August 2016
Volume 57, Issue 10
Open Access
Cornea  |   August 2016
Concomitant Evaluation of a Panel of Exosome Proteins and MiRs for Qualification of Cultured Human Corneal Endothelial Cells
Author Affiliations & Notes
  • Morio Ueno
    Department of Ophthalmology Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Kazuko Asada
    Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Munetoyo Toda
    Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Kazue Nagata
    Toray Industries, Inc., Tokyo, Japan
  • Chie Sotozono
    Department of Ophthalmology Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Nobuyoshi Kosaka
    Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan
  • Takahiro Ochiya
    Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan
  • Shigeru Kinoshita
    Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Junji Hamuro
    Department of Ophthalmology Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Correspondence: Junji Hamuro, Department of Ophthalmology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Hirokoji-agaru, Kawaramachi-dori, Kamigyo-ku, Kyoto 602-8566, Japan; jshimo@koto.kpu-m.ac.jp
Investigative Ophthalmology & Visual Science August 2016, Vol.57, 4393-4402. doi:10.1167/iovs.16-19805
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Morio Ueno, Kazuko Asada, Munetoyo Toda, Kazue Nagata, Chie Sotozono, Nobuyoshi Kosaka, Takahiro Ochiya, Shigeru Kinoshita, Junji Hamuro; Concomitant Evaluation of a Panel of Exosome Proteins and MiRs for Qualification of Cultured Human Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2016;57(10):4393-4402. doi: 10.1167/iovs.16-19805.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose: We elucidate a method to use secreted miRNA profiles to qualify cultured human corneal endothelial cells (cHCECs) adaptable for cell-injection therapy.

Methods: The variations of cHCECs in their composites of heterogeneous subpopulations (SPs) were verified in relation to their surface cluster-of-differentiation (CD) markers. Integrated analysis of micro RNA (miRNA) profiles in culture supernatants (CS) were investigated by 3D-Gene Human microRNA Chips. To validate 3D-Gene results, quantitative real-time PCR was done from numerous cultures with distinct morphology and SP composition. Exosomes and miRNAs in CS also were analyzed.

Results: Secreted miRNA profiles among morphologically-diverse cHCEC SPs proved useful for individual distinction. Candidate miRNAs to discriminate CD44 SPs from those with CD44++ ∼ CD44+++ phenotypes were miRs 221-3p, 1246, 1915-3p, and 4732-5p. The levels of the latter-three miRs decreased dramatically in cHCEC CS without cell-state transition (CST) compared to those of control medium, whereas those from cHCECs with senescence-like CST showed an increase. MicroR184 decreased inversely in parallel with the upregulation of CD44 on cHCECs. CD9+ exosomes were more elevated in cHCEC CS with senescence-like CST than those without CST, indicating the possible import of these extracellular vesicles (EVs) into cHCECs without CST.

Conclusions: Cultured HCECs sharing a CD44 phenotype of matured HCECs may be discriminated by measuring the amount of miRNAs or exosome in CS. Thus, miRNA in CS may serve as a tool to qualify cHCECs. Future detailed analysis of cell-to-cell communication via these EVs might open novel pathways for a better understanding of CST in HCEC cultures.

