May 2005
Volume 46, Issue 5
Free
Cornea  |   May 2005
Human Corneal Endothelial Cell Precursors Isolated by Sphere-Forming Assay
Author Affiliations
  • Seiichi Yokoo
    From the Departments of Corneal Tissue Regeneration and
  • Satoru Yamagami
    From the Departments of Corneal Tissue Regeneration and
  • Yasuo Yanagi
    Ophthalmology, Tokyo University Graduate School of Medicine, Tokyo, Japan.
  • Saiko Uchida
    Ophthalmology, Tokyo University Graduate School of Medicine, Tokyo, Japan.
  • Tatsuya Mimura
    Ophthalmology, Tokyo University Graduate School of Medicine, Tokyo, Japan.
  • Tomohiko Usui
    Ophthalmology, Tokyo University Graduate School of Medicine, Tokyo, Japan.
  • Shiro Amano
    Ophthalmology, Tokyo University Graduate School of Medicine, Tokyo, Japan.
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 1626-1631. doi:10.1167/iovs.04-1263
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      Seiichi Yokoo, Satoru Yamagami, Yasuo Yanagi, Saiko Uchida, Tatsuya Mimura, Tomohiko Usui, Shiro Amano; Human Corneal Endothelial Cell Precursors Isolated by Sphere-Forming Assay. Invest. Ophthalmol. Vis. Sci. 2005;46(5):1626-1631. doi: 10.1167/iovs.04-1263.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. To isolate precursors of human corneal endothelial cells (HCECs) in vitro.

methods. HCECs were subjected to a sphere-forming assay in which spheres floated in serum-free medium containing growth factors. To promote differentiation, the isolated sphere colonies were plated in dishes coated with poly-l-lysine (PLL)/laminin or fetal bovine endothelium extracellular matrix. Marker expression of neural and mesenchymal cells was examined in the sphere colonies and their progenies by immunocytochemistry and/or reverse transcription–polymerase chain reaction (RT-PCR). Adherent differentiated cells from the sphere colonies were evaluated morphologically and functionally.

results. HCECs formed primary and secondary spherical colonies, as shown by sphere-forming assay in vitro. The colonies expressed nestin, β3-tublin, glial fibrillary acidic protein, and α-smooth muscle actin on immunocytochemistry. The progeny, proliferating on extracellular matrix derived from bovine corneal endothelium, but not on PLL/laminin-coated and noncoated dishes, expressed nestin and β3-tublin. These markers were confirmed by RT-PCR. Adherent differentiated cells from the sphere colonies had an HCEC-like hexagonal shape and satisfactory transport activity that is essential in HCECs.

conclusions. These findings indicate that the HCEC contains precursor cells with a propensity to differentiate into HCECs and that these cells can also produce neuronal and mesenchymal cell proteins.

