STEM CELLS AND DEVELOPMENT Volume 23, Number 18, 2014  Mary Ann Liebert, Inc. DOI: 10.1089/scd.2013.0387

Identification and Potential Application of Human Corneal Endothelial Progenitor Cells Susumu Hara,1 Ryuhei Hayashi,1 Takeshi Soma,1 Tomofumi Kageyama,1,2 Thomas Duncan,1 Motokazu Tsujikawa,1 and Kohji Nishida1

The corneal endothelium is believed to be developmentally originated from the periocular mesenchyme via the neural crest. Human corneal endothelial progenitor cells (HCEPs) have been investigated because of their potential availability for the tissue regenerative medicine. However, the existence and the properties of HCEPs have not been elucidated yet. We first established a novel serum-free culture system for HCEPs. The HCEPs highly expressed p75 neurotrophin receptor, SOX9, and FOXC2, and partially retained the properties of neural crest and periocular mesenchyme. Further, we demonstrated that HCEPs had a high proliferative potency, and the differentiated HCEP sheets had corneal endothelial function by using the Ussing chamber system and transplantation to the rabbit cornea. These findings suggest that the HCEPs can be selectively expanded from the corneal endothelium using a specific culture system and will provide cell sheets for corneal regenerative medicine.

Introduction

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he cornea consists of three layers: epithelium, stroma, and endothelium. The corneal endothelium, the most inner part, has a barrier and pump function to prevent water from anterior chamber entering into the stroma and epithelium [1]. Deficiency of the corneal endothelium results in severe corneal edema and visual loss. The deficiency is important because it is quite common situation caused by Fuch’s dystrophy, pseudoexfoliative glaucoma, complication of cataract surgery, and trauma. For example, in the United States, Fuch’s dystrophy is the cause for 80% of corneal transplantations [2]. We and other groups developed cultured human corneal endothelial cell (HCEC) transplantation and resolved corneal deficiency in animal models [3–8]. However, we have still difficulty in amplifying corneal endothelial cells in sufficient numbers, so the cultured HCEC transplantation has few advantages over the conventional corneal transplant. Cultured HCECs can replace current treatment for corneal endothelial deficiencies when the cells can be expanded in sufficient numbers. Many groups have tried to resolve the problem, with many reporting successful amplification of HCECs [9,10]. However to date, no clinical application has been reported. It may be because proliferation is insufficient or in expansion the HCEC barrier and pump function disappears. Presently, procedures for HCEC amplification still have difficulties to overcome. An efficient way to resolve these problems is through the isolation of corneal endothelial stem or progenitor cells. The 1 2

presence of human corneal endothelial progenitor cells (HCEPs) has been reported [11–15], but the features of HCEPs, including markers or proliferative capabilities, have not yet been revealed. Here, we present the isolation of HCEPs using p75 neurotrophin receptor (p75NTR), a marker for neural crest cells, from which corneal endothelial cells derive [16–18]. The progenitors have enough proliferative potential and lineage to produce functional corneal endothelial cells. Using these cells we can generate transplantable corneal endothelial cell sheets, which rescue the phenotype of the corneal endothelium deficiency model.

Materials and Methods Cell isolation, culture, and differentiation All studies adhered to the Declaration of Helsinki. The human corneal tissues used were established research corneas from eye banks (Sight Life). Donor age was 47 – 6 years old, and preservation time was 6.2 – 1.4 days (n = 46). The corneal endothelial density of the corneas used in the present study was more than 1,500 cells/mm2. The Descemet’s membranes were stripped from the corneas using sterile surgical forceps in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies) with 10 mM Y27632 (Wako Pure Chemical Industrials, Ltd). The membranes were then treated with enzyme cell detachment medium (Accutase; Life Technologies) at 37C for 30 min. The cells were seeded at a density of 100–300 cells per cm2 onto

Department of Ophthalmology, Osaka University Graduate School of Medicine, Suita, Japan. Ophthalmic Research and Development Center, Santen Pharmaceutical Co., Ltd., Ikoma-shi, Nara, Japan.