Cultured human corneal endothelial cells (cHCECs) are anticipated to serve as an alternative to donor corneas for the treatment of corneal endothelial dysfunction. We, together with others, have been investigating the possibility of the transplantation of cHCECs in the form of a cell suspension.14 Okumura et al.4 previously reported that the use of Rho-associated protein kinase (ROCK) inhibitor Y-27632 enhances cell proliferation, and Koizumi et al.5 reported the development of a new corneal-cell transplantation method involving the intraocular injection of cHCECs. Although HCECs retain the capacity to proliferate in vitro,6 culturing HCECs for a long period of time is known to be extremely difficult.7 However, the predisposition of cHCECs into a senescence phenotype, epithelial-mesenchymal transition (EMT), and fibroblastic transformation hamper the application of cell-injection therapy in the clinical setting. In fact, cHCECs are neither homogeneous nor stable in their quality, including their surface markers, such as cluster-of-differentiation (CD) antigens, from culture to culture.810 
MicroRNAs (miRNAs or miRs) are noncoding RNAs that function as an endogenous regulator of gene expression. Growing evidence suggests that miRs have a critical role in various biologic processes, such as cell proliferation, development, and differentiation.11,12 The expression of miRNAs is essential in the regulation of many cellular processes, including the formation, maintenance, and remodeling of the extracellular matrix (ECM),13 which is linked to cell-state transition (CST) in cHCECs. This implies the necessity to qualify the cardinal features of cHCECs by practical biochemical terms, to distinguish the unclarified fine distinctions among cHCECs. We have been attempting to apply an assay of protein products in culture supernatants (CS) to distinguish cHCEC subpopulations (SPs); however, the method was limited to only the discrimination of morphologically distinct SPs, and not for SPs differing in fine cell states, despite their seemingly similar morphology. The 3D-Gene miRNA Microarray Platform (Toray Industries, Inc., Tokyo, Japan) and hierarchical clustering revealed a distinct expression pattern of miRs in a comparative analysis of cHCECs with distinct phenotypes, although it is invasive (Ueno M, submitted for publication, 2016). 
MicroRNAs in CS serve as an alternative tool to qualify cHCEC SPs. To date, many previous works reported the critical roles of secreted forms of miRs in relation to a paracrine cell-to-cell communication. In 2007, miRs were confirmed also to exist inside exosomes.14 Exosomes are extracellular vesicles that recently have been recognized as important mediators of intercellular communication, as they carry lipids, proteins, and miRNAs.14 The existence of circulating miRs has been investigated widely as possible tools for the diagnosis and prognosis in a diverse types of diseases,15,16 as well as a use as liquid biomarkers.16 The existence of miRs in a variety of other human body fluids also has been reported.17,18 However, and to the best of our knowledge, no previous studies have dealt with miRs in CS as a biomarker to qualify the CST or heterogeneity of somatic cells in cultures. 
In this current study, we present the first finding that candidate miRs can be used to discriminate CD44 differentiated HCEC SPs from in-differentiated SPs with CD44++ ∼ CD44+++ phenotypes. The expression levels of these miRs decreased dramatically in cHCECs without CST, whereas those of cHCECs with CST showed an evident increase. Of interest, CD9+ (either CD63+ or CD63) exosomes were highly elevated in CS of cHCECs with CST than in those without CST, indicating the possible import of these extracellular vesicles (EVs) into cHCECs without CST. 
Materials and Methods
HCEC Donors
The human tissue used in this study was handled in accordance with the tenets set forth in the Declaration of Helsinki. Human donor corneas were obtained from SightLife, Inc. (Seattle, WA, USA). Informed written consent for eye donation for research was obtained from the next of kin of all deceased donors. All tissues were recovered under the tenets of the Uniform Anatomical Gift Act (UAGA) of the particular state in which the donor consent was obtained and the tissue was recovered. 
All donor corneas were preserved in Optisol GS (Chiron Vision, Inc., Irvine, CA, USA) and imported via international air transport for research purposes. Donor information accompanying the donor corneas showed that they were all considered healthy and absent of any corneal disease, and that all donors had no history of chromosomal abnormality. 
Cell Culture of HCECs
Unless otherwise stated, the HCECs were cultured according to the published protocols, with some modifications.4 Human donor corneas at the distinct ages were used for the experiments. Briefly, the Descemet's membranes with the CECs were stripped from donor corneas and digested at 37°C with 1 mg/mL collagenase A (Roche Applied Science, Penzberg, Germany) for 2 hours. The HCECs obtained from a single donor cornea were seeded in 1 well of a Type-I collagen-coated 6-well plate (Corning, Inc., Corning, NY, USA). The culture medium was prepared according to published protocols. Briefly, basal medium was prepared with OptiMEM I (Life Technologies Corporation, Carlsbad, CA, USA), 8% fetal bovine serum (FBS), 5 ng/mL epidermal growth factor (EGF; Life Technologies), 20 μg/mL ascorbic acid (Sigma-Aldrich Corp., St. Louis, MO, USA), 200 mg/L calcium chloride (Sigma-Aldrich Corp.), 0.08% chondroitin sulfate (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and 50 μg/mL gentamicin. Mesenchymal stem cell (MSC)-conditioned medium was prepared. The HCECs were cultured using MSC-conditioned medium at 37°C in a humidified atmosphere containing 5% CO2, and the culture medium was changed twice per week. The HCECs were passaged at ratios of 1:3 using 10x TrypLE Select (Life Technologies) at 37°C for 12 minutes when they reached confluence. The HCECs at passages 2 through 5 were used for all experiments. In some experiments, SB203580 (a p38 MAP Kinase inhibitor) was added to regulate senescence-like CST. 
Phase Contrast Microscopy
Phase contrast microscopy images ware taken with an inverted microscope system (CKX41; Olympus Corporation, Tokyo, Japan). 
Flow Cytometry Analysis of the cHCECs
Human CECs were collected from the culture dish by TrypLE Select treatment as described above and suspended at a concentration of 4 × 106 cells/mL in FACS buffer (PBS containing 1% BSA and 0.05% NaN3). Next, an equal volume of antibody solution was added and incubated at 4°C for 2 hours. The antibody solutions were as follows: FITC-conjugated anti-human CD26 mAb, PE-conjugated anti-human CD166 mAb, PerCP-Cy 5.5 conjugated anti-human CD24 mAb, PE-Cy 7-conjugated anti-human CD44 (all from BD Biosciences, San Jose, CA, USA), and APC-conjugated anti-human CD105 (eBioscience, Inc., San Diego, CA, USA). After washing with FACS buffer, the HCECs were analyzed by use of a BD FACSCanto II Flow Cytometry System (BD Biosciences). 
3D-Gene Microarray Analysis
RNA Extraction.
Supernatants of cHCECs were lysed by use of QIAzol Lysis Reagent (QIAGEN, Hilden, Germany) and then stored at −80°C until total RNA extraction. MicroRNA was extracted by use of the miRNeasy Mini kit (QIAGEN). The quality of the purified total RNA was analyzed by use of an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). 
miRNA Expression Profiling.
For miR expression profiling, 3D-Gene Human miRNA Oligo Chips (miRBase version 17-21; Toray Industries, Inc.) were used. All of the miRNA derived from 400 uL supernatants were labeled with Hy5 by use of the miRCURY LNA microRNA Power Labeling Kits (Exiqon A/S, Vedbaek, Denmark). The labeled microRNA were individually hybridized onto the surface of the miRNA chips, and the dried miRNA chips then were scanned using a 3D-Gene Scanner 3000 (Toray Industries, Inc.) and analyzed by use of 3D-Gene Extraction Software (Toray Industries, Inc.). 
Normalized Data Processing.
The digitalized fluorescent signals provided by the above-described software were regarded as the raw data. All of the normalized data were globally normalized per microarray, such that the median of the signal intensity was adjusted to 25. 
Quantitative Real-Time PCR (qRT-PCR)
Total RNA was extracted from culture supernatants using the miRNeasy Mini kit (QIAGEN). The cDNA was synthesized using High Capacity cDNA Reverse Transcription kit with RNase Inhibitor (Applied Biosystems, Foster City, CA, USA). The PCR reactions were performed using TaqMan Fast Advanced Master Mix (Applied Biosystems) and TaqMan Gene Expression Assays, Inventoried (Applied Biosystems) under the following conditions: activation of enzyme at 95°C for 20 seconds, 40 cycles of denature at 95°C for 1 second, and annealing/elongation at 60°C for 20 seconds. The StepOnePlus Real-Time PCR System (Applied Biosystems) was used for PCR amplification and analysis. 
ExoScreen
Quantification of CD9+, CD63+, and CD9+CD63+ exosomes in the CSs of HCECs were performed according to the method described by Yoshioka et al.19 
EV Isolation.
Briefly, CSs were harvested after a 48-hour incubation in the above-described culture medium. The collected CSs were centrifuged at 2000g for 10 minutes, then filtered through a 0.22 μm filter (EMD Millipore Corporation, Temecula, CA, USA). The CSs then were used for EV isolation by ultracentrifugation at 110,000g for 70 minutes. The pellets were washed with PBS, ultracentrifuged at 110,000g for 70 minutes, and then resuspended in PBS. The antibodies used for ExoScreen were mouse monoclonal anti-human CD63 antibody and mouse monoclonal anti-human CD9 antibody. 
ExoScreen Assay.
A 96-well half-area white plate was filled with 5 mL of sample, 5 nM biotinylated antibodies, and 50 μg/mL AlphaLISA Acceptor Beads (PerkinElmer, Inc., Waltham, MA, USA) conjugated antibodies. The volume of each reagent was10 μl. The plate then was incubated for 3 hours. Without a washing step, 25 μL of 80 μg/mL AlphaScreen streptavidin-coated donor beads were added. The reaction mixture was incubated for another 30 minutes, and the plate then was read on the EnSpire Alpha 2300 Multilabel Plate Reader (PerkinElmer) using an excitation wavelength of 680 nm and emission detection set at 615 nm. Background signals obtained from PBS were subtracted from the measured signals. 
Exosome Isolation Using Total Isolation Reagent
Culture supernatants were harvested after incubation for 72 to 96 hours in the culture medium. The collected CSs were centrifuged at 1580g for 10 minutes, then filtered through a 0.22 μm filter (EMD Millipore). Mixture of the CS and total exosome isolation reagents from cell culture media (Thermo Fishier Scientific, Inc., Waltham, MA, USA) were incubated at 4°C overnight and centrifuged. The pellets were washed with PBS after centrifugation again at 10,000g for 60 minutes at 4°C, and then resuspended in PBS. 
Immunoblotting of CD9 and CD63 Exosomes
The protein concentration was measured using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Inc.). Equal amounts of protein were separated by electrophoresis on 4% to 12% SDS-PAGE. Transferring on nitrocellulose membrane was performed using iBlot 2 Dry Blotting System (Life Technologies). The blocking and antibodies reaction was performed using the iBind Western System (Life Technologies). The protein bands were made visible by Novex ECL Chemiluminescent Substrate Reagent Kit (Invitrogen, Carlsbad, CA, USA). Luminescence was observed by use of an ImageQuant LAS-3000 (FUJIFILM, Tokyo, Japan) dedicated charge-coupled device (CCD) camera system. 
Statistical Analysis
Student's t-test was used to determine the statistical significance (P value) of the mean values for 2-sample comparisons, and Dunnett's multiple-comparisons test was used to determine the statistical significance for the comparison of multiple sample sets. 
Results
Distinct miR Profiles in CS With Normal and Senescent-Like cHCECs
In this study, we had dealt with the improved cultures of cHCECs nearly without conspicuous EMT, although it is difficult to discriminate the presence of a minuscule amount of EMT among heterogeneous cHCECs. At first, we compared the profiles of miRs between a CD44 SP (effector cell, 66-P4) and 55-P5 with senescent-like CST (Fig. 1A). The senescent-like CST was confirmed on the grounds of elevated SA-β-Gal staining and their phenotypes with CD44++, senescent-type, nonhexagonal cells forming island-like clusters, distinct from CD44 mature, differentiated, hexagonal, cobblestone-shape cells. The cHCECs with the senescent-like CST exhibited cell areas that were nearly twice as large as the latter mature cells.20 The scatter plot depicted in Figure 1B indicates the upregulation of several miRs in 55-P5 compared to 66-P4. The upregulated miRs are summarized in Figure 1C. Of note, some of the miRs present in the control medium were greatly decreased in CS during the 2-day cultures of 66-P4, although no decreases in CS were observed in the cultures of 55-P5. These miRs were miRs-1246, 1273g-3p, and 4732-5p. Simultaneously, other groups of miRs, such as miRs-1273f, 1285-3p, 1972, and 221-3p, were significantly increased in CS of the 55-P5 culture than in the control medium. These two typical changes of miRs in CS also were confirmed by the comparison of 66-P4 with other cHCECs (i.e., 72-P3 and 69-P3) with evident morphologic CST. 
Figure 1
 