Human embryonic stem (ES) cells can be greatly expanded in vitro and differentiate into various clinically functional cell types. Establishment of human ES cell lines has enabled researchers to generate all the tissues of the body. 1 However, ethical problems continue to prevent the development of this new medical approach employing human ES cells. 2 These ethical problems with regard to clinical use of human ES cells have recently encouraged research on adult stem cells (ASCs) and precursor cells, which are regarded as a less controversial alternative for the future of regenerative medicine. Use of ASCs and precursors may lead to new and powerful strategies for tissue regeneration and engineering. 3  
Common features of ASCs are both the capacity for self-renewal and the ability to differentiate into mature effector cells, but precursor cells have a limited self-renewal capacity. In vitro, many ASCs and precursor cells show proliferative potential and form floating clonal colonies that are termed spheres. The sphere-forming assay is a widely used method for the isolation of multipotent ASCs or precursors. 4 5 6 7 These cells have been found in several murine organs, including the central nervous system, 8 9 bone marrow, 10 11 skin, 4 12 corneal limbal tissue, 5 inner ear, 6 and pancreas. 13 In contrast, identification of these cells has been more limited in human tissues because of restricted availability. 4 14 15 16  
The corneal endothelial cell (CEC) is a single layer of flat hexagonal cells that lies on a basement membrane, Descemet’s membrane, 17 and forms a pure cell sheet without any other cell types. The CEC is essential for maintaining corneal transparency. 18 This function is dependent on endothelial regulation of stromal hydration, including the barrier and pump functions of the aqueous humor. 18 19 Damage to the human CEC (HCEC) caused by intraocular surgery, glaucoma, trauma, or congenital corneal disease results in irreversible corneal edema, because there is no or extremely low mitotic activity in the HCEC after birth, which leads to a gradual decrease in the cell population with age. 19 Moreover, proliferation of adult HCECs is not achieved with standard cell culture techniques. 20 However, adult HCECs have been reported to proliferate when cultured with bovine CEC extracellular matrix, 21 and cultured adult HCECs have been used in various experimental systems. 22 23 24 25 The fact that adult HCECs show proliferative activity in vitro in specific conditions suggests the possibility of the existence of precursor cells. Accordingly, we tried to isolate precursors from HCEC specimens and examine them by sphere-forming assay and examined whether the isolated cells would differentiate into different lineages or act to repair the CEC. 
Materials and Methods
Isolation of Sphere Colonies from HCEC
This study was conducted in accordance with the Declaration of Helsinki. Corneas were obtained from the Central Florida Lions Eye Tissue Bank and the Rocky Mountain Lions’ Eye Bank at 4 to 10 days postmortem. The age of the donors was 41 to 78 years. The CEC and Descemet’s membrane were peeled away in a sheet from the periphery to the center of the inner surface of the cornea with fine forceps according to a procedure described previously. 26 The removed epithelium was cut into small pieces approximately 1 to 2 mm in diameter, which were incubated overnight at 37°C in basal culture medium (DMEM-F12, 1:1; Invitrogen, San Diego, CA) with 40 ng/mL basic fibroblast growth factor (bFGF; Sigma-Aldrich, St. Louis, MO), 20 ng/mL epidermal growth factor (EGF; Sigma-Aldrich), 0.02% type IA collagenase (Sigma-Aldrich), B-27 (Invitrogen-Gibco, Grand Island, NY), 100 U/mL penicillin, 100 μg/mL streptomycin, and 250 ng/mL amphotericin B. Cells were collected in tubes covered with poly-2-hydroxyethyl methacrylate (poly-HEME; Sigma-Aldrich), allowed to stand in 0.05% trypsin/EDTA (EDTA) for 10 minutes at 37°C, and then dissociated into single cells by pipetting. After trypsin inhibitor was added (Invitrogen-Gibco), the cells were resuspended in the basal medium. The viability of the isolated HCECs was >90%, as shown by trypan blue staining (Wako Pure Chemical Industries, Osaka, Japan). The number of cells was then determined (Coulter counter; Beckman-Coulter, Hialeah, FL). 
Half of the cells were labeled with a fluorescent cell tracker (CM-DiI; C-7000; Molecular Probes, Eugene, OR), as described elsewhere, 22 to examine the formation of spheres by reaggregation. Then DiI-labeled and unlabeled cells were mixed and seeded at a density of 1 cell/μL (250 cells/cm2), 10 cells/μL (2,500 cells/cm2), 30 cells/μL (7,500 cells/cm2), or 50 cells/μL (12,500 cells/cm2) on 60-mm uncoated dishes containing 5 mL of medium for floating culturing. 27 28 Culturing was conducted in a humidified incubator in an atmosphere of 5% CO2, and 40 ng/mL bFGF and 20 ng/mL EGF were added to the medium every other day. To investigate whether the isolated cells were contaminated with corneal epithelial cells, expression of epithelial markers such as keratins K3 and K12 29 30 was assessed by the reverse transcription–polymerase chain reaction (RT-PCR) before the cells were cultured. Primary culturing was then performed, and the existence of fibroblast-like cells was investigated, to assess contamination by stromal cells. 
After 10 days, only cell clusters with a diameter of >50 μm were counted, to distinguish growing spheres from dying ones. The sphere colonies were stained with FITC-conjugated anti-BrdU antibody (1:100; Roche Diagnostics, Basel, Switzerland) at room temperature (RT) for 60 minutes in the dark. For passaging, primary spheres (day 10) were treated with 0.05% trypsin/0.02% EDTA and dissociated into single cells, after which the cells were added to 24-well culture plates at a density of 10 cells/μL in medium containing primary culture supernatant. The cells were then cultured for a further 10 days in the basal culture medium. 
Adherent Culture of Sphere Colonies
Individual primary cultured spheres (day 10) were transferred to 13-mm glass coverslips coated with 50 μg/mL poly-l-lysine (PLL; Sigma-Aldrich) and 10 μg/mL fibronectin (BD Biosciences, Billerica, MA), as described previously, 28 or to coverslips coated with bovine extracellular matrix (ECM) alone. 21 The latter coating was prepared by primary culture with bovine CECs and removal of the cells with trypsin-EDTA, because the ECM derived from the bovine cells is essential for successful primary culture of HCECs. 21 To promote differentiation, basal medium containing 40 ng/mL bFGF, 20 ng/mL EGF, and 1% bovine serum albumin (BSA), or 2 ng/mL bFGF plus 15% BSA, was used, and adherent culturing proceeded for 7 days. 
Immunocytochemistry