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culture plates coated with 20 mg/mL laminin-511 (BioLamina). The medium was composed of DMEM/Nutrient Mixture F-12 (DMEM/F12; Life Technologies) containing 20% Knockout Serum Replacement (KSR; Life Technologies), 2 mM l-glutamine (Life Technologies), 1% non-essential amino acids (Life Technologies), 100 mM 2-mercaptoethanol (Life Technologies), 50 U/mL penicillin G and 50 mg/mL streptomycin (Life Technologies), and 4 ng/mL basic fibroblast growth factor (bFGF; Wako Pure Chemical Industrials, Ltd). The culture medium was changed every 2–3 days. When the cells reached 70% confluency, they were harvested with Accutase and passaged at ratios of 1:2 to 1:5. The HCEPs were differentiated into mature corneal endothelial cells (differentiated HCEPs) on dishes coated with FNC coating mix (AthenaES) or atelocollagen sheets (KOKEN Co., Ltd). The differentiation medium consisted of low-glucose DMEM (Nikken Biomedical Laboratory) with 10% fetal bovine serum (FBS; Japan Bio Serum) and 50 U/mL penicillin G and 50 mg/mL streptomycin (Life Technologies). The cells were cultured at 37C, atmospheric air/normoxia, and 5% CO2 for 14–28 days. To cultivate HCECs using a conventional method with FBS medium (HCECs-FBS) as a control, we cultured Descemet’s membranes with endothelium via explant culture onto culture dishes coated with FNC coating mix in DMEM containing 10% FBS and 2 ng/mL bFGF [7]. Cells at the proliferation stage were collected and subcultured when they reached 70% confluency, and they were collected again when they reached confluent in the culture.

Immunohistochemistry For cell staining, cells were fixed with methanol at 4C for 30 min. Non-specific absorption was blocked in the samples with 5% normal donkey serum in Tris-buffered saline and permeabilized with 0.3% Triton X-100. The cells were then incubated at 4C for overnight with primary antibodies as follows: p75NTR (1:100; Advanced Targeting Systems), SOX9 (1:100; Abcam), FOXC2 (1:50; Abcam), ZO-1 (1:100; Life Technologies), Na + /K + -ATPase (1:100; Millipore), N-cadherin (1:100; Santa Cruz Biotechnology, Inc.), and Ki67 (1:400; Abcam) in Tris-buffered saline containing 1% normal donkey serum and 0.3% Triton X100. The same concentration of corresponding normal nonspecific immunoglobulin G (IgG) was used as a negative control. The cells were incubated with Alexa Fluor-488-, Alexa Fluor-568-, or Alexa Flour-647-conjugated antimouse, anti-rabbit, or anti-goat IgG (Life Technologies) at room temperature for 2 h. The cells were counterstained with 5 mg/mL Hoechst 33342 (Life Technologies) and observed with a fluorescence microscope (Carl Zeiss). For the corneal flat-mount samples, the tissues were fixed with 4% paraformaldehyde (PFA) at room temperature for 30 min. The tissues were stained with an LSAB plus kit (Dako Japan) or immunofluorescence as described and observed with a microscope (KEYENCE or Carl Zeiss).

Fluorescence-activated cell sorting The Descemet’s membranes containing corneal endothelial cells isolated from donor corneas were enzymatically treated with Accutase at 37C for 30 min. After blocking with phosphate-buffered saline containing 2% bovine serum albumin

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(Sigma-Aldrich), the cells were incubated with phycoerythrinconjugated anti-p75NTR antibody (BD Biosciences) in 2% bovine serum albumin containing phosphate-buffered saline at 4C for 30 min. Stained cells were sorted using a fluorescence-activated cell sorting (FACS) Aria II (BD Biosciences). For staining of Ki67, cells on primary culture were fixed with Cytofix/Cytoperm (BD Biosciences) and stained with fluorescein isothiocyanate-conjugated Ki67 antibody (BD Biosciences). The same concentration of corresponding normal non-specific IgG was used as a negative control. Stained cells were analyzed with a flow cytometer (FACS Verse; BD Biosciences).

Colony-forming assay Cells were seeded at a density of 3,000 cells per well on laminin-511-coated six-well plates. After 13–16 days, the cells were fixed with formalin and stained with Giemsa staining solution (Sigma-Aldrich), and colony formation was assessed under a dissecting microscope. For the distribution of HCEPs, the corneal endothelium was divided into a center region ( < 8.0 mm) and a peripheral region ( > 8.0 mm) using a trephine.

Quantitative reverse transcription–polymerase chain reaction Total RNA was extracted from cells using an RNeasy plus micro kit (QIAGEN, GmbH). Complementary DNAs were synthesized using a SuperScript III first-strand synthesis system (Life Technologies) according to the manufacturer’s protocol. Primers and TaqMan probe mixtures were purchased from Life Technologies (Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/scd). Quantitative polymerase chain reaction (PCR) was carried out with a 7500 Fast Real-Time PCR System (Life Technologies). All assays were run in duplicate of five individual samples. Data were normalized to glyceraldehyde 3-phosphate dehydrogenase expression.