The profiles of secreted miRs in CS of CD44 SP and senescent-like cHCEC. (A) Phase contrast microscopy images. MicroRs were extracted from the CS of either cHCECs (#66, P4, passage 5; donor age, 23 years; donor endothelial cell density [ECD], 3504 cells/mm2) without CST and of which the expression of surface CD44 was absent (CD44) or cHCECs with CST (#55, P5, passage 5; donor age, 49 years; donor ECD, 2699 cells/mm2). (B) The comparison of miR expression profiles between cHCECs #66 (passage 5; donor age, 23 years; donor ECD, 3504 cells/mm2) and #55 by the scatter plots. Y-axis values were indicated as averages of global normalization values. The upper and lower of 2-fold lines are indicated in the scatter plot. (C) Summarization of the upregulated miRs in senescent-like cHCECs. MicroR1246, miR1273g-3p, and miR4732-5p were greatly decreased in the CS of #66 than in the medium control (P < 0.01). MicroR1273f, miR1285-3p, miR1972, and miR221-3p were increased in the CS of #55 than in the CS of #66 (P < 0.01).
Figure 1
 
The profiles of secreted miRs in CS of CD44 SP and senescent-like cHCEC. (A) Phase contrast microscopy images. MicroRs were extracted from the CS of either cHCECs (#66, P4, passage 5; donor age, 23 years; donor endothelial cell density [ECD], 3504 cells/mm2) without CST and of which the expression of surface CD44 was absent (CD44) or cHCECs with CST (#55, P5, passage 5; donor age, 49 years; donor ECD, 2699 cells/mm2). (B) The comparison of miR expression profiles between cHCECs #66 (passage 5; donor age, 23 years; donor ECD, 3504 cells/mm2) and #55 by the scatter plots. Y-axis values were indicated as averages of global normalization values. The upper and lower of 2-fold lines are indicated in the scatter plot. (C) Summarization of the upregulated miRs in senescent-like cHCECs. MicroR1246, miR1273g-3p, and miR4732-5p were greatly decreased in the CS of #66 than in the medium control (P < 0.01). MicroR1273f, miR1285-3p, miR1972, and miR221-3p were increased in the CS of #55 than in the CS of #66 (P < 0.01).
Distinct miR Profiles in the CS of cHCECs From Adult and Newborn Donors
In some cases, we experienced abnormal cHCEC cultures from newborn donors below 2 or 3 months of age. Those cells sometimes showed senescent-like morphologic phenotypes. To confirm this, we established cHCECs from cornea tissues of d4 and d1 donors (i.e., 79-P2 and 85-P0 in Fig. 2A) and analyzed miRs profiles in the CS. Morphologies of cHCECs showed the presence of uniformly enlarged cells in the absence of cluster islands when compared to 66-P4. Accordingly, the scatter plot showed the presence of an abundant number of upregulated miRs in the CS of newborn-derived cHCECs compared to 66-P4 (Fig. 2B). MicroRs markedly reduced in CS of cHCECs 66-P4 and significantly increased in CS of cHCECs 79-P2 and 85-P0, than control medium were summarized (Fig. 2C). Most of them were in common with those selected in Figure 1. Surprisingly, miR184 was significantly upregulated, even in CS of 66-P4 compared to control medium, and the trend was stronger in those of cHCECs 79-P2 and 85-P0. 
Figure 2
 