Immunocytochemical analysis was performed on 10-day spheres and on their progeny in adherent culture on glass coverslips after 7 days. Cells were fixed with methanol (Wako Pure Chemical Industries) in PBS for 10 minutes. After they were washed in PBS, the cells were incubated for 30 minutes with 3% BSA in PBS containing 0.3% Triton × 20 (BSA/PBST) to block nonspecific staining. Then, the cells were incubated for 2 hours at RT with specific primary antibodies (Abs) diluted in BSA/PBST. The following Abs were used: mouse monoclonal anti-vimentin monoclonal antibody (mAb, 1:300; Dako, Glostrup, Denmark), mouse anti-nestin mAb (1:200; BD PharMingen, San Diego, CA), rabbit anti-p75 NTR polyclonal antibody (pAb, 1:200; Promega Corp., Tokyo, Japan), mouse anti-neurofilament 145 mAb (NFM, 1:400; Chemicon, Temecula, CA), rabbit anti β3-tublin pAb (1:2000; Covance Research Products, Denver, PA), rabbit anti-GFAP pAb (1:400; Dako), mouse anti-O4 mAb (1:10; Chemicon), rabbit anti-peripherin pAb (1:100; Chemicon), and mouse anti-αSMA mAb (1:200; Sigma-Aldrich). As a control, mouse IgG (1:1000; Sigma-Aldrich) or normal rabbit serum (1:1000; Dako) was used instead of the primary Ab. After the cells were washed in PBS, they were reacted for 1 hour at RT with the appropriate secondary Abs diluted in BSA/PBST. The secondary Abs were fluorescence-labeled goat anti-mouse IgG (Alexa Fluor 488, 1:200; Molecular Probes) and fluorescence-labeled goat anti-rabbit IgG (Alexa Fluor 594, 1:400; Molecular Probes). Nuclei were counterstained with Hoechst 33342 (1:2000; Molecular Probes). After another wash in PBS, the cells were examined with a laser scanning confocal microscope (Fluoview; Olympus, Tokyo, Japan). When anti-O4 or p75NTR mAb was used, the cell permeabilization step was omitted. 
Preparation of RNA and RT-PCR
Total RNA was isolated from primary sphere colonies and the adherent progeny of sphere colonies with a kit (Isogen; Nippon Gene, Tokyo, Japan), according to the manufacturer’s instructions. First-strand cDNA was synthesized with a reverse transcription system (Promega) and 1 μg total RNA in a 20-μL reaction mixture. Then PCR was performed with Taq polymerase (AmpliTaq Gold; Applied Biosystems, Foster City, CA) in a 50-μL reaction mixture. After incubation at 95°C for 9 minutes, amplification was performed at 94°C for 30 seconds and then at 60°C for 30 seconds with a thermal cycler (I-Cycler; Bio-Rad Laboratories, Hercules, CA). Products were separated on 2% agarose gel and then stained with ethidium bromide. The primer pairs and produce size are shown in Table 1
Measurement of the Pump Function of Progenies from the Sphere Colonies
The pump function of cells derived from the sphere colonies was measured in a Ussing chamber, as reported previously, with some modifications. 22 31 32 33 Collagen sheets were obtained from Nippi Research Institute of Biomatrix (Tokyo, Japan). Suspensions of cells derived from HCEC spheres (5.0 × 106 cells in 1.5 mL of culture medium) were transferred onto circular collagen sheets (10 mm in diameter), and each sheet was placed in one of the wells of a 24-well plate, after which the plates were centrifuged at 1000 rpm (176 g) for 10 minutes to enhance cell attachment. The sheets were then incubated in culture medium for 2 days, after which nonadherent cells and debris were removed. Donor corneas from which the epithelium had been scraped (n = 4), collagen sheets without cells (n = 4), and collagen sheets coated with sphere-derived cells (n = 4) were mounted in the Ussing chamber. 
Results
Isolation of Sphere Colonies from HCEC
To isolate precursor cells from HCEC, we used the sphere-forming assay. Cells were isolated without contamination by corneal epithelial cells, as demonstrated by RT-PCR analysis of corneal epithelial markers (K3 and K12 genes), as well as the characteristic hexagonal shape of cells in primary culture (data not shown). There was almost complete disaggregation into single cells, since counting the percentage of single, double, and triple cells showed that >99% of all cells were single (Fig. 1A) . To determine the appropriate culture conditions, we prepared dishes at a density of 1, 10, 30, and 50 viable cells/μL and stained the cells with the fluorescent cell tracker CM-DiI to investigate the possibility of spheres being formed by reaggregation. No spheres were generated in the cultures with 1 viable cell/μL, but a significant number of spheres were formed at 30 and 50 cells/μL, with some spheres arising from reaggregation as evidenced by DiI-staining. The spheres were divided into DiI-positive and DiI-negative when culturing was performed at 10 cells/μL (Fig. 1B) , indicating that the spheres were derived from proliferation, not from reaggregation of the dissociated cells. After 5 days of culturing, small floating spheres formed, and these spheres grew larger after 10 days, while the nonproliferating cells died and were eliminated (Fig. 1C) . To verify that the increase in colony size was actually due to proliferation, we added the thymidine analogue BrdU at 24 hours before cell fixation. BrdU labeled most of the cells within each sphere on day 10 (Fig. 1D) , indicating that the colonies contained proliferating cells. These results suggest that the sphere colonies arose from single isolated HCECs and that the sphere-forming cells possessed proliferative capacity. When the number of spheres obtained was counted after 10 days of culture, showing that 257 ± 83 spheres (mean ± SD, n = 8) were generated per dish (50,000 cells). In a typical case, 2.5 × 104 cells were isolated from a 10-mm piece of corneal tissue and then generated approximately 130 spheres after 10 days. These spheres were 88.3 ± 15.9 μm in diameter (mean ± SD, n = 35). The replating efficiency decreased dramatically between the primary sphere colonies and secondary colonies. When the primary colonies were trypsinized and incubated in serum-free floating culture, secondary colonies were generated (Fig. 1E) , at approximately 15 ± 1 (n = 3) per dish (10,000 cells). This suggested that HCECs have the capacity for self-renewal of sphere colonies, but this capacity is limited. 
Immunocytochemistry of Sphere Colonies
Fig. 2Ashows a bright-field image of a typical sphere colony. Sphere colonies derived form HCEC did not stain with nonimmunized mouse IgG (Fig. 2D)or normal rabbit serum (Fig. 2G) . The spheres were immunostained for nestin 34 because immature cells within sphere colonies express nestin (as a marker of immature cells). Expression of α-SMA (as a marker of mesenchymal myofibroblasts) and p75 NTR (as a marker of neural crest stem cells) was investigated by immunocytochemistry, because HCECs are derived from the neural crest. The cells in the spheres showed immunoreactivity for nestin (Fig. 2B)and α-SMA (Fig. 2C) , but not for p75 NTR (data not shown). Next, the spheres were immunostained for various neural markers. As a result, spheres were positive for an immature neuronal marker (β3-tubulin, Fig. 2E ) and an astroglial marker (GFAP, Fig. 2F ), but not a mature neuronal marker (NFM), an oligodendroglial marker (O4), or a peripheral nerve neuronal marker (peripherin; data not shown). 
Immunocytochemistry of the Progeny of Individual Spherical Colonies