Alizarin Red staining The cell sheet was stained with 0.2% Alizarin Red S (Wako Pure Chemical Industrials) in 0.9% NaCl (pH 4.2) at room temperature for 5 min and fixed with 4% PFA for 5 min. The stained sheet was examined with a microscope (KEYENCE).

Scanning electron microscopy The cell sheet was fixed with 2% glutaraldehyde (TAAB Laboratories) at 4C overnight. Subsequently, the sample was dehydrated with ethanol and tert-butyl alcohol (Wako Pure Chemical Industrials). Next, the sample was dried in a freeze-drying device ( JFD-320; JEOL Ltd.) and coated with platinum with an autofine coater ( JFCL-1600; JEOL). The sample was observed using a scanning electron microscope ( JSM-6510LA; JEOL) at 5 kV.

Transmission electron microscopy The sample was fixed at room temperature for 3 h in a solution of 2.5% glutaraldehyde and 2% PFA in 0.1 M

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sodium cacodylate buffer (pH 7.2). En-bloc staining in 2% osmium tetraoxide and 0.5% uranyl acetate at room temperature for 1 h was carried out, and the sample was dehydrated through graded alcohols and propylene oxide before being embedded in Araldite resin (TAAB Labora-

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tories). Sections (90-nm thickness) were cut on a ReichertJung Ultracut Microtome. Routinely prepared samples were post-stained at room temperature in 2% aqueous uranyl acetate and Reynolds lead citrate for 5 min. Examination was carried out at 80 kV with a transmission electron

FIG. 1. Isolation of human corneal endothelial progenitor cells (HCEPs) from the corneal endothelium. Cells from the corneal endothelium were cultured with serum-free media at a density of 100 cells/cm2 onto plates coated with laminin511. (A) Phase-contrast images of HCEPs at days 1, 4, 7, 10, and 14. These cells proliferated after day 10. (B) Phasecontrast image of the cultured cells with fetal bovine serum (FBS)–containing medium for 14 days. (C) Expression of p75 neurotrophin receptor (p75NTR, green) and SOX9 (red) as neural crest markers. The cells were counterstained with Hoechst 33342 (blue). Right panel shows an isotype control image as negative control. (D) Image of the colony-forming assay of the proliferated cells of donors (30 and 64 years old) at days 13–16 in vitro. (E) Colony-forming efficiency (CFE) of 30–59 and 60–69 years old (n = 3–6). (F) Colony-forming assay of isolated cells from the center (< 8 mm) and the periphery ( > 8 mm) of the corneal endothelium from the same donors, cultured with adhesion culture for 10–14 days. (G) CFE of center and periphery (n = 5; donor age = 27–59 years old). Flat-mount samples of the human corneal endothelium were examined via immunohistochemistry. p75NTR was localized in the center (H) and periphery (I) of the corneal endothelium. The broken line represents a boundary of transition between the corneal endothelium (ce) and the trabecular meshwork (tm). ( J) Isotype control as negative control of immunostaining. Scale bars = 100 mm (A, B) and 1 mm (H–J). Data are represented as the means – standard deviations (SDs). N.S. = not significant (unpaired Student’s t test, P > 0.05).

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microscope (H7650; Hitachi). Images were obtained with a charge-coupled device camera (Gatan, Inc.).

Measurement of pump function Pump function of corneal endothelial cells and differentiated HCEPs was measured with an Ussing chamber system (Warner Instruments) following a previously described procedure [19,20] with some modifications. Cell sheets with areas of 28.3 mm2 were incubated in Krebs– Ringer solution: 120.7 mM NaCl, 24 mM NaHCO2, 4.6 mM KCl, 0.7 mM Na2HPO4, 0.5 mM MgCl2, and 10 mM glucose (pH 7.4) at 37C. After steady-state levels of the potential difference and the short-circuit current were reached, 1 mM of ouabain (Sigma-Aldrich), a Na + / K + -ATPase inhibitor, was added to the chamber. The data were analyzed with Data Trax 2 (World Precision Instrument, Inc.).