The profiles of secreted miRs in CS of cHCEC from adult and newborn donors. (A) Phase contrast microscopy images. Cultured HCECs were established from the cornea tissues of d4 and d1 newborn donors (i.e., 79-P2 and 85-P0), and the profiles of miRs in the CS were analyzed. Morphologies of cHCECs showed the presence of uniformly enlarged cells compared to 66-P4 in Figure 1A. (B) The miRs were extracted from CS of cHCEC derived from an adult donor (#66 P4) and two newborn donors (#79P2 and #85P0).The comparison of CS miR expression profiles between newborn donor derived cHCECs #79 (P2, 4D, right eye, ECD3803/left eye ECD5255) and #85 (P0, 1D, R3468/L5156) with adult donor-derived cHCEC #66. (C) The upregulated miRs in CS of cHCECs derived from newborn donors were summarized (#66 P4 versus newborn, P < 0.01; #66P4 versus medium, P < 0.01).
Figure 2
 
The profiles of secreted miRs in CS of cHCEC from adult and newborn donors. (A) Phase contrast microscopy images. Cultured HCECs were established from the cornea tissues of d4 and d1 newborn donors (i.e., 79-P2 and 85-P0), and the profiles of miRs in the CS were analyzed. Morphologies of cHCECs showed the presence of uniformly enlarged cells compared to 66-P4 in Figure 1A. (B) The miRs were extracted from CS of cHCEC derived from an adult donor (#66 P4) and two newborn donors (#79P2 and #85P0).The comparison of CS miR expression profiles between newborn donor derived cHCECs #79 (P2, 4D, right eye, ECD3803/left eye ECD5255) and #85 (P0, 1D, R3468/L5156) with adult donor-derived cHCEC #66. (C) The upregulated miRs in CS of cHCECs derived from newborn donors were summarized (#66 P4 versus newborn, P < 0.01; #66P4 versus medium, P < 0.01).
CD44 Expression Levels and miR Profiles
The improved cHCEC cultures were almost devoid of conspicuous EMT, but it retained a senescent-like morphology, as described above. To exclude the interference by senescent-like cHCECs, SB203580 (a p38 mitogen-activated protein [MAP] kinase [MAPK] inhibitor) was added throughout the cultures to regulate senescence-like CST. Under this culture protocol, we succeeded in gaining the cultures a5, a1, and a2 with seemingly no senescent-like morphology (Fig. 3A). However, and of interest, they were endowed with the heterogeneity in the context of the expression of CD44, CD24, and CD26 on their surface (Fig. 3A). Using these cHCECs, RNA were extracted from the corresponding CSs. MicroRs exhibiting distinct expression among these cHCECs (i.e., a5, a1, and a2) were selected using three volcano plots (Fig. 3B). MicroRs exhibiting distinct expression among cHCECs without CST and CD44−/± (i.e., a5 P1) and with CST positive CD44++ or CD44+++ (i.e., a1P3 and a2P2) were selected using three volcano plots. Global normalization values were compared between a1, a5 and a2. Secreted miRs which cleared both conditions of a P value <0.05 and the fold change more than 2 or less than −2 were listed (Fig. 3C). 
Figure 3
 
(A) Cultured HCEC SPs defined by FACS. HCEC of a5 (p1, 19Y, ECD = 4544) were cultured according to the protocol described in text (seeding density was 800 cells/mm2, SB431542 not added), a1 (P3, 10Y, 3671) were cultured under the conditions like a5, and a2 (P2, 55Y, 3131) were cultured similarly, but the seeding cell density was 380 cells /mm2 in the presence of 1 mM SB431542. (B, C) CD44 expression levels and the profile of miR expression. (B) Volcano plots of miR expression. The miRs were extracted from CS of cHCEC derived from three cHCECs donor (a5, a1 and a2). Values are as in Figure 1. (C) The summarized list of secreted miRs distinct among SPs, a5, a1, and a2.
Figure 3
 
(A) Cultured HCEC SPs defined by FACS. HCEC of a5 (p1, 19Y, ECD = 4544) were cultured according to the protocol described in text (seeding density was 800 cells/mm2, SB431542 not added), a1 (P3, 10Y, 3671) were cultured under the conditions like a5, and a2 (P2, 55Y, 3131) were cultured similarly, but the seeding cell density was 380 cells /mm2 in the presence of 1 mM SB431542. (B, C) CD44 expression levels and the profile of miR expression. (B) Volcano plots of miR expression. The miRs were extracted from CS of cHCEC derived from three cHCECs donor (a5, a1 and a2). Values are as in Figure 1. (C) The summarized list of secreted miRs distinct among SPs, a5, a1, and a2.
The variances of miRs expression patterns were classified into six of these three cHCECs (Fig. 4A). The patterns are partly exemplified in Figure 4B. Different from only one miR34a in cHCECs, seven miRs in CS were candidates for discriminating CD44−/±a5 from CD44++a1. Here again, only miR184 significantly inversely decreased in correlation with the intensity of CD44 expression (Fig. 4B). 
Figure 4
 
The expression patterns of miRs. (A) The distinction of miR expression levels among three cHCECs SPs (a5, a1, and a2) were classified into six. The Y-axis corresponds to the relative expression intensity of respective miRs among a5, a1, and a2. Six typical patterns are are shown. (B) The variations of miR expression levels were illustrated for 9 typical miRs among three cHCECs SPs (a5, a1, and a2). Y-axis values were indicated as averages of global normalization values.
Figure 4
 
The expression patterns of miRs. (A) The distinction of miR expression levels among three cHCECs SPs (a5, a1, and a2) were classified into six. The Y-axis corresponds to the relative expression intensity of respective miRs among a5, a1, and a2. Six typical patterns are are shown. (B) The variations of miR expression levels were illustrated for 9 typical miRs among three cHCECs SPs (a5, a1, and a2). Y-axis values were indicated as averages of global normalization values.
Validation of Selected miRs
The results mentioned above clearly indicated that the senescence-like CST, as well as the differentiation in parallel with decreased expression of CD44, can be monitored by miRs secreted into CSs of heterogeneous cHCECs. The candidate miRs selected were used for the validation by qRT-PCR, using 10 different lots of cHCECs. By this validation, miRs 135a-3p, 221-3p, 1246, 1915-3p, and 4732-5p were selected as the candidates to discriminate CD44 effector SPs from those with CD44++ or CD44+++ phenotypes. 
Elevated Production of C9 Exosomes by Senescent-Like cHCECs
To detect EVs by ExoScreen, we selected CD9 and CD63, which are abundant on the surface of EVs and are expressed in numerous cells. Culture supernatants of cHCECs were processed to obtain purified EVs. ExoScreen was able to quantify the amount of CD9+ and CD63+ EVs present in CS in a dose-dependent manner.18 The cHCECs, from which CSs were derived are summarized in Figure 5. 77-P2 and C11-P2 showed morphologically distinct CST, with the latter possibly being a senescence-like CST. As shown in Figure 5B, C11-P2 elicited the production of the highest levels of CD9+ and CD9CD63++ exosomes, while 77-P2 produced CD63+ and CD9CD63++, and 66-P4 without conspicuous CST only of CD63+ exosome. This implies that the CD9+ exosome might have a role in inducing CST during HCEC cultures under our culture conditions. 
Figure 5
 