To investigate whether the progeny of the spheres possesses the characteristics of mesenchymal or neural lineage cells, single spheres from day 10 of culture were transferred onto PLL/laminin-coated glass coverslips in medium containing 1% or 15% FBS, as well as onto bovine ECM-coated culture plates in medium containing 15% FBS. Although spheres adhered to the PLL/laminin-coated glass coverslips, cells migrated from the spheres grown on glass coverslips coated with bovine ECM alone. After 7 days, some of the cells that had migrated from the spheres were double immunostained for nestin and β3-tublin (Fig. 3) , as reported for human scalp tag–derived cells. 4 There was no positive staining of the cells that migrated from the spheres by anti-αSMA, p75NTR, NFM, peripherin, GFAP, or O4 Abs. 
RT-PCR Analysis
RT-PCR was performed to examine the expression of genes for the proteins detected by immunocytochemistry in the spheres and their progeny (Fig. 4) . GAPDH-mRNA was detected in both the spheres and the progeny, but not in the control assay from which the RT reaction was omitted (30 cycles). Nestin, β3-tublin, GFAP, and α-SMA mRNA expression were detected in the spheres and adherent progeny after 35 PCR cycles. However, mRNA for NFM, p75NTR, or peripherin was not found under any cycling conditions (data not shown). Nestin and β3-tublin mRNAs were also detected in the primary cultured HCEC (data not shown). 
Morphology and Pump Function of HCEC Sheets Reconstituted from Spheres
HCECs have no specific markers that provide positive identification. Therefore, we differentiated spheres by incubation with basal medium containing 15% FBS and 2 ng/mL bFGF, to determine whether the differentiated cells had an HCEC-like hexagonal form, and we also tested the pump function, potential differences, and short circuit current of cell sheets in the standard Ussing chamber system, 21 31 32 33 because HCECs should have significant transport activity. 22 Confluent cells derived from spheres had a characteristic hexagonal HCEC-like shape (Fig. 5A) . The mean (±SD) time course of the potential difference (Fig. 5B)and short circuit current (Fig. 5C)in normal donor corneas and in HCEC sheets reconstituted from spheres are shown. The mean potential difference and short circuit current for the sphere-derived sheet after 1, 5, and 10 minutes ranged from 73% to 78%, which was similar to the range for normal donor corneas deprived of their epithelium. These findings suggest that the spheres mainly generated HCEC-like cells with a hexagonal shape and considerable transport activity. 
Discussion
The CEC–Descemet’s membrane complex were excised smoothly from clear corneas while avoiding the inclusion of residual stroma. Neither the cytokeratin-3 nor -12 genes was detected in the HCECs, indicating that the obtained cells were all CEC with no contamination by other corneal cell types. The fluorescent cell tracker CM-DiI was used to assess clonogenic status, because DiI has been used for tracing the development of neural tissue and mitosis of progenitor cells. 35 36 DiI-positive and -negative spheres, but not those with a mosaic pattern, were obtained after culture at a density of 10 cells/μL. Sphere colonies derived from HCECs are produced in viscous basal medium that contains a methylcellulose gel matrix to prevent cell reaggregation, although sphere formation in this medium is lower than in normal medium (Yokoo S, unpublished observation, 2004). These findings indicate that HCECs can generate a significant number of spheres under clonogenic conditions. 
Spheres derived from HCECs showed a high proliferative capacity, as revealed by BrdU staining. Self-renewal potential was indicated by the ability of the progeny of individual spheres to form secondary spheres, but this potential was limited, as evidenced by the failure of third-passage sphere formation. Individual spheres contained cells that expressed markers of the mesenchymal (α-SMA) and neural (GFAP and β3-tubulin) lineages. We could not demonstrate directly that isolated spheres gave rise to HCECs because of the lack of specific markers. However, the characteristic hexagonal morphology and transport activity of HCECs were revealed in the Ussing chamber system, suggesting that the spheres largely give rise to HCEC-like cells with the features of HCECs. These findings indicate that HCEC-derived spheres have the character of HCEC-precursor cells and have a propensity to differentiate into HCEC-like cells. Considering that adult HCEC cannot be cultured without a bovine ECM-coated culture plate and culture medium containing 15% FBS, the fact that some cells proliferated in serum-free and floating conditions with the aid of growth factors may have significance in the field of HCEC biology. 
Although human cells have sometimes been used for detection of ASCs or precursors, 4 37 rodent cells have been used in most of the previous studies. 38 We used human cells in this study, and the cells in the spheres expressed differentiation markers such as α-SMA and GFAP. The self-renewal capacity and multipotentiality of cells from the spheres were limited compared with those in numerous previous reports on rodent tissues. Svendsen et al. 37 concluded that undifferentiated human cells are difficult to maintain in culture. The fact that spherical colonies isolated from the human brain contain heterogenous cells, including mature neurons, is consistent with our findings, suggesting that human tissue-derived spheres tend to differentiate into mature cells during floating culture under serum-free conditions. Moreover, human skin and corneal stromal cells have no self-renewal capacity and have limited proliferative potential after they differentiate. 4 39 Although further modification of the culture conditions for HCEC may promote the growth of undifferentiated precursors, our present findings and previous reports suggest that the self-renewal capacity and multipotentiality of these cells may be restricted in human tissues. 
Corneal transplantation is the most common form of solid tissue transplantation and is performed to improve the vision of patients with corneal damage or disease. More than half of the patients who undergo full-thickness corneal transplantation have a decrease of visual acuity due to CEC deficiency. 40 41 This technique has the potential for severe complications associated with “open-sky” surgery as well as frequently causing high or irregular astigmatism, refractive errors, and suture-related problems. Spheres grown from the HCECs of donor corneas may provide a cell source for repair of HCEC deficiency that may replace conventional full-thickness corneal transplantation. 
We demonstrated that sphere-forming cells derived from the HCEC largely give rise to HCEC-like cells with a hexagonal shape and essential HCEC functions such as pump activity, while some of the cells express mesenchymal (α-SMA) and neural (GFAP and β3-tubulin) markers. Highly proliferative sphere-forming cells derived from HCECs may be useful in the treatment of bullous keratopathy and corneal graft failure. 
 