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Transplantation of progenitor-derived corneal endothelial cells into a rabbit model of corneal endothelial deficiency This study was performed in accordance with the Association for Research in Vision and Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research and was approved by the animal ethics committee of Osaka University. Two female New Zealand white rabbits weighing 2.8–2.9 kg were obtained from Oriental Yeast Co., Ltd. The rabbits were anesthetized intramuscularly with a mixture of ketamine hydrochloride (60 mg/kg; Sankyo Co., Ltd.) and xylazine (10 mg/kg; Bayer). Transplantation was performed on the right eye of each rabbit only. As the control, an atelocollagen sheet without cells was transplanted. The corneas were subjected to cryoinjury (Ophthalmic Cryo; MIRA) to detach them from the center to the periphery. The HCEP sheet was

FIG. 2. Isolation of p75NTRexpressing cells in the human corneal endothelium. (A) Analysis of p75NTR expression in the corneal endothelium (n = 5; donor age = 41– 69 years). Enzymatically dissociated cells from the corneal endothelium were stained with anti-p75NTR antibody and sorted with fluorescenceactivated cell sorting (P1 population: p75NTR-negative fraction; P2 population: p75-positive fraction). (B, C) Each group of 500 sorted cells was cultured for 14 days. Brightfield image shows each population. The p75NTR-positive population, but not the p75NTR-negative fraction, was increased. (D, E) Cells stained with Giemsa stain. (F) The graph reveals the number of cells per well (n = 3; donor age = 16–42 years). (G) Immunostaining of the proliferated p75NTR-positive cells revealed p75NTR protein expression. Data are represented as the means – SDs. *P < 0.05 by unpaired Student’s t test. Scale bars = 100 mm.

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stained with 0.1 mg/mL 1,1¢-dioctadecyl-3,3,3¢,3¢-tetramethylindocarbocyanine perchlorate (DiI; Sigma-Aldrich) and trephined to a diameter of 6 mm. One week after the implementation of this model of cornea endothelial deficiency, the cell sheet was placed into corneal endothelial face in the anterior chamber using a Descemet’s-stripping endothelial keratoplasty device. Air was then injected in the anterior chamber to hold the sheet in place for 3 h. As a control transplantation, we transplanted only the atelocollagen sheet into the rabbit left eye. Corneal thickness was measured with a pachymeter (SP-100; Tomey). The transplanted eyes were observed with a slit-lamp (AIT-20; Topcon). After 4 weeks, the rabbits were euthanized with an intravenous overdose of pentobarbital sodium. The transplanted eye was enucleated and fixed with 4% PFA. The paraffin sections were stained with Hoechst 33342 for observation of DiI-labeled differentiated HCEPs or hematoxylin and eosin for histological observation.

FIG. 3. Expansion of HCEPs. (A) Colocalization of p75NTR (green) with Ki67 (red) was examined with double immunofluorescence staining. Nuclei were labeled with Hoechst 33342 (blue). (B) Positive cells for p75NTR and Ki67 were analyzed with a flow cytometer. (C) HCEPs from 47year-old donor proliferated during four passages. (D) HCEPs were analyzed for p75NTR expression at passages 0–4 (n = 4–5; donor age = 25–47 years). Data are represented as the means – SDs. Scale bars = 100 mm (A) and 200 mm (C). Color images available online at www .liebertpub.com/scd

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Statistical analysis The data are expressed as the mean – standard deviation and were analyzed by Student’s t-test for normally distributed data. All statistics were calculated by JMP 9.0.2 (SAS Institute, Inc.). A P-value < 0.05 was considered statistically significant.

Results Culture and distribution of p75NTR-positive cells from human corneal endothelium HCECs can proliferate with FBS [9]; however, the cultured HCECs’ pump and barrier function and polygonal morphology are adversely affected. This may be caused by humoral factors (ie, chemokines) in the FBS. To avoid this situation, we first seeded corneal endothelial cells in serumfree medium with bFGF at a relatively low density (100