Exosomes, detected by ExoScreen, with CD9 and/or CD63. (A) Secreted EVs were harvested from CS of cHCECs, #66, #77 (P2, 55Y, 2561) and C11 (P2, 26Y, 3321) according to the method described in the text. (B) The exosome species single positive either with CD9 or CD63 and CD9CD63 double positive were quantitated for several lots of CS for #66, #77, and C11. Y-axis values were indicated as averages of global normalization values. CD63: #66, #77 and C11 versus medium, P < 0.01, CD9, #66 and #77 versus c11, P < 0.01, CD9CD63, #66 versus #77, P < 0.01, #66 versus c11, P < 0.01. Three cHCECs were distinct in their CD44 expression levels. #66, CD44−/±, 90.8%; CD44++ ∼ +++, 9.0%; C11,CD44+++, 87.8%, CD44−/±, 4.2%; #77 showed the highest expression of CD44++, but the exact data were missing.
Figure 5
 
Exosomes, detected by ExoScreen, with CD9 and/or CD63. (A) Secreted EVs were harvested from CS of cHCECs, #66, #77 (P2, 55Y, 2561) and C11 (P2, 26Y, 3321) according to the method described in the text. (B) The exosome species single positive either with CD9 or CD63 and CD9CD63 double positive were quantitated for several lots of CS for #66, #77, and C11. Y-axis values were indicated as averages of global normalization values. CD63: #66, #77 and C11 versus medium, P < 0.01, CD9, #66 and #77 versus c11, P < 0.01, CD9CD63, #66 versus #77, P < 0.01, #66 versus c11, P < 0.01. Three cHCECs were distinct in their CD44 expression levels. #66, CD44−/±, 90.8%; CD44++ ∼ +++, 9.0%; C11,CD44+++, 87.8%, CD44−/±, 4.2%; #77 showed the highest expression of CD44++, but the exact data were missing.
Secreted Exosome From cHCECs With Distinctive CD44 Expression
Immunoblotting of EV preparations from CS from distinct cHCECs confirmed the data obtained by ExoScreen. Three cHCECs, a5, a6 (instead of a1), and a2, contain SPs mainly composed of CD44CD24CD26, CD44++CD24±CD26, and CD44+++CD24CD26++ SPs, respectively. Again the expression levels of CD9 and CD63 were comparable between a6 and a2, but the expression levels were lowest in a5 for CD9 and CD63. Seemingly the content of CD9+ and/or CD63+ exosomes paralleled the increase of CD44 expression. Of note, the CS of a5 still contained a trace amount of CD9 exosome (Fig. 6), in contrast with absence in CS of 66-P4 described above. 
Figure 6
 
Immunoblotting of exosomes secreted from cHCECs. Secreted exosomes were harvested from CS of cHCECs, a2, a5, and a6 (P2, 66Y, 3399, CD44++). (B) The expression levels of CD63 and CD9 were evaluated by Western blotting. CD9 and CD63 positive exosome were a2 (CD44+++) and a6 (CD44++). Their expression levels in a5 (CD44) were lowest for CD9 and CD63. cHCEC a6 showed CD44−/±, 58.2%; CD44++, 35.8%; CD44+++, 1.8%).
Figure 6
 
