 0.12 0.82 ± 0.07‡ 0.62 ± 0.14 0.79 ± 0.10§ 0.74 ± 0.11

Cultured hCECs propagated in dual media were found to be larger than their respective counterparts that were cultured in M4 alone at the second passage (P2), *p < 0.01, and the third passage, **p < 0.01. However, cell circularity analysis showed that hCECs expanded using the dual media approach were significantly more polygonal/hexagonal compared to hCECs cultured in M4 alone, which were significantly more heterogeneous in terms of their cellular morphology at the first passage, †p < 0.01, second passage, ‡p < 0.01, as well as the third passage, §p < 0.01. When P2 hCECs expanded in the dual media were plated at a high seeding density of approximately 3,000 per mm2 (dual mediaH), average cell sizes of 828.06 ± 256.20 mm2 and a cell circularity measurement of 0.74 ± 0.11 were obtained.

Signs of such transformations were observed in some of the CECs expanded in M4 from as early as the first round of passage (unpublished observation). Dual Media Culture System to Propagate Human Corneal Endothelial Cells Based on the above observation, we hypothesized that using M4 for cell growth and proliferation and M5 for cell stabilization and maintenance would aid in an increased capacity to cultivate the hCECs while retaining their cellular morphology. Hence, we developed a dual media culture strategy for the propagation of hCECs as depicted in Figure 2. Subsequent comparative studies were performed on independent sets of hCECs isolated from 11 pairs of donor corneas (Table 1; serial numbers 06 to 16), where isolated CECs were divided equally into two culture conditions, using either M4 alone or via the dual media approach. Morphometric Analysis: A Comparison of CECs in M4 Alone Versus CECs in Dual Media hCECs isolated from four pairs of donor corneas (Table 1; serial numbers 06 to 09) were used for the

following analysis and grown for three passages. Over the three passages, it was evident that cells expanded in M4 alone gradually lost their unique hexagonal/polygonal morphology and became vastly heterogeneous by the third passage (Fig. 3). Interestingly, the cellular morphology of hCECs propagated using the dual media was maintained throughout the three passages, and phase contrast microscopy showed that dual media-expanded CECs appeared more homogenous than their respective counterparts propagated in M4 alone (Fig. 3). This observation was confirmed by comparative cell circularity measurements of the two CEC populations from P1 to P3, and CECs grown in M4 alone were significantly more elongated (less polygonal/hexagonal) at confluence across all three passages (Fig. 3, Table 3). It should be noted that cell circularity measurement of CECs from a healthy human corneal endothelium taken from a specular micrograph is 0.82 ± 0.03 (unpublished observation). When confluent P0 hCECs were passaged for further cell expansion, based on a seeding density of 1 × 104 cells per cm2, their cellular sizes became significantly larger at confluence—especially CECs propagated under the dual media approach (Table 3). To investigate if this was a

Figure 2. Schematic diagram depicting the propagation of hCECs using a dual media approach. (A) Procurement—pairs of researchgrade corneas used in this study were procured from Lions Eye Institute for Transplant and Research Inc. (Tampa, FL). Research corneas were preserved and transported in Optisol-GS, and processed within 14 days from preservation. (B) Process and isolation—once received, corneas were processed within 1 day where the hCECs were isolated and plated as passage 0 culture. (C to C¢) Stabilization— isolated hCECs were seeded and allowed to attach and stabilize in M5 media overnight. (C¢ to C¢¢) Proliferation—to promote the proliferation of the adhered CECs, M4 media was utilized throughout the expansion phase. (C¢¢ to D) Stabilization and maintenance—when expanding hCECs became approximately 80% confluent, M5 media was reintroduced for at least 7 days, which aided in the preservation of the cellular morphology of cultivated hCECs. Thereafter, confluent hCECs were dissociated using TrypLE™ Express (TE), seeded at a density of 1 × 104 cells per cm2, and subjected to the same interswitching dual media approach for subsequent passages.

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Figure 3. Cellular morphology and circularity of hCECs cultured over three passages. Representative sets of photomicrographs showing morphology of confluent hCECs propagated in either M4 alone or using the dual media approach and a bar chart comparing cell circularity for both conditions during (A) the first passage, (B) the second passage, and (C) the third passage.

reversible phenomenon, we seeded P2 hCECs (average size of 4,954.47 ± 1,227.25 mm2) (Table 3) at a physiological cell density of 2,000 per mm2. Subsequent cell size measurement of these CECs showed average cell sizes of 828.06 ± 256.20 mm2 and a cell circularity of 0.74 ± 0.11 (dual mediaH; Table 3). Characterization of Cultivated Human Corneal Endothelial Cells at the Third Passage Expression levels of five genes, three known to be expressed by the corneal endothelium [ATPase, Na+/K+

transporting, a 1 polypeptide (ATP1A1), solute carrier family 4, sodium borate transporter, member 11 (SLC4A11), collagen, type VIII, a 1 (COL8A1)] and two not specifically related to the corneal endothelium [thymocyte antigen 1 (THY1; CD90), collagen, type I, a 1 (COL1A1)], were measured using quantitative real-time PCR in four separate sets of hCECs (P3) exposed to M4 alone or cultivated using the dual medium approach (n = 4) (Table 1; serial numbers 10 to 13). The relative expression of each gene in P3 hCECs cultured using the two different approaches were compared and normalized to obtain a