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cells/cm2). To examine the effect of substrates in serum-free culture for HCEPs, HCEPs were cultured on a dish coated with collagen I, collagen IV, FNC coating mix, and laminin511. The result demonstrated that the laminin-511 was able to proliferate the HCEPs more efficiently than the other substrates (Supplementary Fig. S1). Proliferating cells appeared *10–14 days after seeding (Fig. 1A). Conversely, cells cultured in a conventional medium containing FBS and bFGF showed no proliferation in this culture condition (Fig. 1B). The obtained cells exhibited a bipolar, spindle-shaped morphology similar to that of neural crest cells (Fig. 1A). Accordingly, we confirmed the expression of neural crest markers in immunohistochemistry. The proliferating cells expressed neural crest markers p75NTR and SOX9, suggesting that the putative HCEPs retain some features of neural crest, the origin of corneal endothelium (Fig. 1C). The colony-forming efficiency (CFE) in serum-free medium had no significant relationship to donor age (Fig. 1D, E; donor age: 30–59, 0.31 – 0.11%, n = 6 and donor age: 60–69, 0.30 – 0.09%, n = 3. P = 0.875). We also compared the CFE between the center and the periphery of the corneal endothelium from the same donors. Photographs of representative colonies from the center and the periphery are shown in Fig. 1F. Statistical analysis indicated that the ratio of CFE in the periphery (0.44 – 0.17%, n = 5; Fig. 1G) was not significantly different from that in the center (0.64 – 0.10%, n = 5, P = 0.21). This data suggested that HCEPs are present throughout the corneal endothelium. Flat-mount immunohistochemistry of human corneal endothelium revealed that p75NTR-expressing cells were diffusely localized in both the center (Fig. 1H) and the periphery (Fig. 1I) of the corneal endothelium. The number of p75NTR-positive cells was not significantly different

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between the central and peripheral corneal endothelium (Supplementary Fig. S2), but they were specifically enriched at the corneal endothelial side of the transition region between the corneal endothelium and the trabecular meshwork (Fig. 1I).

Isolation of HCEPs in the human corneal endothelium Because p75NTR is a cell surface marker, we tried to isolate HCEPs by FACS. FACS analysis revealed that p75NTR-positive cells were present at 7.0 – 4.9% (n = 5) in the whole corneal endothelium (Fig. 2A). After isolation via FACS, p75NTR-positive and p75NTR-negative cells were cultured in serum-free medium with bFGF on 96-well plates coated with laminin-511. The p75NTR-positive cells proliferated and formed a number of colonies (Fig. 2B, D, and F) but the p75NTR-negative cells did not (Fig. 2C, E, and F). Most of the proliferating p75NTR-positive cells maintained the expression of p75NTR on day 14 (Fig. 2G). These results suggested that p75NTR-positive cells have high proliferative potential in vitro while maintaining p75NTR expression.

HCEP expansion We examined the proliferative capacity of HCEPs. Immunostaining and flow cytometry showed that Ki67positive cells composed 58.3 – 5.9% of the total HCEPs (Fig. 3A, B, n = 4). About 93.5 – 7.1% of these cells expressed p75NTR. The HCEPs underwent several passage in donors younger than 60 years (Fig. 3C), whereas those from donors older than 60 years displayed a lower proliferative capability (Table 1). Finally, the total cell number

Table 1. Passage Number Dependency for Proliferation Capabilities in Human Corneal Endothelial Progenitor Cells and Human Corneal Endothelial Cells Cultured Conventionally with Fetal Bovine Serum

HCEPs

HCECs-FBS

Age (years)

P0

P1

P2

P3

P4

P5

P6

25 30 41 47 50 56 59 60 62 63 69 75 79 27 40 55 63

2.2 2.3 7.9 1.6 10.6 5.4 4.7 2.4 5.8 2.4 0.9 0.4 2.7 0.3 0.3 X 0.3

14.9 28.6 31.5 8.7 X X 6.1 X 2.4 X 1.2 X X 1.1 X

22.3 41.4 189 23.5

57.7 38.1 1,096 53.9

69.3 X X 83.4

165.4

X

29.5

68.9

X

3.6

X

X

X X 2

X

Dissociated cells from the human corneal endothelium were seeded onto culture plates at 100 cells/cm2. When the cells reached 70% confluent, they were re-seeded onto new culture dishes. The data shows the theoretical cell number at which they converted in one corneal endothelium. As a control, the proliferation potency of HCEPs was compared with that of cultured HCECs-FBS via explant culture with FBS and basic fibroblast growth factor. The explant culture used whole tissue of the corneal endothelium from a single donor (· 106 cells/ eye). The X indicates that the cells did not subculture. HCEPs, human corneal endothelial progenitor cells; HCECs-FBS, human corneal endothelial cells cultured conventionally with fetal bovine serum.

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of HCEPs reached *109 cells per eye in a 25-year-old donor. The expression level of p75NTR in HCEPs gradually decreased through each passage (Fig. 3D). These data indicate that HCEPs have higher potency for proliferation compared with that of HCECs-FBS associated with p75NTR expression.

Characterization of HCEPs and differentiated HCEPs We cultured HCEPs in differentiation medium to examine whether they can differentiate into corneal endothelial cells (Fig. 4A–C). The differentiated HCEPs showed uniform corneal endothelial-cell-like, polygonal morphology (Fig.