Immunoblotting of exosomes secreted from cHCECs. Secreted exosomes were harvested from CS of cHCECs, a2, a5, and a6 (P2, 66Y, 3399, CD44++). (B) The expression levels of CD63 and CD9 were evaluated by Western blotting. CD9 and CD63 positive exosome were a2 (CD44+++) and a6 (CD44++). Their expression levels in a5 (CD44) were lowest for CD9 and CD63. cHCEC a6 showed CD44−/±, 58.2%; CD44++, 35.8%; CD44+++, 1.8%).
Discussion
Human CECs expanded in an in vitro culture system can be a mixture of SPs with distinct CST. Cultured cells tend to be inclined toward karyotype changes.8 We defined the SP with CD133, CD105, CD90, CD44, CD26, CD24, and HLA-DR negative and CD166, HLA-ABC, and PD-L1 positive as effector cells, with complete absence of karyotype aneuploidy.21 Recently, advances in circulating miRNA research have indicated that intracellular miRNAs may be released into the circulation during processes accompanying cellular destruction or pathologic injury.22 Thus, circulating miRNAs have the potential to be used as novel biomarkers. 
The secreted miRs can deliver a gene-silencing signal between living cells in vitro and in vivo.23 Of note, discrepant expression of miRs between the tissue and body fluids reportedly has been confirmed in a variety of systems.2426 Consistent with these previous works, miRs selected on the grounds of the presence in CS of HCECS were absolutely different from those selected from 3D genes of cHCECs. MicroR146, 34a, and 378 were the representatives of the latter, and these miRs were not selected from CSs. 
The predisposition of cHCECs into a senescence phenotype, EMT, and fibroblastic transformation is well known. In fact, cHCECs are neither homogeneous nor stable in their quality from culture to culture.810 Cultured HCECs sharing EMT and fibroblastic phenotypes are relatively easy to distinguish on the grounds of their morphology, whereas morphologic distinction of senescent phenotypes is far more difficult. Cellular senescence entails a robust increase in the secretion of numerous mediators, collectively referred to as the senescence-associated secretory phenotype (SASP).27 The chronic senescent cells may act to the detriment of the neighboring cells through inflammatory paracrine signaling through the SASP. The SASP depends upon the activation of p38MAPK,27,28 and blockade of this signaling by low-molecular-weight molecules represses the induction of the SASP.29 A compensatory response to restrain inflammation reportedly resulted in miR-146a/b upregulation in a delayed manner.30 In the beginning of this study, we observed the frequent appearance of senescent-like cHCECs. However, a later continuous addition of p38MAPK-inhibitor SB203580 made it possible to analyze the miRs profiles in the CS of cHCECs without senescence-like CST (a5, a1, a2, and a6). 
CD44 is the key to distinguishing differentiated cHCECs from either in-differentiated or cHCECs with CST.21 CD44 ablation increased metabolic flux to mitochondrial respiration, and concomitantly inhibited entry into glycolysis.31 In 2007, several groups identified the members of the miR34 family as the most prevalent p53-induced miRNAs.32 The miR378 family showed the gradual decrease in parallel with decreases of CD44 expression,21 and several miRs, including miR-378a-5p, also have been shown to be involved in senescence targeting the p53 pathway. Of note, CD44 might be repressed by wild-type p53.33 
It is intriguing that the selected secreted miRs by comparison of 66-P4 versus cHCECs with senescent-like CST or versus cHCECs with distinct CD44 expression levels were mutually not overlapped, except for miR1246, thus implicating the distinct role displayed in the maintenance of culture homeostasis. 
Most of the candidate miRs selected in this study to qualify cHCECs adaptable for cell-injection therapy also are known as the possible biomarkers, such as miR1246 upregulated in colorectal cancer tissues.34 Some of these miRs, (i.e., miR1246, 1273, 1915, 4732, and 346a) are categorized to p53 target-gene–encoded miRs. They also have been shown to have a role in controlling cell growth, apoptosis, senescence, and autophagy in a p53-dependent fashion.35 
p53 is now known to modulate genes involved in senescence36 and to restrain the SASP by blocking p38MAPK activation.28 Pigati et al. reported that the extracellular and intracellular miRNA profiles differed, and suggested the existence of a cellular selection mechanism for miR-1246 release.37 In regard to the secreted miRs, one of the most relevant issues that remains elusive and must be solved is whether these secreted miRs are released from, and act on, cHCECs as free forms or as inclusion molecules in exosomes. 
In regard to our observations, it is worthwhile to mention that the activation of p53 by a stress signal results in enhanced exosome production by those cells.37,38 The observed elevated secretion of exosomes, confirmed in the ExoScreen assay and immunoblotting in CD44++ ∼ +++ SPs of cHCECs (Fig. 6) might be under the regulation of p53. 
The findings of this study demonstrated that cHCECs are composed of a dysregulated expression of a hierarchy of miR clusters, probably due to the presence of CST paralleled with the reduced expression of CD44 and senescence. The assay of miRs secreted in CS, with further extended selection, may distinguish the cell-state-transitioned CD44++ ∼ +++ cHCEC from CD44 effector cells applicable in a cell-injection therapy for corneal endothelial dysfunctions. 
It should be noted that the aim of this present study was to describe new findings, that is, the presence of secreted miRNAs and exosomes in the CS of cHCECs and the effectiveness of these secreted products as practical indices of the quality of heterogeneous cHCECs with or without CST. The additional experiment should be critical to verify the functional reversal of corneal endothelial CST either by the exposure of exosomes or transfection of the miRNAs identified here to further extend the findings in regard to biological significance. Secreted miRNAs were found either in a free form or in an inclusion form in exosomes. We currently are investigating the correct method for the functional reversal by the transmission of multiple species of exosomes and/or miRNAs. 
Acknowledgments
The authors thank Asako Hiraga for technical assistance; Yoko Hamuro, Keiko Takada, and Tomoko Fujita for their secretarial assistance; Satoshi Kondo (Toray Industries, Inc.) for her invaluable assistance in analyzing 3D gene; and John Bush for his thorough and excellent review of the manuscript. 
Supported by The Highway Program for Realization of Regenerative Medicine from MEXT and the Program for the Promotion of Science from MEXT, Japan. 
Disclosure: M. Ueno, None; K. Asada, None; M. Toda, None; K. Nagata, Toray Industries, Inc. (E); C. Sotozono, None; N. Kosaka, None; T. Ochiya, None; S. Kinoshita, None; J. Hamuro, None 
References
Mimura T, Shimomura N, Usui T, et al. Magnetic attraction of iron-endocytosed corneal endothelial cells to Descemet's membrane. Exp Eye Res. 2003 ; 76: 745–751.
Mimura T, Yokoo S, Araie M, Amano S, Yamagami S. Treatment of rabbit bullous keratopathy with precursors derived from cultured human corneal endothelium. Invest Ophthalmol Vis Sci. 2005 ; 46: 3637–3644.
Patel SV, Bachman LA, Hann CR, Bahler CK, Fautsch MP. Human corneal endothelial cell transplantation in a human ex vivo model. Invest Ophthalmol Vis Sci. 2009 ; 50: 2123–2131.
Okumura N, Koizumi N, Ueno M, et al. ROCK inhibitor converts corneal endothelial cells into a phenotype capable of regenerating in vivo endothelial tissue. Am J Pathol. 2012 ; 181: 268–277.
Koizumi N, Sakamoto Y, Okumura N, et al. Cultivated corneal endothelial cell sheet transplantation in a primate model. Invest Ophthalmol Vis Sci. 2007 ; 48: 4519–4526.
Engelmann K, Bohnke M, Friedl P. Isolation and long-term cultivation of human corneal endothelial cells. Invest Ophthalmol Vis Sci. 1988 ; 29: 1656–1662.
Peh GS, Beuerman RW, Colman A, et al. Human corneal endothelial cell expansion for corneal endothelium transplantation: an overview. Transplantation. 2011 ; 91: 811–819.
Miyai T, Maruyama Y, Osakabe Y, et al. Karyotype changes in cultured human corneal endothelial cells. Mol Vis. 2008 ; 14: 942–950.
Okumura N, Hirano H, Numata R, et al. Cell surface markers of functional phenotypic corneal endothelial cells. Invest Ophthalmol Vis Sci. 2014 ; 55: 7610–7618.
Cheong YK, Ngoh ZX, Peh GS, et al. Identification of cell surface markers glypican-4 and CD200 that differentiate human corneal endothelium from stromal fibroblasts. Invest Ophthalmol Vis Sci. 2013 ; 54: 4538–4547.
Bartel DP. MicroRNAs: genomics biogenesis, mechanism, and function. Cell. 2004 ; 116: 281–297.
Croce CM. Calin GA. miRNAs, cancer, and stem cell division. Cell. 2005 ; 122: 6–7.
Rutnam ZJ, Wight TN. Yang BB. miRNAs regulate expression and function of extracellular matrix molecules. Matrix Biol. 2013 ; 32: 74–85.
Valadi H, Ekstrom K, Bossios A, et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007 ; 9: 654–659.
Chim SS, Shing TK, Hung EC, et al. Detection and characterization of placental microRNAs in maternal plasma. Clin Chem. 2008 ; 54: 482–490.
Lawrie CH, Gal S, Dunlop HM, et al. Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large B-cell lymphoma. Br J Haematol. 2008 ; 141: 672–675.
Kosaka N, Iguchi H, Ochiya T. Circulating microRNA in body fluid: a new potential biomarker for cancer diagnosis and prognosis. Cancer Sci. 2010 ; 101: 2087–2092.
Kosaka N, Iguchi H, Yoshioka Y, et al. Secretory mechanisms and intercellular transfer of microRNAs in living cells. J Biol Chem. 2010 ; 285: 17442–17452.
Yoshioka Y, Kosaka N, Konishi Y, et al. Ultra-sensitive liquid biopsy of circulating extracellular vesicles using ExoScreen. Nat Commun. 2014 ; 5: 3591–3598.
Hamuro J, Ueno M, Asada K, et al. Metabolic plasticity in cell state homeostasis and differentiation of cultured human corneal endothelial cells. Invest Ophthalmol Vis Sci. In press.
Hamuro J, Toda M, Asada K, et al. Cell homogeneity indispensable for regenerative medicine by cultured human corneal endothelial cells. Invest Ophthalmol Vis Sci. In press.
Ji X, Takahashi R, Hiura Y, et al. Plasma miR-208 as a biomarker of myocardial injury. Clin Chem. 2009 ; 55: 1944–1949.
Iguchi H, Kosaka N, Ochiya T. Secretory microRNAs as a versatile communication tool. Commun Integr Biol. 2010 ; 3: 478–481.
Tanaka M, Oikawa K, Takanashi M, et al. Down-regulation of miR-92 in human plasma is a novel marker for acute leukemia patients. PLoS One. 2009 ; 4: e5532.
Wang K, Zhang S, Marzolf B, et al. Circulating microRNAs, potential biomarkers for drug-induced liver injury. Proc Natl Acad Sci U S A. 2009; 106: 4402–4407.
Liu H, Zhu L, Liu B, et al. Genome-wide microRNA profiles identify miR-378 as a serum biomarker for early detection of gastric cancer. Cancer Lett. 2012 ; 316: 196–203.
Childs BG, Baker DJ, Kirkland JL, Campisi J, van Deursen JM. Senescence and apoptosis: dueling or complementary cell fates? EMBO Rep. 2014; 15: 1139–1153.
Freund A, Patil CK, Campisi J. p38MAPK is a novel DNA damage response-independent regulator of the senescence-associated secretory phenotype. EMBO J. 2011 ; 30: 1536–1548.
Zhang J, Shen B, Lin A. Novel strategies for inhibition of the p38 MAPK pathway. Trends Pharmacol Sci. 2007 ; 28: 286–295.
Bhaumik D, Scott GK, Schokrpur S, et al. MicroRNAs miR-146a/b negatively modulate the senescence-associated inflammatory mediators IL-6 and IL-8. Aging (Albany NY). 2009 ; 1: 402–411.
Soga T. Cancer metabolism: key players in metabolic reprogramming. Cancer Sci. 2013 ; 104: 275–281.
Cairns RA, Harris IS, Mak TW. Regulation of cancer cell metabolism. Nat Rev Cancer. 2011 ; 11: 85–95.
Godar S, Ince TA, Bell GW, et al. Growth-inhibitory and tumor- suppressive functions of p53 depend on its repression of CD44 expression. Cell. 2008 ; 134: 62–73.
Piepoli A, Tavano F, Copetti M, et al. Mirna expression profiles identify drivers in colorectal and pancreatic cancers. PLoS One. 2012 ; 7: e33663.
Zhang Y, Liao JM, Zeng SX, Lu H. p53 downregulates Down syndrome-associated DYRK1A through miR-1246. EMBO Rep. 2011; 12: 811–817.
Murray-Zmijewski F, Slee EA, Lu X. A complex barcode underlies the heterogeneous response of p53 to stress. Nat Rev Mol Cell Biol. 2008 ; 9: 702–712.
Pigati L, Yaddanapudi SC, Iyengar R, et al. Selective release of microRNA species from normal and malignant mammary epithelial cells. PLoS One. 2010 ; 5: e13515.
Yu X, Harris SL, Levine AJ. The regulation of exosome secretion: a novel function of the p53 protein. Cancer Res. 2006 ; 66: 4795–4801.
Figure 1
 