Table 1.
 
Primers Used for Polymerase Chain Reactions
Table 1.
 
Primers Used for Polymerase Chain Reactions
Gene Forward Primer Reverse Primer Size (bp)
GAPDH 5′-GGTGAAGGTCGGTGTGAACGGA-3′ 5′-TGTTAGTGGGGTCTCGCTCCTG-3′ 223
Nestin 5′-CACCTGTGCCAGCCTTTCTTAA-3′ 5′-CCACCGGATTCTCCATCCTTA-3′ 361
β3-Tublin 5′-TCTCACAAGTACGTGCCTCGA-3′ 5′-TGATGAGCAACGTGCCCAT-3′ 292
NFM 5′-GGAAGAAAAGGATCTCCGAGGA-3′ 5′-CGGATTCGCCTTCTTTCCAT-3′ 339
Peripherin 5′-CCCGTCCATTCTTTTGCCT-3′ 5′-TTGGCCATAAGCCAGGAAA-3′ 324
GFAP 5′-CTGGGCTCAAGCAGTCTACC-3′ 5′-AATTGCCTCCTCCTCCATCT-3′ 429
P75NTR 5′-TGAGTGCTGCAAAGCGTCCAA-3′ 5′-TCTCATCCTGGTAGTAGCCGT-3′ 325
α-SMA 5′-CTGTTCCAGCCATCCTTCAT-3′ 5′-GCTGGAAGGTGGACAGAGAG-3′ 280
CK3 5′-CATCATTGCTGAAGTTGGTGC-3′ 5′-TCTTGGAGCTTGGCATTGG-3′ 291
CK12 5′-TTGTGACAGACTCCAAATCA-3′ 5′-TACTCCAGTTGTCCAGAAGG-3′ 398
Figure 1.
 