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relative fold increase. Results showed that cells cultured using the dual media approach expressed higher levels of ATP1A1 (1.75 ± 0.15-fold*), SLC4A11 (11.94 ± 4.20fold*), and COL8A1 (4.42 ± 2.10-fold**), and cells grown in M4 alone showed higher levels of THY1 (6.77 ± 2.38fold**) and COL1A1 (4.76 ± 1.93-fold**) (*p < 0.01, **p < 0.05) (Fig. 4A). In separate experiments, indirect immunofluorescence showed that P3 hCECs propagated using the dual media approach (Table 1; serial numbers

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14 to 16), seeded at a density of 2,000 cells per mm2, expressed corneal endothelium-associated pump marker Na+K+/ATPase (Fig. 4B), tight junction marker ZO-1 (Fig. 4C), heparin sulfate proteoglycan GPC-4 (Fig. 4D) (11), and cell membrane glycoprotein CD200 (Fig. 4E) (11). More importantly, the staining patterns clearly showed the polygonal shape of cultivated hCECs at the third passage. These results suggested that hCECs propagated using the dual media system retained their unique cellular morphology,

Figure 4. Characterization of cultivated hCECs of the third passage. (A) Quantitative RT-PCR of mRNA from cultures of confluent human CECs at the third passage expanded using M4 alone (green), compared to CECs from the same sets of donors propagated to the third passage under the dual media approach (blue). Fold increase of each gene was calculated and significantly higher expression of adenine triphosphatase (ATPase), Na+/K+ transporting, a 1 polypeptide (ATP1A1), solute carrier family 4, sodium borate transporter, member 11 (SLC4A11), and collagen, type VIII, a 1 (COL8A1) was observed in CECs propagated using the dual media approach, whereas thymocyte antigen 1 (THY1) and COL1A1 expression was significantly higher in CECs expanded in M4 medium only. n = 4, *p < 0.01, and **p < 0.05. Primary CECs were cultivated using the dual media approach to the third passage and characterized for their expression of known markers indicative of the corneal endothelium such as (B) sodium–potassiumtransporting adenosine triphosphatase (Na+K+ATPase), (C) zona occludens 1 (ZO-1), (D) glypican 4 (GPC-4), and (E) cluster of differentiation 200 (CD200), by immunocytochemistry. (F) A representative image of an isotype-matched negative control. (n = 5; scale bar: 50 µm).

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and expressed higher levels of corneal endothelium-specific genes ATP1A1, SLC4A11 and COL8A1, as well as reported cellular markers of the corneal endothelium (11,44). Gaining Insights Into Gene Expression Changes of Human Corneal Endothelial Cells Following Exposure to M5 Medium The polygonal morphology of primary hCECs was consistently retained for each of the three rounds of expansion when the confluent cells grown in the proliferative M4 medium were switched to the maintenance M5 medium for 7 days. In order to better understand the observed morphological changes at the molecular level, high throughput microarray analysis was performed on hCECs isolated from two donors (Table 1; serial numbers 17 and 18) cultivated to the third passage. Here, the global gene expression profile of the confluent CECs expanded in M4 for the first (D17p) and second donor (D18p) were compared to their respective counterparts maintained in M5 medium (D17m and D18m) for a further 7 days. Hierarchical clustering showed that propagated hCECs from the two separate donors cultured in M4 had more similarities in their gene expression profiles than cells that were maintained in M5 for an additional 7 days (Fig. 5A). In all, a total of 1,485 upregulated genes (Fig. 5B) and 1,420 downregulated genes (Fig. 5C) were found. Additionally, the top 20 upregulated and downregulated genes are listed in Tables 4 and 5, respectively. Validation of Microarray Data for Selected Differentially Expressed Genes by QPCR We performed quantitative real-time PCR using three independent sets of cultivated P3 hCECs to validate the microarray results (Table 1; serial numbers 19 to 21). The relative gene expression of five upregulated genes [SLC4A11, angiopoietin-like 7 (ANGPTL7), sodium channel, non-

PEH ET AL.

voltage-gated 1 (SCNN1A), serpin peptidase inhibitor, clade A, member 3 (SERPINA3), and phosphatidylinositol-4-phosphate 5-kinase, type 1 b (PIP5K1B)] and five downregulated genes [interferon-induced transmembrane protein 1 (IFITM1), laminin, g 2 (LAMC2), DIRAS family, GTP-binding RAS-like 3 (DIRAS3), endothelial cell-specific molecule 1 (ESM1), and DNA-damageinducible transcript 4-like (DDIT4L)] selected from the top 20 up- and downregulated list of genes were examined. The fold changes in gene expression were calculated relative to GAPDH, and the results obtained were normalized against cultures in M4 before the 7-day exposure to M5. Results showed that the direction of gene expression of the selected genes was consistent with the microarray results (Fig. 5D). Pathway Analysis The sorted gene list (fold difference >2 and p < 0.05) comparing primary hCECs in proliferative M4 medium and maintained an additional 7 days in M5 media was analyzed by DAVID Functional Annotation Bioinformatics Microarray analysis v6.7 for gene ontology and significant pathways. Differentially expressed genes were engaged to calculate the significant gene annotation and enrichment to reveal the potential biological pathways and functions. Table 6 showed the significant ranking of gene ontology terms by p values (

Propagation of human corneal endothelial cells: a novel dual media approach.

Corneal endothelium-associated corneal blindness is the most common indication for corneal transplantation. Restorative corneal transplant surgery is ...
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