The profiles of secreted miRs in CS of CD44 SP and senescent-like cHCEC. (A) Phase contrast microscopy images. MicroRs were extracted from the CS of either cHCECs (#66, P4, passage 5; donor age, 23 years; donor endothelial cell density [ECD], 3504 cells/mm2) without CST and of which the expression of surface CD44 was absent (CD44) or cHCECs with CST (#55, P5, passage 5; donor age, 49 years; donor ECD, 2699 cells/mm2). (B) The comparison of miR expression profiles between cHCECs #66 (passage 5; donor age, 23 years; donor ECD, 3504 cells/mm2) and #55 by the scatter plots. Y-axis values were indicated as averages of global normalization values. The upper and lower of 2-fold lines are indicated in the scatter plot. (C) Summarization of the upregulated miRs in senescent-like cHCECs. MicroR1246, miR1273g-3p, and miR4732-5p were greatly decreased in the CS of #66 than in the medium control (P < 0.01). MicroR1273f, miR1285-3p, miR1972, and miR221-3p were increased in the CS of #55 than in the CS of #66 (P < 0.01).
Figure 1
 
The profiles of secreted miRs in CS of CD44 SP and senescent-like cHCEC. (A) Phase contrast microscopy images. MicroRs were extracted from the CS of either cHCECs (#66, P4, passage 5; donor age, 23 years; donor endothelial cell density [ECD], 3504 cells/mm2) without CST and of which the expression of surface CD44 was absent (CD44) or cHCECs with CST (#55, P5, passage 5; donor age, 49 years; donor ECD, 2699 cells/mm2). (B) The comparison of miR expression profiles between cHCECs #66 (passage 5; donor age, 23 years; donor ECD, 3504 cells/mm2) and #55 by the scatter plots. Y-axis values were indicated as averages of global normalization values. The upper and lower of 2-fold lines are indicated in the scatter plot. (C) Summarization of the upregulated miRs in senescent-like cHCECs. MicroR1246, miR1273g-3p, and miR4732-5p were greatly decreased in the CS of #66 than in the medium control (P < 0.01). MicroR1273f, miR1285-3p, miR1972, and miR221-3p were increased in the CS of #55 than in the CS of #66 (P < 0.01).
Figure 2
 
The profiles of secreted miRs in CS of cHCEC from adult and newborn donors. (A) Phase contrast microscopy images. Cultured HCECs were established from the cornea tissues of d4 and d1 newborn donors (i.e., 79-P2 and 85-P0), and the profiles of miRs in the CS were analyzed. Morphologies of cHCECs showed the presence of uniformly enlarged cells compared to 66-P4 in Figure 1A. (B) The miRs were extracted from CS of cHCEC derived from an adult donor (#66 P4) and two newborn donors (#79P2 and #85P0).The comparison of CS miR expression profiles between newborn donor derived cHCECs #79 (P2, 4D, right eye, ECD3803/left eye ECD5255) and #85 (P0, 1D, R3468/L5156) with adult donor-derived cHCEC #66. (C) The upregulated miRs in CS of cHCECs derived from newborn donors were summarized (#66 P4 versus newborn, P < 0.01; #66P4 versus medium, P < 0.01).
Figure 2
 