Sphere formation by HCECs. HCECs were disaggregated into single cells, which were plated at a density of 10 viable cells/μL in basal medium (A). More than 99% of the cells were single cells on day 0. (B) DiI-positive and -negative spheres were completely separated after culture at a density of 10 viable cells/μL. (C) The mean (±SD) sphere size was 88.3 ± 15.9 μm on day 10. (D) Each colony was labeled with BrdU on day 10. (E) Secondary spheres generated after the dissociation of primary spheres. Replating efficiency decreased from the primary to secondary sphere colonies.
Figure 1.
 
Sphere formation by HCECs. HCECs were disaggregated into single cells, which were plated at a density of 10 viable cells/μL in basal medium (A). More than 99% of the cells were single cells on day 0. (B) DiI-positive and -negative spheres were completely separated after culture at a density of 10 viable cells/μL. (C) The mean (±SD) sphere size was 88.3 ± 15.9 μm on day 10. (D) Each colony was labeled with BrdU on day 10. (E) Secondary spheres generated after the dissociation of primary spheres. Replating efficiency decreased from the primary to secondary sphere colonies.
Figure 2.
 
Immunocytochemical analysis of a sphere colony. (A) Bright-field image of a typical sphere colony. Immunostaining of the entire sphere on day 10 showed cells that expressed nestin, a marker of immature cells (B). The spheres were immunostained for the mesenchymal myofibroblast marker (α-SMA, C), an immature neuronal marker (β3-tubulin, E), and an astroglial cell marker (GFAP, F), indicating that both mesenchymal and neuronal differentiation occurred under these conditions. Sphere colonies derived from HCEC did not stain with nonimmunized mouse IgG (D) and normal rabbit serum (G). Scale bar, 100 μm.
Figure 2.
 
Immunocytochemical analysis of a sphere colony. (A) Bright-field image of a typical sphere colony. Immunostaining of the entire sphere on day 10 showed cells that expressed nestin, a marker of immature cells (B). The spheres were immunostained for the mesenchymal myofibroblast marker (α-SMA, C), an immature neuronal marker (β3-tubulin, E), and an astroglial cell marker (GFAP, F), indicating that both mesenchymal and neuronal differentiation occurred under these conditions. Sphere colonies derived from HCEC did not stain with nonimmunized mouse IgG (D) and normal rabbit serum (G). Scale bar, 100 μm.
Figure 3.
 
Immunocytochemical analysis of the differentiated cells derived from spheres. The cells are double immunostained by nestin and β3-tubulin, indicating that the colonies contain immature (undifferentiated) cells under these conditions. Scale bar, 200 μm.
Figure 3.
 
Immunocytochemical analysis of the differentiated cells derived from spheres. The cells are double immunostained by nestin and β3-tubulin, indicating that the colonies contain immature (undifferentiated) cells under these conditions. Scale bar, 200 μm.
Figure 4.
 
RT-PCR analysis of cells from the spheres and their progeny. GAPDH gene expression is detected in the sphere colonies and their progeny (30 cycles), but not when reverse transcription is omitted. Nestin, α-SMA, β3-tubulin, and GFAP genes are positive in both the sphere cells and the progeny, but not in total RNA processed without reverse transcription (35 cycles).
Figure 4.
 