FIG. 4. Morphology and gene expression analysis of HCEPs, human corneal endothelial cells (HCECs), and differentiated HCEPs. Phase-contrast images of HCEPs after 14 days (A) and growth-phase HCECs cultured using a conventional method with FBS (HCECsFBS). (B) HCEPs differentiated into matured corneal endothelial cells with medium containing 10% FBS for 4 weeks (C). Differentiated HCEPs were polygonal. (D–M) Expression analysis of the HCEPs (a), growth-phase HCEC-FBS (b), and differentiated HCEPs (c). Each cell was subjected to quantitative real-time reverse transcription– polymerase chain reaction for neural crest markers [(D–H); p75NTR, SOX9, AP-2b, slug, and snail], periocular mesenchymal markers [(I, J); PITX2 and FOXC2], a stem cell marker [(K); nestin], and corneal endothelium markers [(L, M); type VIII collagen alpha 1 and alpha 2 (COL8A1 and COL8A2, respectively)]. Expression of p75NTR (green) and FOXC2 (red) as neural crest markers was colocalized in HCEPs (N) and in vivo [(O): center, (P): periphery]. The cells counterstained with Hoechst 33342 (blue). Data are represented as means – SDs (HCEPs, differentiated HCEPs: n = 5, donor age = 25–47 years; growth-phase HCEC-FBS: n = 5, donor age = 25–68 years). *P < 0.05 by unpaired Student’s t test. Scale bars = 200 mm (A–C) and 100 mm (N).

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4C). For further analysis of HCEPs and differentiated HCEPs, we examined the expression of neural crest, periocular mesenchyme, stem cell, and corneal endothelial markers using real-time PCR. We compared HCEPs (Fig. 4A) and HCECs-FBS at the growth phase (Fig. 4L–M) with differentiated HCEPs (Fig. 4C). The differentiated HCEPs expressed corneal endothelial markers (Fig. 4D–M). Expression levels of corneal endothelial markers revealed that the expression of type VIII collagen alpha 2 chains (COL8A2), but not type VIII collagen alpha 1 (COL8A1), was present at lower levels in HCEPs compared with that in growth-state HCECs-FBS.

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Expression levels of neural-crest-related markers p75NTR, SOX9, and AP-2b in HCEPs were higher than those in growth-phase HCECs-FBS (Fig. 4D–F). Differentiated HCEPs exhibited levels of p75NTR, SOX9, and AP-2b lower than those of proliferating HCEPs. The expression pattern of FOXC2, periocular mesenchymal marker [21], and nestin stem cell marker was similar to that of neural crest markers (Fig. 4J, K). Expression of snail, slug, and PITX2 was not significantly different between HCEPs and HCECs-FBS (Fig. 4G–I). p75NTR expression was colocalized with FOXC2 expression in HCEPs (Fig. 4N) and in the corneal endothelium (Fig. 4O, P). These indicate that HCEPs retain features of neural crest and stem cells and differentiated to HCEC like in differentiation media.

Differentiated-HCEP-derived corneal endothelial cells on an atelocollagen sheet Expression pattern analysis revealed that differentiated HCEPs resemble HCECs-FBS, indicating that the differentiated HCEPs can be used for therapeutic trials. In pursuit of our goal of transplantation of differentiated HCEPs, we investigated and characterized differentiated HCEPs cultured on an atelocollagen sheet as a carrier for corneal endothelial transplantation [4]. The differentiated HCEP-derived cell

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sheet (differentiated HCEP sheet) showed high transparency and cobblestone-like polygonal morphology on phasecontrast microscopy (Fig. 5A, B). Alizarin Red S staining showed tightly packed corneal endothelial cells (Fig. 5C). The cell density of the differentiated HCEP sheet was 2,263 – 215 cells/mm2. These cells also displayed the functional corneal endothelial markers Na + /K + -ATPase, ZO-1, and N-cadherin on the sheets (Fig. 5D–F). Scanning electron microscopy observations showed intercellular junctions and the presence of microvilli on the surface (Fig. 5G). Transmission electron microscopy revealed that monolayer cells with a clear cellular junction including a gap junction were closely attached to the atelocollagen sheet (Fig. 5H).