The profiles of secreted miRs in CS of cHCEC from adult and newborn donors. (A) Phase contrast microscopy images. Cultured HCECs were established from the cornea tissues of d4 and d1 newborn donors (i.e., 79-P2 and 85-P0), and the profiles of miRs in the CS were analyzed. Morphologies of cHCECs showed the presence of uniformly enlarged cells compared to 66-P4 in Figure 1A. (B) The miRs were extracted from CS of cHCEC derived from an adult donor (#66 P4) and two newborn donors (#79P2 and #85P0).The comparison of CS miR expression profiles between newborn donor derived cHCECs #79 (P2, 4D, right eye, ECD3803/left eye ECD5255) and #85 (P0, 1D, R3468/L5156) with adult donor-derived cHCEC #66. (C) The upregulated miRs in CS of cHCECs derived from newborn donors were summarized (#66 P4 versus newborn, P < 0.01; #66P4 versus medium, P < 0.01).
Figure 3
 
(A) Cultured HCEC SPs defined by FACS. HCEC of a5 (p1, 19Y, ECD = 4544) were cultured according to the protocol described in text (seeding density was 800 cells/mm2, SB431542 not added), a1 (P3, 10Y, 3671) were cultured under the conditions like a5, and a2 (P2, 55Y, 3131) were cultured similarly, but the seeding cell density was 380 cells /mm2 in the presence of 1 mM SB431542. (B, C) CD44 expression levels and the profile of miR expression. (B) Volcano plots of miR expression. The miRs were extracted from CS of cHCEC derived from three cHCECs donor (a5, a1 and a2). Values are as in Figure 1. (C) The summarized list of secreted miRs distinct among SPs, a5, a1, and a2.
Figure 3
 
(A) Cultured HCEC SPs defined by FACS. HCEC of a5 (p1, 19Y, ECD = 4544) were cultured according to the protocol described in text (seeding density was 800 cells/mm2, SB431542 not added), a1 (P3, 10Y, 3671) were cultured under the conditions like a5, and a2 (P2, 55Y, 3131) were cultured similarly, but the seeding cell density was 380 cells /mm2 in the presence of 1 mM SB431542. (B, C) CD44 expression levels and the profile of miR expression. (B) Volcano plots of miR expression. The miRs were extracted from CS of cHCEC derived from three cHCECs donor (a5, a1 and a2). Values are as in Figure 1. (C) The summarized list of secreted miRs distinct among SPs, a5, a1, and a2.
Figure 4
 
The expression patterns of miRs. (A) The distinction of miR expression levels among three cHCECs SPs (a5, a1, and a2) were classified into six. The Y-axis corresponds to the relative expression intensity of respective miRs among a5, a1, and a2. Six typical patterns are are shown. (B) The variations of miR expression levels were illustrated for 9 typical miRs among three cHCECs SPs (a5, a1, and a2). Y-axis values were indicated as averages of global normalization values.
Figure 4
 
The expression patterns of miRs. (A) The distinction of miR expression levels among three cHCECs SPs (a5, a1, and a2) were classified into six. The Y-axis corresponds to the relative expression intensity of respective miRs among a5, a1, and a2. Six typical patterns are are shown. (B) The variations of miR expression levels were illustrated for 9 typical miRs among three cHCECs SPs (a5, a1, and a2). Y-axis values were indicated as averages of global normalization values.
Figure 5
 
Exosomes, detected by ExoScreen, with CD9 and/or CD63. (A) Secreted EVs were harvested from CS of cHCECs, #66, #77 (P2, 55Y, 2561) and C11 (P2, 26Y, 3321) according to the method described in the text. (B) The exosome species single positive either with CD9 or CD63 and CD9CD63 double positive were quantitated for several lots of CS for #66, #77, and C11. Y-axis values were indicated as averages of global normalization values. CD63: #66, #77 and C11 versus medium, P < 0.01, CD9, #66 and #77 versus c11, P < 0.01, CD9CD63, #66 versus #77, P < 0.01, #66 versus c11, P < 0.01. Three cHCECs were distinct in their CD44 expression levels. #66, CD44−/±, 90.8%; CD44++ ∼ +++, 9.0%; C11,CD44+++, 87.8%, CD44−/±, 4.2%; #77 showed the highest expression of CD44++, but the exact data were missing.
Figure 5
 
Exosomes, detected by ExoScreen, with CD9 and/or CD63. (A) Secreted EVs were harvested from CS of cHCECs, #66, #77 (P2, 55Y, 2561) and C11 (P2, 26Y, 3321) according to the method described in the text. (B) The exosome species single positive either with CD9 or CD63 and CD9CD63 double positive were quantitated for several lots of CS for #66, #77, and C11. Y-axis values were indicated as averages of global normalization values. CD63: #66, #77 and C11 versus medium, P < 0.01, CD9, #66 and #77 versus c11, P < 0.01, CD9CD63, #66 versus #77, P < 0.01, #66 versus c11, P < 0.01. Three cHCECs were distinct in their CD44 expression levels. #66, CD44−/±, 90.8%; CD44++ ∼ +++, 9.0%; C11,CD44+++, 87.8%, CD44−/±, 4.2%; #77 showed the highest expression of CD44++, but the exact data were missing.
Figure 6
 
Immunoblotting of exosomes secreted from cHCECs. Secreted exosomes were harvested from CS of cHCECs, a2, a5, and a6 (P2, 66Y, 3399, CD44++). (B) The expression levels of CD63 and CD9 were evaluated by Western blotting. CD9 and CD63 positive exosome were a2 (CD44+++) and a6 (CD44++). Their expression levels in a5 (CD44) were lowest for CD9 and CD63. cHCEC a6 showed CD44−/±, 58.2%; CD44++, 35.8%; CD44+++, 1.8%).
Figure 6
 
Immunoblotting of exosomes secreted from cHCECs. Secreted exosomes were harvested from CS of cHCECs, a2, a5, and a6 (P2, 66Y, 3399, CD44++). (B) The expression levels of CD63 and CD9 were evaluated by Western blotting. CD9 and CD63 positive exosome were a2 (CD44+++) and a6 (CD44++). Their expression levels in a5 (CD44) were lowest for CD9 and CD63. cHCEC a6 showed CD44−/±, 58.2%; CD44++, 35.8%; CD44+++, 1.8%).
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×