RT-PCR analysis of cells from the spheres and their progeny. GAPDH gene expression is detected in the sphere colonies and their progeny (30 cycles), but not when reverse transcription is omitted. Nestin, α-SMA, β3-tubulin, and GFAP genes are positive in both the sphere cells and the progeny, but not in total RNA processed without reverse transcription (35 cycles).
Figure 5.
 
Morphology and function of sphere-derived cells. (A) Confluent cells grown in DMEM containing 15% FBS show the characteristic hexagonal shape of HCECs. Time course of changes in the mean (± SD) potential difference (B) and short circuit current (C) in human donor corneas and HCEC-coated collagen sheets reconstituted from spheres. The mean potential difference and short circuit current of the HCEC sheets at 1, 5, and 10 minutes ranged from 73% to 78%, which corresponded to the range for donor corneas denuded of their epithelium, indicating that the HCEC-like cells possessed satisfactory transport activity. After the Na+-K+-ATPase inhibitor ouabain was added to the chambers, the potential difference became 0 mV and the short circuit current was 0 μA in all tests. Squares, diamonds, and triangles respectively indicate (▪) human donor corneas; (♦) HCEC-coated collagen sheets; and (▴) uncoated collagen sheets.
Figure 5.
 
Morphology and function of sphere-derived cells. (A) Confluent cells grown in DMEM containing 15% FBS show the characteristic hexagonal shape of HCECs. Time course of changes in the mean (± SD) potential difference (B) and short circuit current (C) in human donor corneas and HCEC-coated collagen sheets reconstituted from spheres. The mean potential difference and short circuit current of the HCEC sheets at 1, 5, and 10 minutes ranged from 73% to 78%, which corresponded to the range for donor corneas denuded of their epithelium, indicating that the HCEC-like cells possessed satisfactory transport activity. After the Na+-K+-ATPase inhibitor ouabain was added to the chambers, the potential difference became 0 mV and the short circuit current was 0 μA in all tests. Squares, diamonds, and triangles respectively indicate (▪) human donor corneas; (♦) HCEC-coated collagen sheets; and (▴) uncoated collagen sheets.
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Figure 1.
 
Sphere formation by HCECs. HCECs were disaggregated into single cells, which were plated at a density of 10 viable cells/μL in basal medium (A). More than 99% of the cells were single cells on day 0. (B) DiI-positive and -negative spheres were completely separated after culture at a density of 10 viable cells/μL. (C) The mean (±SD) sphere size was 88.3 ± 15.9 μm on day 10. (D) Each colony was labeled with BrdU on day 10. (E) Secondary spheres generated after the dissociation of primary spheres. Replating efficiency decreased from the primary to secondary sphere colonies.
Figure 1.
 
Sphere formation by HCECs. HCECs were disaggregated into single cells, which were plated at a density of 10 viable cells/μL in basal medium (A). More than 99% of the cells were single cells on day 0. (B) DiI-positive and -negative spheres were completely separated after culture at a density of 10 viable cells/μL. (C) The mean (±SD) sphere size was 88.3 ± 15.9 μm on day 10. (D) Each colony was labeled with BrdU on day 10. (E) Secondary spheres generated after the dissociation of primary spheres. Replating efficiency decreased from the primary to secondary sphere colonies.
Figure 2.
 
Immunocytochemical analysis of a sphere colony. (A) Bright-field image of a typical sphere colony. Immunostaining of the entire sphere on day 10 showed cells that expressed nestin, a marker of immature cells (B). The spheres were immunostained for the mesenchymal myofibroblast marker (α-SMA, C), an immature neuronal marker (β3-tubulin, E), and an astroglial cell marker (GFAP, F), indicating that both mesenchymal and neuronal differentiation occurred under these conditions. Sphere colonies derived from HCEC did not stain with nonimmunized mouse IgG (D) and normal rabbit serum (G). Scale bar, 100 μm.
Figure 2.
 
Immunocytochemical analysis of a sphere colony. (A) Bright-field image of a typical sphere colony. Immunostaining of the entire sphere on day 10 showed cells that expressed nestin, a marker of immature cells (B). The spheres were immunostained for the mesenchymal myofibroblast marker (α-SMA, C), an immature neuronal marker (β3-tubulin, E), and an astroglial cell marker (GFAP, F), indicating that both mesenchymal and neuronal differentiation occurred under these conditions. Sphere colonies derived from HCEC did not stain with nonimmunized mouse IgG (D) and normal rabbit serum (G). Scale bar, 100 μm.
Figure 3.
 