Physiological function of HCEP-derived corneal endothelial cells We further analyzed the physiological function of differentiated HCEP sheets with an Ussing chamber system. Pump function was not significantly different between differentiated HCEP sheets and cultured HCEC sheets (n = 4, P = 0.66; Fig. 6A, B). To assess the function in vivo, we also transplanted a differentiated HCEP sheet into a rabbit model of corneal endothelial deficiency via a Descemet’s-stripping endothelial keratoplasty method. After 28 days, the eye with

FIG. 5. Morphology and expression analysis of differentiated HCEPderived cell sheets (differentiated HCEP sheets). (A, B) Macroimage and Phase-contrast image of the differentiated HCEP sheet as a transplant carrier. (C) The differentiated HCEP sheet was stained with Alizarin Red as the corneal endothelial marker. Immunohistochemistry of the differentiated HCEP sheet for Na + /K + -ATPase (D), ZO-1 (E), and N-cadherin (F) was performed to identify functional markers of the corneal endothelium. (G) Scanning electron microscopy showed polygonal cells with few intercellular spaces. (H) Transmission electron microscopy showed that the cells form a monolayer and bind with atelocollagen sheet closely. Scale bars = 50 mm (B–F), 10 mm (G), and 100 nm (H). ac, atelocollagen sheet. Color images available online at www.liebertpub.com/scd

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FIG. 6. In vitro and in vivo physiological function of differentiated HCEP sheets. (A, B) Measurement of pump function in vitro with an Ussing chamber. Short-circuit currents representing Na + /K + -ATPase activity from differentiated HCEP sheets (A) and HCEC sheets were calculated before and after the addition of 1 mM ouabain as an Na + /K + -ATPase inhibitor (differentiated HCEP sheets: n = 4, donor age = 30–64 years; HCEC sheets: n = 4, donor age = 25–63 years). Arrow indicates the point of ouabain addition. (C–H) Anterior segment photographs of rabbit eyes. The rabbit models of corneal endothelial deficiency were prepared through cryo-treatment. Photograph of the rabbit model pretransplantation shows corneal opacity (C); but 4 weeks after transplantation (D, E), the cornea became transparent in the eye in which the differentiated HCEP sheet was transplanted but not the eye in which the atelocollagen sheet (no cells) was used as a control (F–H). The differentiated HCEP sheet attached on the side of the cornea for 28 days after transplantation. 1,1¢-Dioctadecyl-3,3,3¢,3¢tetramethylindocarbocyanine perchlorate-labeled cells were still observed with a fluorescent microscope in the wholemount tissue of the corneas that received differentiated HCEP sheet transplantation (I, J) and in cross-section (K). (L) Hematoxylin and eosin staining shows that the differentiated HCEP sheet attached stably to the host stroma and that stromal swelling was decreased in the differentiated-HCEP-sheet-transplanted rabbit. (M) In the control model, hematoxylin and eosin staining indicates stromal edema and fibrous tissue with fibroblast-like cell infiltration in the posterior stroma. Scale bars = 100 mm (I–K). Data are represented as the means – SDs. N.S. = not significant (unpaired Student’s t test, P > 0.05).

the transplanted differentiated HCEP sheet was optically transparent compared with the pre-transplantation eye (Fig. 6C–E). Corneal thickness recovered to 369 mm from 1,138 mm after transplantation of the differentiated HCEP sheet. Conversely, the control eye showed no change in

corneal thickness (1,030 to 1,013 mm; Fig. 6F–H). DiIlabeled cells were still present on the atelocollagen carrier (Fig. 6I, J) and a cross-section of the cornea (Fig. 6K). The cells were also present on the posterior surface of the carrier in the transplantation rabbit (Fig. 6L). Stromal edema and

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diffuse cell infiltration into the stroma were present in the control eye (Fig. 6M). This indicates that differentiated HCEP sheet has enough corneal endothelium function in vivo and in vitro, to be used as a source for transplant therapies.