Immunocytochemical analysis of the differentiated cells derived from spheres. The cells are double immunostained by nestin and β3-tubulin, indicating that the colonies contain immature (undifferentiated) cells under these conditions. Scale bar, 200 μm.
Figure 3.
 
Immunocytochemical analysis of the differentiated cells derived from spheres. The cells are double immunostained by nestin and β3-tubulin, indicating that the colonies contain immature (undifferentiated) cells under these conditions. Scale bar, 200 μm.
Figure 4.
 
RT-PCR analysis of cells from the spheres and their progeny. GAPDH gene expression is detected in the sphere colonies and their progeny (30 cycles), but not when reverse transcription is omitted. Nestin, α-SMA, β3-tubulin, and GFAP genes are positive in both the sphere cells and the progeny, but not in total RNA processed without reverse transcription (35 cycles).
Figure 4.
 
RT-PCR analysis of cells from the spheres and their progeny. GAPDH gene expression is detected in the sphere colonies and their progeny (30 cycles), but not when reverse transcription is omitted. Nestin, α-SMA, β3-tubulin, and GFAP genes are positive in both the sphere cells and the progeny, but not in total RNA processed without reverse transcription (35 cycles).
Figure 5.
 
Morphology and function of sphere-derived cells. (A) Confluent cells grown in DMEM containing 15% FBS show the characteristic hexagonal shape of HCECs. Time course of changes in the mean (± SD) potential difference (B) and short circuit current (C) in human donor corneas and HCEC-coated collagen sheets reconstituted from spheres. The mean potential difference and short circuit current of the HCEC sheets at 1, 5, and 10 minutes ranged from 73% to 78%, which corresponded to the range for donor corneas denuded of their epithelium, indicating that the HCEC-like cells possessed satisfactory transport activity. After the Na+-K+-ATPase inhibitor ouabain was added to the chambers, the potential difference became 0 mV and the short circuit current was 0 μA in all tests. Squares, diamonds, and triangles respectively indicate (▪) human donor corneas; (♦) HCEC-coated collagen sheets; and (▴) uncoated collagen sheets.
Figure 5.
 
Morphology and function of sphere-derived cells. (A) Confluent cells grown in DMEM containing 15% FBS show the characteristic hexagonal shape of HCECs. Time course of changes in the mean (± SD) potential difference (B) and short circuit current (C) in human donor corneas and HCEC-coated collagen sheets reconstituted from spheres. The mean potential difference and short circuit current of the HCEC sheets at 1, 5, and 10 minutes ranged from 73% to 78%, which corresponded to the range for donor corneas denuded of their epithelium, indicating that the HCEC-like cells possessed satisfactory transport activity. After the Na+-K+-ATPase inhibitor ouabain was added to the chambers, the potential difference became 0 mV and the short circuit current was 0 μA in all tests. Squares, diamonds, and triangles respectively indicate (▪) human donor corneas; (♦) HCEC-coated collagen sheets; and (▴) uncoated collagen sheets.
Table 1.
 
Primers Used for Polymerase Chain Reactions
Table 1.
 
Primers Used for Polymerase Chain Reactions
Gene Forward Primer Reverse Primer Size (bp)
GAPDH 5′-GGTGAAGGTCGGTGTGAACGGA-3′ 5′-TGTTAGTGGGGTCTCGCTCCTG-3′ 223
Nestin 5′-CACCTGTGCCAGCCTTTCTTAA-3′ 5′-CCACCGGATTCTCCATCCTTA-3′ 361
β3-Tublin 5′-TCTCACAAGTACGTGCCTCGA-3′ 5′-TGATGAGCAACGTGCCCAT-3′ 292
NFM 5′-GGAAGAAAAGGATCTCCGAGGA-3′ 5′-CGGATTCGCCTTCTTTCCAT-3′ 339
Peripherin 5′-CCCGTCCATTCTTTTGCCT-3′ 5′-TTGGCCATAAGCCAGGAAA-3′ 324
GFAP 5′-CTGGGCTCAAGCAGTCTACC-3′ 5′-AATTGCCTCCTCCTCCATCT-3′ 429
P75NTR 5′-TGAGTGCTGCAAAGCGTCCAA-3′ 5′-TCTCATCCTGGTAGTAGCCGT-3′ 325
α-SMA 5′-CTGTTCCAGCCATCCTTCAT-3′ 5′-GCTGGAAGGTGGACAGAGAG-3′ 280
CK3 5′-CATCATTGCTGAAGTTGGTGC-3′ 5′-TCTTGGAGCTTGGCATTGG-3′ 291
CK12 5′-TTGTGACAGACTCCAAATCA-3′ 5′-TACTCCAGTTGTCCAGAAGG-3′ 398
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