Discussion Although there have been some studies on corneal endothelial stem/progenitor cells, few report their characteristics. In this study, we have isolated HCEPs and characterized them by FACS using p75NTR, gene expression, and differentiation assays. Further, we have showed that HCEPs have a high proliferative potency and the differentiated HCEP sheets had physiological endothelial function in vitro and in vivo. These findings suggest that the HCEPs can be selectively expanded, and differentiated into functional corneal endothelial cell sheet. In previous reports, the existence of the human corneal endothelial stem/progenitor cells was shown in isolation with sphere-forming culture and with immunostaining of Ki67, nestin, telomerase, and LGR5 of in vivo corneal endothelium [11,15,22]. However, direct evidence of corneal endothelial stem/progenitor cells has not yet been shown. We attempted to isolate HCEPs from human corneal endothelium by FACS. p75NTR is a cell surface marker and has been used to isolate neural-crest-derived stem/progenitor cells from some tissues by FACS [23]. We succeeded in the isolation of the HCEPs, and present data that the fractionated p75NTR-positive cells have high proliferative potential in our culture system. Expression analysis showed that HCEPs expressed not only p75NTR but also the typical neural crest markers, including SOX9 and AP-2b, and a periocular mesenchyme marker, including FOXC2. p75NTR and SOX9 play an important role in the development of neural crest cells [23,24]. FOXC2 is necessary in the periocular mesenchyme for anterior segment development [21]. Nestin is a marker for neural stem cells and neural crest stem cells [25,26]. The precursor cells isolated by the sphere-forming assay expressed nestin, as did HCEPs [11]. These findings suggest that HCEPs partially retain the properties of neural crest cells or periocular mesenchymal cells. Moreover, differentiated HCEPs have a uniform and polygonal morphology. Multipotent stem cells such as neural crest cells typically differentiate into various cells with medium containing FBS, but HCEPs become only corneal endothelial cells under this condition. Thus, HCEPs are regarded as the corneal endothelial progenitor cells that were derived from the corneal endothelium. Our data demonstrate that isolated HCEPs maintain the proliferative capability in the culture system using KSRbased serum-free media with bFGF and laminin-511. In general, KSR-based media with bFGF is used to maintain undifferentiated cells, such as human embryonic stem cells and mesenchymal stem cells [27,28]. As a coating matrix, previous studies have reported HCEC expansion on plates coated with extracellular matrix components, such as collagen, fibronectin, and the extracellular matrix of bovine corneal endothelial cells [9,29–31]. Interestingly, laminin511 used in feeder-free/serum-free culture of human embryonic stem cells [32] is present in Descemet’s membrane

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according to a report by Kabosova et al. [33]. A culture system utilizing laminin-511 can also expand HCEPs. These characteristics suggest that laminin-511 is required in the microenvironment of HCEPs. The HCEPs showed that proliferative potency was very high in comparison with the HCECs cultured by conventional methods. In general, expanding HCECs require high cell density at cell seeding or explant cultures with FBScontaining media. The ratio of Ki67-positive cells in HCEPs was *3–4 times higher than that in cells cultivated using other procedures [34,35]. HCEPs have high proliferation potency, can undergo subculture, and can produce large amount of cell numbers by serial passages if a young corneal donor is used. Surprisingly, the HCEPs from the young donor were able to proliferate to amounts *300 times larger than corneal endothelial cells cultivated by conventional method. However, HCEPs from older donors lacked proliferation potency. This data corresponds to previous reports that show that the proliferative potential decreases with aging [31,36]. Although the expression of p75NTR in HCEPs decreased by each passage, the HCEPs with low p75NTR expression can also differentiate to corneal endothelial cells (data not shown). We successfully differentiated corneal endothelial cells from HCEPs as assessed by the morphology, expression of corneal endothelial markers, and physiological pump function in vitro and in vivo. These findings indicate that HCEPs can be differentiated into HCECs with the expected morphology and physiological function. Corneal endothelial cells are important for regenerative medicine, because corneal endothelial cells have almost no regenerative capacity in vivo. For corneal endothelial dysfunction, various culture methods and transplantation techniques have been attempted with HCEC sheet [3,6,7,9]; however, none have led to regeneration of the corneal endothelium. In many cases, for the treatment of corneal endothelial dysfunction, transplantation using a donor cornea is performed. Functional HCEC sheets that we developed in this study may change the treatment of corneal endothelial diseases. Taken altogether, we showed that p75NTR-expressing HCEPs from human corneal endothelium have highproliferative potency and can be differentiated into functional corneal endothelial cells. In regenerative medicine, stem cell therapy with cells such as keratinocytes and corneal epithelial cells has already begun. Our tissue-engineered HCEC sheets may become available for clinical applications in the near future and lead to successful corneal endothelial regeneration.

Acknowledgments This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Health, Labor and Welfare and from the National Institute of Biomedical Innovation in Japan.

Author Disclosure Statement The authors declare no conflict of interest. The sponsor or funding organization had no role in the design or conduct of this research.

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Address correspondence to: Kohji Nishida, MD, PhD Department of Ophthalmology Osaka University Graduate School of Medicine Rm. E7, 2-2 Yamadaoka Suita, Osaka 565-0871 Japan E-mail: [email protected] Received for publication August 15, 2013 Accepted after revision March 3, 2014 Prepublished on Liebert Instant Online March 3, 2014